M.D. (MICROBIOLOGY) BRANCH IVrepository-tnmgrmu.ac.in/8717/1/200400418sharanya.pdfI owe my gratitude...
Transcript of M.D. (MICROBIOLOGY) BRANCH IVrepository-tnmgrmu.ac.in/8717/1/200400418sharanya.pdfI owe my gratitude...
IDENTIFICATION, CHARACTERISATION AND
ANTIMICROBIAL RESISTANCE PATTERN OF
NON FERMENTING GRAM NEGATIVE BACILLI FROM
VARIOUS CLINICAL ISOLATES
Dissertation Submitted To
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY
CHENNAI
In partial fulfillment of the regulations
For the award of the degree of
M.D. (MICROBIOLOGY)
BRANCH IV
GOVT. KILPAUK MEDICAL COLLEGE
CHENNAI
May 2018
CERTIFICATE
This is to certify that this dissertation entitled “IDENTIFICATION,
CHARACTERISATION AND ANTIMICROBIAL RESISTANCE
PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM
VARIOUS CLINICAL ISOLATES” is the bonafide original work done by
Dr.R.SHARANYA, Post graduate in Microbiology, under my overall
supervision and guidance in the Department of Microbiology, Govt. Kilpauk
Medical College, Chennai, in partial fulfillment of the regulations of The
Tamil Nadu Dr. M.G.R. Medical University for the award of M.D Degree
in Microbiology (Branch IV).
Dr.K.V.LEELA, M.D.,DGO.,
Professor & H.O.D
Department of Microbiology
Govt. Kilpauk Medical College
Chennai-600010
Dr.P.VASANTHAMANI M.D.,DGO.,
The Dean
Govt. Kilpauk Medical College
Chennai-600010.
CERTIFICATE
This is to certify that the dissertation entitled “IDENTIFICATION,
CHARACTERISATION AND ANTIMICROBIAL RESISTANCE
PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM
VARIOUS CLINICAL ISOLATES” is a bonafide research work done by
Dr.R.SHARANYA Post graduate in Microbiology, under my guidance in
the Department of Microbiology, Govt. Kilpauk Medical College, Chennai,
in partial fulfillment of the regulations of The Tamil Nadu
Dr.M.G.R.Medical University for the award of M.D Degree in
Microbiology (Branch IV).
Dr. THYAGARAJAN RAVINDER,M.D.,
Professor
Department of Microbiology
Govt. Kilpauk Medical College
Chennai-600010
CERTIFICATE
This is to certify that this dissertation work titled “IDENTIFICATION,
CHARACTERISATION AND ANTIMICROBIAL RESISTANCE
PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM
VARIOUS CLINICAL ISOLATES” of the candidate Dr.R. SHARANYA
with registration number 201414152 for the award of M.D Degree in
Microbiology (Branch IV). I personally verified the urkund.com website for the
purpose of plagiarism check. I found that the uploaded thesis file contains from
introduction to conclusion pages and result shows 6 percentage of plagiarism in
the dissertation.
Dr. THYAGARAJAN RAVINDER,M.D.,
Professor
Department of Microbiology
Govt. Kilpauk Medical College
Chennai-600010
DECLARATION
I solemnly declare that this dissertation “IDENTIFICATION,
CHARACTERISATION AND ANTIMICROBIAL RESISTANCE
PATTERN OF NON FERMENTING GRAM NEGATIVE BACILLI FROM
VARIOUS CLINICAL ISOLATES” is the bonafide work done by me at the
Department of Microbiology, Government. Kilpauk Medical College and
Hospital, Chennai, under the guidance and supervision of,
Dr.K.V.LEELA, M.D., DGO., Professor & H.O.D of Microbiology,
Dr.THYAGARAJAN RAVINDER, M.D., Professor of Microbiology
Department of Microbiology and Dr .M. KAVITHA.M.D., Associate
Professor, Department of Microbiology Govt. Kilpauk Medical College,
Chennai - 600 010. This dissertation is submitted to The Tamil Nadu Dr.
M.G.R. Medical University, Chennai in partial fulfillment of the University
regulations for the award of Degree of M.D. Branch IV Microbiology
examinations to be held in May 2018.
Place : Chennai.
Date : Dr.R.SHARANYA
ACKNOWLEDGEMENT
My heartfelt thanks and deepest sense of gratitude to
Dr.VASANTHAMANI, M.D.,DGO., Dean, Government Kilpauk Medical
College and Hospital for giving me permission to carry out my dissertation
work and also to avail all the facilities available in the department.
I owe my gratitude to Dr.K.V.LEELA,M.D.DGO, Professor and
H.O.D., Department of Microbiology for her relentless efforts, valuable
advice, excellent guidance and encouragement given to me throughout this
study.
I am immensely grateful to Dr. THYAGARAJAN RAVINDER,
M.D., Professor, and Department of Microbiology for his constant
motivation and guidance extended to me during my study.
My sincere thanks to Dr.M. KAVITHA, M.D., Associate Professor,
Department of Microbiology for her timely advice, guidance and
encouragement in this study.
I extend my sincere thanks to Dr.K.LAVANYA, M.D
Dr.M.SUGANTHI, M.D., Dr.S. HEMALATHA,M.D., Dr.B.RAVICHANDRAN,
M.D., Dr.C.AMUTHA, M.D., Assistant Professors, Department of
Microbiology for their help, support, interest and valuable suggestions.
I also thank all my department colleagues for their timely help,
cooperation and moral support. I express many thanks to all the technical
staffs and other staff members of the Department of Microbiology for their
kind co-operation to carry out this work successfully.
I also extend my thanks to all the patients who participated in my
study. I also thank my family members for their selfless love and moral
support.
SL.NO. TITLE PAGE NO.
1. INTRODUCTION 1
2. AIMS AND OBJECTIVES 4
3. REVIEW OF LITERATURE 5
4. MATERIALS AND METHODS 34
5. RESULTS 51
6. DISCUSSION 69
7. SUMMARY 80
8. CONCLUSION 83
9. ANNEXURES
I) PROFORMA
II) APPENDIX
III) BIBLIOGRAPHY
IV) MASTER CHART
INTRODUCTION
The Nonfermentative gram-negative bacilli are a group of aerobic, non-
sporing bacilli that do not either use carbohydrates as a source of energy or
degrade them through metabolic pathways other than fermentation. Non-
fermentative gram negative bacilli account for ≥ 15% of isolates from most
clinical specimens 1 Hospital acquired infections in the acute care units are major
threat to patient safety.
Even after a decade, four nonfermenting gram-negative bacilli (NFGNB)
continue to be recognised as notorious multidrug-resistant organisms. These are
Pseudomonas aeruginosa, Acinetobacter calcoaceticus-baumannii complex,
Stenotrophomonas maltophilia and Burkholderia cepacia complex (BCC).22
Pseudomonas aeruginosa is implicated in a wide spectrum of nosocomial
infections, including bacteremia, secondary meningitis, wound infection, severe
sepsis, ocular and urinary tract infection2 These organisms seem to have a
remarkable ability to acquire antibiotic resistance genes, to persist in the hospital
environment and to spread easily from patient to patient.2. Acinetobacter causes a
wide variety of illnesses in debilitated and hospitalized patients, especially in the
intensive care units (ICUs)23
. Burkholderia Cepacia Complex, a devastating
pulmonary pathogen in Cystic fibrosis and chronic granulomatous disease (CGD)
patients, has also been reported as a cause of bacteraemia, particularly in patients
with indwelling catheters, urinary tract infection, septic arthritis, peritonitis and
respiratory tract infection22
Antimicrobial resistance is on the rise and it is a major public health
problem across the world, and especially in developing countries like India.
Infections caused by bacterial pathogens with multi drug resistant (MDR),
extremely drug resistant(XDR) and pan drug resistant phenotypes (PDR) are
challenging and difficult to treat. 9 Pseudomonas aeruginosa resistant to
carbapenem, currently the most effective treatment option is being increasingly
reported.
Resistance to carbapenems is often mediated by production of Metallo-
Beta-Lactamase (MBL), a class B type of beta-lactamases that require bivalent
metal ions, usually zinc for their activity.3 Pseudomonas aeruginosa, producing
MBLs, was first reported from Japan in 1991 and since then has been described
from various parts of the world, including Asia, Europe, Australia, South
America, and North America.3. Prompt detection and recognition of the MBLs is
important to implement adequate counter-measures to control the spread of the
organisms bearing these enzymes, and proper treatment of infections caused by
MBL-producing microorganisms.4
MBL production is a significant problem in hospital isolates of
Pseudomonas aeruginosa28
the accurate identification and reporting of MBL-
producing Pseudomonas aeruginosa will aid infection control practitioners in
preventing the spread of these multidrug-resistant isolates. Many phenotyping
methods have performed to search MBL enzymes of Pseudomonas aeruginosa
strains. All these methods are based on the ability of metal chelators, such as
EDTA and thiol-based compounds, to inhibit the activity of MBL.4
Thus,
identification, characterisation and antimicrobial resistance pattern of non
fermenting gram negative bacilli from various clinical isolates and finding MBL
is of prime importance.
AIM
To identify, characterise and detect antimicrobial resistance pattern of non
fermenting gram negative bacilli from various clinical isolates
OBJECTIVES
1. To isolate and speciate the non fermenting Gram negative bacilli
2. To characterise the non-fermenting Gram negative bacilli isolated
3. To find out the antimicrobial resistance pattern of the non-fermenting
Gram negative bacilli isolated.
4. To detect the production of extended spectrum of betalactamases.
5. To detect the acquired metallo betalactamases(MBL) by phenotypic
method of detection.
6. To identify the genes responsible for acquired MBL production
REVIEW OF LITERATURE
Nonfermenting Gram Negative Bacilli (NFGNB) are a group of
taxonomically diverse organisms growing significantly under aerobic conditions.
They all share the common phenotypic feature of failing to acidify the butt of
Triple sugar iron agar (TSI) or Kligler iron agar (KIA) agar or of oxidative-
fermentative (OF) media.7 Nonfermenters are cosmopolitan in their distribution
inhabiting soil, water, plants and animals. Their medical importance derives
principally from their being opportunistic pathogens and clinical diseases they
cause are nosocomial in origin.
Approximately 15% of all gram negative clinical isolates are nonglucose
fermenting gram negative rods. Of these, more than 2/3rds are Pseudomonas
aeruginosa5,8
, Large group of these nonfermenters have undergone confusing
taxonomic changes for many years. New definitions of species and genera using
modern genotyping analysis, together with reliable identification methods have
resulted in a better knowledge of these bacteria and a significantly increased
awareness of their pathogenic role in hospitals and in rare cases of community
acquired infections.5,6
The major genera of nonfermenting Gram negative bacilli
have been classified into atleast 15 families in addition to a number of clinically
important nonfermenters with uncertain taxonomic positions. Medically important
nonfermenters can be grouped on the basis of presence / absence of motility and
the type of flagella present in strains that are motile.6
NFGNB, normally a saprophyte, causes serious infections in
immunocompromised and hospitalized patients especially those admitted to
intensive care units (ICU). These bacteria survive for a long time in the hospital
environment and thereby the opportunities for cross infection between patients are
enhanced.23,40
Because of frequent resistance to aminoglycosides,
fluoroquinolones, ureidopenicillins and third-generation cephalosporins,
carbapenems are important agents for managing these infections. 23,43
Carbapenem
resistance is also being increasingly reported in Pseudomonas aeruginosa and
Acinetobacter baumannii.41,42,
These organisms further worsen the situation by
virtue of their multidrug resistance and thus limit therapeutic options. 30, 31
MOTILE WITH POLAR FLAGELLA
Family : Pseudomonadaceae Family Xanthomonadaceae
Genus Pseudomonas Genus stenotrophomonas
Family Burkholderiaceae Family Sphingomonadaceae
Genus Burkholderia Genus sphingomonas
Genus cupriavidus Family Oceanospirillaceae
Family Comamonadaceae Genus Balneatrix
Genus Comamonas Family Alteromonadaceae
Genus Acidovorax Genus Alishewanella
Genus Delftia Genus Shewanella
Family Caulobacteraceae Family Oxalobacteraceae
Genus Brevundimonas Genus Herbaspirillum
Family Methylobacteriaceae
Genus Methylobacterium
MOTILE WITH PERITRICHOUS FLAGELLA
Family Alcaligenaceae
Genus Achromobacter
Genus Alcaligenes
Genus Bordetella
Family Rhizobiaceae
Genus Rhizobium
Family Brucellaceae
Genus Ochrobactrum
Family Halomonadaceae
Genus Halomonas
NONMOTILE, OXIDASE NEGATIVE
Family Moraxellaceae
Genus Acinetobacter
Family Alcaligenaceae
Genus Bordetella
Organisms Whose Taxonomlc
Position is Uncertain
CDC group NO-1
CDC group EO-5
NONMOTILE, OXIDASE POSITIVE
Family Flavobacteriaceae
Genus Flavobacterium
Genus Bergeyella
Genus Chryseobacterium
Genus Weeksella
Family Sphingobacteriaceae
Genus Sphingobacterium
Family Moraxellaceae
Genus Moraxella
Genus Psychrobacter
Family Neisseriaceae
Genus Neisseria
INITIAL CLUES THAT AN UNKNOWN ISOLATE IS A
NONFERMENTER
Lack of evidence for glucose fermentation.
Positive cytochrome oxidase test.
CHARACTERISTICS OF INDIVIDUAL ORGANISMS
PSEUDOMONADS
The Genus pseudomonas and closely related genera which were formerly
placed in the Genus pseudomonas are referred to as pseudomonads.
Pseudomonads are straight or slightly curved, aerobic, gram negative bacilli
motile by means of polar flagella and utilize glucose and other carbohydrates
oxidatively and are usually cytochrome oxidase positive. 6
Molecular analysis led to revised taxonomic classification and many
species have been reallocated to new genera which includes Burkholderia,
Comamonas, Stenotrophomonas, Ralstonia and Brevundimonas.32
Palleroni
separated pseudomonads into five ribosomal RNA homology groups based on
rRNA-DNA homology studies. Gilardi on the other hand separated pseudomonads
into seven major groups based on phenotypic characteristics
Fluorescent
Stutzeri
Alcaligenes
Pseudomallei
Facilis-delafieldii
Acidovorans
Diminuta
Pseudomonas species included in rRNA group 1 includes 3 groups
Fluorescent group
Stutzeri group
Alcaligenes group
Fluorescent Group
The species within this group are characterized by the production of water-
soluble pigment pyoverdin that fluoresces white to blue-green under UV light.
This group includes Pseudomonas aeruginosa, Pseudomonas fluorescens and
P.putida. Although all 3 species produce pyoverdin, only Pseudomonas
aeruginosa produces the distinctive blue water-soluble pigment pyocyanin.6
Pseudomonas aeruginosa is the species most commonly associated with human
disease.32
there are several reasons for the prominence of Pseudomonas
aeruginosa as a human pathogen.
Its adaptability
Its innate resistance to many antibiotics and disinfectants
Its armoury of putative virulence factors
An increasing supply of patient’s compromised by age, underlying
diseases or immunosuppressive therapy. 32
Pseudomonas aeruginosa produces a characteristic appearance on Blood
agar plate (BAP) and the colonies have an alligator skin appearance and exhibits a
metallic sheen with beta-hemolysis. Rapid identification in culture can be made
by
Typical colony morphology
Production of diffusible pigments
Presence of fruity odour
Positive oxidase.
Pseudomonas aeruginosa infection is prevalent among patients with burns,
cystic fibrosis, acute leukemia, organ transplantation and intravenous drug
addicts.10
Infection commonly occurs at any site where moisture tends to accumulate
tracheotomies, indwelling catheters, burns, external ear and weeping cutaneous
wounds. Pseudomonas aeruginosa also causes urinary tract infections and lower
respiratory tract infections, the later can be severe and life threatening in
immunocompromised patients.6 The organism also causes keratitis. Analysis of
bacterial keratitis reveals that Pseudomonas species is the second most important
cause of bacterial keratitis in India after gram-positive bacteria.78
Pseudomonas aeruginosa produces several substances that are thought to
enhance the colonization and infection of host tissues. These substances, together
with the variety of virulence factors including lipopolysaccharide, exotoxin A,
leucocidin, extracellular slime, proteases, phospholipases and several other
enzymes make Pseudomonas aeruginosa the most clinically significant bacteria
among NFB. 6 An unususal mucoid morphotype of Pseudomonas aeruginosa is
recovered from respiratory secretions of patients with cystic fibrosis which is due
to the production of large amounts of polysaccharide called alginate. The
production of alginate is associated with poor prognosis and high mortality rates
among patients with cystic fibrosis. 33
Pseudomonas fluorescens and P.putida occur in water and soil and may
exist in water sources in hospital environment. Both may exist as normal
pharyngeal flora and are rare opportunistic pathogens. Both the species fail to
grow at 42ᴼC as Pseudomonas aeruginosa. They produce only pyoverdin and not
pyocyanin. Another character in which they differ from Pseudomonas aeruginosa
is that they do not deaminate acetamide.
These two species differ from each other in gelatine hydrolysis where
P.flourescens gives a positive reaction; P.putida gives a negative reaction.6
P.putida has been reported to cause catheter-related sepsis in patients with cancer
and septic arthritis.35
Treatment of Pseudomonas aeruginosa infection is difficult
because it express innate resistance to many antibiotics. An alarming increase in
resistance to various antimicrobial agents has been reported from India and
abroad.80
Increased use of broad–spectrum antibiotics, intubation of respiratory,
gastrointestinal or urinary tract and intravascular catheterization are significant
predisposing factors for development of antibiotic resistance.79
They are found to
be sensitive to aminoglycosides, anti pseudomonal penicillin, fluoroquinolones,
and third generation cephalosporins. Amikacin and ceftazidime were found to be
highly effective.80
The incidence of meropenem–
resistant Pseudomonas
aeruginosa is also increasing among nosocomially infected patients in ICU.
81 The
potential risk factors are previous antimicrobial drug exposure. A growing number
of multidrug resistant (MDR) Pseudomonas aeruginosa
producing metallo
betalactamases (MBL) is also reported. Such strains are resistant to most broad
spectrum beta-lactams, aminoglycosides and fluoroquinolones and the traditional
antipseudomonal antimicrobials.77
The
common form of drug resistance is
mediated by lack of drug penetration (porin mutation and efflux pump) and/or
cabapenem–hydrolysing betalactamases.
Based on molecular studies, carbapenem-hydrolysing enzymes are
classified into four groups A,B,C,D. The metallo betalactamases are enzymes
requiring divalent cations as cofactors for enzyme activity, being inhibited by the
action of a metal ion chelator.70
There are reports of MBL production in
Pseudomonas aeruginosa from various countries like Brazil, Korea, Singapore
and France. MBL was first reported as a zinc dependent enzyme in Bacillus
cereus in mid 1960s. A few decades later, meropenem hydrolyzing
metalloenzymes were found in Aeromonas hydrophila and Bacteroides fragilis.
All these enzymes were produced by chromosomal genes and at first
recorded only from single clinical isolates. In 1991, Japan reported the first
plasmid mediated MBL in Pseudomonas aeruginosa. Apart from Pseudomonas
aeruginosa, other bacteria like Serratia, Klebsiella pneumonia, Escherichia coli,
Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Proteus
vulgaris, P.putida, Acinetobacter and Alcaligenes xylosoxidans were also shown
to produce MBL. These carbapenems may be class B MBL(VIM,IMP) or class D
oxacillinases(OXA-23 to OXA-27) or class A clavulanic acid inhibitory
enzymes(SME,NMC,IMI,KPC). They may be chromosomally or plasmid
mediated and therefore possess a threat of spread of resistance by gene transfer
among GNB. 30
Since carbapenem resistance is mediated by several mechanisms,
cross-resistance is commonly seen among related antibiotics.
Although there are various specific tests to detect the underlying
mechanism of carbapenem resistance, Kirby-bauer disc diffusion test is a simple,
easy to perform and cost-effective test which can be conveniently used to screen
carbapenem resistance. These strains also remain resistant to several other
antibiotics including penicillins, cephalosporins, quinolones, aminoglycosides and
third generation cephalosporins including ceftazidime and cefotaxime.
Thus, they may be ESBL producers as well. These MBLs effectively
hydrolyse all betalactams except Aztreonam in vitro. This disturbing situation
could be attributed to the increased use of antibiotics which has to be controlled
by strict antibiotic policy. Various strategies such as strict infection control
measures, judicious prescribing of antibiotics, antibiotic resistance surveillance
programs and antibiotic cycling must be tried. Therefore, detection of MBL-
producing gram negative bacilli especially Pseudomonas aeruginosa is crucial for
the optimal treatment of patients particularly in critically ill and hospitalized
patients and to control the spread of resistance
PSEUDOMONAS AERUGINOSA
Pseudomonas aeruginosa is the most common organism isolated among
the nonfermenters from the clinical specimens, more often than all other
Pseudomonas species especially in teaching hospitals with more than 500 beds.10
They are ubiquitous organisms widely distributed in nature. They have emerged
as a major hospital pathogens because of their ability to grow in a variety of
environments with minimal nutritional requirements.82
Intensive care units,
Immunosuppressants, invasive procedures and antibiotic usage have provided
opportunities for emergence, persistence and transmission of Pseudomonas
between patients, from patients to staff and to inanimate reservoirs. 11
Many
carriage sites like respiratory tract, genitourinary tract and skin serve as source of
dissemination.12
The virulence is multifactorial including loss of host defence
mechanisms like immunosuppression, loss of mucosal barrier, cellular factors,
toxins elaborated by Pseudomonas aeruginosa like endotoxins, exotoxin A,
enzymes like elastases, alkaline protease and hemolysins are responsible for many
of the systemic manifestations of Pseudomonas disease.12
.In addition, the
colonies of the organism form biofilms within which they are protected from host
defenses and antimicrobial agents and communicate with each other through
complex system of cell to cell signaling called Quorum sensing.The production of
alginate and epithelial cell tropism in cystic fibrosis is associated with poor
prognosis and high mortality.10
In the National Nosocomial Infection Surveillance (NNIS) survey from the
Centres for Disease Control and Prevention (CDC), it is the fourth most common
cause of nosocomial infection and leading cause of hospital acquired infections.It
is the most common cause of wound infection caused by gram negative bacteria
with an isolation rate of upto 62%.Urinary tract infections caused by these
organisms are mostly hospital acquired and isolations range from 12%-30%. It
causes life threatening bacteremia especially in intensive care settings at a rate of
10%. Pseudomonas aeruginosa is the leading cause of pneumonia in ICU patients
with a mortality of 80 -100% Other infections caused by Pseudomonas
aeruginosa are osteochondritis, chronic suppurative otitis media, external ear
infections, meningitis following trauma and surgery, endochondritis and
peritonitis 7
IDENTIFICATION 6
Pseudomonas aeruginosa produces large flat colonies with spreading and
serrated edges witha metallic sheen. Various diffusible pigments are produced like
pyoverdin andpyocyanin. It is betahemolytic on blood agar It produces nonlactose
fermenting colonies on MacConkey agar. They are motile organisms. It is oxidase
positive, catalase positive, indole negative, citrate and urease variable. It oxidizes
glucose in OF media, reduces nitrates to nitrites, arginine is decarboxylated,
acetamide positive, ONPG negative, sensitive to Polymixin B and grows at 42ᴼ C
which differentiates it from Pseudomonas fluorescens and Pseudomonas putida.
Characteristics of fluorescent group
Test Pseudomonas
aeruginosa
Pseudomonas
fluorescens
Pseudomonas
putida
Oxidase + + +
Motility + + +
Pyoverdin + + +
Pyocyanin + - -
OF glucose A A A
Acetamide V + +
Growth at 42c + - -
Nitrate reduction V(74%) V(19%) -
Arginine + + +
+ positive, - negative ,V- variable, A - acid reaction / ( ) numbers in the
parenthesis are % of strains giving positive reactions.
ANTIBIOTIC SENSITIVITY
They are sensitive to semisynthetic penicillins like Piperacillin/Ticaricillin,
third generation cephalosporins (ceftazidime), carbapenems (imipenem and
meropenem), monobactams, aminoglycosides and fluroquinolones.13
It is
intrinsically resistant to ampicillin, amoxycillin and amoxicillin-clavulanic acid
due to an inducible chromosomal AmpC beta lactamase.14
, Multiple resistance in
these organisms is frequent, leading to the development of multidrug and pandrug
resistant Ps.aeruginosa strains caused by mutations & or production of
betalactamases ranging from extended spectrum of betalactamases to
metallobetalactamases.7
ACINETOBACTER BAUMANNI
Acinetobacter are strictly aerobic, gram negative coccobacillary rods,
widely distributed in nature and hospital environments 17,
7 They are second most
commonly isolated nonfermenters in human specimens next to Pseudomonas
aeruginosa with a prevalence of 10% of all gram negative isolates.7 They are
generally considered as nonpathogic but cause serious infections in debilitated
patients. The species most frequently isolated is Acinetobacter baumannii It is
most often responsible for hospital acquired infections.7 They are the most
common gram-negative organisms to be isolated from the hands of medical
personnel.
A study conducted by CDC has reported Acinetobacter baumannii to be
the cause of 1% nosocomial blood stream infections(CDC) A mortality of 17-
46% is associated with nosocomial bacteremia by these organisms.20
Analysis of
data from the NNIS system showed that the proportion of ICU pneumonia
episodes range from 4% -7%.14
These organisms have high rate of colonization of the trachea. Respiratory
tract is the most common site for Acinetobacter baumannii infections in ICU with
a mortality rate approaching 70%.12
Traumatic wounds, burns and postoperative surgical site infections are also
common with multidrug resistant strains being observed.16
Several reviews have described these organisms in 2-6% of nosocomially
acquired urinary tract infections. 16,
17
IDENTIFICATION6
Colonies are translucent to opaque, convex and entire with a diameter
between 0.5 and 2mm. It produces nonlactose fermenting colonies on MacConkey
agar with a pinkish tint. It is oxidase negative, nonmotile, catalase positive, citrate
positive and urease negative. It oxidizes glucose and 10% lactose and dextrose in
OF media. It does not reduce nitrates to nitrites. It deaminates arginine,
acetamide negative, ONPG negative and grows at 44ᴼC.
Characteristics of Acinetobacter
TEST A.baumannii A,iwoffi
Oxidase - -
Motility - -
Growth on Macconkey + +
OF glucose A -
Nitrate reduction - -
Citrate + v
10% Lactose + -
+ positive , - negative ,A – acid reaction.
ANTIBIOTIC SUSCEPTIBILITY 16
,6,14
They are universally resistant to penicillin, ampicillin and
chloramphenicol. They show variable susceptibility to second and third generation
cephalosporins. Recently extended spectrum of betalactamases and
carbapenemase resistance is reported in nosocomial infections.
PSEUDOMONAS FLUORESCENS
P.fluorescens is a psychrophilic organism which favours its presence in
blood products. Outbreaks of bacteremia, respiratory tract infections in cystic
fibrosis patients, wound infections, urinary tract infections and rare cases of
community acquired pneumonia have been reported. They behave as opportunistic
pathogens in immunocompromised patients.6
IDENTIFICATION 6
Colonies are large with spreading edges forming nonlactose fermenting
colonies on MacConkey agar and hemolytic colonies on blood agar. It is oxidase
positive,catalase positive, motile, oxidizing glucose, deaminating arginine,
reducing nitrates to nitrites, ONPG negative, acetamide negative, sensitive to
polymyxin B and do not grow at 42ᴼC.
STENOTROPHOMONAS MALTOPHILIA
Originally classified as Pseudomonas maltophilia, it is an obligate aerobe
and an ubiquitous organism It is an emerging opportunistic pathogen. It is the
third most common encountered nonfermenter in clinical laboratory next to
Pseudomonas and Acinetobacter.6
It is an important nosocomial pathogen associated with substantial
morbidity and mortality especially in immunosuppressed patients.It is one among
the most common causes of wound infections due to trauma. It is frequently
isolated from patients with ventilatory support in ICU. It is an important pathogen
in cystic fibrosis patients. It produces proteolytic enzymes, deoxyribonucleases,
ribonucleases, hemolysins, hyaluronidase and mucinase etc. which contribute to
its severity in immunosuppressed patients. The rate of infections caused by
Stenotrophomonas maltophilia is increased in recent years and are being isolated
from wound infections, bacteremia, pneumonia, urinary tract infections,
meningitis and peritonitis. A significant feature of Stenotrophomonas maltophilia
is its ability to adhere to plastics and form bacterial films (biofilms).
Stenotrophomonas maltophilia has been identified on the surfaces of materials
used in intravenous (i.v.) cannulae, prosthetic devices, dental unit waterlines, and
nebulizers 96,97,98,99,100,101,102
IDENTIFICATION6
Colonies formed are pale yellow / lavender green with good growth on
Blood agar and MacConkey agar. It is oxidase negative, motile, catalase positive,
indole negative, citrate variable, urease negative. It oxidizes glucose and maltose,
decarboxylates lysine, ONPG positive, with variable nitrate reduction.
Characteristics of Stenotrophomonas maltophilia and Burkholderia
cepacia complex
Test
Stenotrophomonas
maltophilia
Burholderia
cepacia complex
Oxidase - +(93)
Motility + +
Growth on Mac conkey agar + +
OF glucose A Weak A
Nitrate reduction V(42%) V(37%)
Nitrate to gas - -
Lysine + +
Polymyxin B S R
+ positive , - negative , V-variable , A – acid reaction / ( ) numbers in the
parenthesis are % of strains giving positive/ S- Susceptible / R-Resistant
ANTIBIOTIC SUSCEPTIBILITY
Therapy for Stenotrophomonas maltophilia infections is problematic
because of the broad antibiotic resistance that typifies this organism. The most
active agents are trimethoprimsulphamethoxazole, colistin and quinolones. Like
other nonfermenters it is intrinsically resistant to many common antibiotics like
aminoglycosides, carbapenems and many betalactam agents.6
BURKHOLDERIA CEPACIA
It is a motile free living phytopathogen identified as both endemic and
epidemic nosocomial pathogen.Its detection rates are low, in the range of 1%-16%
of clinical samples.It belongs to rRNA group Ie. It produces virulence factors like
proteases, lipases, exopolysaccharides and lipopolysaccharides.
A few case reports have described serious infections, including severe
pneumonia, invasive otitis and sepsis in cystic fibrosis patients. Diabetes mellitus
is a potential risk factor for development of infections.by Burkholderia cepacia .6ia
Burkholderia cepacia is also an important pathogen among patients with chronic
granulomatous disease. Like other nonfermenters, it can contaminate disinfectant
solutions The major importance of this organism lies in its role as opportunistic
agent of pneumonia in cystic fibrosis patients seeded in sputum samples.
Burkholderia cepacia complex is ambiguously reported as a non-fermenting
Gram-negative bacilli (NFGNB). Hence, there is a need for molecular
confirmation of Burkholderia cepacia complex.84
Burkholderia cepacia complex
has emerged as a serious nosocomial pathogen worldwide, due to its high intrinsic
resistance to most antibiotics, acquired resistance to fluoroquinolones and
antiseptics, besides its ability to survive in the environment for prolonged periods
with limited nutrition.23
Members of Burkholderia cepacia complex family are the
most common contaminants of many finished pharmaceutical products and
environment in which pharmaceutical products are manufactured.85
Burkholderia
cepacia complex survives, multiplies and may persist for long periods in moist
hospital environment, including detergent solutions and intravenous (IV)
fluids83,86
The spectrum of infections by these organisms includes wound infections,
bacteremia, UTI, pneumonia, meningitis, peritonitis, and endocarditis.
IDENTIFICATION6
Colonies are smooth and glistening, forming non-lactose fermenting
colonies on MacConkey agar and yellow pigmented colonies on blood agar. It is
weakly oxidase positive, catalase positive, motile, oxidizes all sugars,
decarboxylates lysine, ONPG negative, acetamide negative and resistant to
Polymixin B. Nitrate reduction is variable.
ANTIBIOTIC SUSCEPTIBILITY
As with other nonfermenters intrinsic antibiotic resistance typifies
Burkholderia cepacia and greatly complicates treatment. Trimethoprim-
sulfamethoxazole has historically been the drug of choice. Most active agents are,
ceftazidime, meropenem, ciprofloxacin and other quinolones.
ANTIBIOTIC SUSCEPTIBILITY 14
,6
It is sensitive to antipseudomonal penicillins like Piperacillin, betalactam
agents and carbapenems. It is resistant to penicillins.
WEEKSELLA VIROSA 6
Flavobactericeae comprises indole positive organisms like
Chyseobacterium, Empedobacter, Spingobacterium and Weeksella.6
Weeksella
virosa are associated with urinary tract infections.34
IDENTIFICATION6
Weeksella virosa form yellow colonies on blood agar. They are oxidase
positive and nonmotile, form indole, citrate variable and urease negative, do not
oxidizes glucose and maltose. They are nitrate negative. Weeksella is sensitive to
penicillin and polymyxin B.
SHEWANELLA PUTREFACIENS 87,88
Shewanella is a marine bacteria rarely implicated as a human pathogen.
Two species of importance are Shewanella algae and Shewanella putrefaciens. It
is an oxidase-positive, hydrogen sulphide producing Gram negative bacilli. It was
infrequently recovered from clinical specimens probably because of inadequate
processing of non-fermenting oxidase-positive gram-negative bacilli.87
Most
human isolates of S. putrefaciens occur as part of a mixed bacterial flora, clouding
their clinical significance. However, a number of monomicrobic illnesses due to S.
putrefaciens have been documented and include bacteremia, soft tissue infections,
and otitis media .93, 94, 95
IDENTIFICATION
Isolates that were motile, had oxidative metabolisms, were oxidase and
catalase positive, ornithine decarboxylase positive, and DNase positive, and
produced H2S on triple sugar iron slants within 72 h of incubation were identified
as belonging to the phenospecies S.putrefaciens.All other reactions were
uniformly positive for the Shewanella strains studied. 6
The biovar and biotype of
each isolate were determined according to Gilardi90
and Weyant et al91
respectively, based on acid production from sucrose and maltose, growth on SS
agar, and growth in the presence of 6.5% NaCl. Species designations were
determined by the criteria of Nozue et al. 92
by using the following tests:
hemolysis on sheep blood agar, growth at 42°C, growth on nutrient agar
containing 6.5% NaCl, growth on SS agar, and acid production from maltose and
l-arabinose.
Methods for Identification Using Automated Identification Systems
The Vitek Legacy System
The Vitek Legacy System (BioMérieux), , has also been used with success
in the identification of the nonfermenters most frequently encountered in the
clinical laboratory.
The Vitek 2 System
The original Vitek 2 card for gram-negative bacteria identification has been
redesigned to improve the identification of fermenting and nonfermenting bacilli.
The new card contains 47 tests (26 that had been included in the previous card and
21 new tests), compared with 41 in the established Vitek 2 ID-GNB card. The
database for the new card has been expanded to 159 taxa compared with only 101
for the original Vitek 2 card.
The Microscan Walkaway-96, Walkaway-40, and Autoscan-4 Systems
These three systems (manufactured by Beckman Coulter, West
Sacramento, CA),all have an extensive database that includes many species of
NFBs. Tenover and colleagues1064 evaluated the Walkaway-96 (formerly called
the autoSCAN-W/A) for its ability to identify 310 well-characterized non–
glucosefermenting gram-negative bacilli. In their study, two types of
identification panels were tested: the dried colorimetric Neg ID type 2 panel
(DCP) and the rapid fluorometric Neg ID panel (RFP). Problems in identifying
relatively common nonfermentative bacilli, such as Pseudomonas fluorescens, P.
putida and Stenotrophomonas maltophilia were reported with the DCP panel. The
researchers reported better results with the RFP panels.The RFP panels were
available as early as 2 hours; thus, if an organism cannot be identified, additional
biochemical tests can be inoculated on the same day, and less time is lost in
identifying the organisms.
The Sensititre AP80 System6
The Sensititre AP80 Identification panels (TREK Diagnostic Systems,
Cleveland, OH) can be inoculated and incubated offline and then read in the
Sensititre Autoreader, or can be inoculated and placed in the ARIS (Automated
Reading and Incubation System) Instrument. The AP80 panel identifies gram-
negative bacilli as early as 5 hours,
The Phoenix System
The Phoenix Automated Microbiology System (Becton Dickinson
Microbiology Systems) is a fully automated, identification and antimicrobial
susceptibility test system. 6
Methods for Identification Using Molecular Systems
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry
An overview of this new technology and modern applications in the
clinical microbiology laboratory are described in a recent review. Overall
performance of MALDI-TOF MS has been reported to be significantly better than
commercially available systems for identification of the NFBs although overall
performance is still less than satisfactory. 44, 45, 46, 6
Discrepancies were refereed with 16S rRNA sequencing or whole genome
sequencing (WGS) using Illumina’s MiSeq technology. Correct identification to
the species level for Bruker RUO, Vitek RUO, and Vitek IVD was 62.1%, 48%,
and 54.3%, respectively.
Both systems gave a low number (<5%) of incorrect IDs; however, the
ability to identify NFBs correctly to species level was low for both systems.
Improvements are needed in the databases used for identification of NFBs with
both systems for accurate identification of NFBs to the species level.
16S rRNA Gene Sequencing
Due to the poor performance of commercially available systems for the
identification of NFBs and sometimes less than satisfactory performance of
MALDI-TOF MS to identify NFBs at the species level, laboratories have
increasingly turned to sequencing methods such as 16S rRNA gene sequencing to
determine the identification of clinically relevant isolates. 50,51,6
16S rRNA is a
component of the 30S small subunit of prokaryotic ribosomes.
16S rRNA gene sequences contain hypervariable regions that can provide
species-specific signature sequences useful for identification of bacteria. As a
result, 16S rRNA gene sequencing has become prevalent in medical microbiology
as a rapid and inexpensive alternative to phenotypic methods of bacterial
identification.
Phenotypic testing was performed by conventional phenotypic and
commercial methods in use at each of the participating laboratories and included
the Vitek or API 20NE systems (bioMèrieux, Durham, NC) or the MicroScan
system (Beckman Coulter, Sacramento, CA). 52,49,6
Using 16S rRNA sequencing,
92% of the isolates were assigned to species level and 8% to genus level (100%
combined). Using API 20 NE, 54% of the isolates were identified to the species
level, and 7% to the genus level (61% combined), and 39% of the isolates could
not be identified. For Vitek-2, 53% could be identified to the species level, 1% to
the genus level (54% combined), and 46% could not be identified.48,6
Resolution of 16S rRNA Gene Sequencing.
Although 16S rRNA gene sequencing is highly useful in regards to
bacterial classification, it has low phylogenetic power at the species level and
poor discriminatory power for some genera. With the NFB this is particularly true
for members of the Burkholderia cepacia complex, the Acinetobacter
calcoaceticus–Acinetobacter baumannii complex, and some members of the
genus Pseudomonas, the genus Achromobacter, the genus Bordetella, and the
genus Ralstonia.47, 48, 6
As for any identification method, limitations for 16S rRNA gene
sequencing exist and students and laboratorians should be aware of these pitfalls
when using gene sequencing for bacterial identification in the diagnostic
laboratory.47, 49, 6
ANTIBIOTIC SUSCEPTIBILITY6
They are resistant to aminoglycosides, third generation cephalosporins,
Imipenem and erythromycin. They are sensitive to ciprofloxacin and
betalactamase inhibitors.
INTRINSIC RESISTANCE15
ANTIMICROBIAL
AGENT
Am
ipic
illi
n
Pip
erci
llin
Tazo
bct
um
efo
tax
ime
Cef
ipim
e
Azt
reo
na
m
Mer
op
enem
Po
lym
ixin
B
Am
ino
gly
cosi
de
Co
trim
ox
azo
le
ceft
ria
xo
ne
chlo
ram
ph
enic
ol
Acinetobacter
baumannii
R R R
Burkholderia
cepacia complex
R R R R R R R R
Pseudomonas
aeruginosa
R R R R R
Stenotrophomonas
maltophila
R R R R R R R
MULTIDRUG RESISTANCE IN NONFERMENTING GRAM NEGATIVE
BACILLI
Nonfermenting Gram Negative Bacilli pose a particular difficulty for
healthcare community because they represent the problem of multidrug resistance
to the maximum6. They are resistant to three or more drugs and important
members of this group are Pseudomonas aeruginosa, Acinetobacter baumannii,
Stenotrophomonas maltophilia and Burkholderia cepacia 21
. They use several
mechanism of resistance including intrinsic and rapidly acquired resistance.
Intrinsic resistance is due to relative impermeability of outer membrane proteins
compared to that of other gram negative bacteria (ten fold times lower). Efflux
system also contributes to intrinsic resistance Acquired resisitance is by
mutational changes and acquisition of exogenous genetic material. Lastly
resistance may also develop during therapy turning an initially susceptible isolate
into a resistant one.13
Pseudomonas aeruginosa exhibits multidrug resistance to 4
antibiotic classes -ceftazidime, imipenem, gentamicin, and a fluroquinolone. The
increase in multidrug resistant strains suggests that therapy with compounds like
polymyxinB or colistin must be considered.14
A report in Germany revealed
multidrug resistant profiles in Acinetobacter to drugs like cefepime, ciprofloxacin
and amikacin 14
Stenotrophomonas maltophilia and Burkholderia cepacia are associated
with intrinsic drug resistance. Multidrug antibiotic resitance negatively affects
outcomes of the patients.14
. Intrinsic includes over-expression of efflux pumps
(mexAB, mexCD, mexEF and mexXY), chromosomal hyper ampC producers and
loss of porins (OprD); extrinsic includes acquisition of resistance genes such as
extended spectrum beta-lactamases (ESBLs; blaSHV, blaTEM, blaVEB, blaPER and
blaOXA types) and carbapenemases (blaGES, blaKPC, blaIMP, blaSPM, blaVIM and
blaNDM)9
EXTENDED SPECTRUM OF BETALACTAMASES
ESBL are a group of betalactamases which share the ability to hydrolyse
third generation cephalosporins and are inhibited by clavulanic acid. They are
plasmidcoded. Carbapenems are treatment of choice for serious infections due to
ESBL producing organisms. ESBLs in nonfermenters are Ambler class A. These
enzymes are SHV type, TEM type, TEM 1 and 2, CTX-M type, OXA- type ,
PER- type, VEB, BES – types and others. Screening tests for ESBL producers are
disk diffusion and dilution susceptibility testing methods. The phenotypic
confirmatory tests for ESBL production are
1. Cephalosporin / clavulanate combination disks15
2. E tests 15,21
CARBAPENEMASES AND METALLOBETALACTAMASES
Carbapenemases are betalactamases with versatile hydrolytic capacities.
They have the ability to hydrolyze penicillins, cephalosporins, monobactams, and
carbapenems. Bacteria producing these betalactamases may cause serious
infections in which the carbapenemases activity renders many betalactams
ineffective. They are members of molecular class A, B and D betalactamases.
Class A and D have serine based hydrolytic mechanisms while class B are
metallobetalactamases that contain zinc in the active site. Class D carbapenemases
consist of OXA type betalactamases frequently detected in Acinetobacter
baumanni. The metallobetalactamases belong to IMP, VIM, SPM, GIM and SIM
families and have been detected primarily in Pseudomonas aeruginosa.
Nonfermenters especially Pseudomonas aeruginosa and Acinetobacter baumannii
have acquired metallobetalactamases through genetic elements (plasmids/
transposons) and Scan be transmitted to other bacteria. These enzymes confer
resistance to all carbapenems (Imipenems, Meropenems, Ertapenems), all
betalactams, aminoglycosides and quinolones. The dissemination is thought to be
driven by regional consumption of ESBLs. Stenotrophomonas maltophilia is
naturally resistant to imipenem and meropenem because of chromosomally
mediated carbapenemase production.19
The families and subgroups of
carbapenemases known till now are IMP-1&2, VIM-1&2, SPM-1, GIM-1, and
SIM-1.
IMP was first discovered in Ps.aeruginosa in Japan.and this has spread to
other gram negative bacteria and reports show their detection in Acinetobacter
baumannii, Serratia and Klebsiella. Currently IMP family members number upto
18 in the published literature. The second dominant group of acquired MBLs is
the VIM type enzymes. It was first described in Verona Italy, from Pseudomonas
aeruginosa isolate. This family currently consists of 14 members and seen mostly
in Pseudomonas aeruginosa. It has dubious distinction of being the most reported
metallo-beta-lactamase worldwide. These genes are easily transferred on mobile
elements among species. While considered by some to be rare, reports of their
occurrence have increased.
DETECTION OF CARBAPENEMASES
1. Raise in MIC of carbapenems in the range of 8 >128 μgm / ml.
2. Microbiological test with inhibitors:
a. Disc approximation test with EDTA
b. Combined disc method: Imipenem with EDTA 18
c. E test strips with Imipenem and Imipenem EDTA combination
d. Modified Hodge test 18
Of these tests, studies conducted showed that both combined disc test and
E test were more sensitive and equally effective for MBL detection.
MOLECULAR METHOD
In this study, PCR was used to determine the gene for MBL production in
Pseudomonas aeruginosa isolates that were resistant to carbapenems PCR was
done using primers specific for MBL genes.
Cell lysates of the isolates were used as DNA template for colony lysate
PCR. Around 5 – 10 colonies were suspended in 100ml of Milli Q water & boiled
for 5 minutes. It is then centrifuged at 10,000 rpm for 10 minutes. The supernatant
provided templates for PCR reactions.
Forty amplification cycles were performed with an automated thermocycler
according to the following format: Initial denaturation for 5 min at 94ᴼC, 30
cycles of DNA denaturation for 30 s at 94.c, annealing for 30 s at 55.c and
extension for 1.5 min at 72ᴼc. The final cycle was followed by an additional 5 min
at 72ᴼc to complete partial polymerizations. Amplified products were run using
horizontal 1.5 % agarose gel electrophoresis. The gel was visualized using a UV
transilluminator. The amplified PCR products and 100 base pair DNA molecular
markers were seen as bright fluorescent bands.
INTERPRETATION
A 261 bp corresoponds to VIM and 587 bp corresponds to IMP
gene.specific oligonucleotides.
MATERIALS & METHODS
STUDY PERIOD
This cross sectional study was conducted from January 2015 to January 2016
PLACE OF STUDY
Govt Kilpauk Medical College and Hospital, Chennai.
ETHICAL CONSIDERATION
The study was approved by our Institutional Ethical Committee and Ethical
clearance was obtained
STATISTICAL ANALYSIS
All statistical analyses were carried out using SPSS for Windows. Odds
ratios (ORs) and 95% confidence intervals (CIs) were calculated. P values were
calculated using the chi-square test. A p value of < 0.05 was considered
significant.
SAMPLE
A total of 200 nonfermenting bacteria isolated from various clinical
specimens like pus, urine, blood, bronchoalveolar lavage, endotracheal aspirations
and cerebrospinal fluids collected from both outpatients and inpatients of Govt
kilpauk Medical college and hospital, Chennai were studied.
SAMPLE PROCESSING
The samples were processed according to standard procedures. The
collected samples were subjected to direct Gram stain and all specimens were
inoculated onto nutrient agar, 5% sheep blood agar and MacConkey’s agar
medium. Urine samples were also inoculated onto Cystine Lactose Electrolyte
Deficient agar (CLED) in addition.
All the catalase positive, oxidase positive and negative, nonlactose
fermenting colonies on Mac Conkey agar were provisionally identified by colony
morphology and pigment production. They were inoculated in Triple sugar iron
(TSI) agar slope. The colonies which failed to acidify the TSI agar were
considered as nonfermenters and subjected to the following tests.(annexure)
Motility, Indole, Citrate, Urease, Nitrate reduction, growth at 42ᴼ c and 44ᴼc,
Sensitivity to Polymyxin B and following special biochemical tests and grouped
according to P.C.Schreckenberger scheme6
TESTS USED FOR IDENTIFICATION OF NON-FERMENTERS6.
Positive Cytochrome Oxidase Reaction
Any colony of a GNB growing on blood agar or any other primary
isolation media that is cytochrome oxidase positive can be suspected of belonging
to NF group. The cytochromes are iron-containing hemoproteins that act as the
last link in the chain of aerobic respiration by transferring electrons (hydrogen) to
oxygen, with the formation of water.6
The cytochrome oxidase test uses certain reagent dyes, such as p-
phenylenediamine dihydrochloride, that substitute for oxygen as artificial electron
acceptors. In the reduced state, the dye is colorless; however, in the presence of
cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized,
forming indophenol blue:
1. Positive control: Pseudomonas aeruginosa
2. Negative control: Escherichia coli
The test is commonly performed by one of two methods:
1. The direct plate technique, in which two to three drops of reagent are
added directly to isolated bacterial colonies growing on plate medium; and
2. The indirect paper strip procedure, in which either a few drops of the
reagent are added to a filter paper strip or commercial disks or strips
impregnated with dried reagent are used. The tetramethyl derivative of p-
phenylenediamine is recommended because the reagent is more stable in
storage and is more sensitive to the detection of cytochrome oxidase and is
less toxic than the dimethyl derivative. In either method, a loopful of
suspected colony is smeared into the reagent zone of the filter paper.
Bacterial colonies having cytochrome oxidase activity develop a deep blue
color at the inoculation site within 10 seconds. Any organism producing a blue
color in the 10- to 60-second period is considered negative.6
Lack Of Evidence For Glucose Fermentation
Acid produced by NFs are considerably weaker than mixed acids derived
from fermentative bacteria, thus the pH in fermentation test media in which a NF
is growing may not drop sufficiently to convert the pH indicator. The initial clue
that an unknown organism is a NF is usually the lack of acid production in either
Triple sugar iron (TSI) or Kligler iron agar(KIA) media, manifested as an alkaline
slant and an alkaline deep.6
Motility6
The hanging drop preparation may be more accurate in detecting motility
of NFGNB. A loopful of 6 to 24 hr, actively growing broth culture that has been
incubated at 37ᴼC is placed in the center of No-1 coverslip that is inverted and
suspended over the concavity of depression slide. True motility must be
differentiated from Brownian movement. Motile bacteria show directional
movement and change in position relative to each other; when Brownian
movement is the cause of motion, they maintain the same relative position.
Motility B medium with tetrazolium also used for demonstrating motility of
NFGNB. Flagellar stains can also be used to demonstrate motility.
Pigment Production6
Pseudomonas produces water-soluble and diffusible pigments like
fluorescein (pyoverdin), pyocyanin, pyorubin, pyomelanin that discolor the
culture media. “Tech” and “Flo” media were developed to enhance the formation
of water-soluble pigments pyoverdin and pyocyanin. These media have special
peptones and an increased concentration of magnesium and sulfate ions to
enhance pigment production. Pigment production also enhanced by growing the
organism in gelatin, potato or milk-containing media and by incubating them at 25
–30ᴼC.
Nitrate Reduction6
The ability of the organisms to reduce nitrate to nitrite is an important
characteristic used in the identification and speciation of many microorganisms.
Organisms demonstrating nitrate reduction have the capability of extracting
oxygen from nitrate to form nitrite and other reduction products. The presence of
nitrite in the test medium is detected by the addition of alpha-naphthylamine and
sulphanilic acid which leads to the development of red color. If red color do not
develop, either nitrate has not been reduced or reduction is beyond the nitrite stage
to the formation of other compounds or to nitrogen gas (denitrification). The
appearance of red color on addition of small quantity of zinc dust indicates the
residual presence of nitrate, denoting a negative test; absence of color indicates
nitrate has been reduced beyond nitrite, indicating the original test was positive.
Denitrification of Nitrates And Nitrites6
Certain nonfermenters have the capability of reducing nitrate or nitrite or
both to gaseous nitrogen. Nitrate-nitrite broth with an inverted Durham tube may
be used. Because the media contains no carbohydrate, any gas that is formed is
derived from nitrate or nitrite, indicating a positive reaction.
Indole Production6
An enriched tryptophan –containing media, usually heart infusion broth
may be needed. Because only small quantities of Indole are formed by some NFs,
extraction of culture media by layering a small quantity of xylene or chloroform
on the surface may be helpful. The appearance of fuchsia red color at the surface
of medium with the reagent (kovac or Ehrlich reagent) indicates indole formation
and a positive test. One organism, Delftia acidovorans, produces a distinctive
“pumpkin orange” indole reaction owing to the formation of anthranilic acid
rather than indole from tryptophan.
Citrate Utilisation6
A well isolated colony is picked from the surface of a primary inoculation
plate and inoculated as a single streak on the slant surface of Simmon’s citrate
medium and incubated at 35ᴼC for 24 to 48 hours. Development of blue color
indicates a positive test.
Hydrolysis Of Urea6
Christensen’s urea agar slants used. Bacterial species like Bordetella
bronchiseptica produce a red color change within 4hours; weak reactors may
require up to 48 hours
SPECIAL BIOCHEMICAL TESTS USED FOR IDENTIFICATION
OF NON FERMENTERS
1. HUGH – LEIFSON OXIDATION - FERMENTATION MEDIUM
6
Two tubes were required for the test, each inoculated with the unknown
organism, using a straight needle stabbing the medium three to four times half
way to the bottom of the tube. One tube of each pair was covered with a 1cm layer
of sterile mineral oil (or) melted paraffin, leaving the other open to the air. Both
tubes were incubated at 35ᴼC and examined daily for several days.
In case of oxidative metabolism, yellow color appears along the upper one
fourth of the medium and in the tube where no oil overlay was done. In case of
fermentative organisms yellow color develops in both the tubes.
CONTROL
Glucose fermentation: Escherichia coli
Glucose oxidation: Pseudomonas aeruginosa
Non saccharolytic: Alcaligenes species.
2. DECARBOXYLATION OF LYSINE, ARGININE ORNITHINE 6
Decarboxylases are a group of specific enzymes which react with carboxyl
portion of aminoacid forming alkaline reacting amines. The reaction is
decarboxylation. Each enzyme is specific for Lysine, Arginine and Ornithine.
Amino Acid Positive Control Negative Control
Lysine Enterobacter aerogenes Enterobacter cloacae
Ornithine Enterobacter cloacae Klebsiella pneumoniae
Arginine Enterobacter cloacae Enterobacter aerogenes
Procedure
From a well-isolated colony of the test organism previously recovered on
primary isolation agar, inoculate two tubes of Moller decarboxylase medium, one
containing the amino acid to be tested and the other to be used as a control tube
devoid of amino acid. Overlay both tubes with sterile mineral oil to cover about 1
cm of the surface and incubate at 35°C for 18–24 hours.
Conversion of the control tube to a yellow color indicates that the organism
is viable and that the pH of the medium has been lowered sufficiently to activate
the decarboxylase enzymes. Reversion of the tube containing the amino acid to a
blue-purple color indicates a positive test owing to the formation of amines from
the decarboxylation reaction
3. O–NITROPHENYL β - D GALACTOPYRANOSIDE 6
A dense suspension of the test organism grown in TSI agar was prepared in
saline.About 1 drop of toluene was added to the suspension and ONPG disc was
added to the suspension and incubated at 37ᴼC b-galactosidase producing
organism show yellow color after 1 hour or 18-24 hours incubation.
.
4. GELATIN LIQUEFACTION TEST6
Gelatin breakdown can be demonstrated by incorporating it in a buffered
nutrient agar, growing the culture and then flooding the medium with tannic acid
that differentially precipitates either gelatin or its breakdown products.causing
opacity in the medium with clear zones around gelatin-liquefying colonies
ANTIBIOTIC SENSITIVITY15
Antibiotic susceptibility pattern was done on Mueller Hinton Agar by
Kirby- Bauer disc diffusion method as recommended by Clinical and Laboratory
Standards Institute(CLSI).Himedia discs were used for disc diffusion testing.
Antibiotic Discs Contents
Amikacin - 30µg
Gentamicin - 10µg
Cephotaxime - 30µg
Ceftazidime - 30µg
Cefepime - 30µg
Ciprofloxacin - 5µg
Ofloxacin - 5µg
Piperacillin - 100µg
Piperacillin – Tazobactum 100/10µg
Imipenem - 10µg
Meropenem - 10µg
Aztreonam - 30µg
colistin - 10µg
polymyxin - 300 U
The control strains used were E.coli ATCC 25922 and Pseudomonas
aeruginosa ATCC 27853 Overnight broth culture compared to 0.5 McFarland’s
was used as inoculum. After incubation at 37ᴼC for 16-18 hrs, zone of inhibition
was noted. Results were interpreted according to CLSI standard.
Multidrug resistant (MDR) isolates of the nonfermenters were estimated
MDR isolate was defined as resistant to three or more drugs of therapeutic
relevance.
DETECTION OF EXTENDED SPECTRUM OF β -LACTAMASES 15
All nonfermenters that were resistant to cefotaxime and or ceftazidime
were tested for Extended Spectrum of β-Lactamases.by the following methods:
Phenotypic confirmation test with Cephalosporin / clavulanate combination
disks.15, 75
This was done as recommended by CLSI guidelines. Mueller Hinton Agar
plates were swabbed with test organism having the turbidity equivalent to 0.5 Mc
Farland’s standard. Aseptically cefotaxime disk (30µg), cefotaxime – clavulanic
acid (30µg/10µg) ceftazidime (30µg) & ceftazidime clavulanic acid (30µg/10µg)
were placed on surface of agar. The plates were incubated at 35ᴼC for 16-18 hours
and diameter of zone of inhibition produced was recorded. A 5mm increase in
zone diameter for combination disc than that when tested alone confirmed the
presence of ESBL production. ATCC Escherichia coli 25922 & Klebsiella
pneumoniae ATCC 700603 were used as negative and positive control
respectively.
DETECTION OF ESBL BY E-TEST15
It is a plastic drug – impregnated strips, one end of which contains a
gradient of ceftazidime (MIC test range 0.5 to 32 μg/ml) and the other with a
gradient of ceftazidime plus a constant concentration of clavulanate (4 μg/ml).
Similar strips were used for cefotaxime and cefotaxime / clavulanate. A 0.5 Mc
Farland turidity standard of the organism was inoculated as a lawn culture on
Mueller Hinton agar. E strips were placed on the agar surface and plate were
incubated at 35°C for 16-18 hours.
INTERPRETATION
A > 8 fold reduction in cephalosporin MICs in the presence of clavulanate
is taken as positive for ESBL.
Amp C disk test: AmpC disk test was also done for the meropenem resistant
strains for detection of AmpCβ-lactamases. On a MHA plate, lawn culture of
E.coli ATCC 25922 was made from an overnight culturesuspension adjusted to
0.5 McFarland standard12. A 30μg cefoxitin disk was kept on the surface of the
agar. A blank disk (6 mm in diameter, Whatmann filter paper no.1) was moistened
with sterile saline and inoculated with a few colonies of the test strain. The
inoculated disk was then placed beside the cefoxitin disk almost touching it. The
plate was incubated overnight at 37ᴼC. A flattening or indentation of the cefoxitin
inhibition zone in the vicinity of the disk with test strain was interpreted as
positive for the production of AmpC β-lactamase. An undistorted zone was
considered as negative54
SCREENING FOR METALLOBETALACTAMASE PRODUCTION
Screening for metallobetalactamse production was done in isolates of
nonfermenters that were resistant to Imipenem and or Meropenem. Due to
intrinsic resistance to carbapenems mediated by resident MBL production, isolates
of Stenotrophomonas maltophilia were not considered eligible for MBL
screening. The methods used were:
MODIFIED HODGE TEST 18
The indicator organism, Escherichia coli ATCC 25922, at a turbidity of 0.5
McFarland Standard was used to inoculate the surface of a Mueller - Hinton agar
plate supplemented with zinc sulfate at a concentration of 70μgm/ml.and the test
strain was heavily streaked from the centre to the plate periphery. After the plate
was allowed to stand for 15 min at room temperature, 10 mg Imipenem disk was
placed at the center and the plate was incubated overnight. The presence of a
distored inhibition zone was interpreted as a positive result for carbapenem
hydrolysis screening.
IMIPENEM – EDTA DOUBLE DISC SYNERGY TEST 18
Test organism was adjusted to the 0.5 Mc Farland Standard and used to
inoculate Muller Hinton agar plates. 10mg Imipenem disk was placed on the plate
and a blank filter paper disk was place at a distance of 10mm (edge to edge). To
the blank disk, 10ml of 0.5M EDTA solution (1,900 mg of disodium salt,
dihydrate) was added. After overnight incubation, the presence of even a small
synergistic zone was interpreted as positive.
IMIPENEM – EDTA COMBINED DISK TEST 75
Test organisms of 0.5 Mc Farland Standard were inoculated onto plates of
Mueller– Hinton agar. A 0.5M EDTA solution was prepared. Two 10mg
Imipenem discs and Meropenem discs were placed on the plate. 5ml of EDTA
solution was added to one of the disc. The inhibition zones of the Imipenem and
Imipenem + EDTA discs were compared after 16-18 hrs of incubation at 37ᴼC.
An increase in inhibition zone of ≥ 7mm in combined disc than imipenem disc
alone was considered as positive
MBL E-test
The E Test MBL strip containing a double sided seven-dilution range of
IPM (4 to 256 μg/mL) and IPM (1 to 64 μg/mL) in combination with a fixed
concentration of EDTA has been reported to be the most sensitive format for
MBL detection. The E-test was done according to manufacturer's instructions.
MIC ratio of IP (Imipenem)/IPI (Imipenem-EDTA) of >8 or >3 log 2 dilutions
indicates MBL production24
DETERMINATION. OF METALLOBETALACTAMASE GENE BY PCR
METHOD
When the presence of a carbapenemase is suspectd, PCR is the fastest way
to determine which family of betalactamase is present. Ultimately the
identification of the betalactamase gene requires sequencing of the entire coding
region.
Molecular identification of AntibioticResistance Gene
PureFast Bacterial DNA minispin purification kit [Kit contains Lysozyme,
Lysozyme digestion buffer, Proteinase-K, Binding buffer, Wash Buffer-1, Wash
Buffer-2, Spin columns with collection tube and elution buffer. 2X ReDdye PCR
Master Mix, Agarose gel electrophoresis consumables and blaKPC Primers are
used.
2X Master Mix:
It contains 2U of Taq DNA polymerase, 10X Taq reaction buffer, 2mM
MgCl2, 1μl of 10mM dNTPs mix and RedDye PCR additives.
Agarose gel electrophoresis:
Agarose, 50X TAE buffer, 6X gel loading buffer and Ethidium bromide
are used.
PCR:
Ready to use VIM gene Primer mix - 5μl/reaction
Product size: 480bp
Bacterial DNA Purification
1. 1ml of overnight culture centrifuged at 6000rpm for 5min
2. Supernatant discarded
3. Pellet is suspended in 0.2ml PBS.
4. 180μl of Lysozyme digestion buffer and 20μl of Lysozyme [10mg/ml]
added.
5. Incubated at 37C for 15min.
6. 400μl of Binding buffer, 5μl of internal control template and 20μl of
Proteinase K added,
Mixed well by inverting several times.
7. Incubate at 56ºC for 15min.
8. Added 300μl of Ethanol and mixed well.
9. Transferred entire sample into the PureFast® spin column. Centrifuged for
1 min. Discard the flow-through and place the column back into the same
collection tube.
10. Added 500μl Wash buffer-1 to the PureFast® spin column. Centrifuge for
30-60 seconds and discard the flow-through. Place the column back into
the same collection tube.
11. Added 500μl Wash buffer-2 to the PureFast® spin column. Centrifuge for
30-60 seconds and discard the flow-through. Place the column back into
the same collection tube.
12. Discard the flow-through and centrifuge for an additional 1 min. This step
is essential to avoid residual ethanol.
13. Transferred the PureFast® spin column into a fresh 1.5 ml micro-
centrifuge tube.
14. Added 100μl of Elution Buffer to the center of PureFast® spin column
membrane.
15. Incubate for 1 min at room temperature and centrifuge for 2 min.
16. Discard the column and store the purified DNA at -20°C. Quality and
Quantity of extracted DNA is checked by loading in 1% agarose gel and
5μl of extracted DNA is used for PCR amplification.
PCR Procedure:
1. Reactions set up as follows;
Components Quantity
RedDye PCR Master mix 10μl
Ready to use - VIM gene primer mix 5μl
Purified Bacterial DNA 5μl
1. Total volume 20μl
2. Mixed gently and spin down briefly.
3. Place into PCR machine and program it as follows;
Initial Denaturation: 94ºC for 5 min
Denaturation: 94ºC for 30sec
Annealing: 58ºC for 30sec 35 cycles
Extension: 72ºC for 30sec
Final extension: 72º C for 5 min
Loading:
1. Prepared 2% agarose gel. [2gm of agarose in 100ml of 1X TAE buffer]
2. Run electrophoresis at 50V till the dye reaches three fourth distances and
observe the bands in UV Transilluminator.
Agarose gel electrophoresis:
1. Prepared 2% agarose. (2gm agarose in 100ml of 1X TAE buffer and
melted using micro oven)
2. When the agarose gel temperature was around 60ºC, added 5μl of Ethidium
bromide.
3. Poured warm agarose solution slowly into the gel platform.
4. Kept the gel set undisturbed till the agarose solidifies.
5. Poured 1XTAE buffer into submarine gel tank.
6. Carefully placed the gel platform into tank. Maintained the tank buffer
level 0.5cm above than the gel.
7. PCR Samples are loaded after mixed with gel loading dye along with 10μl
100bp DNA Ladder. [100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp,
800bp, 900bp, 1000bp and 1500bp]
8. Run electrophoresis at 50V till the dye reaches three fourth distance of the
gel.
9. Gel viewed in UV Transilluminator and observed the bands pattern.
RESULTS
Table:1 Age Distribution of Patients (n=200)
Age group No of patients Percentage
0-10 7 3.5
11-20 19 9.5
21-30 47 23.5
31-40 57 28.5
41-50 26 13
51-60 28 14
>60 16 8
Two hundred nonfermenting gram negative bacilli were isolated from all
age groups, it ranged from less than one year to 80 years, the youngest was 1 year
old child and the oldest was of 80 years age. the Table 1( Agewise distribution )
gives higher incidence of infection by NFGNB was seen in the age group of 31-40
years (28.5%) followed b 21-30 years age group(23.5%)
Table: 2. Gender wise distribution (n=200)
As per our study in table -2, the total numbers of males were 121(60.5%)
and females were 79(39.5%). The male to female ratio was 3.1:1.
Gender Number Percentage
Male 121 60.5%
Female 79 39.5%
46%
27%
9%
11%
7%
Sample Distribution (n=200)
Pus(wound infection)
Urine
Blood
Sputum
Body Fluids
0
10
20
30
40
50
60
Age Distribution of Patients (n-200)
No of patients Percentage
0-10 11-20 21-30 31-40 41-50 51-60 >60
TABLE 1
TABLE : 2
Table:3. Sample Distribution (n=200)
According to this table, most samples were obtained from pus 93
samples(46.5%) followed by urine53 samples(26.5%),Blood 18((9%) sputum
22(11%) and body fluids 14(7%)
Table: 4. Wardwise Distribution (n=200)
Sample Total No Percentage
Pus(wound
infection)
93 46.5%
Urine 53 26.5%
Blood 18 9%
Sputum 22 11%
Body Fluids 14 7%
Speciality No of cases Percentage
Burns Ward 73 36.5
Otorhinolaryngology 34 17
Surgery 22 11
Chest Medicine 19 9.5
Medicine 15 7.5
Intensive care unit 13 6.5
Urology 9 4.5
Labour Ward 9 4.5
Nephrology ward 6 3
As per the table 4(wardwise distribution), most samples are obtained from
burns ward 73(36.5%) followed by otorhinolaryngology 34 (17%), Surgery
22(11%), Medicine 15(7.5), chest Medicine 19(9.5%), Intensive care unit
13(6.5%), Urology 9(4.5%), Labour ward 9(4.5%) Nephrology 6(3%)
Table: 5. Risk Factors Associated With Infections By Nonfermenters (n=200)
According to table no 5 (Risk factors associated with infections by
nonfermenters) Diabetes Melitus 26 (13%) Road traffic accident (contaminated
wound) 21(10.5), Indwelling Catheter 13(6.5%), Intensive care unit stay
13(6.5%), Chronic renal disease 6(3%) Post renal transplantation 2(1%)
Risk Factor Number Percentage
Diabetes Melitus 26 13
Post operative wound infection 21 10.5
Indwelling catheter 13 6.5
Intensive care unit stay 13 6.5
Chronic renal disease 6 3
Post renal transplantation 2 1
0
10
20
30
40
50
60
70
80
Wardwise Distribution (n=200)
No of cases
Percentage
Table : 4
Table:5
0
5
10
15
20
25
30
Risk Factors Associated With Infections By Nonfermenters (n=200)
Number
Percentage
Table: 6. Grouping of Non Fermenting Gram Negative Bacilli (n=200)
According to Table no 6 (Grouping of Non fermenting gram negative
bacilli) Oxidase positive and motile is 129(64.5%) which includes Pseudomonas
aeruginosa Pseudomonas fluorescens, Burkholderia cepacia, Shewanella
putrefaciens, oxidase positive and non motile is 2(1%) includes Weeksella virosa,
Oxidase negative and motile is 3(1.5%) includes Stenotrophomonas maltophilia,
Oxidase negative and nonmotile is 66(33%) includes Acinetobacter baumannii,
Acinetobacter lwoffii
Group Number Percentage
Oxidase positive and Motile 129 64.5%
Oxidase positive and non motile 2 1%
Oxidase negative and Motile 3 1.5%
Oxidase negative and Nonmotile 66 33%
Total 200 100%
Table:7. Identification of Non Fermenting Gram Negative Bacilli Isolated
(n=200)
As per the table 7, predominant organism is Pseudomonas aeruginosa
117(58.5%) followed by Acinetobacter baumannii 60(30%), Pseudomonas
fluorescens 8(4%), Acinetobacter iwoffi 6(3%) Burkholderia cepacia 3(1.5%)
and Stenotrophomonas maltophilia 3(1.5%) and Weeksella virosa 2(1%).
Clinical Isolates Number Percentage
Pseudomonas aeruginosa 117 58.5%
Acinetobacter baumaunnii 60 30%
Pseudomonas fluorescens 8 4%
Acinetobacter lwoffii 6 3%
Burkholderia cepacia 3 1.5%
Stenotrophomonas maltophilia 3 1.5%
Weeksella virosa 2 1%
Shewanella putrefaciens 1 0.5%
Table: 8. Drug Susceptibility of Pseudomonas Aeruginosa (n=117)
According to Table no8, Pseudomonas aeruginosa shows 100% sensitive to
colistin, it shows higher sensitivity Pipercillin tazobactem 115(98.29%),
Imipenem 100(98.29%), Amikacin 81(69.23%), Ceftazidime 73(62.39%),
Gentamicin51 (43.58%), Ciprofloxacin 44 (3.60%), Levofloxacin 43 (36.75%),
Cefoperazone sulbactam 39 (33.3%), Cotrimoxazole 37(31.62%)
Drug Susceptible Percentage of susceptibility
Gentamicin 51 43.58%
Amikacin 81 69.23%
cotrimoxazole 37 31.62%
Ceftazidime 73 62.39%
Cefoperazone
sulbactam
39 33.3%
Pipercillin
tazobactem
115 98.29%
Imipenem 100 85.47%
Ciprofloxacin 44 37.60%
levofloxacin 43 36.75%
Colistin 117 100%
Table: 9. Drug Susceptibility of Acinetobacter Baumannii (n=60)
According to Table no 9, Acinetobacter baumannii shows 100% sensitive
to colistin(60), it shows higher sensitivity Pipercillin tazobactem 53(88.33%),
Imipenem 64(90%), Amikacin 48(80%), Ceftazidime 40(66.6%),
Gentamicin34(56.66%), Ciprofloxacin 40(66.6%), Levofloxacin 30(50%),
Cefoperazone sulbactam 40(66.6%), Cotrimoxazole 32(53.33%)
Drug Susceptible Percentage of susceptible
Gentamicin 34 56.66%
Amikacin 48 80%
Cotrimoxazole 32 53.33%
Imipenem 54 90%
Ceftazidime 40 66.6%
Ceftriaxone 52 86.6%
Cefoperazone
sulbactam
40 66.6%
Pipercillin
tazobactem
53 88.33%
Ciprofloxacin 40 66.6%
Levofloxacin 30 50%
Colistin 60 100%
Table: 10. Drug Susceptibility of other NFGNB
According to Table no 10, Drug susceptibility of different NFGNB, All
Burkholderia cepacia and Stenotrophomonas maltophilia shows sensitive to
cotrimoxazole and resistant to Amikacin. Weeksella virosa and Shewanella
putrefaciens shows sensitive to almost all drugs
Organisms Pseudomonas
fluorescens
(n=8)
Acinetobacter
lwoffii (n=6)
Burkholderia
cepacia (n=3)
Stenotrop
homonas
maltophilia (n=3)
Weeksella
virosa
(n=2)
Shewanella
putrefaciens
(n=1)
Amikacin 5 4 0 0 2 1
Ceftazidime 7 6 3 3 2 1
cotrimoxazole 3 2 3 3 1 0
Pipercillin
tazobactem 8 6 3 3 2 1
Imipenem 8 6 3 3 2 1
Ceftriaxone 6 4 2 2 2 1
Colistin 8 6 3 3 2 1
Table:11 Multidrug Resistant Nonfermenting gram negative bacilli (>2 drugs
resistant) (n=200)
According to the Table no 11, Multi drug Resistant among NFGNB that is
resistant to more than two drugs. Among 117 Pseudomonas aeruginosa ,86
organisms shows multi drug resistant, 49 MDR in Acinetobacter baumannii, 6
MDR in Pseudomonas fluorescens,5 MDR in Acinetobacter lwoffi, 1 MDR in
case of Burkholderia cepacia and Stenotrophomonas maltophilia each, 1MDR in
Shewanella putrefaciens and no MDR in Weeksella virosa
Organisms Total No MDR
Pseudomonas aeruginosa 117 86
Acinetobacter baumaunnii 60 49
Pseudomonas fluorescens 8 6
Acinetobacter lwoffii 6 5
Burkholderia cepacia 5 1
Stenotrophomonas maltophilia 3 1
Weeksella virosa 2 0
Shewanella putrefaciens 1 1
0
20
40
60
80
100
120
140
Multidrug Resistant Nonfermenting gram negative bacilli
(>2 drugs resistant) (n=200)
Total No
MDR
Table :10
Table :11
0
1
2
3
4
5
6
7
8
9Drug Susceptibility of Other - NFGNB
Pseudomonas fluorescens (n=8) Acinetobacter lwoffii (n=6)
Burkholderia cepacia (n=3) Stenotrophomonas maltophilia (n=3)
Weeksella virosa (n=2) Shewanella putrefaciens (n=1)
1. Amikacin
2. Ceftazidime
3. Cotrimoxazole
4. Pipercillin
Tazobactem
5. Imipenem
6. Ceftriaxone
7. Colistin
1 2 3 4 5 6 7
Table: 12 ESBL producers in NFGNB (n=200)
Clinical Isolates Total no ESBL Production
Pseudomonas aeruginosa 117 24
Acinetobacter baumaunnii 60 12
Pseudomonas fluorescens 8 2
Acinetobacter lwoffii 6 0
Burkholderia cepacia 5 0
Stenotrophomonas
maltophilia
3 0
Shewanella putrefaciens 1 0
Total 200 38
Table: 12
Chi-Square Test Value p-Value
Fisher's Exact Test 0.328 0.899
According to Table no 12, ESBL producers in non fermenting gram
negative bacilli, Twenty four of 117 Pseudomonas aeruginosa are ESBL
producers , twelve of 60 Acinetobacter baumannii, two of 8 Pseudomonas
fluorescens, no ESBL producers Acinetobacter lwoffii, Burkholderia cepacia,
Stenotrophomonas maltophilia , Shewanella putrefaciens. p value is insignificant
( p value>0.05) ie insignificant association between organisms and ESBL
production
Table: 13.ESBL Production in Sample (n=200)
Table 13
Chi-Square Test Value p-Value
Fisher's Exact Test 9.617 0.039
According to Table no 13, Sample wise ESBL producers, 18 ESBL
producers are from Pus sample, 5 from Urine sample, 4 from Blood sample, 9
from Sputum sample, 2 from Body fluids. p value is significant (p value<0.05) ie
significant association between samples and ESBL production
Sample Total No ESBL Percentage
Pus 93 18 19.3
urine 53 5 9.4
Blood 18 4 22.2
Sputum 22 9 40.9
Body Fluids 14 2 14.2
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5
ESBL Production in Sample (n=200)
Total No ESBL Percentage
1. Pus 2. urine 3. Blood 4. Sputum 5.Body Fluids
0
50
100
150
200
250
1 2 3 4 5 6 7 8
ESBL producers in NFGNB (n=200)
Total no ESBL Producer
1.Pseudomonas aeruginosa 2. Acinetobacter baumaunnii 3. Pseudomonas fluorescens 4. Acinetobacter lwoffii 5. Burkholderia cepacia 6.Stenotrophomonas maltophilia 7. Shewanella putrefaciens 8. Total organism
Table :12
Table :13
Table:14. MBL screening by Imipenem Resistance (n=200)
Clinical Isolates Total no Carbapenem
resistant Isolates
Percentage
Pseudomonas aeruginosa 117 17 14.5
Acinetobacter baumaunnii 60 8 13.3
Pseudomonas fluorescens 8 0 0
Acinetobacter lwoffii 6 0 0
Burkholderia cepacia 2 0 0
Stenotrophomonas maltophilia 1 0 0
Shewanella putrefaciens 1 0 0
Table 14
Chi-Square Test Value p-Value
Pearson Chi-Square 0.047 0.829
According to Table no 14 carbapenem resistant isolates in NFGNB,
seventeen of 117 Pseudomonas aeruginosa are imipenem resistant, eight of 60
isolates of Acinetobacter baumannii are imipenem resistant. No imipenem
resistant in Pseudomonas fluorescens, Acinetobacter lwoffii, Burkholderia
cepacia, Stenotrophomonas maltophilia, Shewanella putrefaciens. p value is
insignificant (p value>0.05) ie insignificant association between organisms and
carbapenem resistance.
Table: 15. MBL production in Non fermenters by Imipenem EDTA E Strip
Clinical Isolates Total no Carbapenem
resistant Isolates
MBL
Pseudomonas
aeruginosa
117 17 8
Acinetobacter
baumaunnii
60 8 5
Table 15
Chi-Square Test Value p-Value
Fisher's Exact Test 0.250 0.912
According to Table no 15,
MBL production in Non fermenters by
Imipenem EDTA E Strip ,Eight MBL producers (6.83%) in 17 Imipenem resistant
Pseudomonas aeruginosa (8MBL/117 total Pseudomonas aeruginosa), five MBL
producers (8.33%) in 8 Imipenem resistant Acinetobacter baumannii (8 MBL/ 60
total Acinetobacter baumannii). No MBL producers in other organisms
(Pseudomonas fluorescens, Acinetobacter lwoffii, Burkholderia cepacia,
Stenotrophomonas maltophilia, Shewanella putrefaciens) . p value is insignificant
(p value>0.05)) ie insignificant association between organisms and MBL
production.
117
175
60
8
8
0
20
40
60
80
100
120
140
Total no Carbapenam resistant Isolates
MBL
MBL production in Non fermenters by Imipenem EDTA
E Strip
Pseudomonas aeruginosa
Acinetobacter baumaunnii
Table :14
Table :15
0
20
40
60
80
100
120
140
1 2 3 4 5 6 7
MBL screening by Imipenem Resistance (n=200)
Total no Carbapenem resistant Isolates Percentage
1.Pseudomonas aeruginosa 2. Acinetobacter baumaunnii 3. Pseudomonas fluorescens 4. Acinetobacter lwoffii 5. Burkholderia cepacia 6.Stenotrophomonas maltophilia 7.Shewanella putrefaciens
Table:16. Comparison of MBL detection by different methods
Clinical Isolates Modified
Hodge Test
Double disc
synergy test
Combined
disc test
Imp EDTA
E strip
Pseudomonas
aeruginosa (n=17)
5 7 8 8
Acinetobacter
baumaunnii (n=8)
3 3 4 4
Table:16
Chi-Square Test Chi-Square Test Value p-Value
Pseudomonas
aeruginosa (n=17) Pearson Chi-Square 1.457 0.692
Acinetobacter
baumaunnii (n=8) Fisher's Exact Test 0.688 0.999
According to Table no 16, Comparison of MBL detection by different
methods such as Modified Hodge test, Double disc synergy test (DDST),
combined disc diffusion test (CDDT), Imipenem EDTA E strip method. p value is
insignificant (p value>0.05) for both Pseudomonas aeruginosa and Acinetobacter
baumaunnii ) ie insignificant association between organisms and MBL
production.
Table: 17.MBL production in samples (n=200)
Sample Total No MBL Percentage
Pus 93 9 9.6
urine 53 2 3.7
Blood 18 0 0
Sputum 22 1 4.5
Body Fluids 14 0 0
Table:18
Chi-Square Test Value p-Value
Fisher's Exact Test 3.014 0.496
According to Table no 17, sample wise distribution of MBL producers, 9
MBL producers from Pus sample, 2 from Urine sample and one from sputum
sample. No MBL producers in blood, body fluids sample. p value is insignificant
(p value>0.05) ie insignificant association between samples and MBL production.
Table: 18. Result of PCR in MBL Pseudomonas aeruginosa (n=8)
MBL Gene Total No of Cases Percentage
Positive 4 50%
Negative 4 50%
According to table 18, four of 8 (50%) MBL-positive isolates were
confirmed to be positive for MBL by PCR
Table: 19. Pattern of MBL Gene in Pseudomonas Aeruginosa n=8
Type of MBL gene Total number cases Percentage
Bla-VIM 4 50%
Bla-IMP - 0%
According to Table no 19, pattern of MBL gene in Pseudomonas
aeruginosa, Four of 8 (50%) MBL-positive isolates were confirmed to be positive
for MBL for the gene Bla VIM by PCR and no Bla IMP was found in the 8 MBL
producers
Table:20. AmpC βlactamase production in NFGNB
Clinical Isolates Total no Amp C Production
Pseudomonas
aeruginosa
117 52
Acinetobacter
baumaunnii
60 18
Pseudomonas
fluorescens
8 0
Acinetobacter lwoffii 6 0
Burkholderia cepacia 5 0
Stenotrophomonas
maltophilia
3 0
Shewanella putrefaciens 1 0
Total 200 70
Table -20
Chi-Square Test Value p-Value
Person Chi-Square 3.461 0.063
According to Table no 20, Amp C producers in NFGNB, Fifty two Amp C
producers (44.4%) in (Pseudomonas aeruginosa52/117 )and 18 AmpC
producers(30%) in Acinetobacter baumannii (18/60). No Amp C producers on
other NFGNB. p value is insignificant (p value>0.05) ie insignificant association
between organisms and Amp C production.
Table:17
Table:20
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5
Total No MBL Percentage
1. Pus 2. urine 3. Blood 4. Sputum 5.Body Fluids
0
50
100
150
200
250
1 2 3 4 5 6 7 8
Amp C βlactamase production in NFGNB
Total no Amp C Producer
1.Pseudomonas aeruginosa 2. Acinetobacter baumaunnii 3. Pseudomonas fluorescens 4. Acinetobacter lwoffii 5. Burkholderia cepacia 6.Stenotrophomonas maltophilia 7.Shewanella putrefaciens8. Total organism
Table:21. Amp C β lactamase producers in samples
Sample Total No Amp C
producers
Percentage
Pus 93 36 38.7
Urine 53 21 39.6
Blood 18 4 22.2
Sputum 22 6 27.2
Body Fluids 14 3 21.4
Table-21
Chi-Square Test Value p-Value
Fisher's Exact Test 3.789 0.438
According to Table no 21, Sample wise distribution of Amp C producers,
maximum Amp C producers seen in urine 21(39.6%), followed by Pus 36(38.7%),
sputum 6(27.2%), blood 4(22.2%) and body fluids 3(21.4%).p value is
insignificant (p value>0.05 ) ie insignificant association between samples and
Amp C production.
DISCUSSION
Two hundred isolates of non fermenting gram negative bacilli were taken
from various clinical samples like pus, urine, blood, sputum, body fluids and
evaluated for their role in infections in hospitalised patients including the
characteristics of their drug resistance.
Two hundred nonfermenting gram negative bacilli were isolated from all
age groups, study gives higher incidence of infection by NFGNB was seen in the
age group of 31-40 years (28.5%) followed b 21-30 years age group(23.5%)
which is in contrast with Usha Kalawat, Sumathi I, Jaya Prada, Satish Kumar
Reddy et al where 61-70 year age group(19%) and 51- 60 year age group(18%)
are common26
. This may be due to the reason that in our study most of the cases
are from burns ward(73/200), NFGNB infections are more common in the age
groups 21-30 years(23.5%) and 31-40(28.5%) years which is in concordance with
the study Enayatollah kalantar et al where 24.7% seen in 21-30 age groups and
19.8% seen in 31-40 age group 65.
The total numbers of males were 121(60.5%) and females were 79(39.5%).
The male to female ratio was 3.1:1. It shows higher incidence in male (60.5%)
compared to females(39.5%) which is in concordance with P. A. Shiny1, S.
Rajendran et al 25
.
Non fermenting gram negative bacilli obtained from various clinical
samples were collected, most samples were obtained from pus 93 samples(46.5%)
followed by urine53 samples(26.5%),Blood 18((9%) sputum 22(11%) and body
fluids 14(7%) which is concordance with study N sinha, J Agarwal , S srivastava,
M singh et al where maximum isolates pus 52/140 samples(37.14%) followed by
blood 32/140 samples(22.85%) and urine 19/140(13.57%)36
and also according to
N Ozkalay Yilmaz1, N Agus
1, E Bozcal
2, A Uzel
2 et al, Strains were dominantly
isolated from the pus (n: 16, 42.1%), also blood cultures (n: 10, 26.3%), urinary
tract (n: 6, 15.8%) and tracheal aspirate specimens (n: 6, 15.8%)4
Most samples are obtained from burns ward 73(36.5%) followed by
otorhinolaryngology 34 (17%), Surgery 22(11%), Medicine 15(7.5), chest
Medicine 19(9.5%), Intensive care unit 13(6.5%), Urology 9(4.5%), Labour ward
9(4.5%) Nephrology 6(3%) in contrast to N sinha, J Agarwal , S srivastava, M
singh et al where maximum cases are from ICU(22.14%) followed by
paediatrics(20.71%), neurosurgery(15.71%) and general surgery wards (12.85%)36
and also in the study N Ozkalay Yilmaz1, N Agus
1, E Bozcal
2, A Uzel
2 et
al,Bacterial strains were isolated mainly from intensive care unit (n: 18, 47%),
general surgery (n: 12, 32%) and internal medicine (n: 8, 21%).4
Most common risk factors associated with infections by nonfermenters
were Diabetes Melitus 26( 13%) followed by Post op wound infection, 21(10.5),
Indwelling Catheter 13(6.5%), Intensive care unit stay 13(6.5%), Chronic renal
disease 6(3%) Post renal transplantation 2(1%), which is in concordance with
Sherertz .R.J and F A Sarubbi 1983 et al where Post op wound infection infection
rate is 2.08%, Intensive care unit stay (22.9%) (especially Lower respiratory
tract) 53
which is also in concordance with Meharwal SK , Taneja N, Sharma SK,.
et al, where Postoperative wound infection accounts for 42.6%, chronic renal
failure(5.3%), indwelling wrinary catheter (2.6%), Diabetes mellitus(1.3%), Post
renal transplant(8.1%)54
Grouping of Non fermenting gram negative bacilli were Oxidase positive
and motile organisms were 129(64.5%) which includes Pseudomonas aeruginosa,
Pseudomonas fluorescens, Burkholderia cepacia, Shewanella putrefaciens ,
oxidase positive and non motile organisms were 2(1%) includes Weeksella virosa,
Oxidase negative and motile organisms were 3(1.5%) includes Stenotrophomonas
maltophilia, Oxidase negative and nonmotile organisms were 66(33%) includes
Acinetobacter baumannii, Acinetobacter lwoffii
Most predominant organism was Pseudomonas aeruginosa 117(58.5%)
followed by Acinetobacter baumannii 60(30%), Pseudomonas fluorescens
8(4%), Acinetobacter iwoffi 6(3%) Burkholderia cepacia 3(1.5%) and
Stenotrophomonas maltophilia 3(1.5%) and Weeksella virosa 2(1%). Which is in
contrast with S Arora, V Gautam, P Ray at al, 18% (1781/9662) grew NFGNBs.
Acinetobacter spp. (62%) was the most common followed by Pseudomonas
aeruginosa (18%), Burkholderia cepacia complex (5%) and S. maltophilia (3%).
12% (221/1781) of the NFGNBs could not be identified. 29ia
and in concordance
with ziad et al,
Pseudomonas aeruginosa (72.9%%) was the most common
followed by Acinetobacter spp (25.3%), S. maltophilia(1.8%)
Pseudomonas aeruginosa shows 100% sensitive to colistin, it shows
higher sensitivity Pipercillin tazobactem 115(98.29%), Imipenem 100(98.29%),
Amikacin 81(69.23%),Ceftazidime 73(62.39%), Gentamicin51(43.58%),
Ciprofloxacin 44(3.60%), Levofloxacin 43(36.75%), Cefoperazone sulbactam
39(33.3%), Cotrimoxazole 37(31.62%) which is in concordance with B Behera1, P
Mathur2, A Das
3, A Kapil
3, V Sharma
1 et al of the 91 isolates of Pseudomonas
aeruginosa, 64 (70%) were resistant to ceftazidime, 68 (75%) to piperacillin, 54
(59%) to piperacilin/tazobactam, 58 (63%) to ticarcillin/clavulanic acid, 75 (82%)
to cefoperazone, 67(74%) to amikacin, 74 (81%) to cefepime, 65 (71%) to
levofloxacin, and 72 (79%) to ciprofloxacin by the disc diffuse ion (CLSI)
method.24
Acinetobacter baumannii shows 100% sensitive to colistin(60), it shows higher
sensitivity Pipercillin tazobactem 53 (88.33%), Imipenem 64 (90%), Amikacin
48(80%),Ceftazidime 40 (66.6%), Gentamicin34 (56.66%), Ciprofloxacin 40
(66.6%), Levofloxacin 30 (50%), Cefoperazone sulbactam 40 (66.6%),
Cotrimoxazole 32 (53.33%) which is in concordance with Reza Mirnejad ,
Somayeh Vafaei et al where highest antibiotic resistance in 100 isolates of the
Acinetobacter baumannii was related to antibiotics namely: cefepime (100%),
ceftriaxone (95%), amikacin (95%), imipenem (76%), piperacillin - tazobactam
(70%), meropenem (69%), gentamicin (63%), tobramycin (56%), tetracycline
(51%), ampicillin - sulbactam (49%) and the lowest resistance to polymyxin B73
Drug susceptibility of different NFGNB, all Burkholderia cepacia and
Stenotrophomonas maltophilia shows sensitive to cotrimoxazole (3/3) and
resistant to Amikacin (0/3) each which is in concordance with Malavalli
Venkatesh Bhavana, Sangeetha Joshi et al where Burkholderia cepacia shows
100% sensitive to cotrimoxazole and Stenotrophomonas maltophilia shows 90%
sensitive to cotrimoxazole. Cotrimoxazole exhibited very good susceptibility
against both the isolates. According to the SENTRY Antimicrobial Surveillance
Program, which monitors the predominant community-acquired and nosocomial
pathogens including their antimicrobial resistance globally, cotrimoxazole was
found to have an excellent susceptibility against both these organisms. In the
Indian scenario, Gautam et al. from North India have reported susceptibility rates
of 75%–80% among Burkholderia cepacia complex isolates and 70%–90%
among Stenotrophomonas maltophilia isolates to cotrimoxazole.72
As per a study
conducted in a tertiary care centre of coastal Karnataka by Chawla et al., 86.7%
susceptibility to cotrimoxazole for Stenotrophomonas maltophilia was seen.71
Weeksella virosa and Shewanella putrefaciens shows sensitive to almost all drugs
Multi drug Resistant among NFGNB that is resistant to more than two
drugs. Among 117 Pseudomonas aeruginosa ,86 organisms shows multi drug
resistant, 49 MDR in 60 Acinetobacter baumannii, 6 MDR in Pseudomonas
fluorescens,5 MDR in Acinetobacter lwoffi, 1 MDR in case of Burkholderia
cepacia and Stenotrophomonas maltophilia each, 1MDR in Shewanella
putrefaciens and no MDR in Weeksella virosa. This is higher than the studies in
India reported previously in the year 1998 (Vijaya et al) and 2000.(Veenu et al) at
31% & 62% respectively and comparable to the study .by Patwardhan et el in
2008 who showed >68% of Pseudomonas isolates and >90% Acinetobacter
isolates were multidrug resistant.60,59,58
While Stenotrophomonas maltophilia is
intrinsically resistant to antibiotics, Pseudomonas aeruginosa & Acinetobacter
baumannii acquire resistance by many different mechanism like Extended
spectrum of betalactamases (ESBL) and Metallobetalactamases(MBL). This is of
concern as efficacious antimicrobial options are limited.61
The present study
showing Acinetobacter as more multidrug resistant than Pseudomonas correlated
with the studies by Tanya et al (53%and49%) and Homer et al(62% and 58%)56, 57
ESBL producers in non fermenting gram negative bacilli, Twenty four of
117 Pseudomonas aeruginosa are ESBL producers, twelve of 60 Acinetobacter
baumannii, two of 8 Pseudomonas fluorescens, no ESBL producers in
Acinetobacter lwoffii, Burkholderia cepacia, Stenotrophomonas maltophilia,
Shewanella putrefaciens. While Stenotrophomonas maltophilia and Burkholderia
cepacia show intrinsic resistance , ESBL production by Pseudomonas aeruginosa
and Acinetobacter baumannii is significant 64
All the ESBL producers were
multidrug resistant. Maximum sensitivity among the ESBL producers was seen
with Imipenem (82%) followed by piperacillin – Tazobactam (60%) as seen in
the studies conducted by Brauers et al and Freshteh et al62,63
Sample wise ESBL producers, 18 ESBL producers are from Pus sample, 5
from Urine sample, 4 from Blood sample, 9 from Sputum sample, 2 from Body
fluids which is in concordance with Shahanara Begum Md Abdus Salam et al,
where out of 31 ESBL producers, 18 ESBL producers from pus and swab,12 from
urine and 8 from sputum74
Carbapenem resistant isolates in NFGNB, seventeen of 117 Pseudomonas
aeruginosa are imipenem resistant, eight of 60 isolates of Acinetobacter
baumannii are imipenem resistant. No imipenem resistant in Pseudomonas
fluorescens, Acinetobacter lwoffii, Burkholderia cepacia, Stenotrophomonas
maltophilia, Shewanella putrefaciens. This carbapenem resistant was tested with
imipenem in our study, in which out of 17 imipenem resistant screening for MBL
production, only 7 were found to be MBL producers phenotype method and in
that only 4 turned to be positive by genotype method (4 blaVIM, no bla IMP ) by
PCR shows that imipenem resistant screening test is not that sensitive for MBL
production detection . This is in concordance with S Buchunde et al where Out of
326 Pseudomonas aeruginosa strains studied, 63 (19.3%) were resistant to
meropenem, 63 (19.3%) to CAZ and 58 (17.8%) to Imipenem (screen-test
positive). IPM failed to pick up five isolates that were found resistant to
meropenem and Ceftazidime. meropenem DDST and Ceftazidime DDST
confirmed all the 63 screen-test positives, while Imipenem DDST 88.9% of the
screen-test positives. Using criteria by Yong D et al. 69
for Disc potentiation test
(DPT-1), meropenem confirmed 100% screen-test positive isolates, Imipenem
85.7% and Ceftazidime 76.2% ( p>0.05). However, when criteria given by
Hemlatha et al. 70
for Ceftazidime DPT was used (DPT-2), Ceftazidime was able
to confirm additional 11 (17.4%) isolates, thereby raising the positivity of
Ceftazidime DPT to 59 (93.7%). All the 63 screen-test positive isolates, including
those not picked by Imipenem, were found to be positive for the presence of bla
using PCR.68
MBL production in Non fermenters by Imipenem EDTA E Strip ,Eight
MBL producers(6.83%) in 17 Imipenem resistant Pseudomonas aeruginosa
(8MBL/117 total Pseudomonas aeruginosa), five MBL producers(8.33%) in 8
Imipenem resistant Acinetobacter baumannii(8 MBL/ 60 total Acinetobacter
baumannii). No MBL producers in other organisms (Pseudomonas fluorescens,
Acinetobacter lwoffii, Burkholderia cepacia, Stenotrophomonas maltophilia ,
Shewanella putrefaciens. Out of 117 total Pseudomonas aeruginosa isolates, 17
were resistant for Imipenem and screened for MBL production. 8 were positive
for MBL by both DDST and MIC reduction test which is in concordance with G
Agrawal et al, out of 174 total isolates, 18 were resistant for Imipenem and
screened for MBL production. 14 were positive for MBL by both DDST and MIC
reduction test66
Higher MBL production is seen in Acinetobacter baumannii (8.33%) than
Pseudomonas aeruginosa (6.83%) which is in concordance with s John el al
where higher MBL production is seen in Acinetobacter baumannii (27.7%) than
Pseudomonas aeruginosa (14.8%)64.
Comparison of MBL detection by different methods such as Modified
Hodge test, double disc synergy test (DDST), combined disc diffusion test
(CDDT), Imipenem EDTA E strip method. In Pseudomonas aeruginosa, among
17 Imipenem resistant isolates MBL producers are identified by Modified Hodge
test 5, Double disc synergy test (DDST) 7, combined disc diffusion test (CDDT)
8, Imipenem EDTA E strip method 8. In case of Acinetobacter baumannii, MBL
producers are identified by as Modified Hodge test 3, Double disc synergy test
(DDST) 3, combined disc diffusion test (CDDT) 4, Imipenem EDTA E strip
method 4 . This is in concordance with B Behera1, P Mathur
2, A Das
3, A Kapil
3, V
Sharma et al Of the 63-imipenem resistant isolates, 56 isolates were tested for
MBL production. Forty-eight of these 56 isolates shows positive in the combined
disc test, whereas 36 isolates gave positive result by DDST. MBL E test was done
in 30 isolates (in 26 of which, both DDST and combined disc method
demonstrated MBL production and four isolates were randomly selected from the
12 which gave a positive combined disc result and negative DDST result36.
Sample wise distribution of MBL producers, 9 MBL producers from Pus
sample, 2 from Urine sample and one from sputum sample. No MBL producers in
blood, body fluids sample. This is in concordance with N Ozkalay Yilmaz et al,
where among 38 imipenem resistant isolates, maximum isolated from pus sample
16(42.1%), blood culture 10(26.3%), urinary tract 6(15.8%) and tracheal aspirate
6(15.8%) and majority of MBL production is from pus sample4
According to our study,the MBL enzyme was found positive in 8 of 17
(47.05%) IMP resistant Pseudomonas aeruginosa isolates by DDST and Imipenem
EDTA E strip. And according to table 18, Four of 8 (50%) MBL-positive isolates
were confirmed to be positive for MBL by PCR. According to N Ozkalay
Yilmaz1, N Agus
1, E Bozcal
2, A Uzel et al, MBL enzyme was found positive in 27
of 38 (71.05%) IMP resistant Pseudomonas aeruginosa isolates by DDST. Eight
of 38 (21.05%) MBL-positive isolates were confirmed to be positive for MBL by
PCR. 28,4
Pattern of MBL gene in Pseudomonas aeruginosa, Four of 8 (50%) MBL-
positive isolates were confirmed to be positive for MBL for the gene Bla VIM by
PCR and no Bla IMP was found in the 8 MBL producers where is in concordance
with AK Pragasam1, S Vijayakumar
1, YD Bakthavatchalam
1, A Kapil
2, BK Das et
al in which among the class B carbapenemases (MBLs), blaIMP, blaVIM, blaNDM
was identified except blaSPM. Within that, blaVIM were detected in 24%–57%,
blaNDM were 8%–19% and blaIMP were 4%–5%, respectively.9
and also this is
concordance with M Purohit1, DK Mendiratta
1, VS Deotale
1, M Madhan
2, A
Manoharan2, P Narang et al, we found bla-VIM MBL gene only in 7 (16.28%) of
the 43 screen test positive isolates. None of our isolates showed presence of bla-
IMP gene39
AmpC producers in NFGNB, Fifty two AmpC producers (44.4%) in
(Pseudomonas aeruginosa 52/117 )and 18 Amp producers(30%) in Acinetobacter
baumannii(18/60). No AmpC producers on other NFGNB. Higher incidence of
AmpC production is seen in Pseudomonas aeruginosa (44.4%) than
Acinetobacter baumannii (30%) which is in contrast with with Noyal et al where
Higher incidence of AmpC production is seen in Acinetobacter baumannii
(67.4%) than Pseudomonas aeruginosa (46.9%)55
Samplewise distribution of Amp C producers, maximum Amc C producers
seen in urine 21(39.6%), followed by Pus 36(38.7%), sputum 6(27.2%), blood
4(22.2%) and bodyfluids 3(21.4%) which is in concordance with S Upadhyay1, S
Mishra1, MR Sen
1, T Banerjee
1, A Bhattacharjee et al where Strains harbouring
chromosomal and plasmidic AmpC gene (i.e., n0 = 64) were predominantly
cultured from pus ( n = 26), urine ( n = 13), burn wound ( n = 09), blood ( n = 06),
endotracheal tube (ETT) aspirates ( n = 07) and other body sites/fluids ( n = 03).36
SUMMARY
Two hundred nonfermenting gram negative bacilli were isolated from all
age groups, The total numbers of males were 121(60.5%) and females were
79(39.5%). The male to female ratio was 3.1:1.
Most samples were obtained from pus 93 samples (46.5%) followed by
urine 53 samples (26.5%). Most samples are obtained from burns ward
73(36.5%) followed by otorhinolaryngology 34 (17%), Surgery 22(11%).
Risk factors associated with infections by nonfermenters are Diabetes
Mellitus 26 ( 13%), Post surgical wound infection 21(10.5).
Most predominant organism is Pseudomonas aeruginosa 117(58.5%)
followed by Acinetobacter baumannii 60(30%), Pseudomonas fluorescens
8(4%), Acinetobacter lwoffi 6(3%) Burkholderia cepacia 3(1.5%) and
Stenotrophomonas maltophilia 3(1.5%) and Weeksella virosa 2(1%).
Pseudomonas aeruginosa shows higher sensitivity Pipercillin tazobactem
115(98.29%), Imipenem 100(85.47%), Amikacin 81(69.23%),Ceftazidime
73(62.39%) and least to Cotrimoxazole 37(31.62%). It shows 100%
sensitive to colistin
Acinetobacter baumannii shows higher sensitivity Pipercillin tazobactem
53(88.33%), Imipenem 64(90%), Amikacin 48(80%),Ceftazidime
40(66.6%). It shows 100% sensitive to colistin (60),
Drug susceptibility of different NFGNB are that all Burkholderia cepacia
and Stenotrophomonas maltophilia shows sensitive to cotrimoxazole and
resistant to Amikacin. Weeksella virosa and Shewanella putrefaciens
shows sensitive to almost all drugs
Multi drug Resistant among NFGNB that is resistant to more than two
drugs. Among 117 Pseudomonas aeruginosa ,86 organisms shows multi
drug resistant, 49 MDR in Acinetobacter baumannii, 6 MDR in
Pseudomonas fluorescens,5 MDR in Acinetobacter lwoffi, 1 MDR in case
of Burkholderia cepacia, Stenotrophomonas maltophilia and Shewanella
putrefaciens
ESBL producers in non fermenting gram negative bacilli, Twenty four of
117 Pseudomonas aeruginosa are ESBL producers, twelve of 60
Acinetobacter baumannii, two of 8 Pseudomonas fluorescens, no ESBL
producers in other NFGNB.
Carbapenem resistant isolates in NFGNB are seventeen of 117
Pseudomonas aeruginosa are imipenem resistant, eight of 60 isolates of
Acinetobacter baumannii are imipenem resistant. No imipenem resistant in
other NFGNB.
MBL production in Non fermenters by Imipenem EDTA E Strip , Eight
MBL producers (6.83%) in 17 Imipenem resistant Pseudomonas
aeruginosa (8MBL/117 total Pseudomonas aeruginosa), five MBL
producers(8.33%) in 8 Imipenem resistant Acinetobacter baumannii (8
MBL/ 60 total Acinetobacter baumannii). No MBL producers in other
organisms
Comparison of MBL detection by different methods such as Modified
Hodge test , Double disc synergy test (DDST), combined disc diffusion
test(CDDT), Imipenem EDTA E strip method were done and found
Combined disc diffusion test (CDDT) and Imipenem EDTA E strip method
were more sensitive for MBL screening than Modified Hodge test , Double
disc synergy test(DDST) .
Four of 8 (50%) MBL-positive isolates were confirmed to be positive for
MBL by PCR for the gene Bla VIM by PCR and no Bla IMP was found in
the 8 MBL producers.
AmpC producers in NFGNB are Fifty two Amp C producers(44.4%) in
(Pseudomonas aeruginosa52/117 )and 18 Amp C producers(30%) in
Acinetobacter baumannii(18/60). No Amp C producers on other NFGNB.
Early diagnosis of Nonfermenting gram negative bacilli and administration
of specific antibiotic therapy, based on the antibiogram of the isolated
pathogen will reduce the complication as well as the emergence of drug
resistance, there by hasten the recovery of patients. This will lead to better
outcome and effective patient care
CONCLUSION
In our study, 200 nonfermenting gram negative bacilli were isolated from
various clinical samples. Higher incidence of infection by NFGNB was
seen in the age group of 31-40 years with male preponderance
Maximum samples were obtained from pus samples especially from Burns
ward and the maximum risk factors associated with infections by
nonfermenters are Diabetes Mellitus.
Most predominant organism is Pseudomonas aeruginosa followed by
Acinetobacter baumannii
Pseudomonas aeruginosa and Acinetobacter baumannii shows higher
sensitivity to Pipercillin tazobactem and Imipenem. Stenotrophomonas
maltophilia and Burkholderia cepacia shows sensitive to cotrimoxazole
and resistant to Amikacin. Weeksella virosa and Shewanella putrefaciens
shows sensitive to almost all drugs. All NFGNB were 100% sensitive to
colistin.
In Pseudomonas aeruginosa, Acinetobacter baumannii ESBL producers,
MBL producers and Amp C producers were seen.
MBL-positive isolates were confirmed to be positive for the gene Bla VIM
and no Bla IMP.
The present study showed emerging resistance of Nonfermenting gram
negative bacilli especially Pseudomonas aeruginosa and Acinetobacter
baumannii which may lead to increase in morbidity & mortality that can
be controlled by strict enforcement of antibiotic policy coupled with strict
adherence to infection control measures to prevent further emergence and
spread of antibiotic resistance.
PROFORMA
Name: Dept / Ward:
Age/Sex IP.No / OP No:
Address: Lab No:
Occupation: Date of Admission:
Present Complaints: Diagnosis:
Past History & Treatment:
Type of Clinical Sample:
Microscopic Finding:
Gram Staining:
Culture Characteristics:
Blood Agar:
MacConkey Agar:
CLED Agar:
Biochemical Characteristics:
Antibiotic Susceptibility Pattern:
Signature of Investigator Signature of Guide
APPENDIX
PEPTONE WATER
Peptone - 10g
Sodium chloride - 5g
Distilled water - 1 litre
Dissolve the ingredients in warm water, adjust the pH to 7.4 -7.5 and filter.
Distribute as required and autoclave at 121 degree Celsius for 15 mins.
BLOOD AGAR
Sterile sheep blood - 50 ml
Peptone - 10 g
Beef extract - 3g
Sodium chloride - 5 g
Distilled water - 1000 ml
Autoclave the nutrient agar base at 121º C for 15 minutes and blood with sterile
precautions and distribute in Petri dishes.
MAC CONKEY AGAR
Peptic digest of animal tissue - 17g
Proteose peptone - 3g
Lactose - 10g
Bile salts - 1.5g
Sodium chloride - 5g
Neutral red - 0.03g
Agar - 15g
Distilled water - 1000 ml
Final pH at (25º C) 7.1±0.2.
Suspend 51.53 grams in 1000 ml of distilled water. Heat to boiling to
dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure
(121ºC) for 15 minutes. Mix well and pour into Petri dish plates.
NUTRIENT AGAR
Peptic digest of animal tissue - 5g
Beef extract - 1.5g
Yeast extract - 5g
Agar - 15g
Distilled water - 1000ml
Dissolve the contents in water and mix by heating. Autoclave at 121° C for
15 minutes. Adjust pH to 7.4 + 0.2. Pour 20-25 ml of 9 cm diameter. Petri dishes
to give 4 mm thickness
1. OXIDASE TEST 6
This test is mainly done to differentiate organisms lacking cytochrome
oxidase enzyme, mainly the members of enterobactericeae. This enzyme helps in
the transfer of electrons to oxygen, with formation of water. The dye tetramethyl
paraphenylene diamine dihydrochloride is substituted for oxygen as artificial
electron acceptor. In the reduced state, it is colorless and in the presence of
cytochrome oxidase and oxygen it forms indophenol, which is purple. Strips
impregnated with dried reagents were used. The colony was taken on a wooden
stick and smeared onto the strip. The appearance of purple color within 10 sec.
was taken as positive.
2. CATALASE TEST 6
Organisms’ possessing the enzyme catalase splits hydrogen peroxide into
water and oxygen. The evolution of oxygen appears as bubbles. Tube catalse test
was performed 3ml of 3% Hydrogen peroxide was taken in a test tube. The colony
of the organism to be tested was taken on a glass rod and introduced into the tube.
Appearance of brisk effervescence was taken as positive.
3. MOTILITY6
Motility was done by hanging drop method. A drop of saline with the test
organism was placed on the coverslip. The hanging drop slide is inverted over the
coverslip where wax had been applied on the corners. The slide is turned quickly
so that drop is in centre of the concavity. The edge of the drop is focused on the
low power and high power objectives. Motility was observed.
4. INDOLE TEST6
Indole is a benzyl pyroll which is a metabolic product of tryptophan.
Bacteria which possess tryptophanase hydrolyse trytophan to indole. The
organism was inoculated in peptone water medium, incubated at 370C for 18-24
hours, 1ml of zylene was added and Erlichs’reagent (Para dimethyl amino
benzaldehyde) was added drop by drop. Formation of fuchsia red ring was
positive. It is a red complex of indole and paradimethylaminobenzaldehyde.
5. TRIPLE SUGAR IRON MEDIUM 6
Triple sugar iron medium was taken and the organism was stabbed to the
butt as well streaked on the surface. It was incubated for 18-24hours at 370C, and
then looked for the presence of growth and change in pH Growth with no change
in PH (slant and butt) indicated the organism to be nonfermenting.
6. CITRATE UTILIZATION TEST 6
Sodium citrate is a salt of citric acid seen in metabolism in Krebs cycle.
Some bacteria utilize citrate as the sole source of carbon and it is detected by the
production of alkaline by products Christensen’s citrate media was used. The
organisms were streaked on the surface of the slant and incubated at 370C for 18-
24hrs. Development of deep blue color of the medium with growth was taken as
positive.
8. UREASE TEST 6
Urea is a diamide carbonic acid which when hydrolyzed releases ammonia
and carbon dioxide. Urease is an enzyme when present hydrolyses urea and
release ammonia changing the medium to alkaline, PH.
The organism was streaked on the surface of the slant and incubated at
370C for 18-24 hours. Development of magenta pink of the medium along with
growth was taken as positive.
9. NITRATE REDUCTION TEST 6
The capacity of an organism to reduce nitrates to nitrites is shown by this
test. The presence of nitrate in the medium is detected by addition of a
naphthalamine and sulphanilic acid.
The organism was inoculated in nitrate broth and incubated at 370C for 18-
24 hours and observed for gas production by Durham’s tube. One ml each of
reagents a naphthalamine & sulphanilic acid were added simultaneously and
looked for the development of red color.Development of red color was taken as
positive and when it was negative zinc dust was added. When the red color
developed after adding zinc the test was taken as negative because it indicated the
presence of residual nitrates.
10. GROWTH AT 42ᴼC 6
The organism was plated in nutrient agar plate and incubated at 420C for
18-24 hours and looked for growth. Presence of uniform growth indicated positive
results.
11. GROWTH AT 44ᴼC 6
The organism was plated in nutrient agar plate and incubated at 440C for
18-24 hours and looked for growth. Presence of uniform growth indicated positive
results.
12. POLYMYXIN B SENSITIVITY 6
The organism was plated on nutrient agar plate and a polymyxin B disc of
300U was kept. It was incubated at 370C for 18-24 hrs and looked for zone of
inhibition
SPECIAL BIOCHEMICAL TESTS USED FOR IDENTIFICATION OF
NON FERMENTERS
1. HUGH – LEIFSON OXIDATION - FERMENTATION MEDIUM6
Peptone: 2g
Sodium Chloride: 5g
D-Glucose: 10g
Bromothymol blue: 0.03g
Agar: 3.0g
Dipotassium Phosphate: 0.3g
Distilled water: 1 litre
pH: 7%
Medium was sterilized by autoclaving. After cooling the medium to 450C,
filter sterilized carbohydrate solution was added to get a final concentration of 1%
Carbohydrates used for the study were glucose, maltose, xylose, fructose and
mannitol. Of medium was poured as a butt and without a slant into tubes. Two
tubes were required for the test, each inoculated with the unknown organism,
using a straight needle stabbing the medium three to four times half way to the
bottom of the tube. One tube of each pair was covered with a 1cm layer of sterile
mineral oil (or) melted paraffin, leaving the other open to the air. Both tubes were
incubated at 350C and examined daily for several days.
In case of oxidative metabolism, yellow color appears along the upper one
fourth of the medium and in the tube where no oil overlay was done. In case of
fermentative organisms yellow color develops in both the tubes.
CONTROL
Glucose fermentation: Escherichia coli
Glucose oxidation: Pseudomonas aeruginosa
Non saccharolytic: Alcaligenes species.
2. DECARBOXYLATION OF LYSINE, ARGININE & ORNITHINE 6
Decarboxylases are a group of specific enzymes which react with carboxyl
portion of aminoacid forming alkaline reacting amines. The reaction is
decarboxylation. Each enzyme is specific for Lysine, Arginine and Ornithine.
INGREDIENTS
Yeast extract: 5g
Peptone: 5g
Glucose: 0.5g
Pyridoxal: 5mg
Bromocresol Purple: 5ml
Cresol Red: 2.5ml
Distilled water: 1 ltr.
All aminoacids were added individually to a final concentration of 1% pH
adjusted at 6.0 & autoclaved at 15lbs for 15 minutes.
PROCEDURE
The organism was inoculated in four tubes. One having the basal medium
without aminoacid for control. Other three tubes having lysine, arginine and
ornithine each. All tubes were overlaid with liquid paraffin. All were incubated at
370C for 24 hours.
The control tube turned yellow indicating that the organism is viable and
the test medium turning blue purple indicating positive result.
3. O–NITROPHENYL β - D GALACTOPYRANOSIDE 6
Reagent
Sodium Phosphate buffer 1M pH 7.0 – 5ml
O – Nitrophenyl β - D – galactopyranoside – 80mg
Distilled water – 15ml.
A dense suspension of the test organism grown in TSI agar was prepared in
saline.About 1 drop of toluene was added to the suspension and 0.2ml of ONPG
solution was added to the suspension and incubated at 370C β -galactosidase
producing organism show yellow color after 1 hour or 18-24 hours incubation.
CONTROL
Positive Control – Escherichia coli
Negative control – Proteus species.
4. ACETAMIDE AGAR
Ingredients
Magnesium Sulphate: 0.2g
Ammonium dihydrogen Phosphate: 1g
Pottasium monohydrogen phosphate: 1g
Sodium Chloride: 5g
Acetamide: 10g
Bromothymol blue solution: 6.4ml
Agar: 15g
Final pH: 6.9
Distilled water: 1 litre
The ingredients are mixed and pH adjusted to 6.9, dispensed into screw cap
tubes and sterilized at 1210C for 15 min. The medium was allowed to cool in a
slant. The slant was inoculated with a portion of isolated colony and incubated at
370C overnight and was observed for color change. Tubes with negative result
were further incubated for 7days.
Control
Positive control: Pseudomonas aeruginosa
Negative Control: Stenotrophomonas maltophilia
5. GELATIN LIQUEFACTION TEST 6
Gelatin breakdown can be demonstrated by incorporating it in a buffered
nutrient agar, growing the culture and then flooding the medium with mercuric
chloride that differentially precipitates either gelatin or its breakdown products
causing opacity in the medium with clear zones around gelatin-liquefying colonies
CONTROL
Positive control: Pseudomonas aeruginosa
Negative Control: Stenotrophomonas maltophilia
PCR Primers and procedure
Two sets of primers were used for multiplex PCR. The primers used are
given below
IMP FORWARD – 5’ CTA CCG CCG CAG CAG AGT CTT TG -3’
REVERSE 5’-AAC CAG TTT TGC CTT ACC AT-3’
VIM FORWARD 5’- AGT GGT GAG TAT CCG ACA G -3’
REVERSE 5’-ATG AAA GTG CGT GGA GAC -3
PCR was conducted with 1μl of boiled bacterial suspensions, 0.5μl of each
primer, 2μl 5mM of dNTP, 2.μl 10mM Tris-HCL (pH 8.3), 0.75 μl 50mM Mgcl2
and 2U of Taq DNA polymerase in a total volume of 20μl
ABBREVIATIONS
NFGNB - Nonfermenting gram negative bacilli
NF - Nonfermenters
MDR - Multidrug resistant
PDR - Pandrug resistant
XDR - Extremely drug resistant
ESBL - Extended Spectrum of Betalactamases
MBL - Metallo betalactamases
ONPG - O–Nitrophenyl β - D Galactopyranoside
ATCC - American Type Culture Collection
CAPD - Chronic Ambulatory Peritoneal Dialysis
CFU - Colony Forming Units.
CLED - Cystine Lactose Electrolyte Deficient
CLSI - Central Laboratory Standards Institute
ELISA - Enzyme Linked Immunosorbent Assay
E- TEST - Epsilometer Test
FDA - Food and Drug Administration (United States
USFDA)
ICU - Intensive Care Unit
OPD - Out Patient Department
MALDI –TOF - Matrix Assisted Laser Desorption/Ionisation –
Time of Flight.
DDST - Double Disc Synergy Test
CDDT - Combined Disc Diffusion Test
DPT - Disc Potentiation Test
MHA - Mueller Hinton Agar
MIC - Minimum Inhibition Concentration
PCR - Polymerase Chain Reaction
PFGE - Pulsed-Field Gel Electrophoresis
SPSS - Statistical Package for Social Science
KEY TO MASTER CHART
M - Male patient
F - Female patient
IP - Inpatient
OP - Outpatient
LBW - Labour Ward
ICU - Intensive Care Unit
ENT - Otorhinolaryngology
UTI - Urinary tract infection
DM - Diabetes Mellitus
RTA - Road Traffic Accident
P - Positive
N - Negative
S - Susceptible
R - Resistant
AMP - Ampicillin
AMX - Amoxicillin
CN - Cephalexin
CTR - Ceftriaxone
CAZ - Ceftazidime
AK - Amikacin
GEN - Gentamicin
CIP - Ciproflaxacin
CFS - Cefoperazone Sulbactam
COT - Cotrimoxazole
PIT - Piperacillin tazobactam
CX - Cefoxitin
IMP - Imipenem
LE - Levofloxacin
NIT - Nitrofurantoin
LZ - Linezolid
CL - Colisitin
ESBL - Extended spectrum of betalactamases
MBL - Metallobetalactamases
Amp C - AmpC betalactamases
BIBLIOGRAPHY
1. Kharangate NV, Pinto MJ, et al Characterization of Nonfermenters from
Clinical Samples , J Assoc Physicians India 2001 Mar 49:324-6.
2. K Prashanth, SK Singh, R Kanungo, S Sharma, P Shashikala, S Joshi, S
Jayachandran et al Correlations between genotyping and antibiograms of
clinical isolates of Pseudomonas aeruginosa from three different south
Indian hospital Indian J Med Microbiol 2010 28(2) :130-137
3. A Manoharan, S Chatterjee, D Mathai, SARI Study Group Detection and
characterization of metallo beta lactamases producing Pseudomonas
aeruginosa Indian J Med Microbiol. 2010 28(3):241-244
4. N Ozkalay Yilmaz, N Agus, E Bozcal, A Uzel et al Prevalence and molecular
characterisation of metallo-beta-lactamase producing strains of imipenem-
resistant Pseudomonas aeruginosa in Turkey. Indian J Med Microbiol.
2014 :32(3): 349-350
5. Jonathen Cohen, William G.Powderly, Infectious Diseases, 2004 Infectious
Disease 11 nd edition: Elsevier Limited. Mosby Publications.
6. Koneman EW, Allen SD, Jande WM. et al 1997 Colour atlas and text book
diagnostic Microbiology. Lippincott 6th
edition; Chapter 7 p 309-392.
7. Murray PR,. Baron EJ, Jorgensen JH, et al 2007 Manual of Clinical
Microbiology. Washington, DC: ASM Press;9th
edition chapter 48,49,50p
734-802.
8. Rossolini GM and Mantengoli E. 2005 Treatment and control of severe
infections caused by multiresistant Pseudomonas aeruginosa. Clin Microbiol
Infect; 11(Suppl. 4): 17–32
9. AK Pragasam, S Vijayakumar, YD Bakthavatchalam, A Kapil, BK Das2, P
Ray et al Molecular characterisation of antimicrobial resistance in
Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and
2015 collected across India Indian J Med Microbiol 2016: 34(4): 433-441
10. Jene Teng, Cheng-Yi Chen, et al Pandrug-Resistant Acinetobacter
baumannii Causing Nosocomial Infections in a University Hospital, Taiwan
Emerging Infectious diseases 2002 Aug 8( 8):827-32
11. Veenu, Rama S, Arora DR et al Isolation and susceptibility pattern of non
fermenting Gram negative bacilli from clinical samples Indian J Med
Microbiol 1999;17(1):14-7.
12. Mandell G, Bennett JE, Dolin R. Principles and practice of infectious
diseases. Churchill Livingstone 5th edition;2:239-41
13. Nicolas Troillet, Matthew H. Samore, and Yehuda Carmeli et al Imipenem-
Resistant Pseudomonas aeruginosa: Risk Factors and Antibiotic
Susceptibility Patterns Clinical Infectious Diseases 1997;25:1094-8
14. John E Mc Gowen et al Resistance In nonfementing gram-negative bacteria:
Multidrug resitance to the maximum. American Journal Of Infection Control
2006 :34 (5) 1
15. Clinical and Laboratory Standard Institute.2016. Performance standards for
antimicrobial susceptibility testing; Seventeenth informational supplement,
CLSI M100-S17, Vol.27No1 Wayne, PA: Clinical and Laboratory Standard
Institute.2016.
16. Anton Y. Peleg, Harald Seifert, and David L. Paterson. Acinetobacter
baumannii: Emergence of a Successful Pathogen 2008 Clin Microbiol Rev.
21(3): 538–582
17. Arora U, Jaitwani J. 2006 Acinetobacter spp: An emerging pathogen in
neonatal septicemia in Amritsar. Indian J Med Microbiol;24:81.
18. Lee. k , Y. Chong, H. B. Shin , et al Modified Hodge and EDTA-disk
synergy tests to screen metallo-β-lactamase-producing strains of
Pseudomonas and Acinetobacter species Clinical Microbiology &
Infection:7(2) 88- 2001.
19. Gladstone P, Rajendran P, Brahmadathan KN et al Incidence of carbapenam
resistant nonfermenting gram negative bacilli from patients with respiratory
infections in the intensive care units 2005 Indian J Med Microbiol 23:189-91
20. Elizabeth T.S. Houang, Y.W .Chu. C.M. et al Epidemiology and Infection
Control Implications of Acinetbacter spp in Hong Kong 2001 Journal Of
Clinical Microbiology :24(3):228-234.
21. David L. Paterson et al The Epidemiological Profile Of Infections with
Multidrug Resistant Pseudomonas aeruginosa and Acinetobacter species.
Clinical InfectiousDiseases, 43:S43-8 2006
22. V Gautam, L Singhal, P Ray et al Burkholderia cepacia complex: Beyond
pseudomonas and acinetobacter Indian J Med Microbiol.2011 Jan 29(1):4-
12
23. M Sinha1, H Srinivasa et al Mechanisms of resistance to carbapenems in
meropenem- resistant Acinetobacter isolates from clinical samples 2007
Indian J Med Microbiol: 25(2): 121-125
24. B Behera, P Mathur, A Das, A Kapil, V Sharma An evaluation of four
different phenotypic techniques for detection of metallo-β-lactamase
producing Pseudomonas aeruginosa 2008 Indian J Med Microbiol :
26(3):233-237
25. P. A. Shiny1, S. Rajendran, Y. Lakshmi Sarayu et al A Study on Isolation
and Antibiotic Sensitivity Testing of Pseudomonas aeruginosa Isolated from
Patients with Respiratory Tract Infection with Special Reference to
Phenotypic and Genotypic Characterization of Extended Spectrum Beta
Lactamases (ESBL) Jun 2016 Indian J Med Microbiol:23(2): 122-126
26. Usha Kalawat, Sumathi I, Jaya Prada, Satish Kumar Reddy, Abhijit
Chaudhury, KK Sharma Speciation of oxidase‑positive non‑fermentative
gram‑negative bacilli December 2013 DOI: 10.4103. 2249-5975.119802
27. A Manoharan1, S Chatterjee1, D Mathai1, SARI Study Group2 et al
Detection and characterization of metallo beta lactamases producing
Pseudomonas aeruginosa Indian J Med Microbiol 2010 : 28 (3) : 241-244.
28. N Ozkalay Yilmaz, N Agus, E Bozcal, A Uzel et al Prevalence and
molecular characterisation of metallo-beta-lactamase producing strains of
imipenem-resistant Pseudomonas aeruginosa in Turkey Indian J Med
Microbiol 2008 : 26(3) : 243-245
29. S Irfan, A Zafar, D Guhar, T Ahsan, R Hasan et al Metallo-β-lactamase-
producing clinical isolates of Acinetobacter species and Pseudomonas
aeruginosa from intensive care unit patients of a tertiary care hospital Indian
J Med Microbiol 2012 : 30 ( 4) :485-486
30. S Arora, V Gautam, P Ray et al Changing susceptibility patterns of
nonfermenting Gram-negative bacilli Indian J Med Microbiol 2012: 30(4):
485-486
31. P Gladstone, P Rajendran, KN Brahmadathan et al Incidence of carbapenem
resistant nonfermenting gram negative bacilli from patients with respiratory
infections in the intensive care units Indian J Med Microbiol 2005:23(3):
189-191
32. Goossens H et al Susceptibility of multi-drug-resistant Pseudomonas
aeruginosa in intensive care units: results from the European MYSTIC study
group. Clin Microbiol Infec . 2003; 9 : 980-3.
33. Greenwood Text book of Medical Microbiology 16th edition,
34. Gilligan PH et al: Microbiology of air way disease in patients with cystic
fibrosis. Clin Microbiol Rev. Vol 4: 35 – 51, 1991.
35. Jonathen Cohen, William G.Powderly, Infectious Diseases, 2004 Infectious
Disease 2nd edition: Elsevier Limited .Mosby Publications.
36. Mac Farlane et al: Septicemia and Septic arthritis due to P.putida in a
neutropenic Patients 1991 J Infec: 23: 346 – 347,.
37. N sinha, J Agarwal, S srivastava, M singh et al. Analysis of carbapenam
resistant Acinetobacter from a tertiary care setting in North India, Indian J
Med Microbiol.(2013)3(1):60-63.
38. S Upadhyay, S Mishra, MR Sen, T Banerjee, A Bhattacharjee et al Co-
existence of Pseudomonas-derived cephalosporinase among plasmid
encoded CMY-2 harbouring isolates of Pseudomonas aeruginosa in north
India.Indian J Med Microbiol 2013:31(3): 257-260.
39. B Behera, P Mathur, A Das, A Kapil, V Sharma et al An evaluation of four
different phenotypic techniques for detection of metallo-β-lactamase
producing Pseudomonas aeruginosa. Indian J Med Microbiol. 2008 :26 (3) :
233-237
40. M Purohit, DK Mendiratta, VS Deotale, M Madhan, A Manoharan, P
Narang et al Detection of metallo-β-lactamases producing Acinetobacter
baumannii using microbiological assay, disc synergy test and PCR 2012
Indian J Med Microbiol. : 30(4): 456-461
41. .Bergogne-Berezin E, Towner KJ et al Acinetobacter species as nosocomial
pathogens: Microbiological, clinical and epidemiological features. Clin
Micro Rev 1996; 9 :148-65.
42. Lee K, Lim YS, Yong D, Yum JH, Chong Y et al Evaluation of the Hodge
test and the Imipenem-EDTA double disk synergy test for differentiating
metallo-β -lactamase-producing isolates of Pseudomonas spp. and
Acinetobacter spp. J Clin Microbiol 2003; 41 :4623-9.
43. Manikal VM, Landman D, Saurina G, Oydna E, Lal H, Quale J et al
Endemic carbapenem-resistant Acinetobacter species in Brooklyn, New
York: Citywide prevalence, inter-institutional spread and relation to
antibiotic usage. Clin Infect Dis 2000; 31: 101-6.
44. Yong D, Lee K, Yum JH, Shin HB, Rossolini GB, Chong Y et al Imipenem-
EDTA disk method for differentiation of metallo-β-lactamase-producing
clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin
Microbiol 2002; 40: 3798-801.
45. Lee K, Ha GY, Shin BM, Kim JJ, Kang JO, Jang SJ, et al. Metallo-beta-
lactamase-producing Gram-negative bacilli in Korean Nationwide
Surveillance of Antimicrobial Resistance group hospitals in 2003: Continued
prevalence of VIM-producing Pseudomonas spp. and increase of IMP-
producing Acinetobacter spp. Diagn Microbiol Infect Dis 2004; 50: 51-8.
46. Degand N, Carbonnelle E, Dauphin B, et al. Matrix-assisted laser desorption
ionization-time of flight mass spectrometry for identification of
nonfermenting gram-negative bacilli isolated from cystic fibrosis patients. J
Clin Microbiol 2008;46:3361–3367.
47. Gopaul KK, Koylass MS, Smith CJ, et al. Rapid identification of Brucella
isolates to the species level by real-time PCR based single nucleotide
polymorphism (SNP) analysis. BMC Microbiol 2008;8:86.
48. Goral S, Anderson B, Hager C, et al. Detection of Rochalimaea henselae
DNA by polymerase chain reaction from supperative nodes of children with
cat-scratch disease. Pediatr Infect Dis J 1994;13:994–997.
49. Berger P, Papazian L, Drancourt M, et al. Amoeba-associated
microorganisms and diagnosis of nosocomial pneumonia. Emerg Infect Dis
2006;12:248–255.
50. Koylass MS, King AC, Edwards-Smallbone J, et al. Compaative
performance of SNP typing and the “Bruce-ladder” in the discrimination of
Brucella suis and Brucella canis. Vet Microbiol 2010;149:450–454.
51. Shenoy S, Kavitha R, Laxmi V, et al. Septic arthritis due to Actinobacillus
actinomycetemcomitans. Indian J Pediatr 1996;63:569–570.
52. Shepard CW, Daneshvar MI, Kaiser RM, et al. Bordetella holmesii
bacteremia: a newly recognized clinical entity among asplenic patients. Clin
Infect Dis 2004;38:799–804.
53. Catry B, Baele M, Opsomer G, et al. tRNA-intergenic spacer PCR for the
identification of Pasteurella and Mannheimia spp. Vet Microbiol
2004;98:251–260.
54. Sherertz .R.J and F A Sarubbi 1983 A three-year study of nosocomial
infections associated with Pseudomonas aeruginosa. J Clin Microbiol.;
18(1): 160–164
55. Meharwal SK , Taneja N, Sharma SK,. et al 2002 Complicated nosocomial
UTI caused by nonfermenters. Indian J Urol 18:123-8
56. M.J.C. Noyal, G.A. Menezes, B.N. Harish, S. Sujatha & S.C. Parija et al
Simple screening tests for detection of carbapenemases in clinicalisolates of
nonfermentative Gram-negative bacteria Indian J Med Res June 2009:192:
707-712
57. Tanya Strateva, Vessela Ouzounova-Raykova, Boyka Markova,Albena
Todorova, et al 2007 Problematic clinical isolates of Pseudomonas
aeruginosa from the university hospitals in Sofia, Bulgaria: current status of
antimicrobial resistanceand prevailing resistance mechanisms Journal of
Medical Microbiology, 56, 956–963
58. Homer C Tien , Anthony Battad , Elizabeth A Bryce , et al Multi-drug
resistant Acinetobacter infections in critically injured Canadian forces
soldiers BMC infectious diseases 2007:7:95
59. Patwardhan,.R.B. P.K. Dhakephalkar, K.B. Niphadkar et al A study on
nosocomial pathogens in ICU with special reference to multiresistant
Acinetobacter baumannii harbouring multipleplasmids 2008 Indian J Med
Res 128:178-187
60. Veenu, Rama S, Arora DR. Isolation and susceptibility pattern of non
fermenting Gram negative bacilli from clinical samples. 1999 Indian J Med
Microbiol;17(1):14-7.
61. Vijaya D, K.Bavani S, Veena M, 2000 Prevalence of nonfermenters in
clinical specimens:54:87-91 Indian journal Of medical sciences :54(3) : 87-
91
62. John E Mc Gowen, 2006, Resistance In nonfementing gram-negative
bacteria:Multidrug resitance to the maximum. American Journal Of Infection
Control : 34 (5) : 1
63. Brauers J , U. Frank, M. Kresken et al Activities of various β-lactams and β-
lactam/β-lactamase inhibitor combinations against Acinetobacter baumannii
and Acinetobacter DNA group 3 strains 2005clinical microbiology and
infection:11( 1): 24–30
64. Reshteh Shahcheraghi, Vajiheh-Sadat et al. Prevalence of ESBLs Genes
Among Multidrug-Resistant Isolates of Pseudomonas aeruginosa Isolated
from Patients in Tehran 2009 Microbial Drug Resistance, 15(1): 37-39
65. S John1, R Balagurunathan et al Metallo beta lactamase producing
Pseudomonas aeruginosa and Acinetobacter baumannii. Indian J Med
Microbiol.. 2011 : 29 (3) : 302-304
66. Enayatollah kalantar1, 2, vahideh Torabi, Himen Salimizand, Fariborz
Soheili, Rashid Ramezanzadeh. Incidence and SusceptiblitybPattern of
Metallo-Beta Lactamase Producers Among Pseudomonas aeruginosa
isolated from Burns Patients at Kurdistan Province J J Microbiol
2012:5(3s);507-510
67. G Agrawal, RB Lodhi, UP Kamalakar, RK Khadse, SV Jalgaonkar Study of
metallo-β-lactamase production in clinical isolates of Pseudomonas
aeruginosa. Indian J Med Microbiol. 2008 26 (4): 349-351
68. S Buchunde , Dk Mendiratta et al Comparison of disc and MIC reduction
methods with polymerase chain reaction for the detection of metallo-β-
lactamase in Pseudomonas aeruginosa 2012:30(2):170-174
69. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-
EDTA Disk Method for Differentiation of Metallo-beta-lactamase-producing
Clinical isolates of Pseudomonas spp and Acinetobacter spp. J Clin
Microbiol 2002;40:3798-801.
70. Hemalatha V, Sekar U, Kamat V et alDetection of metallo betalactamase
producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med
Res 2005;122:148-52.
71. Chawla K, Vishwanath S, Munim FC et al Nonfermenting Gram-negative
Bacilli other than Pseudomonas aeruginosa and Acinetobacter Spp. Causing
Respiratory Tract Infections in a Tertiary Care Center. J Glob Infect Dis
2013;5:144-8.
72. Gautam V, Kumar S, Kaur P, Deepak T, Singhal L, Tewari R, et al.
Antimicrobial susceptibility pattern of Burkholderia cepacia complex &
Stenotrophomonas maltophilia over six years (2007-2012). Indian J Med Res
2015;142:492-4.
73. Reza Mirnejad 1, Somayeh Vafaei et al Antibiotic resistance patterns and
the prevalence of ESBLs among strains of Acinetobacter baumannii isolated
from clinical specimens spaces 2013: 2013 Article ID jgmi-00002, 8 Pages
74. Shahanara Begum Md Abdus Salam et al Detection of extended spectrum β-
lactamase in Pseudomonas spp. isolated from two tertiary care hospitals in
Bangladesh. BioMed Central doi. 10.1186. 156-0500-6-7
75. Yoo Chul Lee1, Byung Jun Ahn1, Jong Sook Jin1, et al 2007 Molecular
Characterization of Pseudomonas aeruginosa Isolates Resistant to All
Antimicrobial Agents, but Susceptible to Colistin, in The Journal of
Microbiology :22(3): 358-363
76. David L. Paterson and Robert A. Bonomo 2005 Extended-Spectrum s-
Lactamases: a Clinical Update Clinical Microbiology Reviews: 18(4): 657-
686
77. Agarwal et al Antimicrobial resistance profile of Pseudomonas aeruginosa
producing metallo Beta lactamases Nov 2006 Indian Journal of Med Res
124: 588 – 590
78. S.Smitha et al Susceptibility trends of Pseudomonas species fromcorneal
ulcers. 2005 Indian J Med Microbiol: Vol 23 (3): 168 – 171
79. James J.Rahal et al Nosocomial antibiotic resistance in multiple gram
negative species – experience at one hospital with squeezing the resistance
balloon at multiple sites 2002 Clinical Infectious Diseases:22(3)499 – 503,
80. B.S.Nagoba et al: Invitro susceptibility of Pseudomonas aeruginosa to
different antibiotics 1997 Indian J Med Microbiol:15 (4) : 185 – 186..
81. Anthony D.Harris et al : Risk factor for imipenem resistant Pseudomonas
aeruginosa among hospitalized patients 2002. Clinical Infectious
Diseases:34 : 340 -345.
82. Gales,A.C. Jones,R.N Turnidge,.J et al 2001 Characterization of
Pseudomonas aeruginosa Isolates: Occurrence Rates, Antimicrobial
Susceptibility Patterns, and Molecular Typing in the Global SENTRY
Antimicrobial Surveillance Program, 1997–1999 Clinical Infectious
Diseases; 32(2):146–55
83. Swapna Mali1, Lona Dash1, Vikas Gautam2, Jayanthi Shastri1, Sunil
Kumar2 An outbreak of Burkholderia cepacia complex in the paediatric unit
of a tertiary care hospital 2017 Indian J Med Microbiol 35(2):216-220.
84. Gautam V, Ray P, Vandamme P, Chatterjee SS, Das A, Sharma K, et al.
Identification of lysine positive non-fermenting gram negative bacilli
(Stenotrophomonas maltophilia and Burkholderia cepacia complex) 2009.
Indian J Med Microbiol;27:128-33.
85. Torbeck L, Raccasi D, Guilfoyle DE, Friedman RL, Hussong D.
Burkholderia cepacia: This decision is overdue. PDA J Pharm Sci Technol
2011;65:535-43.
86. Vial L, Chapalain A, Groleau MC, Déziel E. The various lifestyles of the
Burkholderia cepacia complex species: A tribute to adaptation. Environ M
crobiol 2011;13:1-12.
87. Khashe S, Janda JM. Biochemical and pathogenic properties of Shewanella
alga and Shewanella putrefaciens. J Clin Microbiol 1998;36:783-7.
88. R Nath, L Saikia, G Choudhury, PP Das. Isolation of Shewanella algae from
rectal swabs of patients with bloody diarrhoea. 2011 . Indian J Med
Microbiol;:29 (4):422-425
89. Shideh Khashe and J. Michael Janda et al .Biochemical and Pathogenic
Properties of Shewanella alga and Shewanella putrefaciens J. Clin.
Microbiol. March 1998 vol. 36 no. 3 783-787
90. Gilardi G. L.et al (1985) Cultural and biochemical aspects for identification
of glucose-nonfermenting gram-negative rods. in Nonfermentative gram-
negative rods. edition Gilardi G. L. (Marcel Dekker, Inc. New York, N.Y),
pp 17–84.
91. Weyant R. S., Moss C. W., et al (1995) Identification of unusual pathogenic
gram-negative aerobic and facultatively anaerobic bacteria (Williams &
Wilkins, Baltimore, Md), 2nd edition.
92. Nozue H., Hayashi T.,et al (1992) Isolation and characterization of
Shewanella alga from human clinical specimens and emendation of the
description of S. alga Simidu et al., 1990, 335. Int. J. Syst. Bacteriol.
42:628–634.
93. Brink A. J., van Straten A., van Rensburg A. J.et al(1995) Shewanella
(Pseudomonas) putrefaciens bacteremia. Clin. Infect. Dis. 20:1327–1332.
94. Butt A. A., Figueroa J., Martin D. H.et al 1997 Ocular infection caused by
three unusual marine organisms. Clin. Infect. Dis. 24:470.
95. Chen Y.-S., Liu Y.-C., Yen M.-Y., et al 1997 Skin and soft-tissue
manifestations of Shewanella putrefaciens infection. Clin. Infect. Dis. 25:225–
229
96. Joanna S. Brooke et al Stenotrophomonas maltophilia: an Emerging Global
Opportunistic Pathogen Jan 2012 Clin. Microbiol. Rev. : 25(1): 2-41
97. Denton M, et al. Stenotrophomonas maltophilia contamination of nebulizers
used to deliver aerosolized therapy to inpatients with cystic fibrosis. 2003 J.
Hosp. Infect. 55:180–183.
98. Hutchinson GR, et al Home-use nebulizers: a potential primary source of
Burkholderia cepacia and other colistin-resistant, Gram-negative bacteria in
patients with cystic fibrosis. 1996. J. Clin. Microbiol. 34:584–587.
99. Lai C-H et al. Central venous catheter-related Stenotrophomonas maltophilia
bacteraemia and associated relapsing bacteraemia in haematology and
oncology patients. 2006. Clin. Microbiol. Infect. 12:986–991.
100. Lidsky K, Hoyen C et al 2002. Antibiotic-resistant Gram-negative organisms
in pediatric chronic-care facilities. Clin. Infect. Dis. 34:760–766.
101. Metan G, Hayran M, Hascelik G, et al. 2006. Which patient is a candidate for
empirical therapy against Stenotrophomonas maltophilia bacteraemia An
analysis of associated risk factors in a tertiary care hospital. Scand. J. Infect.
Dis. 38:527–531.
102. O'Donnell MJ, Tuttlebee CM et al Bacterial contamination of dental chair
units in a modern dental hospital caused by leakage from suction system
hoses containing extensive biofilm. 2005. J. Hosp. Infect. 59:348–360.
S.NO sex Age Lab
no IP.No Risk factor ward sample Organism AMP AMX CN CTR CAZ AK GEN CIP CFS COT PIT CX IMP LF CL AmpC ESBL MBL
PCR -
Genotype
for MBL
Bla-
VIM
Bla-
IMP
1 F 42 1606 7888 ENT Pus Pseudomonas
aeruginosa
S R R R S S S R R S S S S R S N N N
2 F 21 1610 176 Burns Pus Acinetobacter
baumannii
R R R S S S S S S S S S S S S N N N
3 F 24 1615 9072 ENT Pus Pseudomonas
aeruginosa
S R R R S S S R S R S S S S S N N N
4 M 43 1616 9247 Chest
Medicine
Sputum Pseudomonas
aeruginosa
S R R R S R S R S R S R S R S P N N
5 F 29 1618 9296 Burns Pus Acinetobacter
baumannii
R S R S S S R S S R S R S S S P N N
6 M 61 1619 9252 ICU stay IMCU Blood Pseudomonas
aeruginosa
S R R S S R S R R R S R S S S P N N
7 M 34 1621 9016 Burns Pus Pseudomonas
fluorescens
S S S S S S S S S S S S S S S N N N
8 F 13 1623 8975 ICU stay IMCU Blood Pseudomonas
aeruginosa
S R R R S R R R R S S R S S S P N N
9 M 44 1624 9122 RTA Surgery Pus Acinetobacter
baumannii
R S R S S R S S R S R R S R S P N N
10 M 29 1625 9022 RTA Surgery Pus Pseudomonas
aeruginosa
S R R R S S R R S R S R S R S P N N
11 M 16 1626 8028 DM, ICU stay IMCU Blood Acinetobacter
baumannii
S S R S S S S R S S S S S S S N N N
12 M 23 1637 8765 RTA Surgery Pus Pseudomonas
fluorescens
S S S S S S S S S S S S S S S N N N
13 M 46 1648 9072 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
S R R R R R R R S R S R S S S P N N
14 M 55 1650 9321 ICU stay IMCU Blood Acinetobacter
baumannii
R R R R R S R S R R S R S R S P N N
15 F 8 1651 9302 RTA Surgery Pus Pseudomonas
aeruginosa
S R R R S S R R R S S R R R S P N N
16 M 72 1663 9147 DM Medicine Ascitic fluid Acinetobacter
baumannii
R R R S S R S S S S S R R S S P N N
17 M 9 1667 9446 Urology Urine Pseudomonas
aeruginosa
R R R S R S R R S R S R S S S P P N
18 M 48 1670 9209 ICU stay IMCU Blood Pseudomonas
aeruginosa
R R R R S S R R R S S R S S S P N N
19 F 24 1676 8358 Indwelling
catheter
Urology Urine Pseudomonas
aeruginosa
R R R R S R R R R R S R S R S P N N
20 M 18 1690 9761 Chest
Medicine
Sputum Acinetobacter
baumannii
S S R S S S S S S S S S S S S N N N
21 F 56 1697 8527 DM, ICU stay IMCU Blood Pseudomonas
aeruginosa
R R R R R S R R S R S R R R S P P N
22 M 41 1700 9702 RTA Surgery Pus Pseudomonas
fluorescens
R S R S S S S S S S S S S R S N P N
23 M 43 1567 9929 Burns Pus Pseudomonas
aeruginosa
R S S S S S S S S S S R S S S P N N
24 M 80 1710 8542 Indwelling
catheter
Urology Urine Acinetobacter
baumannii
R R R S R S R R S S R S S R S N P N
25 M 29 1714 9748 RTA Surgery pus Pseudomonas
aeruginosa
R R R R S S R R R S R R S S S P N N
26 M 5 1721 7315 Indwelling
catheter
Urology Urine Acinetobacter
baumannii
R S R S S R S S R R S R S S S P N N
27 M 29 1748 75636 RTA Surgery Pus Pseudomonas
aeruginosa
R R R R S S R R S R S R S R S P N N
28 M 44 1751 9945 Medicine Pleuralfluid Acinetobacter
baumannii
R S R R S S S S S S S S S R S N N N
29 F 7 1764 9801 Burns urine Acinetobacter
baumannii
R R R S S R R R S S S S S S S N N N
30 M 22 1767 505/15 Burns urine Pseudomonas
aeruginosa
R R R S R S R R S R S R S S S P P N
31 M 65 1769 10308 Burns urine Pseudomonas
aeruginosa
R R R R S R R R R S S R S R S P N N
32 F 53 1772 10279 RTA Surgery Pus Acinetobacter
baumannii
S R S S R S S S S R S R S S S P P N
33 F 8 1776 9774 Chronic renal
disease
Medicine Ascitic fluid Pseudomonas
aeruginosa
R R R R S S R R R R S R S S S P N N
34 F 49 1780 41427 DM, ICU stay IMCU Pus Pseudomonas
aeruginosa
R R R R S S R R R R S R R R S P N P N N N
35 F 23 1784 10353 ICU stay IMCU Pus Pseudomonas
aeruginosa
R R R S S S R R S R S S S R S N N N
36 F 55 1785 8204 Indwelling
catheter
Burns urine Pseudomonas
aeruginosa
R R R R R R R R S S S S S S S N P N
37 M 11 1787 9133 Burns urine Acinetobacter
baumannii
R S R S S S S R S R R S S R S N N N
38 M 3 1793 10205 Burns urine Pseudomonas
aeruginosa
R R R R S S R R R R S S S S S N N N
39 M 25 1801 8692 Burns urine Acinetobacter
baumannii
R S R R S S R S S S S S S S S N N N
40 F 19 1808 100634 RTA Surgery urine Pseudomonas
aeruginosa
R R R R S R R R R R S S S R S N N N
41 F 45 1813 10048 Indwelling
catheter
Urology urine Acinetobacter
baumannii
R R R S S R S S R R S S S R S N N N
42 M 67 1815 9989 Surgery urine Pseudomonas
aeruginosa
R R R R R R R R R S S S S S S N P N
43 F 6 1816 10353 Chest
Medicine
Sputum Acinetobacter
baumannii
S S S S R S R R S S S R S S S P P N
44 M 51 1824 10440 DM Chest
Medicine
Sputum Acinetobacter
baumannii
R R R S S S S S S R S S S R S N N N
45 F 26 1834 10601 DM, ICU stay IMCU Pus Acinetobacter
lwoffii
S S S S S S S S S S S S S S S N N N
46 M 13 1836 9900 Medicine Pleuralfluid Pseudomonas
aeruginosa
R R R R S R R R S R S S S R S N N N
47 M 22 1840 10666 DM, ICU stay IMCU Pus Pseudomonas
aeruginosa
R R R R S R R R S R S S R S S N N N
48 M 57 1850 10509 Burns Pus Acinetobacter
baumannii
S R S S S R S S S R S S S S S N N N
49 F 17 1859 91443 Burns urine Acinetobacter
baumannii
R S R R R S R R R S S R S R S P P N
50 M 48 1875 11046 Indwelling
catheter
Burns urine Pseudomonas
aeruginosa
R R R R R S R R R S S S S S S N P N
51 M 55 1890 11228 ENT Pus Pseudomonas
aeruginosa
R R R R R S R R S R S S S R S N P N
52 M 27 1924 8743 ICU stay IMCU Blood Acinetobacter
lwoffii
S S S S S S S S S S S S S S S N N N
53 F 13 1943 DM ENT Pus Pseudomonas
aeruginosa
R R R R S R R R R R S S S S S N N N
54 F 55 1953 11220 Burns Pus Pseudomonas
fluorescens
R R R R S S S R R R S S S S S N N N
55 M 41 1958 11275 Chronic renal
disease
Nephro urine Acinetobacter
baumannii
S S R S S S R S S R S S R S S N N N
56 F 12 1960 11548 ENT Pus Pseudomonas
aeruginosa
R R R R S R R R S S S S S R S N N N
57 F 29 1974 Burns Pus Acinetobacter
baumannii
R R S S S R S S S R S S S R S N N N
58 M 28 1983 12013 ENT urine Pseudomonas
aeruginosa
R R R R S R R R R R S S S S S N N N
59 F 44 1994 12043 Medicine Pleuralfluid Acinetobacter
baumannii
R S R S R S R R S S S R S S S P N N
60 M 23 2001 11611 RTA Surgery Pus Pseudomonas
aeruginosa
R R R R R S R S S R S S R R S N P N
61 M 47 2024 9893 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R R S S R R S S R S S S P N N
62 M 49 2033 11952 Surgery Pus Pseudomonas
aeruginosa
R R R R S S R R S R S R S R S P N N
63 F 59 2034 12104 Burns Pus Acinetobacter
baumannii
S R R S S S R S R R S S S R S N N N
64 M 43 2036 11117 Burns Pus Acinetobacter
baumannii
R R S S S R S S S R S S S S S N N N
65 M 29 2040 12105 ENT Pus Pseudomonas
aeruginosa
R R R R S R R R S R S R S S S P N N
66 M 47 2043 12334 Indwelling
catheter
Nephro urine Acinetobacter
baumannii
R S R R S S R R S S S S R S S N N P N N N
67 M 32 2050 12708 Urology urine Pseudomonas
aeruginosa
R R R R S R R S R R S R S R S P N N
68 M 46 2065 91026 Indwelling
catheter
Urology urine Pseudomonas
aeruginosa
R R R R S S R S R S S S S R S N N N
69 M 52 2066 12080 Indwelling
catheter
Urology urine Pseudomonas
aeruginosa
R R R R R S R S S R S R S R S P P N
70 F 36 2067 12043 Burns Pus Acinetobacter
baumannii
S R R S R S R S S R R R S R S P N N
71 M 37 2068 12334 Medicine Pleuralfluid Pseudomonas
aeruginosa
R R R R S R S R R R S S S S S N N N
72 F 42 2081 12104 DM ENT Pus Pseudomonas
aeruginosa
R R R R S R S R R S S R R S S P N P N N N
73 F 51 2100 11927 Burns Pus Acinetobacter
baumannii
R R S S S R R S R R S S S R S N N N
74 M 69 2108 12257 Urology urine Pseudomonas
aeruginosa
R R R R S R S S R S S S S R S N N N
75 F 31 2117 12830 Burns urine Pseudomonas
aeruginosa
R R R R R R S R R S S S S R S N P N
76 M 22 2118 12823 Chronic renal
disease
Nephro urine Acinetobacter
baumannii
R R R S S S S S S S S R S S S P N N
77 M 47 2119 12104 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R S R S R R R S R S S S P N N
78 M 67 2124 471/15 Burns Pus Acinetobacter
baumannii
S S R S R S R R S R S S S R S N P N
79 M 53 2125 12145 ENT Pus Pseudomonas
aeruginosa
R R R R S R R S R S S S S R S N N N
80 F 24 2126 12134 ENT Pus Pseudomonas
aeruginosa
R R R R S R R R R R S R S R S P N N
81 F 44 2128 12167 Burns Pus Acinetobacter
baumannii
R R S R S S R S S S S R R R S P N P N N N
82 F 33 2130 12105 Burns Pus Acinetobacter
baumannii
S R R S S S S S R R S S S S S N N N
83 M 70 2131 12146 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R R R R R R R S R S R S P N N
84 F 55 2132 12147 ENT Pus Pseudomonas
aeruginosa
R R R R S R R S R S S S R S S N N P P P N
85 F 27 2133 12149 Burns Pus Acinetobacter
baumannii
R S R S R S R R S R S R S R S P P N
86 M 41 2134 12165 Indwelling
catheter
Burns urine Pseudomonas
aeruginosa
R R R R S R R R R R S S S S S N N N
87 F 28 2135 12164 Chest
Medicine
Sputum Acinetobacter
baumannii
R S S S S R R S R S S S S R S N N N
88 M 43 2137 12161 Burns Pus Acinetobacter
baumannii
S R R R R S S S S S S R S S S P N N
89 F 75 2139 12169 DM Medicine Pleuralfluid Pseudomonas
aeruginosa
R R R S S R R R R S S R S R S P N N
90 F 57 2140 12157 Burns Pus Acinetobacter
baumannii
R R R S R S S R R R R S R R S N N P N N N
91 M 44 2143 12198 Chronic renal
disease
Nephro urine Acinetobacter
baumannii
S S S S S R R S R S S S S R S N N N
92 M 21 2145 12164 ENT Pus Pseudomonas
aeruginosa
R R R R S R S S R R S S S R S N N N
93 M 37 2146 12178 ENT Pus Pseudomonas
aeruginosa
R R R R R R S R R R S S S S S N P N
94 M 42 2147 12189 Burns Pus Acinetobacter
baumannii
R S R S R S S S S R S S S S S N P N
95 M 59 2148 12187 RTA Surgery urine Pseudomonas
aeruginosa
R R R R R R S R S R S R S S S P P N
96 F 39 2150 12168 Chest
Medicine
Sputum Acinetobacter
lwoffii
R R S R S S S R S R S S S S S N N N
97 M 69 2152 12197 ENT Blood Pseudomonas
aeruginosa
R R R R S R S R R R S S R R S N N P P P N
98 M 58 2154 12179 DM, ICU stay IMCU Blood Acinetobacter
baumannii
S R R S R S S R S S S R S R S P N N
99 M 26 2156 12181 RTA Surgery Pus Pseudomonas
aeruginosa
R R R S R R R R R R S R S R S P N N
100 F 45 2157 12183 Burns Blood Acinetobacter
baumannii
S R R S S S R S R S S S R R S N N P N N N
101 M 31 213 9893 RTA Surgery Pus Pseudomonas
aeruginosa
R R R R S R R R R R S S S S S N N N
102 M 23 137 11611 Burns Pus Acinetobacter
baumannii
R S S S S S S S R R R R S S S P N N
103 F 81 159 11952 Surgery urine Pseudomonas
aeruginosa
R R R R S R S R S R S S S R S N N N
104 F 51 165 12104 Burns Pus Acinetobacter
baumannii
S R R S R R S R S S S S S R S N P N
105 F 49 168 1117 Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R S R R S S R R S R S S P N N
106 F 16 185 12105 Burns Pus Acinetobacter
baumannii
R R R S S S R S R S S R S R S P N N
107 M 22 192 12334 Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R R S R S R R S S R S S N P N
108 M 34 207 12708 Burns Pus Acinetobacter
baumannii
S S S S S S S S S R S S R S S N N N N N N
109 F 53 698 91026 Burns Blood Pseudomonas
aeruginosa
R R R R S S R R R R S S S S S N N N
110 F 36 221 12080 Burns Blood Acinetobacter
baumannii
R R R S R S S R R S S S S R S N P N
111 M 12 262 12043 RTA Surgery Pus Pseudomonas
aeruginosa
R R R S R S R R S R S R S R S P P N
112 F 25 273 11927 RTA Surgery urine Pseudomonas
aeruginosa
R R R R R S R S R R S S S R S N P N
113 M 32 286 12257 Burns Pus Pseudomonas
fluorescens
S S S R R S S S S R S S S S S N N N
114 M 24 296 12830 Burns urine Pseudomonas
aeruginosa
R R R R S S R S R R S S S R S N N N
115 M 11 310 12823 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R S R S R S S R S R R S S P N P N N N
116 F 21 311 12104 RTA Surgery Pus Pseudomonas
aeruginosa
R R R R R S R S R R S S S R S N P N
117 F 83 322 471/15 RTA Surgery Pus Pseudomonas
aeruginosa
R R R R R S R S R S S S S R S N N N
118 F 18 324 13158 Burns urine Pseudomonas
aeruginosa
R R R S S S R S S R R R S R S P N N
119 F 33 325 11568 RTA Surgery Pus Pseudomonas
aeruginosa
R R R R S S R S R R S S S S S N N N
120 M 27 329 13297 Burns Pleuralfluid Acinetobacter
baumannii
S R R S S S S S S S S R S S S P N N
121 F 33 330 12982 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R R S S S R R S R S R S P P N
122 F 12 338 13473 ENT Pus Pseudomonas
aeruginosa
R R R S R S S S S R S S S R S N N N
123 F 31 342 119730 Burns urine Pseudomonas
aeruginosa
R R R R R S S S R S S S R R S N P N
124 M 11 354 13066 ENT Pus Pseudomonas
aeruginosa
R R R R S S S S R R S R S R S P N N
125 M 65 358 13312 DM ENT Pus Pseudomonas
aeruginosa
R R S S S S S S R R S S S S S N N N
126 M 39 359 588416 Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R R S S R S R S S S R S N N N
127 M 23 362 1922 DM Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R R S S S R S S R S R S P N N
128 F 39 365 13020 Burns Pus Acinetobacter
baumannii
R R R S S S R S R R S S S R S N N N
129 M 38 369 2977 DM ENT Pus Pseudomonas
aeruginosa
R R R S R S S R R S S S R R S N P P P P N
130 M 26 379 3726 Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R R R S S S S S S S R S S S P N N
131 F 38 41 2832 Burns Pus Acinetobacter
baumannii
S S S S R S S R S S S S S S S N N N
132 M 34 432 304246 ENT Pus Pseudomonas
aeruginosa
R R R R S S S R R R S S S R S N N N
133 M 35 437 577744 Medicine Pleuralfluid Acinetobacter
baumannii
R R R S S S S S S S S S S S S N N N
134 F 23 439 661 ENT Pus Pseudomonas
aeruginosa
R R R S R S S S R S S S S R S N N N
135 M 62 454 58 burns Pus Acinetobacter
baumannii
R R S S S S R R R R S S S R S N N N
136 M 35 494 85 Burns urine Pseudomonas
aeruginosa
R R S R S S S R S R S R S R S P N N
137 M 33 517 799 burns Pus Acinetobacter
lwoffii
R R R S S S S S R S S S S R S N N N
138 F 29 520 288 DM Medicine Pleuralfluid Pseudomonas
aeruginosa
R R R S R S S R R R S S S S S N P N
139 F 32 527 43801 ENT Pus Pseudomonas
aeruginosa
R R R R S S S S R S S R S R S P N N
140 M 31 531 705 burns Pus Pseudomonas
fluorescens
R R R S S R S R R R S S S R S N N N
141 M 37 541 584601 Indwelling
catheter
Burns urine Pseudomonas
aeruginosa
R R R S R S S R S R S S R R S N N N
142 M 22 543 15423 Burns urine Pseudomonas
aeruginosa
R R R R S S S R R R S R S R S P N N
143 F 38 547 939 ENT Pus Pseudomonas
aeruginosa
R R R R S S S S R S S S S S S N N N
144 F 39 1172 698 burns Pus Stenotrophomonas
maltophilia
R S S S S S S S S S S S S S S N N N
145 F 23 1240 381 ENT Pus Pseudomonas
aeruginosa
R R R S R S S R S R S R S R S P P N
146 F 38 1318 575077 Burns urine Pseudomonas
aeruginosa
R R R R R S S S R R S S S R S N P N
147 M 56 1337 18781 DM ENT Pus Pseudomonas
aeruginosa
R R R R S S S R R S S S S R S N N N
148 F 34 1358 1547 burns Pus Pseudomonas
fluorescens
R R R S S R R S S R S S S R S N N N
149 M 22 1367 2100 DM Medicine Pleuralfluid Pseudomonas
aeruginosa
R R R S S S S R S R S S R S S N N P P P N
150 M 56 132 590167 burns Pus Acinetobacter
baumannii
S R R S R S S R S S R S S S S N P N
151 M 88 1447 2268 Burns urine Pseudomonas
aeruginosa
R R R R S S S R R R S R S R S P N N
152 F 14 1470 2518 burns urine Acinetobacter
baumannii
R S S S S S S S S S S S S R S N N N
153 M 18 1511 2225 ENT Pus Pseudomonas
aeruginosa
R R R R S S S R R S S S S R S N N N
154 F 33 1568 27281 Burns urine Pseudomonas
aeruginosa
R R R R R S S R S R S R S R S P N N
155 F 23 163 5875 burns urine Acinetobacter
baumannii
S R R S S S R S R R S S S R S N N N
156 F 31 1788 2339 Medicine Ascitic fluid Acinetobacter
baumannii
R R R S R S S R S S S S S S S N N N
157 F 58 5 50827 RTA Surgery Pus Pseudomonas
aeruginosa
R R R S R S S S R R S S S S S N P N
158 F 33 9 585581 burns Pus Pseudomonas
fluorescens
S S S S S S S S S R S S S S S N N N
159 M 27 16 591920 Chest
Medicine
Sputum Pseudomonas
aeruginosa
R R S R S S S S R S S R S R S P N N
160 M 39 50 1948 RTA Surgery Pus Acinetobacter
baumannii
R R R R S S S S S R S S R R S N N N
161 F 19 71 16 Indwelling
catheter
Burns urine Pseudomonas
aeruginosa
R R R R S S S S R R S R S R S P N N
162 M 51 94 1948 burns Pus Acinetobacter
baumannii
S S R R S S S S S R S S S R S N N N
163 F 35 107 1622 Surgery Pus Pseudomonas
aeruginosa
R R R S S S S S R S S S S R S N N N
164 M 25 109 1518 Indwelling
catheter
Nephro urine Acinetobacter
baumannii
R S R S S S R S R R S S S S S N N N
165 F 11 122 51227 DM, ICU stay IMCU Ascitic fluid Pseudomonas
aeruginosa
R R R R R S S S S R S R S S S P N N
166 F 33 41 1869 Chest
Medicine
Sputum Acinetobacter
baumannii
R S R S R S S R S R S S S R S N P N
167 M 56 57 2588 Burns urine Pseudomonas
aeruginosa
R R R R S S R R R S S R R S N P N
168 F 28 65 52978 Chronic renal
disease
Nephro urine Acinetobacter
baumannii
S S R S S S R S S S S S R S S N N N
169 M 32 66 49886 Medicine Sputum Acinetobacter
baumannii
R R R S S S S S R S S S S R S N N N
170 M 38 72 3115 Burns Pus Pseudomonas
aeruginosa
R s R S S S S S R R S R R R S P N P N N N
171 F 36 73 50421 Burns urine Pseudomonas
aeruginosa
R R R R S S S S S S S S S R S N N N
172 F 22 77 3007 Burns Pus Shewanella
putrifaciens
S S S S S S S S S R S S S S S N N N
173 F 34 81 3106 Post renal
transplantation
Nephro Urine Burkholderia
cepacia
R S R R S R S S S S S S S S S N N N
174 M 65 82 1788 Burns Pus Stenotrophomonas
maltophilia
R S R R S S S S R S S S S R S N N N
175 M 34 118 3763 DM Medicine Sputum Pseudomonas
aeruginosa
R R R R S S S R R R S S S S S N N N
176 M 22 127 2564 Burns Pus Pseudomonas
aeruginosa
R s R R S S S S R R S R S R S P N N
177 M 55 128 51030 Burns Pus Acinetobacter
lwoffii
R R R R S R R R S R S S S S S N N N
178 M 27 129 3103 Burns urine Pseudomonas
aeruginosa
R R S S R S S S S S S S S R S N N N
179 M 51 115 3949 Post renal
transplantation
Nephro urine Weekseilla virosa S S S S S S S S S R S S S S S N N N
180 F 32 221 2924 Chronic renal
disease
Nephro urine Weekseilla virosa S S S S S S S S S S S S S S S N N N
181 M 31 276 5443 DM Medicine Sputum Pseudomonas
aeruginosa
R R R R S S S R R R S R S R S P N N
182 M 32 229 38117 Burns Pus Pseudomonas
aeruginosa
R s R S S S S S R R S R S S S P N N
183 F 24 321 5699 Burns Urine Burkholderia
cepacia
R S R S S R R R S S S S S R S N N N
184 M 58 333 6110 ENT Pus Pseudomonas
aeruginosa
R R R R S S S S S S S S S R S N N N
185 M 33 376 1252 ENT Pus Pseudomonas
aeruginosa
R R S R R S S R R R S S S R S N N N
186 M 22 399 2464 Chest
Medicine
Blood Pseudomonas
aeruginosa
R s R R R S S S R R S S S R S N N N
187 M 36 367 169 Burns Blood Pseudomonas
aeruginosa
R R R R S S S R S S S S S S S N N N
188 M 37 397 472 ENT Pus Pseudomonas
aeruginosa
R R R R S S S S R R S S S R S N N N
189 F 37 323 44320 Burns Urine Burkholderia
cepacia
R S R S S R S S S S S S S S S N N N
190 M 39 334 472 Burns Pus Pseudomonas
aeruginosa
R R R R S S S R R S S S S R S N N N
191 M 39 367 44320 ENT Pus Pseudomonas
aeruginosa
R s R R S S S S S R S S S R S N N N
192 M 45 345 7219 Burns Pus Stenotrophomonas
maltophilia
R S R S S S S R R S S S S R S N N N
193 F 38 390 2225 ENT Pus Pseudomonas
aeruginosa
R R R S R S S R R R S S S S S N N N
194 M 34 312 8207 Burns urine Pseudomonas
aeruginosa
R s R R S S S R R S S S S R S N N N
195 M 35 311 3857 Burns Pus Acinetobacter
lwoffii
S S S S S R R S S S S S S R S N N N
196 M 33 344 51810 RTA Surgery Ascitic fluid Pseudomonas
aeruginosa
R R S R R S S S S R S S S R S N N N
197 M 31 370 52626 ENT Pus Pseudomonas
aeruginosa
R R R R S S S S R S S S S S S N N N
198 M 32 307 1125 ENT Pus Pseudomonas
aeruginosa
R s R R S S S R R R S S S R S N P N
199 M 37 309 5043 Burns Blood Acinetobacter
baumannii
R S R S R S R R S R S S S S S N P N
200 M 55 366 319 Burns Blood Pseudomonas
aeruginosa
R R R R R S S R R R S S S R S N N N