A 35 year old male patient presents with an abscess over the jaw.

What are pyogenic bacteria?
These are bacteria that induce pus formation upon infection.

Name some pyogenic bacteria.
Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Hemophilus influenzae etc.

How is the specimen collected from abscesses?
If the abscess is closed, pus can be collected in a sterile container after incision and drainage. Alternatively, the pus can either be aspirated with needle and syringe especially if anaerobic cultures are to be performed. In case of open abscess, pus can collected from the depth of the lesion after superficial cleaning. The material collected (pus) in swab must be kept in a sterile test tube and transported immediately to the laboratory.

Which are the investigations performed?
A smear is prepared on slide and stained with Gram's stain. The specimen is inoculated into Blood agar and incubated at 37^oC overnight. Following identification of the pathogen, antibiotic susceptibility of the isolate is performed.

What is the Gram stain observation?
Plenty of pus cells along with Gram positive cocci in typical grape like clusters seen. This observation is consistent with the morphology of Staphylococcus.

Describe the growth seen on Blood agar.
Smooth, circular, convex, butyrous, golden-yellow pigmented colonies that are beta-hemolytic are seen.

Which is the other beta hemolytic bacterium?
Beta hemolytic Streptococci

Which are the other pigmented bacteria?
Psedomonas aeruginosa, Micrococcus luteus, Serratia marcesens, Chromobacterium violaceum and some chromogenic Mycobacteria produce pigmented colonies.

How do you identify this bacterium?
A Gram stained smear showing gram positive cocci in grape-like clusters suggests Staphylococcus. Further, a positive catalase test differentiates it from Streptococci. Positive mannitol fermentation and  positive tube coagulase test confirms it as Staphylococcus aureus.

What is the significance of coagulase test?
Coagulase test is a pathogenicity test. It helps to identify the pathogenic species of Staphylococcus, namely S. aureus.

Which are the different types of coagulase test?
There are two types of coagulase test: slide coagulase and tube coagulase. Slide coagulase test is useful in detecting bound coagulase while tube coagulase test is useful in detecting free coagulase. While slide coagulase test is used for screening isolates, tube colagulase is used for confirmation.

How is slide coagulase test performed?
Two milky suspensions of the test isolate in saline are made on either side of a glass slide. One is marked as test and the other as control. To the suspension marked test, a loopful of undiluted rabbit plasma is added and mixed well. Immediate appearance of clumps indicates a positive slide coagulase test. The suspension marked control should not show any auto-agglutination.

How is tube coagulase performed?
Three test tubes are taken and marked test, positive control and negative control. Around 0.5 ml of overnight growth of test bacteria in broth is taken in test tube labeled test, 0.5 ml of known S. aureus suspension in test labeled positive and 0.5 ml of sterile broth is taken in tube labeled negative. To the tubes labeled test and positive 0.5 ml of 1 in 10 diluted rabbit plasma is added while same volume of sterile broth is added to tube labeled negative. The tubes are incubated at 37°C and observed at hourly interval for clotting of plasma. A positive test in indicated by clotting (gelling) of plasma, which does not flow when the tube is inverted. There should be no gelling in tube labeled negative.

Are there any other specific tests to identify S. aureus?
Other tests such as phosphatase test and tellurite reduction do help in identifying S. aureus but they are not specific. DNase test or thermonuclease test is more specific.

Which are the other infections produced by this isolate?
S. aureus is known to produce superficial infections such as boils, folliculitis, carbuncle, furuncle, stye and wound infections. They are responsible for deep infections like pharyngitis (less common), post-viral pneumonia, endocarditis, pyelonephritis, bacteremia etc. the toxin mediated diseases include food-poisoning, Toxic shock syndrome and Scalded skin syndrome. They are important nosocomial pathogens.

In what way the superficial lesions produced by S. aureus differ from those produced by Streptococci?
Streptococcal lesions tend to be spreading (due to hyaluronidase) while lesions produced by S. aureus tend to be limiting. The pus in streptococcal lesions tends to be more serous (due to breakdown of DNA by DNase) than in Staphylococcal lesions.

Which are the important virulence factors of S. aureus?
S. aureus is known to produce various surface factors, toxins and enzymes that helps in its pathogenicity. Important enzymes are coagulase, fibrinolysin, lipase, hyaluronidase, and DNase. Important toxins are hemolysins, enterotoxins, TSST-1 and exfoliative toxin. Cell wall peptidoglycan and teichoic acid too confer some degree of virulence.

How is this condition treated?
After the pus is drained, the patient must be put on suitable antibiotics. The choice of antibiotics depends on the results of antibiotic susceptibility test.

What is MRSA?
It stands for methicillin resistant S. aureus. An altered penicillin binding protein (PBP), which has low affinity to penicillins, renders them resistant to penicillinase resistant penicillins such as methicillin, oxacillin and nafcillin. These strains are resistant to all penicillins and cephalosporins and are multi-drug resistant. Serious infections by MRSA strains are currently treated using vancomycin. They are important nosocomial pathogens these days.

How are Staphylococci resistant to penicillins?
Majority of Staphylococci are resistant to penicillins owing to the production of penicillinase (also known as beta-lactamase) enzymes that inactivate penicillins. Other mechanism includes production of altered penicillin binding protein (PBP) with reduced affinity to the penicillins. This mechanism is seen in MRSA. Low-level resistance to vancomycin too has emerged among these isolates.

How is Staphylococcus classified?
Staphylococcus were initially classified on the basis of their pigment production into three: S. aureus (golden yellow pigment), S. citreus (lemon yellow pigment) and S. albus (non-pigmented; white). Since pigment production is not consistent and certain strains of S. aureus may not be pigmented, this system is not of much use. Practical way of classifying Staphylococcus is by way of coagulase test; coagulase positive isolates are usually S. aureus whereas coagulase negative isolates are called coagulase negative Staphylococcus.

What are coagulase negative Staphylococci?
Species of Staphylococcus other than aureus, which are coagulase negative are called coagulase negative Staphylococcus (CoNS). These reside as commensal on human or animal skin and were regarded as contaminants. Its role in human infections is increasing, especially as a nosocomial pathogen. S. epidermidis, S. saprophyticus, and S. intermedius are some of the CoNS. Formation of biofilms help some species to colonise readily. S. saprophyticus is known to cause UTI among sexually active young women ("honeymoon cystitis"). It is identified by its characteristic resitance to novobiocin (disk). Methicillin resistance also occurs among CoNS.

How are hospital strains identified?
Most of the hospital strains are resistant to multiple antibiotics. Healthcare-acquired methicillin-resistant Staphylococcus aureus, or HA-MRSA strains are mostly positive for Panton Valentine gene. Strains causing outbreaks in the hospital can be typed by several phenotypic and genotypic methods. One of the phenotypic methods include phage typing.