Identification of any microbe will follow a process of 3 steps:
- Other tests
We will attempt to deal with each in turn.
Sometimes Gram (+) and Gram (-) organisms can be seen in the same film. We have covered Gram variability before, but just remember that the most common cause is that of antibiotic useage. It is the peptidoglycan that allows Gram (+) organisms to retain the iodine dye. Staphylococci form a characteristic "bunch of grapes" appearance on the Gram film. This occurs because as the staphs divide by binary fission, they do so in 3 planes, in contrast to streps which divide in a single plane, resulting in long chains. The Gram stain will give a suggestion of the possible genus of the organism, but when reporting a Gram film, they are GPCs. Do not make any suggestion of what they may (or may not) be.
Staphylococcus spp. on blood agar:
- 1-3mm long (comparatively large colonies)
- Low convex
- buff or gold coloured
- +/- β-haemolysis
Staphylococcus spp. grow better on the aerobic plate. They will have more colourful and larger colonies than the anaerobic plate. This is in contrast to Streptococci, which grow better in anaerobic conditions. Whilst in the incubator, staphs don't require CO2 to grow, whilst streps do.
Staphylococcus spp. will grow on any media. They are salt tolerant, as most organisms are killed by high [NaCl].
"Salt agar" is 7-10% salt containing broth and agar or salt cooked meat broth. Staphylococcus aureus has the ability to ferment mannitol resulting in acidification. Indicator media containing phenol red turns Staphylococcus aureus colonies yellow (it turns pink in alkaline conditions). CoNS don’t usually ferment mannitol, which aids ID.
When growing staphs on MacConkey’s agar, the colonies are pink due to the neutral red indicator (pink in acid, colourless in alkaline)
With CLED, the bromothymol blue indicator turns yellow in acid & blue in alkaline.
“Sab” plates used in mycology kill off all the bacteria to allow fungi to grow.
Baird-Parker agar (with cipro):
This medium contains lithium chloride and tellurite to inhibit the growth of accompanying microbial flora, whereas pyruvate and glycine selectively stimulate the growth of staphylococci. Staph colonies show two characteristic features when grown in this opaque medium (opaque, because of its egg-yolk content)
- characteristic zones and rings are formed as a result of lipolysis and proteolysis
- reduction of tellurite to tellurium produces a black colouration.
- The egg-yolk reaction and tellurite reduction are usually found to occur together with a positive coagulase reaction and can thus serve as an index for the latter.
- This will not differentiate a MSSA from a MRSA.
Initially the media contained cipro, but this was removed as many CA-MRSA are cipro (S).
We plated out three different organisms on a variety of media. We used the "Oxford Staph" (the positive control), MRSA & Staphylococcus epidermidis (negative control). The results are shown below:
1) MRSA on Blood Agar:
2) MRSA on Baird-Parker:
3) MRSA on Chocolate Agar:
4) MRSA on Chromogenic Agar:
5) MRSA on CLED agar:
6) MRSA on MSA:
7) Oxford Staph on Blood Agar:
8) Oxford Staph on Baird-Parker:
9) Oxford Staph on Chocolate Agar:
10) Oxford Staph on Chromogenic Agar:
11) Oxford Staph on CLED:
12) Oxford Staph on MSA:
13) Staphylococcus epidermidis on Blood Agar:
14) Staphylococcus epidermidis on Baird-Parker:
15) Staphylococcus epidermidis on Chocolate Agar:
16) Staphylococcus epidermidis on Chromogenic Agar:
17) Staphylococcus epidermidis on CLED:
18) And finally a DNAse test (with the HCL added showing precipition):
Identification of MRSA:
Chromogenic agar containing 8mg/l oxacillin resulting in a pink colony. The mechanism is a secret!
For complete ID:
Reculture with a 10mg cefoxitin disc, if diameter <22mm, it is MRSA.
Some S. aureus strains have borderline resistance to oxacillin with MICs between 4 and 16 mg/L and are mecA negative. The mechanism of resistance for these strains is believed to be hyper-production of beta-lactamase. The acronym BORSA has been used, meaning borderline oxacillin resistant S. aureus. These cannot grow in the presence of cefoxitin.
Accurate detection of oxacillin/methicillin resistance can be difficult due to the presence of two subpopulations (one susceptible and the other resistant) that may coexist within a culture. All cells in a culture may carry the genetic information for resistance but only a small number can express the resistance in vitro. This phenomenon is termed heteroresistance and occurs in staphylococci resistant to penicillinase-stable penicillins, such as oxacillin.
Heteroresistance is a problem for clinical laboratory personnel because cells expressing resistance may grow more slowly than the susceptible population. This is why isolates being tested against oxacillin, methicillin, or nafcillin should be incubated at 35° C for a full 24 hours before reading (this ↑↑gene expression. The breakpoints for S. aureus are different from those for coagulase-negative staphylococci (CoNS).
Biochemical tests (1): Catalase [uses 3% H2O2]
Catalase: Staphs (+), Streps (-)
Method: Staph produce catalase: H2O2→H2O + O2.
- Drop of H2O2 on a slide
- Smear colonies onto a coverslip – watch for immediate vigorous bubbles
- +ve control: Oxford Staph
- -ve control: Group A Strep (GAS)
- 1ml H2O2 in a tube
- Pick colony with a wooden stick
- Watch for immediate vigorous bubbles
REMEMBER to pick the colonies from a non-blood agar plate, as blood contains catalyse and can give a false positive. Choc agar is OK.
Biochemical tests (2): Coagulase [rabbit/sheep/horse plasma]
- Two forms
- Bound (clumping factor)
- Free (coagulase)
- Converts fibrinogen in plasma to a fibrin (clot)
- Plasma → Fibrin clot
- Need a thick suspension with a large innoculum
- +ve: Oxford Staph
- -ve: Staphylococcus epidermidis
- Check saline alone for auto-agglutination
- Auto-agglutination: discard test
- -ve slide test: do a tube test
- ? not a CoNS
- Dilute plasma 1:10
- Incubate @ 37oC
- Check @ 1/2hr, 1hr, 2hrs, up to 4 hrs – keep checking as clot may lyse due to streptokinase – if this happens REPEAT
- If no clot @ 4hrs – incubate overnight to be sure
- Citrate consuming organisms give a false +ve
It is important to keep the reagents citrate free, as some organisms can use citrate → auto-activation of the clotting pathway. This is true of Gram (-) bacteria (Citrobacter, Enterobacter & Enterococcus faecalis).
Coagulase positive organisms:
- Staphylococcus aureus
- Staphylococcus intermedius (dog bites)
- positive for both clumping factor and free coagulase
- Staphylococcus lugdunensis
- positive for clumping factor
- Slide (+) & tube (-)
- TUBE (-) DEFINES A CoNS, despite (+) for clumping factor
- Erysipelothrix spp.
- Gram (+) bacilli
- Endocarditis, cellulitis & septicaemia
- ↑growth with 5-10% CO2
- Coagulase (+), catalyse (-)
- α-haemolysis on blood agar
- Rx: penicillin, macrolides & clindamycin. E. rhusiopathiae ® to vanc
Coagulase negative organisms:
- Staphylococcus epidermidis – slime production
- Staphylococcus lugdunensis – ornithine decarboxylase
- Staphylococcus haemolyticus – β-haemolysis
- Staphylococcus saprophyticus – resistant to 5µg novobiocin (< 15mm zone diameter)
Confirmation then occurs by plating on a DNase plate. If a Staph is present, HCL added to the media precipitates this on day 2, clearing around the colony as DNA is digested.
- Heat stable thermonuclease
- Broth heated prior to insertion in the agar.
- Suspended colonies put into wells to allow diffusion, the indicator turns blue around each well (TNase+).
- Found in Staphylococcus aureus
- Binds to the Fc of IgG
- Better than a slide test
- Coloured clumps appear as protein A is found
API Staph: good for speciation
Gene Tests: e.g. PVL, mecA
- Phage typing (4 classes)