Identification of Other Streptococcus Species: Streptococcus General Methods

Section II.

Identification of the Gram-Positive, Catalase Negative Cocci Genera

The following genera are identified in Streptococcus Reference Laboratory. There are three major reasons for the increase in number of genera that clinical microbiologist must identify. First the genetic studies by taxonomists have clarified the relationship of some genera. The enterococci and lactococci were split from the Streptococcus genus. DNA homology studies have indicated that these two genera are separate and distinct entities. In addition the vagococci were split from the Lactococcus genus on the same basis. The tetragenococci were split from the Pediococcus genus because it too is genetically different from the other members of the genus.

The Leuconostocs and pediococci were considered non-pathogenic until the mid 1980s. It is believed that the increase in use of vancomycin caused the increase in isolation of species of these two genera. All species of Leuconostoc and Pediococcus genera are intrinsically resistant to vancomycin. The vancomycin will eliminate all susceptible strains allowing the resistant strains to colonize the genitourininary tract.

Refer to the List of Prokaryotic names with Standing in Nomenclature (LPSN) for the list of streptococcal species names for each of the genera listed below.

Gram stain-Positive Catalase-Negative Coccus and Coccoid-rod Form Genera

• Streptococcus
• Abiotrophia
• Aerococcus
• Alloiococcus
• Dolosicoccus
• Dolosigranulum
• Enterococcus
• Eremococcus
• Facklamia
• Gemella
• Globicatella
• Granulicatella
• Helcococcus
• Ignavigranum
• Lactococcus
• Leuconostoc
• Oenococcus
• Pediococcus
• Tetragenococcus
• Vagococcus

Related Genera, Coccobacillary to Rod-like

• Carnobacterium
• Weissella

Table 1 lists the currently recognized genera of facultatively anaerobic gram positive cocci.

Once it has been determined that the bacteria in question is a gram-positive, catalase-negative coccus, the next step is to determine to what genera the strain belongs. The genera that this laboratory identifies includes: Enterococcus, Leuconostoc/Weissella, Streptococcus, Pediococcus, Tetragenococcus, Aerococcus, Helcococcus, Vagococcus, Lactococcus, Abiotrophia, Granulicatella, Globicatella sanguinis, Dolosicoccus paucivorans. Alloiococcus, Dolosigranulum, Facklamia, and Ignavigranum species.

Initial separation into the appropriate genera is accomplished by determining the physiologic characteristics listed in Table 1. The extent to which each bacterial strain is tested is dependent upon the source of the strain. If the strain is from a normally sterile-site then all the tests listed below should be applied. There are some instances where all the tests do not need to be applied; i.e., the beta-hemolytic streptococci and the pneumococci. There are specific tests that clinical microbiologist perform when these pathogens are suspected. These situations and tests will be discussed in the section on streptococci.

Table 1. Phenotypic characteristics of facultatively anaerobic, catalase-negative, gram-positive cocci

^a Cell arrangement in gram strain: ch, chains; cl, clusters; t, tetrads.

^b VAN, vancomycin susceptibility screening test; GAS, gas production in MRS broth; BE, hydrolysis of esculin in the presence of bile; PYR, production of pyrrolidonylarylamidase; LAP, production of leucine aminopeptidase; NaCl, growth in broth containing 6.5% NaCl; 10oC and 45°C, growth at 10°C and 45°C; HEM, hemolytic activity on Trypticase soy 5% sheep blood agar. +, 85% or more of the strains are positive: -, 15% or less of the strains are positive; V+, variable positive (50 to 84% of the strains are positive); V-, variable negative (16 to 49% of strains are positive).

^cEnterococcus group includes all Enterococcus species, Vagococcus species and some Lactococcus species.

^d Some strains of lactococci and vagococci grow very poorly at 45°C.

^eLeuconostoc and Weisella are often coccobacillary, sometimes appearing rod like in chains.

^f All strains of S. bovis and approximately 10% of viridans streptococci are bile-esculin positive.

^g All strains of S. pyogenes and most strains of S. porcinus and S. iniae are PYR positive. Other streptococci are all negative

^h Nutritional variant streptococci are now identified in two different genera Abiotrophia and Granulicatella

ⁱ Unusual strep/genera includes species of streptococci usually found in animals and Globicatella sanguinis and Dolosicoccus paucivorans.

^jAerococcus species includes A. viridans, A. urinae, A. sanguicola, and A. urinehominis.

^k Salt tolerant Gemella-like bacteria include Alloiococcus, Dolosigranulum, Facklamia, and Ignavigranum species.

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Streptococcus

In spite of the splitting of this genus into the streptococci, enterococci and lactococci it still remains a very diverse group of bacteria. The different species are found in every sort of human infection as well as being a commensal organism in the oral cavity and genitourinary tract. As stated above there are specific situations when all the tests listed in Table 2 do not have to be determined. The first situation is when s-hemolysis is observed. This is most notable with throat swabs. s-hemolysis is such a good indicator among the catalase negative gram positive cocci that after determining that the unknown isolate is gram positive, catalase negative, and ß-hemolytic you can apply the tests listed in the Table 2 for streptococcal species identification to determine what species of Streptococcus is present. The remaining tests in Table 2 do not have to be determined. The second situation occurs when the clinical microbiologist is determining whether or not S. pneumoniae is present in various body fluids. If the unknown strain is catalase negative, gram positive, and ß-hemolytic then the tests used to identify pneumococci can be applied (Table 5). In all other situations all the tests listed in Table 2 must be performed to determine if the unknown strain is a Streptococcus. All streptococci are; vancomycin sensitive, do not form gas from MRS broth, produce LAP, do not grow at 10ΕC, and are non motile. Among the streptococci, group A streptococci, S. porcinus, S. iniae, and nutritionally variant streptococci are PYR positive. In the case of group A streptococci the PYR test is an excellent test for the presumptive identification of group A streptococci. The non-beta hemolytic streptococci (viridans, and non-enterococcal group D) do not grow in 6.5% NaCl broth; but some of the beta-hemolytic strains may grow in the broth. The bovis strains of streptococci usually grow at 45ΕC while the viridans streptococcal strains usually do not grow. The ß-hemolytic strains exhibit variable growth patterns at 45ΕC. The streptococci can be α, ß, or non hemolytic. The hemolysis reaction in blood agar plates is used to help differentiate the streptococcal species.

Enterococcus

Genetic evidence that the S. faecalis and S. faecium was sufficiently different from the other members of the Streptococcus genus was provided by Schleifer and Kilpper-Balz in 1984. It has been several years since this proposal and it is generally accepted that the genus Enterococcus is valid and used in the published literature. Since 1984, 25 other species have been proposed to be included in the Enterococcus genus. Genetic evidence for these proposals has been provided by DNA-DNA and DNA-rRNA hybridizations as well as reverse transcriptase sequencing of 16S ribosomal RNA. The enterococci are among the most ubiquitous bacteria known. They are found in the environment, human and other animal genitourinary tracts, and in most foods. These bacteria are not very invasive but are significant opportunists. The enterococci are usually sensitive to vancomycin but recently many strains have acquired resistance and in some cases the majority of strains identified may be vancomycin resistant because of nosocomial transmission of the strains. The resistance to vancomycin does not interfere with the identification of the strains because only the Leuconostocs and pediococci are intrinsically resistant to vancomycin but all species of these two genera are PYR negative. Thus the only strains that are vancomycin resistant and PYR positive are the enterococci. Only rarely do enterococcal strains (less than 0.5%) produce gas from MRS broth. Nearly all strains produce LAP, grow in 6.5% NaCl, and grow at 10ΕC and 45ΕC. Some strains of E. avium or E. raffinosus do not grow at 10ΕC or produce LAP but this is not consistent or predictable. Some species are motile. Most strains are α or non-hemolytic; occasionally ß-hemolytic strains of E. faecalis or E. durans are identified.

Lactococcus

The transfer of the species of lactic streptococci formally known as group N streptococci to the genus Lactococcus was made in l985. The species included in this genus are thought to be nonpathogenic for man. In fact L. lactis (S. lactis) and L. cremoris (S. cremoris) are used in the dairy industries and are included in foodstuffs. We have identified several strains of lactococci from human sources but the clinical significance of these strains has not been evaluated.

The lactococci, formally called the lactic group of streptococci or the group N streptococci, were not thought to be pathogenic for man. Many of the lactococcal strains are used in the manufacturer of foods, such as cheese. However, several confirmed cases of infections including endocarditis have been attributed to lactococcal strains. The natural habitat of lactococci is thought to be the environment. The physiologic characteristics of the lactococci is very similar to the enterococci and most of the lactococcal strains associated with human infections in our culture collection now identified as lactococci were initially identified as enterococci. One of the criteria previously used to differentiate between the enterococci and lactococci was the inability of lactococci to grow at 45ΕC. However, with the inclusion of new species into the Lactococcus genera, i. e., L. garvieae, some strains of lactococci grow at 45ΕC. All the lactococci are: vancomycin sensitive, do not produce gas in MRS broth, grow at 10ΕC, and are nonmotile. All strains give positive LAP reactions and most strains give positive PYR reactions, however, some L. lactis strains are negative. Variable reactions are observed in the 6.5% NaCl tolerance test. None of the strains are ß-hemolytic.

Vagococcus

Vagococcal strains have rarely been isolated from human infections. The vagococcal strains were split from the Lactococcus genus. These organisms were previously known as motile lactococci. In fact, only the motility characteristic of the vagococci differentiates the vagococci from the lactococci. These bacteria are included in the identification of the enterococcus because of phenotypic similarities.

Leuconostoc

Like the lactococci the Leuconostocs were once not thought to cause human infections. However, there are many reports of human infections caused by different Leuconostoc species. Species of the Leuconostoc genus are the only catalase negative gram positive cocci that produce gas from MRS broth. As stated earlier all strains are intrinsically resistant to vancomycin. All strains of Leuconostocs are negative in PYR and LAP tests. The combination of reactions, vancomycin resistance, and negative PYR and LAP is an excellent indicator of Leuconostoc identification. No other catalase negative, gram positive cocci has these characteristics. Like the lactococci, Leuconostocs grow at 10ΕC but very poorly if at all at 45ΕC. Some strains grow in 6.5% NaCl broth while others do not. None of the strains are motile and none are ß-hemolytic.

Pediococcus

Like the Leuconostocs the pediococci are intrinsically resistant to vancomycin. They too were thought to be nonpathogenic for humans but there are several reports indicating that this is changing. The Pediococcus strains appear very similar to the viridans streptococci on blood agar media and can be easily misidentified as viridans streptococci. All strains of pediococci tested have been resistant to vancomycin and all strains of viridans streptococci have been sensitive to vancomycin. Pediococci do not form gas in MRS broth, they are PYR negative but LAP positive, some strains grow in 6.5% NaCl broth while others do not. Most strains grow at 45ΕC but not at 10ΕC. All strains are nonmotile and appear α-hemolytic on blood agar plates

Globicatella

This new genus of gram positive cocci has just recently been described. The origin of this genus is from a collection of viridans-like streptococci that most closely resembled Streptococcus uberis. What makes Globicatella distinct from the viridans streptococci is that all the Globicatella strains were PYR positive, LAP negative and grow in broth containing 6.5% NaCl while all viridans species are PYR negative, LAP positive and fail to grow in 6.5% NaCl. All strains identified to date have been sensitive to vancomycin, PYR positive, LAP negative, grow in 6.5% NaCl broth, do not grow at 10ΕC or 45ΕC, are nonmotile, and are α-hemolytic.

Tetragenococcus

This genus contains only one species. T. halophilus was previously identified as Pediococus halophilus. The tetragenococci differ from the pediococci by vancomycin resistance. The pediococci are vancomycin resistant and the tetragenococci are vancomycin sensitive. Other characteristics are similar.

Gemella and Gemella-Like

These bacteria grow very poorly on blood agar plates and will often take 48 h to grow. These bacteria may also resemble the viridans streptococci; occasionally the tetrads are not formed and only pairs and short chains are observed in the gram stain. One species was previously identified as a streptococci, G. morbillorum. On blood agar plates some of the strains give an wide-zone alpha hemolytic reaction after extended incubation. Identifying these bacteria is difficult and an extended set of physiologic characteristics may have to be determined before final identification is possible. These bacteria are characteristically negative in most tests listed in Table 1. Some strains are positive in both the PYR and LAP tests but other strains may fail to give a positive reaction in either. Salt tolerant Gemella-like genera include the Alloiococcus, Dolosigranulum, Facklamia, and Ignavigranum .

Aerococcus

The Aerococcus sp. are sensitive to vancomycin, do not form gas from glucose, and are generally PYR positive but are LAP negative. All strains grow in broth containing 6.5% NaCl but do not grow at 10ΕC or 45ΕC. Strains are non motile and are strongly α-hemolytic. The strains grow well on blood agar media and form colonies somewhat smaller than the enterococci but larger than the viridans streptococci after overnight incubation.

Alloiococcus

Only one species of this genus is presently known, A. otitidis. These bacteria have been isolated from the ear fluids of children with otitis. Like the Gemellae this bacterium is difficult to grow. Often 2 to 3 days are necessary for growth to develop on rabbit blood agar plates. Alloiococci are susceptible to vancomycin, do not form gas in MRS broth and are PYR and LAP positive. They are differentiated from the Gemellae by the 6.5% NaCl test. Alloiococci grow in 6.5% broth but Gemellae do not. These bacteria do not grow at 10ΕC or 45ΕC or thioglycolate broth, are not motile and are non-hemolytic on blood agar plates.

Helcococcus

Two species of this genus are presently known. H. kurzii is the only one isolated from humans. These bacteria have been isolated from wound infections. Like the Alloiococci and Gemellae these bacteria grow very slowly on blood agar media. The physiologic characteristics of helocococci are similar to the aerococci in that they are PYR positive, LAP negative, and grow in broth containing 6.5% NaCl. These bacteria grow more slowly and are not α-hemolytic on blood agar while the aerococci grow readily and are α-hemolytic on blood agar. Like the Aerococci these strains are vancomycin sensitive, do not form gas in MRS broth and fail to grow at 10ΕC and 45ΕC. All isolates have been non-motile.

Nutritionally Variant Streptococcus

Nutritionally variant streptococci (NVS) can be identified by demonstrating that the strain requires pyridoxal or grows on an agar plate only when a bacteria that satellites is present. In addition to this requirement, NVS also give positive PYR tests, which helps to differentiate these strains from viridans streptococci.

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Identification of Streptococcal Species

The ß-Hemolytic Streptococci

The most convenient way to begin to identify the streptococci is to determine the hemolysis of the bacteria on blood agar plates. As mentioned earlier the techniques for determining hemolysis is described in detail in; Isolation and Identification of streptococci, Part 1. Collection, transport, and determination of hemolysis, Annex 1. Once the streptococci are divided into ß-hemolytic and non-ß-hemolytic categories differentiation into species, groups, and categories can be made. The identification of most ß-hemolytic strains is performed by determining the antigenic characteristics of the culture; but, identification of the non-ß-hemolytic strains is performed by determining antigenic and physiologic characteristics of the culture

Group A streptococci: Lancefield’s group A Streptococcus is also known as Streptococcus pyogenes. Identification is confirmed by demonstrating the presence of the group A antigen on the streptococcal cells. All S. pyogenes have group A antigen; but, not all streptococci with group A antigen are S. pyogenes. Some strains of S. anginosus and S. dysgalactiae subsp. equisimilis may also have group A antigen. Non-pyogenes strains grow more slowly and form smaller colonies than do S. pyogenes strains. If the ß-hemolytic colonies appear small and growth is delayed or carbon dioxide is required for growth or the group A strain is PYRase negative the microbiologist should suspect that the strain may be S. anginous or S. dysgalactiae subsp. equisimilis, regardless of group reaction. When this occurs the strain should be tested for voges-proskauer (VP) reaction. See Table 2, for correct identification.

Group B streptococci: Lancefield’s group B streptococci is also known as S. agalactiae. Like group A streptococci, identification is confirmed by demonstrating that the streptococcal cells contain group B antigen. Since the group B antigen is not identified with any other streptococcal strain the terms Lancefield group B and S. agalactiae are synonymous.

Group C streptococci: The ß-hemolytic streptococci that contain group C antigen are shown in table on page 30. The group C antigen is found with several different species and the S. anginous group of bacteria, Table 2.

Group G streptococci: The ß-hemolytic streptococci with group G antigen have not had an official taxonomic name. Some have suggested that these strains be called S. canis but this has not gained approval officially or in practical use. ß-hemolytic streptococci with group G antigen should be reported simply as Lancefield’s group G streptococci.

Group F streptococci: S. anginosus is used here to report streptococcal strains that may or may not have group antigen A, C, F, or G antigens. Most of these strains have group F antigen, the next most frequently identified strain will have no group antigen, and only rarely will an anginosus strain have group A, C, or G antigen. All these strains are VP positive and they are the only ß-hemolytic strains to have this characteristic. Technically some of these strains may be S. intermedius or S. constellatus. The DNA-DNA homology studies have shown that ß-hemolysis did not help to differentiate these strains, this characteristic was found among all 3 species. The majority of these strains will require additional carbon dioxide in the atmosphere for growth on blood agar plates. Often growth is not apparent until 48 h of incubation. Again for convenience the ß-hemolytic-VP positive strains should be reported as ß-hemolytic, S. anginosus, group ? (insert group reaction or none).

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Other ß-Hemolytic Streptococci

There are other ß-hemolytic streptococci but they are rarely found in human infections. Some of the strains are associated with infections of swine and they have specific group antigens. Using DNA-DNA homology the taxonomist have suggested that those ß-hemolytic strains with group E, P, U, V, and other experimental group antigens be called S. porcinus. ß-hemolytic strains with group L antigen have been associated with avian sources (chicken), it is suggested that these strains be called S. dysgalactiae. ß-hemolytic strains with group M antigen are not well studied and there is no suggested taxonomic name for these strains. A ß-hemolytic strain with a yet to be named group antigen (suggested X) isolated from fresh water dolphins, and occasionally in human infections, is called S. iniae. This bacterium may be submitted as a group A streptococci because it may react with the group A antibody in the latex slide agglutination assay. It is also PYRase positive but is not sensitive to bacitracin. Whenever a latex agglutination group A positive bacterium is submitted that is not sensitive to bacitracin the group reaction should be confirmed using the Lancefield extraction procedure.

Some investigators have reported that strains with group R antigen can be ß-hemolytic. Using 5% sheep blood agar plates we have been unable to demonstrate that these strains are ß-hemolytic. These strains are α-hemolytic on blood agar plates containing 5% sheep blood. Strains with group R, S, and T antigens are very similar to each other physiologically and the taxonomist have suggested that these strains should be called S. suis. The strains are found commonly in swine and may be transmitted to man. There are several reports of farmers and abattoir workers being infected with group R (S. suis type II) streptococci. Identification of these bacteria is difficult without having knowledge that the infecting strain may be related to nonhuman sources. Demonstration of the group R, S, and T antigens with specific antisera is also difficult. It is suggested that if group R streptococci is the suspected agent in an infection the cells may have to be extracted with the formamide extraction technique in order to extract the group antigen. The formamide extraction technique is described in Annex 2 (Part II of Isolation and identification of streptococci).

Note: S. phocae is maltose positive (data not shown)

The serological and physiological tests listed in Table 2 can be used to identify nearly all of the ß-hemolytic streptococci isolated from human infections.

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Table 2. Identification of the â-hemolytic streptococci.

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Table 3. Differentiation of gram-positive cocci, atypical variants of group A streptococcci.

Hem, hemolysis on 5% sheep blood agar plates; Bac, bacitracin sensitivity; S, sensitive; R, resistant; VP, Voges-Proskauer reaction; β-gal, β-galactosidase reaction; β-glu, β-glucoronidase reaction; Inu, Raf, and Rib, acid formation in inulin, raffinose and ribose broth, += ∃85% of strains positive; – = #15% or less positive; v = variable reactions (16-85% positive).

Non-hemolytic variants of S. pyogenes are well documented. The non-beta hemolytic varieties of S. anginsous group are found more commonly in human infections than the beta hemolytic strains. The non-beta hemolytic varieties of these species are also included in the viridans streptococci identification tables.

In all likelihood beta-hemolytic varieties of the S. anginosus group (S. anginosus, S. constellatus, S. intermedius) can be found in human infections.

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Table 4. Identification of the Streptococcus anginosus group

¹ Motile variant described in reference 14.
Data in table compiled from references 14, 65-67, 129-135. +, positive reactions ≥92% of strains, -, positive reaction ≤8% of strains, v, variable reactions positive in 8 to 91% of strains.

* Test found on the STREP ID32 Test Strip, bioMerieuex

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The Non-ß-hemolytic Streptococci (ß) and Nonhemolytic

The non-ß-hemolytic streptococci can be divided into 6 species and two groups with simple bacteriologic tests. In some instances where only the S. pneumoniae strains are suspected only optochin and bile solubility tests need be determined. Pneumococcal surveillance cultures, pneumococcal epidemiologic investigation cultures, and non-sterile site isolates (sputum) are cultures that are examined only for pneumococci. If the tests for optochin susceptibility and bile solubility are negative then the report can simply be no pneumococci present.

The non-ß-hemolytic streptococci can be identified to the species or group level by the tests listed in Table 5.

Streptococcus pneumoniae: S. pneumoniae cultures are α-hemolytic on blood agar medium. They can be identified and differentiated from the viridans streptococcal species by their susceptibility to optochin and bile solubility. Viridans streptococci: most strains are α-hemolytic on blood agar media, are usually neither susceptible to optochin or bile soluble. On occasion, some strains of viridans streptococci are susceptible to optochin or partially soluble in bile, but rarely will a culture of viridans streptococci be positive in both tests. Cultures suspected of being pneumococci isolated from systemic sources (non-respiratory) that are optochin susceptible and bile soluble but fail to serotype should be tested with the GenProbe pneumococcus probe.

All pneumococcal cultures should be serotyped by the Quellung reaction, with CDC produced typing antisera, see instructions below and Annex 3. Positive Quellung reactions are considered definitive identification of pneumococci.

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Table 5. Identification of nonbeta hemolytic gram-positive cocci in chains.

¹ Abbreviations: Antigen; pn, pneumococcal typing antiserum or Omni serum, letters, Lancefield group antigen; Opt, optochin; BS, bile solubility; BE, bile-esculin reaction; Na, growth in 6.5% NaCl broth; Pyr, pyrrolidonyarlyamidase reaction; Esc, hyrolysis of esculin; Vp, Voges-Proskauer reaction; Man, Sbl, Tre, acidification of mannitol, sorbitol and trehalose broths; St, hydrolysis of starch; Dx, production of extracellular polysaccharide. See footnote in Table 2 for positive and negative reactions.

Note that the only way to differentiate S. suis from viridans streptococci is with serologic typing.

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Table 6. Shows key test in the differentiation of the virdidans streptococci. Most species can only be identified to viridans species group. The VP test aids in the identification and differentiation of the viridans streptococcal species and is a key reaction for the S. anginosus group. The urea test is particularly useful in the identification of Streptococcus salivarius.

Table 6. Identification of major groups of viridans Streptococcus species.

B. Nutritionally variant streptococci: Abiotrophia and Granulicatella.

Nutritionally variant streptococci (NVS) can be identified by demonstrating that the strain requires pyridoxal or grows on an agar plate only when a bacteria that satellites is present. In addition to this requirement, NVS also give positive PYR tests, which helps to differentiate these strains from viridans streptococci. Two genera of NVS have been proposed Abiotrophia and Granulicatella.

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Table 7. Identification of Abiotrophia and Granulicatella species¹.

¹ All strains are positive for PYR and LAP and sensitive to vancomycin; negative reactions for gas production in MRS broth, growth in 6.5% NaCl or at 10 and 45°C. No reaction on bile-esculin medium. All strains require pyridoxal for growth and satillite around staphylococcus culture on blood agar plates.

² Abbreviations: Pul, Suc, Tag, Tre, acid production from pullulan, sucrose, tagatose, & trehalose broth respectively; Hip, hyrolysis of hippurate; Arg, deamination of arginine; α-gal, production of α-galactosidase; β-glu, production of β-glucouronidase; β-gal, production of -β-galactosidase + = 85% or more of the strains positive, – = 15% or less than the strains positive, v = variable reactions (16 to 84% positive)

C. Identification of Enterococci and Vagococci

The enterococci are gram positive cocci, occur in singles, pairs, and short chains. Cells are sometimes coccobacillary when gram stains are prepared from agar plate growth. Cells are more oval and in chains when gram stains are prepared from thioglycolate broth. The enterococci are facultatively anaerobic and optimum growth occurs at 35C. Most strains grow at 10C and 45C. All strains grow in broth containing 6.5% NaCl and hydrolyze esculin in the presence of 40% bile salts (bile-esculin medium). Motility is observed with some species. Enterococci hydrolyze pyrrolidonyl-ß-naphthylamide (PYR), the exceptions to this are E. cecorum, E. columbae, and E. saccharolyticus. Most strains produce leucine aminopeptidase (LAP). Some strains belonging to Group I enterococci give negative LAP tests. Enterococci do not contain cytochrome enzymes but on occasion the catalase test is positive. A pseudo catalase is sometimes produced and a weak effervescence is observed in the catalase test. Nearly all strains are homofermentive, gas is not produced and lactic acid is the end product of glucose fermentation. Most strains produce a cell-wall associated glycerol teichoic acid antigen that is identified as the streptococcal group D antigen. Detection of the group D antigen is sometimes difficult and depends upon the extraction procedure and the quality of the antiserum used.

Presumptive identification of a catalase negative gram positive cocci as an Enterococcus or Vagococcus can be accomplished by demonstrating that the unknown strain is vancomycin sensitive, PYR and LAP positive, and grows in 6.5% NaCl and at 45C.

Species Identification

Once established that the unknown catalase-negative gram-positive coccus is an Enterococcus the tests listed in Table 8 can be used to identify the species. The species into 5 Groups based on the reactions in acid formation in mannitol, sorbitol, and sorbose broths and hydrolysis of arginine.

Group I consists of E. avium, E. malodoratus, E. raffinosus, E. pseudoavium, E. saccharolyticus, E. gilvus, and E. pallens PNS-E3 and Vagococcus lutrae. These species form acid in all three of the aforementioned Carbohydrate broths but do not hydrolyze arginine.

Group II consists of E. faecalis, E. faecium, E. casseliflavus, E. mundtii, and E. gallinarum E. haemoperoxidus, Lactococcus sp., PNS-E2. These species form acid in mannitol broth, hydrolyze arginine, but fail to form acid in sorbose broth and give variable reactions in sorbitol broth.

Group III consists of E. durans, E. hirae, E. dispar, E. porcinus, E. ratti, E. faecium, and E. faecalis variant and E. villorum. These species hydrolyze arginine but do not form acid in mannitol, sorbitol or sorbose broths.

Group IV contains E. sulfureus, E. asini, and E. cecorum, E. phoeniculicola, V. fessus, V. carniphilus, V. salmonarium, and PNS E1. These species are sorbose negative and not hydrolyze arginine.

Group V consists of E. casseliflavus, E. gallinarum, E. faecalis, and E. columbae, E. hermanniensis, E. moraviensis, E. canis, V. fluvialis, and V. lutrae.

The pigmentation test aids in the identification of E. casseliflavus, E. mundtii, E. pullins, E. gilvus and E. sulfureus. These enterococci produce a yellow pigment that can be detected on several different media.

The pyruvate utilization test aids in the differentiation of E. faecalis and E. faecium. This test is also used to help differentiate between E. faecalis variant strains and E. hirae.

The tellurite tolerance test aids in the differentiation of E. faecalis and E. faecium.

E. haemoperoxidus is variable in the mannitol reaction and may be in group II or III.

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Table 8. Phenotypic characteristics used for the identification of Enterococcus species and some physiologically related species of other gram-positive cocci

^a Abbreviations and symbols: MAN, mannitol; SOR, sorbose; ARG, arginine; ARA, arabinose; SBL, sorbitol; RAF, raffinose; TEL, 0.04% tellurite; MOT, motility; PIG, pigment; SUC, sucrose; PYU, pyruvate; MGP, methyl–D-glucopyranoside; +, 90% or more of the strains are positive; -, 90% or more of the strains are negative; V, variable (11 to 89% of the strains are positive);

^b Phenotypic characteristics based on data from type strains.

^c Late positive (3 days incubation or longer).

^d Occasional exceptions occur (<3% of strains show aberrant reactions).

^e Weak reaction

Additional reactions Group III

^a Abbreviations: LM, litmus milk, A acid, C clot formation; PYU, pyruvate utilization; HIP, hippurate hydrolysis; TEL, tolerance to 0.04% tellurite; ARA, GYL, MGP, RAF, SUC, TRE, XYL acid formation in broth containing 1% arabinose, glycerol, methyl α-D-glucopyranoside, raffinose, sucrose, trehalose, xylose respectively; + or – or v/number, interpretation/percent positive;+ = 85% or more of the strains positive, – = 15% or less than the strains positive, v = variable reactions (16 to 84% positive)

^bE. villorum has similar phenotypic characteristics but has not been tested in conventional tests.

^c Mannitol-negative variants.

D. Identification of Lactococcus species

Lactococcus strains identified to the genus level by the tests listed in Table 1. Most of the lactococci resemble the enterococci in presumptive tests (BE, NaCl, PYR, LAP). The majority of strains give positive reactions in all four tests and are different from the enterococci by failing to grow at 45ΕC, not having characteristics identical to any Enterococcus species, and failing to react with the AccuProbe Enterococcus test.

Identification of the Lactococcus species is accomplished by performing the tests listed in Table 9. The majority of lactococcal isolates identified from human sources resemble L. lactis or L. garvieae. An examination of the information provided in Table 9 will show that it is not always possible to differentiate between these two species by phenotypic characteristics.

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Table 9. Differentiation of Lactococcus species

Acid formation in: Lac=lactose, Man=mannitol, Raf=raffinose, Arg=deamination of arginine, PYR=pyrrolidonylarylaminadase, and VP=Voges-Proskauer, + = >90% positive, – = <10% positive, v = 60-90% strains positive

D. Identification of Gemella species

Gemella species are identified to the genus level by the tests listed in Table 1. The Gemella species can be differentiated by the tests listed in Table 10. The acid from mannitol and sorbitol tests should be performed as previously described except the incubation period may have to be longer (up to 10-14 days). Because of the slow growth on blood agar plates by the Gemella species, these strains may be confused with the nutritionally variant streptococci. In such cases, it is necessary to perform the satillitism test to confirm the identity of NVS. In some cases the species cannot be determined by the phenotypic characteristics listed in Table 10. If this occurs the culture should be reported as a Gemella species, not further identified.

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Table 10. Differentiation of Gemella species

Mal, Man, Sbl, and Suc positive means acid production from maltose, mannitiol, sorbitol, and sucrose. VP, Voges-Proskauer reaction

E. Identification of Pediococcus and Tetragenococcus species.

Among the pediococci, only P. acidilactici and P. pentosaceus have been identified from human sources. Only one strain of Tetragenococcus, T. halophilus has been identified from humans.

Identification of the pediococci is accomplished by demonstrating the unknown strain to be vancomycin resistant, PYR negative, LAP positive, and does not form gas from glucose in MRS broth (Table 1).

The majority of strains of pediococci isolated from humans have been bile-esculin positive, deaminated arginine and have streptococcal group D antigen (in Lancefield extracts). All strains have failed to form acid in lactose broth, and most have grown very slowly if at all in NaCl broth. These reactions are similar to Streptococcus anginosus and Streptococcus equinus. Vancomycin resistance has not been identified in any strain of streptococci. Thus the vancomycin screening test should prevent this mis-identification.

Tetragenococcus resemble the Pediococcus except in their sensitivity to vancomycin.

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Table 11. Identification of Pediococcus and Tetragenococcus species. Pediococcus sp. are vancomycin resistant and Tetragenococcus is vancomycin sensitive.

Van= Vancomycin, S=sensitive, R= resistant, Mal = maltose, Ara= arabinose, Str = starch, Suc = sucrose, NaCl = 6.5% NaCl broth, Tre= trehalose, Arg= arginine, CRS = Chromogenic substrates either glucopyronidase or glucosaminidase + = >90% positive, – = <10% positive, v = 60-90% strains positive (-) most strains negative (+) most strains positive P. damnosus, P. dextrinicus, P. urinequi, and P. paruulus are not found in human infections, so far.

F. Identification of Leuconostoc and Weisella species

Leuconostoc
Like the lactococci, the Leuconostoc were once not thought to cause human infections. However, there are many reports of human infections caused by different Leuconostoc species. The Leuconostoc genus is the only catalase-negative gram-positive cocci that produce gas from MRS broth, are vancomycin resistant, PYR and LAP negative (Table 1).

Like the lactococci, Leuconostocs grow at 10C but very poorly if at all at 45C. Some strains grow in 6.5% NaCl broth while others do not. None of the strains are motile and none are ß-hemolytic.

Four Leuconostoc species, L. mesenteroides, L. citreum, L. lactis, and L. pseudomesenteroides have been isolated from humans. The reactions listed in Table 12 can be used to identify the species. L. citreum hydrolyzes esculin, produces slime on 5% sucrose agar and does not form acid in raffinose or melebiose broths. L. lactis does not hydrolyze esculin, does not form slime on 5% sucrose agar but does form acid in raffinose and melebiose broths. These strains may be confused with S. sanguinis biotype II (now classified as S. oralis) phenotypically with the exception of vancomycin susceptibility and production of gas from glucose in MRS broth. Both L. mesenteroides and L. pseudomesenteroides hydrolyze esculin, form slime on 5% sucrose agar, form acid in raffinose and melebiose broths. The latter two species are differentiated by growth in 6.5% NaCl broth and reaction in Litmus milk. L. mesenteroides grows in NaCl broth and may form weak acid (pink color) but does not clot in litmus milk. L. pseudomesenteroides gives the opposite reactions.

L. paramesenteroided has been moved to the genus Weissella.

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Table 12. Differentiation of Leuconostoc and Weissella species Key reactions: Vancomycin resistant, LAP and PYR negative, gas from MRS broth, growth in 6.5% NaCl broth variable, bile esculin reaction variable, and growth more common at 10C than at 45C.

Dex=extracellular polysaccharide (slime) production on 5% sucrose agar + = >90% positive, – = < 10% negative, v = 60-90% strains +

G. Identification of Aerococcus species, Helcococcus kunzi, Dolosigranulum pigrum, Tetragenococcus solitarius

The Aerococcus, Helcoccus, Dolosigranulum and Tetragenococcus all have the cellular arrangement of clusters and tetrads. All will grow in 6.5% NaCl with the exception of A. sanguicola and A. christensenii.

Table 13. Identification of Aerococcus species, Helcococcus kunzi, Dolosigranulum pigrum, Tetragenococcus solitarius

Refer to previous tables for explanation of tests and results.

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H. Identification of Globicatella species

There is only one species in the genus Globicatella. Globicatella sanguinis closely resembles the aerococci, streptococci, and enterococci phenotypically. The major differentiating characteristic between Globicatella and the aerococci is the cellular arrangement of the cells in the Gram stain. Globicatella forms chains while the aerococci form tetrads and clusters. The colonial morphology of Globicatella strains most closely resembles the viridans streptococci. However, these strains are readily distinguished with a negative leucine aminopeptidase reaction (LAP) and growth in the presence of 6.5% NaCl. The viridans streptococci are pyrridonyl arylamidase (PYR) negative, LAP positive and do not grow in the presence of 6.5% NaCl. The enterococci are PYR and LAP positive and grow at 10ΕC. None of the Globicatella isolates grew at 10ΕC or gave positive LAP reactions.

The biochemical characteristics of the most recently identified strains of A. viridans (28 strains) E. avium (28 strains) and S. uberis (9 strains, all nonhuman) were compared to the 28 strains of G. sanguinus. A. viridans is biochemically very similar to G. sanguinis (Table ). The colony morphologies are very similar on TSA with 5% sheep blood agar and both are LAP negative and grow in 6.5% NaCl. The cellular arrangement from growth in broth can be used to differentiate the two as A. viridans is arranged in clusters and tetrads, and G. sanguinis is arranged in short chains. Fermentation of inulin can be useful as the majority of G. sanguinis strains are positive and A. viridans strains are negative. The other key reactions for bile esculin, esculin and hippurate are of limited value in separating these two species since the PYR is a variable reaction for G. sanguinis and the bile esculin, esculin, and hippurate tests are variable reactions for Aerococcus viridans.

The LAP test is also useful in distinguishing enterococci from Globicatella strains. E. avium is further identified by positive pyruvate and sorbose tests and negative inulin and raffinose tests. G. sanguinis has the reverse reactions. The two species are phenotypically similar in the PYR, bile esculin, growth in 6.5% NaCl and at 45ΕC, hydrolysis of esculin and hippurate reactions, and acid production from lactose, maltose, mannitol, sorbitol, sucrose, and trehelose.

S. uberis has been included in the identification scheme with viridans streptococci for a number of years. The incorporation of the PYR and LAP tests in the identification scheme, as well as molecular studies have confirmed that many of the strains that are now Globicatella were previously reported as Streptococcus uberis-like. The key test for differentiation of the streptococci from Globicatella is the positive LAP reaction. S. uberis is also negative for esculin hydrolysis in the presence of bile, and positive for growth at 10ΕC. The majority of strains for both species are PYR positive, grow in 6.5% NaCl, hydrolyze esculin and hippurate, and produce acid from inulin, lactose, maltose, mannitol, raffinose, ribose, sorbitol, sucrose, and trehelose. Acid is not produced from glycerol and sorbose for both species.

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Table 14. Phenotypic differences between G. sanguinis, A. viridans, S. uberis, and E. avium.

+, 85% or more of the strains positive; -, 15% or less of the strains positive; V, variable reaction 16 to 84% of the strains positive; ( ), first number in the parenthesis indicates the total number of strains giving strong or weakly positive reactions. Biochemicals were incubated at 37ΕC for 14 days.
67

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Table 15. Phenotypic Characteristics of Dolosigranulum pigrum, Ignavigranum ruoffiae, Facklamia sp. and Alloiococcus otitidis.

See Table 1 for general characteristics of these bacteria. Tests are performed as described elsewhere, see index.

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Table 16. Potentially useful tests included in the Rapid ID-32 system for differentiating unusual gram positive cocci.

¹ Abbreviations; Adh, arginine hydrolysis; β-gar, β-galactosidase; α-gal, α-galactosidae; Man, mannitol; Sbl, sorbitol;
Tre, trehalose; Sac, saccharose; Dary, d-arabitol; Appa, alanine-phenylalanine-proline arylamidase, β-gal,

² Top number is percent of strains positive, bottom + or – is interpretation for identification.

³ Type strain only, no human isolates tested.