None of the commercial identification systems has been found to accurately identify all species of viridans streptococci or enterococci.

Comments Regarding Specific Organisms

Useful characteristics for differentiation among catalase negative, gram-positive cocci are shown in Tables 1 and 2. Organisms that may be weakly catalase positive, such as Rothia mucilaginosa (formerly Stomatococcus mucilaginosus), or coccobacillary, such as Lactobacillus, are included in these tables.

Table1. Differentiation of Catalase-Negative, Gram-Positive Coccoid Organisms Primarily in Chains

Table2.  Differentiation of Catalase-Negative, Gram-Positive, Coccoid Organisms Primarily in Clusters or Tetrads

Table2. Differentiation of Catalase-Negative, Gram-Positive, Coccoid Organisms Primarily in Clusters or Tetrads—cont’d

The cellular arrangement and the type of hemolysis are important considerations in identification (Figure 1). If the presence of hemolysis is uncertain, the colony should be moved aside with a loop and the medium directly beneath the original colony should be examined by holding the plate in front of a light source.

Fig1. Differentiation of gram-positive cocci. 

A screening test for vancomycin susceptibility is often useful for differentiating among many alpha-hemolytic cocci. All streptococci, aerococci, gemellas, lactococci, and most enterococci are susceptible to vancomycin (any zone of inhibition), whereas pediococci, leuconostocs, and many lactobacilli are typically resistant (growth up to the disk). Other useful tests listed in Tables 1 and 2 includes leucine aminopeptidase (LAP) and pyrrolidonyl arylamidase (PYR), which are commercially available as disks.

Leuconostoc produces gas from glucose in MRS broth; this distinguishes it from all other genera, except the lactobacilli. However, unlike Leuconostoc spp., lactobacilli appear as elongated bacilli when Gram stained from thio glycollate broth. Several organisms (e.g., Leuconostoc, Pediococcus, Lactococcus, Helcococcus, Globicatella, Tetragenococcus, Streptococcus urinalis, and Aerococcus viridans) will show growth on bile-esculin agar and in 6.5% salt broth; this is the reason these two tests no longer solely can be used to identify enterococci.

Serologic grouping of cell wall carbohydrates (Lance field classifications) has classically been used to identify species of beta-hemolytic streptococci. The original Lancefield precipitin test is now rarely performed in clinical laboratories. It has been replaced by either latex agglutination or coagglutination procedures available as commercial kits. Serologic tests have the advantage of being rapid, confirmatory, and easily performed on one or two colonies. However, they are more expensive than biochemical screening tests.

The PYR and hippurate or CAMP tests can be used to identify groups A and B streptococci, respectively. However, use of the 0.04-U bacitracin disk is no longer recommended for S. pyogenes, because groups C and G streptococci are also susceptible to this agent. S. pyogenes is the only species of beta-hemolytic streptococci that will give a positive PYR reaction.

S. agalactiae is able to hydrolyze hippurate and is positive in the CAMP test. The CAMP test detects production of a diffusible, extracellular protein that enhances the hemolysis of sheep erythrocytes by Staphylococcus aureus. A positive test is recognized by the appearance of an arrowhead shape at the juncture of the S. agalactiae and S. aureus streaks (Figure 2). Occasionally, non–beta hemolytic strains of Streptococcus agalactiae may be encountered, but identification of such isolates can be accomplished using the serologic agglutination approach. Enterococci can also be hippurate hydrolysis positive.

Fig2. Positive CAMP reaction as indicated by enlarged zone  of hemolysis shaped like a tip of the arrow, S. agalactiae intersecting  with S. aureus streak line. 

Table 3 shows the differentiation of the clinically relevant beta-hemolytic streptococci. Minute beta hemolytic streptococci are all likely to be of the S. anginosus group; a positive Voges-Proskauer test and negative PYR test identify a beta-hemolytic streptococcal isolate as such.

Table3. Differentiation of the Clinically Relevant Beta-Hemolytic Streptococci

Suspicious colonies thought to be S. pneumoniae must be tested for either bile solubility or susceptibility to optochin (ethylhydrocupreine hydrochloride). The bile solubility test is confirmatory and is based on the ability of bile salts to induce lysis of S. pneumoniae. In the optochin test, which is presumptive, a filter paper disk (“P” disk) impregnated with optochin is placed on a blood agar plate previously streaked with a lawn of the suspect organism. The plate is incubated at 35° C for 18 to 24 hours and read for inhibition. S. pneumoniae produce a zone of inhibition, whereas viridans streptococci grow up to the disk. A newly discovered organism, Streptococcus pseudopneumoniae, may interfere with appropriate interpretation  of the optochin disk test. S. pseudopneumoniae are resistant to optochin (zone ≤14 mm) when they are incubated under increased CO2, but are susceptible to optochin (zone >14 mm) when they are incubated in ambient atmosphere. Therefore, optochin disk tests should be incubated under 5% CO2 and all tests should be confirmed by a bile solubility test. Unfortunately, the commercial molecular probe for S. pneumoniae, AccuProbe Pneumococcus (Gen-Probe, San Diego, California), does not discriminate between S. pneumoniae and S. pseudopneumoniae. Because the pathogenic potential of S. pseudopneumoniae is currently unknown, it is important to differentiate it from S. pneumoniae, a known pathogen. Serologic identification of S. pneumoniae is also possible using coagglutination or latex agglutination test kits.

Once S. pneumoniae has been ruled out as a possibility for an alpha-hemolytic isolate, viridans streptococci and enterococci must be considered. Figure 3 outlines the key tests for differentiating among the viridans streptococci. Carbohydrate fermentation tests are performed in heart infusion broth with bromcresol purple indicator. Although alpha-hemolytic streptococci are not often identified to species, there are cases (i.e., endocarditis, isolation from multiple blood cultures) in which full identification is indicated. This is particularly true for blood culture isolates of S. bovis that have been associated with gastrointestinal malignancy and may be an early indicator of gastrointestinal cancer. S. bovis possesses group D antigen that may be detected using commercially available typing sera. However, this is not a definitive test, because other organisms (e.g., Leuconostoc) may also produce a positive result.

Fig3. Differentiation of clinically relevant viridans streptococcal groups. S. mitis group includes S. mitis, S. sanguinis, S. parasanguinis, S. gordonii, S. oralis, and S. cristatus. S. mutans group includes S. mutans and S. sobrinus. S. anginosus group includes S. anginosus, S. constellatus subsp. constellatus, and S. intermedius. S. salivarius group includes S. salivarius and S. vestibularis. β-gal, Beta-galactosidase; PYR, pyrrolidonyl arylamidase; R, resistant; S, sensitive; +, positive; =, negative. (Compiled from Collins MD, Hutson RA, Falsen E, et al: An unusual Streptococcus from human urine, Streptococcus urinalis sp. nov, Int J Syst Evol Microbiol 50:1173, 2000; Poyart C, Quesne G, Trieu-Cuot P: Taxonomic dissection of the Streptococcus bovis group by analysis of manganese-dependent superoxide dismutase gene (sodA) sequences: reclassification of "Streptococcus infantarius subsp. coli" as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis biotype II.2 as Streptococcus pasteurianus sp. nov, Int J Syst Evol Microbiol 52:1247, 2002; and Schlegel L, Grimont F, Collins MD, et al: Streptococcus infantarius sp. nov, Streptococcus infantarius subsp. infantarius subsp. nov, and Streptococcus infantarius subsp. coli subsp. nov, isolated from humans and food, Int J Syst Evol Microbiol 50:1425, 2000.)

Except for species not usually isolated from humans (E. saccharolyticus, E. cecorum, E. columbae, and E. pallens), all enterococci hydrolyze PYR and possess group D antigen. A flowchart that may be used to identify enterococcal species is shown in Figure 4. Identifying the species of enterococcal isolates is important for understanding the epidemiology of antimicrobial resistance among isolates of this genus and for managing patients with enterococcal infections. Most clinical laboratories identify Enterococcus spp. presumptively by demonstrating that the isolate is PYR and LAP positive and that it grows at 45° C and in 6.5% NaCl. However, the recent discovery of Streptococcus urinalis presents a problem in this regard. S. urinalis and the commonly isolated Enterococcus spp. exhibit identical reactions in the four tests listed here and only differ in the ability to grow at 10° C (S. urinalis cannot).

Fig4. Species identification of clinically relevant enterococcal and enterococcal-like isolates. =, Signifies a negative result. 

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