Nonfermenting gram negative bacteria.pptx12

Nonfermentering

Gram Negative

Bacteria Dr.T.V.Rao MD

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Growing Importance of Non-Fermenting Gram Negative Bacteria

Non fermenting Gram Negative Bacteria are

complex group of Bacteria with few defined

characteristics, Many times discarded in

Diagnostic Microbiology as Contaminants. The

emerging challenges associated with Antibiotic

resistance is a concern to Physicians, All Medical

Microbiologists should update the Knowledge and

improve the Diagnostic facilities in the Laboratories

for better care of the patients

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Nonfermenters can Cause Medically

Important Infections

Nonfermenters are found in nature as

inhabitants of soil and water and as harmless

parasites on the mucous membranes of man

and other animals.

Nonfermenters can cause disease when they

colonize and subsequently infect

immunocompromised individuals or when they

gain access to a normally sterile body site

through trauma.

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Nonfermenters are Ill defined

Nonfermenters only comprise a small

percentage of the total clinical isolates, but

they require more effort for identification.

Classification

No family designation

Includes many genera whose names are

continually changing

By definition they do not ferment glucose

Morphology and cultural characteristics

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Most Common Non-Fermentative

Gram-Negative Bacteria

Pseudomonas aeruginosa (most common)

Acinetobacter species (second most common)

Stenotrophomonas maltophilia (third most common)

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Other Clinically Prevalent

Gram-Negative Non Fermenters

Pseudomonas

stutzeri

Burkholderia

cepacia

Burkholderia

pseudomallei

Moraxella

Achromobacter

xylosoxidans

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Non-Fermentative

Gram-Negative Bacteria

Nonfermentative for glucose (TSI =

alkaline over no reaction)

Oxidative for glucose (Hugh-Leifson

O-F glucose positive)

Asaccharolytic for glucose (Hugh-

Leifson O-F glucose negative)

Cytochrome oxidase positive or

negative

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Hugh-Leifson OF versus

TSI Medium

TSI AGAR SLANT Total protein = 2.6 g%

Total carbohydrate = 2.1 g%

Protein to carbohydrate (w/w) = 1.2

OF BROTH MEDIUM

Total protein = 0.2 g%

Total carbohydrate = 1.0 g%

Protein to carbohydrate (w/w) = 0.2

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Growth of Gram-Negative Non-Fermenters on TSI Agar Slants

Non-fermentative gram-

negative bacteria grow

abundantly within 16-18

hours of inoculation on

the surface of TSI agar

slants.

Non-fermentative gram-

negative bacteria neither

grow in nor acidify the

deep of TSI slants.

Growth of Oxidative Non-Fermenters in

Hugh-Leifson Broth Growth with acidification of broth in Hugh-Leifson tube not sealed by mineral oil (oxidative tube)

No growth in Hugh-Leifson tube sealed by a layer of mineral oil (fermentative tube)

Substrates utilized: glucose, lactose, maltose, xylose, Mannitol, sucrose, adonitil, dulcitol

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Growth of Asaccharolytic Non-Fermenters in Hugh-Leifson OF Broth

Growth without

acidification but with

Alkalinization of

broth in Hugh-

Leifson tube not

sealed by mineral oil

(oxidative tube)

No growth in Hugh-

Leifson tube sealed

by a layer of mineral

oil (fermentative

tube)

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The CDC scheme of

identification The CDC scheme of identification separates organisms into 8

groups based on: Growth versus no growth on Mac

Oxidase test results

O/F results

Further testing might include:

Motility (by polar flagella)

Nitrate reduction or denitrification

Urease production

Esculin hydrolysis

Indole – use Ehrlichs rather than Kovacs reagent because Ehrlichs is more sensitive

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Classification of Pseudomonads1

Burkholderia cepacia (family Burkholderiaceae)

rRNA Group II

Cytochrome oxidase +, OF glu +, motile, polymyxin B resistant

Burkholderia pseudomallei (family Burkholderiaceae)

rRNA Group II

Cytochrome oxidase +, OF glu +, motile, polymyxin B resistant

Stenotrophomas maltophilia (family Xanthomonadaceae)

rRNA Group V

Cytochrome oxidase –, OF glu +, OF maltose ++, motile, polymyxin B susceptible

1Pseudomonads are separated into five taxonomically

distinct ribosomal RNA homology groups.

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Classification of Non-

Pseudomonads Acinetobacter baumannii (family Moraxellaceae)

Cytochrome oxidase –, OF glu +, OF lactose ++, non-motile

Moraxella (family Moraxellaceae) Cytochrome oxidase +, OF glu – (asaccharolytic),

non-motile Achromobacter xylosoxidans (family Alcaligenaceae)

Cytochrome oxidase +, OF glu +, OF xylose +, motile

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Biochemical Tests to be performed for

Identification Rapid decarboxylation reactions

Pigment production

growth in cetramide

Phenylalanine deaminase

Growth at 420 C

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Virulence Factors Virulence factors that are extracellular products (Pseudomonas aeruginosa)

Expression is under control of two component signal transduction, quorum sensing systems.

When the bacteria detects a critical concentration of an auto inducer released by the organism, a signal transduction cascade will trigger the expression of these products:

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Pseudomonas aeruginosa

Pseudomonas aeruginosa is an

opportunistic pathogen, meaning that it exploits some break in the host defenses to initiate an infection. In fact, Pseudomonas aeruginosa is the epitome of an opportunistic pathogen of humans. The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defenses are compromised in some manner

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Characters of Pseudomonas

aeruginosa

Pseudomonas aeruginosa

is a Gram-negative rod

measuring 0.5 to 0.8 µm by

1.5 to 3.0 µm. Almost all

strains are motile by means

of a single polar flagellum.

The bacterium is ubiquitous

in soil and water, and on

surfaces in contact with soil

or water. Its metabolism is

respiratory and never

fermentative, but it will grow

in the absence of O2 if NO3

is available as a respiratory

electron acceptor.

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Colony characters differ

P. aeruginosa isolates may produce

three colony types. Natural isolates

from soil or water typically produce a

small, rough colony. Clinical

samples, in general, yield one

or another of two smooth

colony types. One type has a fried-

egg appearance which is large, smooth,

with flat edges and an elevated

appearance. Another type, frequently

obtained from respiratory and urinary

tract secretions, has a mucoid

appearance, which is attributed to the

production of alginate slime. The

smooth and mucoid colonies are

presumed to play a role in colonization

and virulence.

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Virulence Factors

Elastolytic proteases Elastin is a constituent of lung tissue and blood vessels. The damage caused by the elastotytic proteases causes an

inflammatory reaction that compromises the host and aids in the dissemination of the organism.

Alkaline proteases These proteases may degrade complement and IgA, thus

hindering the immune response. Exotoxin A (iron limitation also contributes to inducing its expression) which is the most toxic product produced by Pseudomonas aeruginosa. It is cytotoxic for eukaryotic tissue culture cells and lethal for many

mammals (LD50 in mice= 60-80 ng.). The mechanism of action is to interfere with protein synthesis by

ADP-ribosylation of elongation factor 2. The liver is a prime target for this toxin.

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Virulence Factors

Exotoxin S – ADP-ribosylates vimentin, a structural component of the host cell, and GTP-binding proteins

Phospholipase C – a hemolysin that may be involved in the breakdown of phospahtidyl choline, a major surfactant of the lung, leading to pulmonary collapse.

Leukocidin

Pyocyanin- a secreted pigment that is toxic due to its involvement in the generation of reactive oxygen intermediates (superoxide radical and hydrogen peroxide)

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Virulence Factors Virulence factors – (P. aeruginosa ) cell surface: Both pilin and non-pilus adhesions are important for

attachment

LPS – endotoxin

Iron capturing ability

Flagella

Alginate synthesis Forms a viscous gel around the bacteria

May function as an adhesion and may also function to prevent phagocytosis

Antimicrobic resistance – due to outer membrane changes

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Stenotrophomonas maltophilia:

Natural Habitats

Widely distributed including moist hospital environments (respiratory therapy equipment)

Colonizer of human respiratory tract in a hospital setting

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Modes of Infection

Colonization of

hospital patients by

environmental

sources

Introduction of

organisms into

normally sterile sites

by medical

instrumentation

(similar to

Acinetobacter)

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Stenotrophomonas maltophilia

Stenotrophomonas

maltophilia (second

most frequently

isolated NF)

Is part of the transient

Normal flora of

hospital patients and

causes a wide variety

of nosocomial

infections

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Microbiological Properties

Short to medium-size, straight

gram-negative rods

Glucose oxidizer (OF glu +) with

occasional negative strains

(~15%)

Strong maltose oxidizer (OF mal

+) (100%) (more intense than OF

glu + reaction)

Colonies on sheep blood agar

rough and lavender-green with

ammonia odor

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Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an aerobic,

nonfermentative, Gram-negative bacterium. It is an uncommon

bacterium and human infection is difficult to treat. Initially

classified as Pseudomonas maltophilia, S. maltophilia was also

grouped in the genus Xanthomonas before eventually becoming

the type species of the genus Stenotrophomonas in 1993.[

S. maltophilia are slightly smaller (0.7–1.8 × 0.4–

0.7 micrometers) than other members of the genus. They are

motile due to polar flagella and grow well on MacConkey agar

producing pigmented colonies. S. maltophilia are catalase

positive, oxidase negative (which distinguishes them from most

other members of the genus) and have a positive reaction for

extracellular DNase.

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Cytochrome oxidase negative

Positive for DNase (unlike most other glucose-oxidizing gram-negative bacilli)

Positive for lysine decarboxylase (unlike most other glucose-oxidizing gram-negative bacilli)

Resistant to most antibiotics except trimethoprim-sulfamethoxazole

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S. Maltophilia Pathogenesis S. maltophilia frequently colonizes breathing tubes such as

endotracheal or tracheostomy tubes, the respiratory tract and

indwelling urinary catheters. Infection is usually facilitated by

the presence of prosthetic material (plastic or metal), and the

most effective treatment is removal of the prosthetic material

(usually a central venous catheters or other device). The

growth of S. maltophilia in microbiological cultures of

respiratory or urinary specimens is therefore sometimes difficult

to interpret and not a proof of infection. If, however, it is grown

from sites which would be normally sterile (e.g., blood), then it

usually represents true infection.

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Burkholderia pseudomallei

Burkholderia

pseudomallei (also

known as Pseudomonas

pseudomallei) is a Gram-

negative, bipolar, aerobic,

motile rod-shaped

bacterium. It infects

humans and animals and

causes the disease

melioidosis. It is also

capable of infecting

plants.

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B. pseudomallei is not fastidious and will grow on a large variety of

culture media (blood agar, MacConkey agar, EMB, etc.). Ashdown's

medium (or Burkholderia cepacia medium) may be used for selective

isolation.] Cultures typically become positive in 24 to 48 hours (this

rapid growth rate differentiates the organism from B. mallei, which

typically takes a minimum of 72 hours to grow). Colonies are

wrinkled, have a metallic appearance, and possess an earthy odour.

On Gram staining, the organism is a Gram-negative rod with a

characteristic "safety pin" appearance (bipolar staining). On sensitivity

testing, the organism appears highly resistant (it is innately resistant

to a large number of antibiotics including colistin and gentamicin) and

that again differentiates it from B. mallei, which is in contrast,

exquisitely sensitive to a large number of antibiotics.-

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Colonies are wrinkled, have a metallic

appearance, and possess an earthy

odour. On Gram staining, the

organism is a Gram-negative rod with

a characteristic "safety pin"

appearance (bipolar staining). On

sensitivity testing, the organism

appears highly resistant (it is innately

resistant to a large number of

antibiotics including colistin and

gentamicin and that again

differentiates it from B. mallei, which

is in contrast, exquisitely sensitive to

a large number of antibiotics.-

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Other Non Fermenters

Acinetobacter Is found in soil and water and as part of the skin NF

Is a common colonizer and less commonly a cause of nosocomial infections

Chryseobacterium meningosepticum Occasionally found causing meningitis and septicemia

Moraxella M. lacunata causes conjunctivitis and keratitis in the

malnourished alcoholic population

Burkholderia – two species are true pathogens

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Pseudomonas stutzeri:

Microbiology

Cytochrome-oxidase positive gram-negative

rods forming distinctive dry, wrinkled colonies

(1-6 mm) on blood agar

Key reactions: OF glucose + and OF lactose –,

arginine dihydrolase –, ability to grow in 6.5%

NaCl broth, gas from nitrate, and no growth

with cetrimide (growth of P. aeruginosa

cetrimide-resistant)

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Burkholderia cepacia:

Natural Habitats and Clinical

Infections Soil and environmental water

Unpasteurized dairy products

Contaminated respiratory therapy equipment, disinfectants, medications, and mouthwash

Nosocomial pathogen causing bacteremia (most often associated with indwelling vascular catheters and polymicrobial), respiratory infections (ventilator-associated pneumonia), septic arthritis, urinary tract infections

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Microbiology Bright pink colonies on MacConkey agar after 4

days of incubation due to lactose oxidation Positive for lysine decarboxylation (genomovar

I) (DNase negative, vs. Stenotrophomonas maltophilia that is DNase positive)1

Saccharolytic with OF glu + and OF xyl + (100%), OF lac + and OF suc + (91%) (acidify slant and deep of TSI slant after 4-7 days be oxidation of glucose, lactose, and sucrose)

ONPG + 1Among non-fermentative gram-negative

bacteria, only B. cepacia and S. maltophilia lysine positive

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Burkholderia cepacia: Use of

Selective Agar Pseudomonas cepacia agar (PCA): selective containing crystal violet, polymyxin B, and bacitracin; differential with B. cepacia forming a pink-red color due to pyruvate metabolism.

Utilized to recover B. cepacia from cystic fibrosis sputum

Isolation from PCA by single colony pick and ID by Vitek-2 but ~15% misidentification

Confirmation of Vitek-2 ID by manual identification (? Role of PCR for ID of genomovariants)

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Clinical Infections Second leading cause of bacteremia and third most common cause of pneumonia in chronic granulomatous disease of childhood

Chronic pneumonia in cystic fibrosis (3-7%) with rapid decline in lung function, transmissibility of infection via close personal contact (nosocomial spread), and poor outcome with lung transplantation

“Cepacia Syndrome” Rapid and fatal clinical deterioration with necrotizing granulomatous pneumonia

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Clinical Infections Skin and soft tissue

infections with burn or surgical wounds, in soldiers with prolonged foot immersion in water

Isolation from blood cultures of multiple patients over short period of time should be investigated for “pseudo bacteremia” (contaminated infusion or disinfectant fluid)

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Microbiology Burkholderia cepacia complex (BCC): nine genomic species (genomovars) including B. cepacia (genomovars I)

DNA PCR and microarray technology under development for laboratory identification

Cytochrome-oxidase positive gram-negative rods forming smooth, round, opaque, and yellow colonies (genomovar I) on blood agar

Wet, runny, and mucoid colonies when recovered for cystic fibrosis sputum (requires at least 3 days for appearance)

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Acinetobacter Species:

Natural Habitats

Widely distributed

including the hospital

environment

Able to survive on

moist and dry

surfaces including

human skin

More frequently

colonizing than

infecting

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Acinetobacter Species: Modes of

Infection

Colonization of hospital patients by

environmental sources

Introduction of organisms into normally sterile

sites by medical instrumentation (intravenous

or urinary catheters, endotracheal tubes or

tracheostomies, respiratory care equipment) in

debilitated hospital patients (antibiotic

treatment, surgery, intensive care units,

surgery)

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Acinetobacter Species: Types of

Infectious Disease

Nosocomial infections of the respiratory tract, urinary tract, and wounds (including catheter wounds) often with progression to bacteremia

Sporadic cases of ambulatory peritoneal-dialysis related peritonitis, endocarditis, meningitis, arthritis, and osteomyelitis

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Acinetobacter Species:


Gram-negative coccobacillary rods occurring singly and in Neisseria-like pairs

Oxidize rather than ferment D-glucose

(OF glucose +)

Neither oxidize nor ferment D-glucose

(OF glucose –)

A. baumannii complex/OF glu + OF lac +, non-hemolytic

A. lwoffii/OF glu – OF lac –, non-hemolytic

A. haemolyticus/OF glu – OF lac –, β-hemolysis on sheep blood agar

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Acinetobacter:

Genomospecies

Twenty-one different Genomospecies based on DNA-DNA hybridization

Genomospecies 1, 2, 3, and 13: A. calcoaceiticus-baumanii complex (A. baumanii1)

Genomospecies 8/9: A. lwoffi2

Genomospecies 4: A. haemolyticus3

1Saccharolytic, non-hemolytic 2Non-Saccharolytic, non-hemolytic 3Non-Saccharolytic, β-hemolytic

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Moraxella: Natural Habitats

and Clinical Infections1 Saprophytic on human skin and mucous membranes

Most frequently isolated species by culture M. nonliquefaciens is a component of normal respiratory flora

Ocular pathogens (conjunctivitis, keratitis) and unusual causes of invasvie infection (meningitis, bacteremia, endocarditis, and arthritis)

1Excludes Moraxella catarrhalis (identified in the laboratory using Neisseria protocols)

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Moraxella: Microbiology

Cytochrome-oxidase positive gram-negative or gram-variable Neisseria-like diplococci, forming small (0.5-1mm) colonies on blood agar (24-48 hr), smooth, translucent to semi opaque in appearance, occasional strains show pitting of agar

Non-motile, indole negative, and asaccharolytic

Species identification generally not performed because given the similarity of pathogenic signficance of all species

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Pseudomonas stutzeri: Natural

Habitats and Clinical Infections

Environmental sources (especially aqueous) as

with Pseudomonas aeruginosa

Bacteremia and meningitis reported in

immunosuppressed individuals

Pneumonia in alcoholics

Endophthalmitis following cataract surgery and

bacteremia due to contaminated hemodialysis fluid

(iatrogenic infections)

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Other Non Fermenters B. pseudomallei

Causes melioidosis, a disease seen primarily in southeast Asia where it is a normal inhabitant of soil and water.

The disease is acquired through contamination of wounds or via inhalation or ingestion.

The disease may range from unapparent, to chronic or acute pulmonary infection, to overwhelming septicemia with multiple abscesses in many organs

B. mallei Causes glanders in equines.

Humans occasionally acquire the disease by contact with infected nasal secretions of equines, through abrasions and occasionally through inhalation.

Used to be a problem in the military when horses where used.

The disease may manifest as a chronic pulmonary disease, as a form characterized by multiple abscesses of the skin, subcutaneous tissue, and lymphatic's (Farcy), or as an acute, fatal septicemia.

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Nonfermenting gram negative bacteria.pptx12

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