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Timestamp: 2019-04-21 18:25:43+00:00

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Unlike gram-negative rods, it can be very difficult to sort out the identification of gram-positive cocci and rods. Many kits for staphylococcal identification have proved to be less sensitive than desired, and new DNA studies indicate that we have misidentified many streptococci. Gram-positive rods have been difficult to identify because there are hundreds of named species and thousands of genotypes or biochemical variants found in the environment and the normal microbiota of the human body, including skin, mucosal membranes, oropharynx, and genitourinary and gastrointestinal tracts. Thus, it is not within the scope of this handbook to identify all isolates, but to detect and identify the known pathogenic microorganisms in the human biosphere and to limit other identifications to those bacteria that are involved in disease from invasively collected specimens. The figures and tables that follow are designed to rapidly determine the agents of infection and to provide guidance for when to perform a kit identification or pursue other microorganisms. Because of the increasing microbial diversity and emergence of common pathogens having rare or unique phenotypic characteristics and the identification of new pathogens with incompletely defined phenotypes, more laboratories are relying on a combination of phenotypic and genotypic methods to report an accurate identification of many bacteria, so indications where molecular identification using DNA target sequencing may be useful are included ( 8 , 32 ).
Flowchart for identification of catalase-positive, gram-positive cocci.
Flowchart for identification of catalase-negative, gram-positive cocci, either beta-hemolytic or nonhemolytic, with morphology of group B streptococci.
Flowchart for identification of gram-positive, catalase-negative cocci that are not beta-hemolytic. R, resistant; S, susceptible; Pos, positive; Neg, negative.
Flowchart for identification of PYR-positive, catalase-negative, gram-positive cocci. R, resistant; S, susceptible; VRE, vancomycin-resistant enterococci; MGP, methyl-α-D-glucopyranoside.
Identification scheme for gram-negative diplococci; also see Table 3.18.2-1 .
Identification scheme for gram-negative rods that do not grow on BAP aerobically in 5% CO2. CYE, charcoal-yeast extract agar.
Identification scheme for gram-negative rods that grow on BAP with 5% CO2 but do not grow well on MAC in 48 h.
Identification scheme for gram-negative rods that grow on BAP and MAC.
Identification scheme for gram-negative rods that grow well on BAP and MAC and are not identified from Fig.3.18.2-4.
Identification scheme for gram-negative rods that grow on BAP and MAC and are not identified by Fig. 3.18.2-4 and 3.18.2-5.
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a Symbols and abbreviations: +, greater than 90% of strains positive in 48 h; −, greater than 90% of strains negative; V, results are between 90 and 10% positive; −+, most strains are negative but rare positive strains exist; NA, not applicable or available; R, resistant; S, susceptible; Bacit, bacitracin; Poly B, polymyxin B; coag, coagulase; SAG, staphylococcal protein A or clumping factor agglutination; VP, Voges-Proskauer; Novo, novobiocin. Data are from references 2 , 9 , 16 , 20 , 21 , 26 , 31 , and 40 . Catalase can be weak for Rothia.
b PYR data are for broth test. Weak positive results with S. aureus ATCC 29213 and ATCC 25923 occur with the disk test, suggesting that this test is unreliable to separate S. aureus from S. intermedius (M. York, personal communication).
c Includes related taxa. The genus Micrococcus has been divided into additional genera, including Kytococcus and Kocuria.
a Abbreviations: ARG, hydrolysis of arginine; MAN, acid production from mannitol; SOR, acid production from sorbitol; +−, most strains positive but rare negative strains exist. See Table 3.18.1-1 , footnote a for other abbreviations and symbols. Strains do not always produce glucans; test is only useful if positive. Commercial kits for identification of streptococci are helpful to resolve variable reactions. Data are extrapolated from reference 46 . Also see references 10 , 14 , 35 , and 41 .
b Only the most commonly isolated species are included within each group; the other species within each group are listed here. S. mutans group species (S. downei, S. ferus, S. macacae, and S. hyvaginalis) have been found only in animals. The S. salivarus group includes S. thermophilus, which has been identified only from dairy products. S. bovis group species (S. equnius, S. gallolyticus, S. infantarius, and S. alactolyticus) are all human pathogens and are frequently isolated from blood cultures. S. mitis group species (S. infantis and S. peroris) have rarely been isolated from clinical specimens ( 22 , 41 ).
a All species grow well on BAP and in 6.5% NaCl and are PYR, bile-esculin, and LAP positive. PYR-negative species E.cecorum, E.columbae, and E. saccharolyticus are not included and have not been isolated from humans. PYR-positive strains E. malodoratus, E. pseudoavium, E. asini, and E. sulfureus (H2S+) also are not listed since they have not been isolated from humans. E. gilvus and E. pallens have been described to occur in humans but are extremely rare. Abbreviations: MGP, methyl-α-D-glucopyranoside; D, different reactions in references. See footnote a to Tables 3.18.1-1 and 3.18.1-2 for other abbreviations and symbols. Table adapted from references 15 , 36 , and 44 . Also see references 27 , 28 , and 43 .
b Motility is done in 0.5 ml of BHI or TSB incubated at 30°C for 2 h.
c Pigment (yellow) is observed by swabbing a blood agar plate incubated at 35°C in 5% CO2 for 24 to 48 h and observing swab for bright yellow color (+).
d Lactose-negative asaccharolytic E. faecalis exists.
a CAT, catalase production; NaCl, grow thin broth containing 6.5% NaCl; 10°C and 45°C, grow that 10 and 45°C, respectively (for the latter, use campylobacter incubator if heat block not available). Abbreviations for cell arrangement in Gram stain: CL, clusters; T, tetrads; CH, chains; W, weak. Large colonies are approximately 1 mm; small colonies are about the size of viridans group streptococci. See Table 3.18.1-1 , footnote a, for other abbreviations and symbols. Tables adapted from references 13 , 24 , 29 , and 35 . Also see reference 9 .
b See reference 8 for guidelines on when DNA target sequencing may be useful for identification. In particular, partial sequencing of the 16S rRNA gene provides excellent identification to the genus and species levels for some of these gram-positive cocci. Abiotrophia/Granulicatella can be separated only by molecular identification.
a Van, vancomycin; MRS, gas production in MRS broth. See footnote a to Table 3.18.1-4a and Table 3.18.1 for other abbreviations.
a Data from references 3 , 7 , 12 , and 33 . Once G. vaginalis, Arcanobacterium, Weissella, and E. rhusiopathiae are ruled out, either call “Anaerobic gram-positive rod” or do anaerobic identification kit. GPR, gram-positive rods; SPS, sodium polyanethol sulfonate. See footnote a to Tables 3.18.1-1 and 3.18.1-2 for other abbreviations and symbols. See Table 3.18.1-9 for catalase-positive Actinomyces.
b See reference 8 for guidelines on when DNA target sequencing may be useful for identification. In particular, partial sequencing of the 16S rRNA gene and/or the rpoB gene for some genera provides excellent identification of most of these gram-positive rods.
a If motile and yellow or positive for esculin and/or gelatin, report as “Motile coryneform, not Corynebacterium spp.” If of clinical significance, use kit (e.g., Coryne API or RapID Coryne) or send to reference laboratory. Other yellow Corynebacterium spp. (C. aurimucosum, C. falsenii, and C. sanguinis) are rarely isolated or associated with human disease. Data are from references 17, 18, 39, and 42; also see section 6. See footnote a to Tables 3.18.1-1 , 3.18.1-2 , and 3.18.1-3 for abbreviations and symbols.
a B. cereus, B. thuringiensis (insect pathogen used in horticulture), B. mycoides (rhizoids or hairy projections in agar), and B. anthracisare included in the B. cereus group. If organism is motile with spores, penicillin resistant, hemolytic, with cells greater than 1 µm in diameter, and/or lecithinase positive, report as “Bacillus cereus group, not B.anthracis.” Otherwise, if organism is motile, with spores, but nonhemolytic and/or lecithinase negative, report as “Bacillus, not B. anthracis or B. cereus.” Data are from references 25 and 45 . Spores can be induced by growing on urea, bile-esculin agar, or an agar plate with vancomycin disk or at 45°C. Spores can be proved by heating broth culture to 80°C for 10 min and subculturing to BAP. Viable colonies indicate that spores survived the heating. See Table 3.18.1-1 , footnote a, for other abbreviations and symbols.
c Submit any nonmotile, spore-forming strain to designated higher reference laboratory to rule out B. anthracis, regardless of the penicillin susceptibility.
d Kurthia (diameter, 0.8 to 1.2 µm) organisms are motile and nonhemolytic and do not produces pores ( 17 ).
a Data are from references 17 and 18 . Other urease-positive or -variable species of less clinical significance include C. durum, C. falsenii, C. singulare, C. sundsvallense, and C. thomssenii, which are CAMP and reverse-CAMP negative, are not lypophilic, and ferment glucose. See Table 3.18.1-1 , footnote a, for abbreviations and symbols.
c Submit to reference laboratory for diphtheria toxin testing. C. pseudotuberculosis is associated with sheep handlers.
d Respiratory pathogen; not able to acidify maltose, ribose, or trehalose.
e C. riegeliiis rarely isolated but has been found in urine and other body sites ( 17 ). It is able to acidify maltose.
f Urinary pathogen; multiresistant to antimicrobials.
a Usually irregular rods. Identify only if clinically significant, but all can be pathogens ( 17 , 18 ). The important tests are fermentation (use Andrade&apos;s base or cysteine Trypticase agar), CAMP, and Gram stain, with careful reading of Gram stain morphology. Coryneform identification kits can be helpful. For abbreviations and symbols, see Table 3.18.1-1 , footnote a.
b See reference 8 for guidelines on when DNA target sequencing may be useful for identification. In particular, partial sequencing of the 16S rRNA gene and/or the rpoB gene for some genera provides excellent identification of most of these gram-positive rods. Molecular identification is a more accurate method for identification and separation of the heterogeneity within Actinomyces spp. ( 19 , 30 ).
a Other species that are rare are not listed (see references 17 and 18 ), including some CAMP test-positive species. Some Corynebacterium organisms have black-pigmented colonies. For identification of species in this table, the combination of CAMP test, lipophilism, O/129 disk, and commercial kits for corynebacteria should be used if identification is clinically important. For abbreviations and symbols, see footnote a to Tables 3.18.1-1 and 3.18.1-9 .
b See reference 8 for guidelines on when DNA target sequencing may be useful for identification. In particular, partial sequencing of the 16S rRNA gene and/or the rpoB gene for some genera provides excellent identification of most Corynebacterium spp. ( 23 ).
d Submit to reference laboratory for diphtheria toxin testing.
a Abbreviations for tests: PRO, prolyl iminopeptidase; ONPG, o-nitrophenyl-β-D-galactopyranoside; BGAL, β-galactosidase; GLUT, ∂-glutamyl-aminopeptidase. Reactions are from package inserts, the website http://www.CDC.gov/ncidod/dastlr/gcdir/neident/index.html, and references 10 , 18 , and 32 . Polymyxin B can be substituted for colistin; alternatively, resistance to these agents can be determined by growth or lack of growth on Thayer-Martin or other selective agar with colistin or polymyxin B. Results for sugars are in cysteine Trypticase agar.
c Abbreviations and symbols for results: +, greater than 90% of strains positive in 48 h; −, greater than 90% of strains negative; V, results are between 90 and 10% positive; + −, most strains are positive but a few are known to be negative, resulting in critical misidentifications if other tests are not also performed; R, 90% of strains resistant or no zone around disk; S, 90% of strains susceptible or zone around disk.
d Does not usually grow on selective media for N. gonorrhoeae. N. subflava and N. flavescens colonies are yellow; N. subflava is the only species other than N. meningitidis to be GLUT positive.
e Nutrient or MH agar or TSA without blood at 35°C.
f See procedure 3.17.7. Do not read after time period in package insert, as this delay may result in false-positive reactions. Many Moraxella spp. and Acinetobacter spp. are butyrate positive. Isolate must be a diplococcus for identification of M. catarrhalis to be accurate.
g N. polysaccharea strains were previously identified as nontypeable strains of N. meningitidis. Strains of N. polysaccharea may also have been misidentified previously as N. subflava because their ability to produce polysaccharide from sucrose was not determined (http://www.cdc.gov).
a Data for table from reference 31 , p. 218–219. See footnote c of Table 3.18.2-1 for abbreviations and symbols. Haemophilus aphrophilus and Haemophilus paraphrophilus have been reclassified as a single species on the basis of multilocus sequence analysis ( 23 ), Aggregatibacter aphrophilus, which includes V factor-dependent and V factor-independent isolates.
b See reference 9 for guidelines on when DNA target sequencing may be useful for identification. In particular, partial sequencing of the 16S rRNA gene provides excellent identification for some genera of oxidase-positive, gram-negative diplococci ( 25 ).
c Hemolysis demonstrated on horse, sheep, or rabbit blood agar. Note that hemolytic strains may grow on BAP without staphylococcal dot even though they require V factor.
d Test is performed in 1% lactose in phenol red broth base (BD Diagnostic Systems) supplemented with hemin and NAD (10 mg of each per liter; Sigma Chemical Co.) (reference 22 , p. 628).
a See reference 26 . Indole is done with Kovács at 48 h directly on plate. For yellow colonies, Ehrlich&apos;s method may be needed. CDC group II-g grows on MAC but is a non-glucose oxidizer. See Table 3.18.2-8 for indole-positive, fermenting rods that grow on MAC. Data are from reference 31. (+), greater than 90% of strains positive in 3 to 7 days; F, glucose fermenting in TSI or KIA or Andrade&apos;s glucose broth or other sugar fermentation medium; n-o, nonoxidizer in glucose OF medium and no reaction in fermentation medium; O, oxidizer in glucose OF medium; Fs, addition of rabbit serum to Andrade&apos;s or other glucose fermentation medium may be required to demonstrate fermentation; gas, gas from either glucose or nitrate, depending on the test. Results for carbohydrates are in OF medium for oxidizers and Andrade&apos;s or rapid sugar agar for fermenters. See footnote c to Table 3.18.2-1 for other abbreviations and symbols.
b See reference 9 for guidelines on when DNA target sequencing may be useful for identification. In particular, 16S rRNA gene partial sequencing separates P. multocida and P. canis, the two species not commonly isolated. C. hominis may also be distinguished from C. valvarum ( 13 ).
c Three species are now recognized in the Dysgonomonas genus, including D. capnocytophagoides (formerly CDC DF-3), D. gadei, and D. mossii ( 15 , 20 ). All resemble Capnocytophaga in their growth characteristics.
d All strains except Balneatrix alpica are nonmotile.
e See Table 3.18.2-6 to separate from Empedobacter brevis, which is yellow and indole positive and grows on MAC.
f Decarboxylase − indicates negative reaction for lysine, arginine, and ornithine.
g Primarily animal pathogens; rarely isolated from humans.
h See reference 29 . C. valvarum is a newly described species that may cause endocarditis ( 4 ). Some isolates may be indole negative.
a All are nonmotile except B. vesicularis. Data are from references 28 and 31 . See footnote c to Table 3.18.2-1 and footnote a to Table 3.18.2-3 for abbreviations and symbols.
b See reference 9 for guidelines on when DNA target sequencing may be useful for identification.
a For indole-positive strains, see Table 3.18.2-3 . All strains nonmotile, except as noted, but even with those, motility is difficult to demonstrate. See Table 3.18.2-4 for catalase-variable rods. Data are from references 28 and 31 . W, weak reaction; NA, not applicable or available. See footnote c to Table 3.18.2-1 and footnote ato Table 3.18.2-3 for other abbreviations and symbols.
c If Brucella is suspected, all plates should be tape-sealed and further testing should be done in a BSL 2 cabinet. Presumptive Brucella isolates that are confirmed to be indole positive, catalase positive, oxidase positive, and urease positive should immediately be referred to the state health laboratory or to the CDC (see procedure 3.4.2 and reference 7 ).
a For indole reaction, Ehrlich&apos;s method may be needed. See Table 3.18.2-4 for yellow-pigmented, catalase-negative Eikenella and Capnocytophaga. Data are from references 26 , 28 , and 31 . See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-3 and 3.18.2-5 for abbreviations and symbols.
a Motility is best at 22°C. All are indole negative except Balneatrix. Data are from references 3 , 26 , 28 , and 31 . − +, most strains are negative but a few are known to be positive. See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-3 and 3.18.2-5 for other abbreviations and symbols.
a See references 2 and 16 .
b 1% salt may be required for sugar and decarboxylase reactions. All strains ferment glucose on TSI or KIA. CDC group II-g grows on MAC and is indole positive but is a non-glucose oxidizer; see Table 3.18.2-3 . Data are from references 17 , 22 , and 31 . See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-5 and 3.18.2-7 for abbreviations and symbols.
c Resistant V. cholerae strains have been isolated in India. Confirmation of arginine-negative Aeromonas may be needed to prevent misidentifications. Testing with O/129 is key to prevention of misidentification of V. fluvialis and V. vulnificus as Aeromonas spp.
d Data on file at University of California, San Francisco. The importance of using both media is to get growth on at least one plate in order to observe the O/129 and other disk susceptibility and to observe that Aeromonas and Plesiomonas do not grow with salt added. Some Vibrio organisms do not grow on MH agar without salt added, and some do not grow with 4% salt.
e The combination of polymyxin B resistance and indole positivity is associated with nonpigmented strains. The combination of sucrose negativity and growth on MH agar separates this organism from V. fluvialis. C. violaceum is lysine, maltose, and mannitol negative, which separates it from Aeromonas.
f See reference 9 for guidelines on when DNA target sequencing may be useful for identification.
a Reactions of Yersinia are faster at room temperature. Data are from references 12 and 30 . See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-3 and 3.18.2-7 for abbreviations and symbols.
b Use rapid urea test method to increase sensitivity. See procedure 3.17.48.
c See reference 9 for guidelines on when DNA target sequencing may be useful for identification.
a Reactions from references 28 and 31 . All strains are catalase positive. For oxidase-negative, yellow-pigmented organisms, see Table 3.18.2-7 . See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-3 and 3.18.2-5 for abbreviations and symbols.
a See reference 21 .
b Data are from references 14 and 22 . PYR data are from reference 19 . All strains are arginine negative. See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-3 and 3.18.2-5 for abbreviations and symbols.
c Ralstonia mannitolytica, formerly known as Ralstonia pickettii bv. 3, is unique among the Ralstonia organisms in that it is mannitol positive. R. mannitolytica is nitrate negative. R. pickettii is nitrate positive.
d Oxidase reactions may be slow, up to 30 s.
e See reference 9 for guidelines on when DNA target sequencing may be useful for identification.
b All strains are motile and indole negative. Also see Table 3.18.2-5 for nonmotile, gram-negative rods that are MAC variable. Verify that strains are nonfermenting rods using TSI or KIA. For fermenting rods, see Table 3.18.2-8 . Data are from references 19 , 22 , 27 , and 31 and from G. L. Gilardi, unpublished identification tables. SS, salmonella-shigella. See footnote c to Table 3.18.2-1 and footnote a to Tables 3.18.2-3 , 3.18.2-5 , and 3.18.2-7 for other abbreviations and symbols for reaction key.
c B. mallei can have similar reactions but is nonmotile, has no odor, and does not produce gas from nitrate.
d To separate Alcaligenes faecalis from other related nonoxidizers: Ralstonia gilardii is nitrite negative; nonyellow Myroides is urea and PYR positive but nonmotile and polymyxin B resistant, and Gilardi rod group 1 is nonmotile and PDA positive.

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