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Citron DM. Appelbaum PC.: How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay? Clinical Infectious Diseases. 16 Suppl 4:S435-8, 1993 Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive. Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory. Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician. Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified. Group-or genus-level identifications may suffice in other instances. The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents. Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of myonecrosis and gas gangrene,potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin).

Staphylococcus aureus, particularly methicillin resistant strains (MRSA), have represented a likely target for molecular development, particularly in blood cultures. As more institutions implement patient screening protocols for MRSA, replacement of routine culture methods with molecular assays has gained increasing attention.PNA-FISH assays provide for the definitive identification of Staphylococcus aureus from positive blood culture vials. Peptide nucleic acid fluorescent in-situ hybridization is a relatively straight forward procedure that does not involve amplification and has limited equipment requirements. Procedurally it is easy to perform with minimal hands on time.PNA is a synthetic imitator of a nucleic acid sequence in which the backbone is a pseudopeptide rather than a sugar. PNA behaves similarly to DNA and will bind to complementary nucleic acid strands. A PNA probe is constructed, utilizing a complementary, hybridizing sequence for a known nucleic acid target sequence. The probe is typically bound to a fluorescent protein as a means of visualizing/detecting the target. In one commercially available method, once a blood culture vial demonstrates gram-positive cocci in clusters, a drop of the blood culture broth is added to fixation solution on a slide. Heat or methanol is used to fix the smear. After fixation, probe that targets species-specific ribosomal RNA is added to the smear, which is then cover-slipped.Slides are then incubated at 55oC. Post incubation, slides are immersed in a preheated wash solution and coverslips gently removed. After incubation in the wash solution, smears are air dried; a drop of mounting medium is added and the slide is cover-slipped again.The slides are examined with a fluorescent microscope, utilizing specific filters. Green fluorescing cocci in clusters are identified as Staphylococcus aureus. This identification would be available, depending on the routine identification system utilized, potentially 24 hours earlier than the norm.A significant number of blood cultures that demonstrate gram-positive cocci in clusters yield coagulase negative staphylococci (CNS), which represent potential contaminants, rather than significant infection. What is the significance of differentiating blood cultures that contain S. aureus from those that are growing CNS in a much earlier timeframe?Studies have shown that IF the differentiation of CNS from S. aureus is effectively communicated to clinicians and pharmacy/antimicrobial stewardship teams, active assessment can occur utilizing defined exclusion criteria for those patients whose cultures yielded CNS rather than S. aureus. In scenarios where contamination rather than infection is indicated, vancomycin can be discontinued earlier, and length of hospital stay is also shortened. Reduced antibiotic exposure, reduced risk of development of resistance, and reduced cost are all potential benefits.

A clinical isolate is presumptively identified as Staphylococcus aureus by means of several simple procedures :Gram Stain : Gram-positive cocci, occurring singly, in pairs or "bunches of grapes"Catalase test: Staphylocci are catalase-positive, distinguishes them from Streptococci which are catalase-negative.Coagulase test: S. aureus is coagulase-positive.DNAse Test – S. aureus is DNAse-positiveHeat stable endonuclease – S. aureus is positiveThere are also many commercial kits available for identification of S. aureus based on latex agglutination.

Gram stain: gram-positive cocci in singles, pairs, or chains; cells can be ovoid to coccobacillaryColony morphology: on blood agar after 24 hours of incubation, colonies are nonhemolytic or alpha hemolytic (rare strains may be beta hemolytic), and approximately 1 to 2 mm in diameter.Catalase: negativePresumptive identification: Growth on bile esculin agar and in 6.5% salt broth are two characteristics that have commonly been used to identify Enterococcus to the genus level. A positive esculin in combination with a positive PYR reaction is another approach to presumptive identification.Species identification: E. faecalis and E. faecium are usually easily identified by most commercial systems. Successful identification of the other species on these systems may vary. With respect to vancomycin intermediate or resistant strains, two key characteristics are motility and pigment. E. casseliflavus is both motile and possesses a yellow pigment; E. gallinarum is also motile but non-pigmented. E. faecalis and E. faecium demonstrate neither characteristic.

Bacteria may have a very distinctive appearance when viewed on a Gram-stained smear. Distinct characteristics are not as evident on direct smears as they are on Gram stains of colonies and/or stains from broth cultures, but these shapes can be identified on direct smears. Cocci: Round or oval Bacilli: Rod-shapedGram-positive cocci (GPC): Singly and in clusters (May indicate staphylococci) In pairs and chains (May indicate streptococci)GPC can be seen in clusters and chains in image 1 Image 1

Gram-negative cocci that occur in pairs with their adjacent sides flattened, giving them a "coffee bean" appearance, are typical of the genus Neisseria. The presence of intracellular gram-negative diplococci on a smear made from a purulent urethral discharge from a male can be confirmatory of the diagnosis of gonorrhea. In this case, cultures may not even be needed unless susceptibilities are required. However, if the genital specimen is from a female (cervical specimen), the presence of gram-negative diplococci is not specific enough to confirm the diagnosis, and a culture or other confirmatory testing must be performed. Organisms such as Acinetobacter sp. and Moraxella sp. may mimic the appearance of N. gonorrhoeae and can lead to false-positive results.Direct smears read specifically for the presence of Neisseria gonorrhea should include a direct reference to gram-negative intracellular diplococci.

Bacteria may also be present, especially during a urinary tract infection. This view shows bacteria as solid gray rods or cocci. Since bacteria may also be a contaminant in specimens remaining at room temperature, or due to an unclean catch, caution must be observed in reporting bacteria. If 20 organisms per high power field (HPF) are seen, the bacteria are considered to be clinically significant.