laboratory diagnosis of staphylococcus

LABORATORY DIAGNOSIS

OF STAPHYLOCOCCUS

MADE BY:

SHALINI BISHT

Saturday, February 18, 2017 1

LABORATORY DIAGNOSIS

Sample collection and Transportation

Direct smear Microscopy

Culture

Biochemicals

Typing of Staphylococcus aureus

Antibiotic Sensitivity Testing (AST)


SAMPLE COLLECTION

Type of sample depends on the site of

infection.


Infection Specimen

Suppurative lesion Pus, wound swab

Respiratory infection Sputum

UTI Mid stream urine

PUO, Bacteremia Blood

Food poisoning Feces, Vomitus, food

Carriers Nasal and perianal swab

DIRECT SMEAR MICROSCOPY

Staphylococcus appear as GPC

measuring 0.5-1.5 microns

Occur singly, in pairs, short chains or

clusters

Present within and outside PMNs

Reporting of direct smears:

◦ quantitation of cell types and

microorganisms

◦ Eg. Many pus cells along with moderate

number of GPC seenSaturday, February 18, 2017 4

CULTURE

• Specimens are inoculated onto the suitable media.

Plates incubated for 18-24 hour at 37°C.

On nutrient agar

Colonies are golden yellow and opaque with smooth

glistening surface, 2-4 mm in diameter, circular, convex,

shiny & easily emusifiable.

(Most strains produce non diffusible Golden yellow

pigment)

Nutrient Agar slope

◦ Confluent growth, Oil paint appearance.Saturday, February 18, 2017 6

Blood agar

◦ Colonies similar to those on Nutrient Agar

◦ Colonies are beta-hemolytic

Liquid medium

◦ Uniform turbidity

MacConkey Agar

◦ Small pink colonies due to Lactose

fermentation


Selective media

MSA – 1% Mannitol + 7.5% NaCl +

phenol red

Salt milk agar – 6.5% NaCl + 10%

skimmed milk

Ludlam’s medium – Lithium chloride

and tellurite


BIOCHEMICAL REACTIONS

•Catalase : positive

•Coagulase test : positive

•Oxidase : negative

(Except S.sciuri group i.e., S.sciuri, S.lentus,

S.vitulinus)

•Ferment glucose, lactose, maltose, sucrose and

mannitol, with production of acid but no gas

•Indole : negative

•MR test : positive

•VP test : positive

•Gelatin liquefaction : positive

•Phosphatase : positive

•DNA-ase : positiveSaturday, February 18, 2017 11

Catalase test •Done to distinguish staphylococci from

streptococci (catalase negative)


Coagulase test•Done to distinguish pathogenic strain (S.aureus)

from non-pathogenic strains.

•2 Methods of coagulase detection are:

(1) Slide coagulase test : detects bound

coagulase

(2) Tube cogulase test : detects free coagulase

(other coagulase +ive staphylococci are

S.intermedius, S.hyicus)


Gelatin liquefactionPrinciple: this test is used to determine the ability

of an organism to produce proteolytic enzyme

(gelatinase) that liquefies the gelatin.


Methyl Red testPrinciple: this test detects the production of

sufficient acid during fermentation of glucose by

bacteria and sustained maintenance of ph below

4.5


Voges-Proskauer (VP) testPrinciple: the test depends upon the production

of acetoin from pyruvic acid in the media. In the

presence of alkali & atmospheric oxygen, acetoin

is oxidised to diacetyl which reacts with alpha

naphthol to give red color.


DNA Hydrolysis•Principle : this test is used to determine the ability of an organism to hydrolyze DNA. Green color of the medium is due to DNA-methyl green complex. If the organism growing on the medium hydrolyzes DNA, the green color fades & the colony is surrounded by a colorless zone.


Phosphatase testPrinciple: Staphylococci are grown on nutrient agar containing sodium phenolphthalein diphosphate and incubated overnight at 37°C. The plate is exposed to ammonia vapours. The pink color of the colonies indicate a positive result.


S.aureus v/s CONS


BACTERIOPHAGE TYPING


Epidemiological purpose to trace

source of infection.

Useful in outbreaks like food poisoning

in a community.

Typing methods:

◦ Phenotypic-bacteriophage typing:

staphylococci are typed based on their to

bacteriophages.

◦ Molecular typing: DNA finger-printing,

ribotyping, PFGE etc.


Bacteriophage typing

Method

◦ Test strain inoculated as lawn culture on NA.

◦ Drops of routine test dose of known set of

different phages are spot inoculated &

incubated.

◦ Zone of lysis will be produced in those areas

where test strain is susceptible to phages

applied.

◦ If strain lysed by phages 29, 52A, 79, but not

other phages; it is designated as phage type

29/52A/79

◦ National Reference Centre: MAMC, New

Delhi


ANTIBIOTIC SENSITIVITY TESTING

This is important as staphylococci develop

resistance to drugs readily.


Why AST has become a

necessity ??

Bacteria have the ability to develop resistance

following repeated or subclinical doses, so more

advanced antibiotics are required to overcome

them.

Antibiotic sensitivity test: A laboratory test

which determines how effective antibiotic

therapy is against a bacterial infections.

Testing will assist the clinicians in the choice of

drugs for the treatment of infections.

Helps in the local pattern of antibiotic

prescribing.Saturday, February 18, 2017 26

Methods of AST•Performed on MHA by Kirby-Bauer Disc diffusion method.

•Following antibiotics are employed for staphylococcus:

•Amoxyclav

•Clindamycin

•Cefoxitin

•Ciprofloxacin

•Erythromycin

•Gentamicin

•Linezolid

•Levofloxacin

•Penicilin-G

•Vancomycin

•TeicoplaninSaturday, February 18, 2017 27

Kirby-Bauer method

1. Dry the agar plates (MHA) & label them.

2. Dip a sterile swab into the broth and express any excess moisture by pressing the swab against the side of the tube.

3. Swab is streaked as a lawn (lawn culture) onto a Mueller-Hinton agar (in 3 directions to ensure confluence).

4. The anitibiotic(s) disk will be placed onto the MHA plate.

5. The plate is incubated and is examined for resistance and sensitivity pattern the following day.


MRSA

•Methicillin-resistant S. aureus.

•First reported in 1960s.

•May colonize mucosal or epithelial surfaces, (common : anterior nares)

•Nosocomial pathogen.

•Shows Resistant to penicillins, cephalosporins, carbapenems, monobactams.

•Vancomycin resistance is rare – so far

•Hospital-acquired (HA MRSA)

•Community-acquired cases now (CA MRSA)


Predisposing factors for

MRSA

•Prolonged & repeated hospitalization

•Indiscriminate use of antibiotics

•Intravenous drug abuse

•Presence of indwelling medical devices


MECHANISM

•MRSA contains the mecA gene which is responsible

for the production of an altered plasma (cell)

membrane-bound enzyme, penicillin-binding protein

2a (PBP- 2a.)

•The altered PBP 2a while able to perform its cell-wall

synthesis function, has a lower affinity and does not

bind to beta-lactam antibiotics

•Thus, the presence of the mecA gene confers

resistance to all beta-lactam antibiotics such as

methicillin.


• Vancomycin remains the drug of choice for

treatment of infections caused by MRSA,

although it is intrinsically less active than the

antistaphylococcal penicillins.

•Combinations of vancomycin with ss-lactam

antibiotics may be synergistic in vivo against

MRSA strains, including those with intermediate

susceptibility to vancomycin.

•Given the increasing prevalence of MRSA in

hospitals and in community settings, alternative

approaches are needed for treatment of

infections caused by MRSA.Saturday, February 18, 2017 32

Preventive measures

•Isolation & treatment of MRSA patients.

•Detection of carriers among hospital staff, their

isolation & treatment.

•Avoid indiscriminate usage of antibiotics.

•Following strict aseptic technique


•HAND WASHING STILL CONTINUES TO REDUCE

SEVERAL INCIDENCES OF MRSA SPREAD IN

HEALTH CARE


Detection of MRSA

•MRSA is determined by disc diffusion test using

cefoxitin (30µg) disc on MHA with 2% NaCl & 104

cfu/ml inoculum and incubated at 33-35°C for 24

hour.

•As per CLSI guidelines inhibition zone of </= 21

mm was taken to be MRSA.


Coagulase Negative

Staphylococci

Two species of coagulase negative

Staphylococci can cause human infections:

1. Staphylococcus epidermidis

2. Staphylococcus saprophyticus


S. epidermidis:

•It is a common cause of stitch abscesses.

•It has predilection for growth on implanted foreign bodies such as artificial valves, shunts, intravascular catheters and prosthetic appliances leading to bacteremia.

•In persons with structural abnormalities of urinary tract, it can cause cystitis.

•Endocarditis may be caused, particularly in drug addicts.


S.saprophyticus:

•It causes urinary tract infections, mostly in sexually active young women.

•The infection is symptomatic and may involve the upper urinary tract also.

•Men are infected much less often.

•It is one of the few frequently isolated CoNS that is resistant to Novobiocin


Other coagulase negative staphylococci:

•S.haemolyticus

•S.saprophyticus

•S.warneri,

•S.hominis,

•S.epidermidis

•S.caprae

•S.lugdunensis


laboratory diagnosis of staphylococcus

Health & Medicine

Transcript of laboratory diagnosis of staphylococcus