Development of a Flow Chart for Identification of Gram-Positive Anaerobic Cocci in the Clinical Laboratory

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2007, p Vol. 45, No /07/$ doi: /jcm Copyright 2007, American Society for Microbiology. All Rights Reserved. Development of a Flow Chart for Identification of Gram-Positive Anaerobic Cocci in the Clinical Laboratory Yuli Song, 1 * Chengxu Liu, 1 and Sydney M. Finegold 2,3,4 Research Service 1 and Infectious Diseases Section, 2 Veterans Administration Medical Center West Los Angeles, and Department of Medicine 3 and Department of Microbiology, Immunology, and Molecular Genetics, 4 University of California Los Angeles School of Medicine, Los Angeles, California Received 8 September 2006/Returned for modification 24 October 2006/Accepted 1 November 2006 Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of organisms that are isolated from clinical specimens more often than any group of anaerobic bacteria except Bacteroides species, yet many strains are still difficult or impossible to identify in the diagnostic laboratory. In this study, a total of 124 strains, including 13 reference strains of GPAC species and 111 isolates that had been recovered from clinical specimens previously and identified by 16S rrna gene sequencing, were subjected to biochemical characterization. Based on the results, a short biochemical scheme that involves the minimum essential biochemical tests for accurate identification of clinically important GPAC was developed. Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of organisms that form part of the normal endogenous flora of humans. They are isolated from clinical specimens more often than any group of anaerobic bacteria except Bacteroides species, yet many strains are still difficult or impossible to identify in the diagnostic laboratory (2, 4, 5). As a result, very little work has been done on the clinical importance of individual species of GPAC. The classification of the GPAC is not sound and has been confused by many loosely defined taxa. Peptoniphilus asaccharolyticus and Anaerococcus prevotii, the two butyrate-producing species commonly reported from human pathological material, have long been recognized as genetically heterogeneous (5). In a previous study (10), we evaluated 16S rrna gene sequencing as a tool for accurate identification of GPAC and noted the problems with sequences for GPAC present in a public database that may not be evident to all users and would lead to misidentification. While 16S rrna gene sequencing provides a powerful and reliable identification protocol for GPAC, it is not yet standardized and feasible for many clinical laboratories. Most clinical laboratories still rely on identification based on phenotypic tests or, more commonly, do not even attempt identification to species level. For routine identification of clinical isolates, ease of testing and total time required for completion are important. It has been shown that the GPAC species can be discriminated by routine phenotypic methods when these are supported by molecular data (7). In this study, in order to clarify the key features for identification of the recognized species of GPAC, and in order to detect groups of strains that might constitute undescribed species, we have studied a collection of clinical strains of GPAC and compared them with a reference collection that included most of the recognized type strains of GPAC. Using 16S rrna gene * Corresponding author. Mailing address: Wilshire Blvd., Room E3-237, Bldg. 304, VA Medical Center West Los Angeles, Los Angeles, CA Phone: (310) , ext Fax: (310) yulis1@yahoo.com. Published ahead of print on 29 November sequencing as a standard, we developed a biochemical scheme for accurate identification of this group of bacteria. MATERIALS AND METHODS Bacterial strains and culture conditions. The bacterial strains used in this study included 13 GPAC reference strains and 111 isolates that were previously recovered from clinical specimens and identified by 16S rrna gene sequencing in our previous study (Table 1). The breakdown of 111 clinical strains by 16S rrna gene sequencing was as follows: 95 strains had a sequence with a relatively high similarity ( 98.0%) to the type strain of an established species. The other 16 strains represent newly recognized taxa, which, for the present, have been assigned to groups I, II, and III. Six strains (group I) shared a sequence similarity of 97.2% with the type strain of Peptoniphilus harei. Four strains (group II) shared relatively high sequence similarity (97.2%) with P. asaccharolyticus strain GIFU 7717 (GenBank accession no. D14145) but very low sequence similarity ( 90%) with P. asaccharolyticus type strain CCUG 9988; again, the most closely related type strain was P. harei (93% sequence similarity). Six strains (group III) had sequences similar to that of an uncultured Clostridium bacterial clone from human wounds (GenBank accession no. DQ169836), and the most closely related described species was Anaerococcus lactolyticus (approximately 96.5% sequence similarity). All strains were cultured overnight on Brucella blood agar (Anaerobe Systems, Morgan Hill, CA) at 37 C under a gas phase of N 2 (86%), H 2 (7%), and CO 2 (7%). Bacterial characterization. The strains were characterized biochemically by using a combination of conventional tests as described in the Wadsworth anaerobe manual (4) and the commercially available biochemical kit Rapid ID 32A (biomérieux, Marcy L Etoile, France). The commercial biochemical kit was used according to the manufacturer s instructions, and the results were graded using a color chart supplied by the manufacturer. Results were graded as negative, weakly positive, or strongly positive; if they were weakly or strongly positive, they were recorded as positive. The conventional tests included prereduced, anaerobically sterilized (PRAS) biochemicals (Anaerobe Systems, Morgan Hill, CA) and gas-liquid chromatography (GLC) for metabolic end products of glucose metabolism. A ph of 5.5 in the PRAS tubes was interpreted as positive fermentation, a ph of 5.6 to 5.8 as weakly positive fermentation, and a ph of 5.9 as negative fermentation. RESULTS Key phenotypic characteristics to differentiate GPAC. A total of 124 strains, including 13 reference strains and 111 clinical isolates, which were identified based on sequence analysis of an approximately full length (1,400-bp) 16S rrna gene in our previous study (10), were subjected to phenotypic characterization. Table 2 summarizes the differential characteris- 512

2 VOL. 45, 2007 IDENTIFICATION OF GPAC BY A BIOCHEMICAL SCHEME 513 TABLE 1. List of type strains and clinical isolates of GPAC species used in this study Species and strain No. Type strains and clinical isolates with high sequence similarity ( 98%) to the type strains of an established species A. lactolyticus CCUG T... 1 Clinical isolates... 3 A. octavius CCUG T... 1 A. prevotii CCUG T... 1 Clinical isolates... 4 A. tetradius CCUG T... 1 Clinical isolate... 1 A. vaginalis CCUG T... 1 Clinical isolates...12 F. magna CCUG T... 1 Clinical isolates...15 P. asaccharolyticus CCUG 9988 T... 1 ATCC Clinical isolates... 2 P. harei CCUG T... 1 Clinical isolates...21 P. ivorii CCUG T... 1 P. lacrimalis CCUG T... 1 Clinical isolate... 3 P. anaerobius CCUG 7835 T... 1 Clinical isolates...10 P. micros CCUG T... 1 Clinical isolates...16 P. stomatis clinical isolates... 8 Clinical isolates with low sequence similarity ( 98%) to the type strains of an established species Group I clinical isolates... 6 Group II clinical isolates... 4 Group III clinical isolates... 6 tics of the tested gram-positive anaerobic coccal species, and Fig. 1 is a flow chart developed to show the key distinguishing tests. The tests included in the flow chart often gave strong reactions; other tests not shown in the flow chart but included in Table 2 can be used for confirmation, if needed. Peptostreptococcus anaerobius and Peptostreptococcus stomatis are the only gram-positive anaerobic cocci that give a zone of inhibition of 12 mm in diameter around a sodium polyanethol sulfonate (SPS) disk. Although P. stomatis resembles P. anaerobius phenotypically, it can be distinguished from P. anaerobius by being proline arylamidase (proline AMD) negative. Peptostreptococcus micros also exhibits a zone of inhibition with SPS; however, the zone is usually 12 mm in diameter. A. prevotii and Anaerococcus tetradius can be distinguished from other SPSresistant species of GPAC by production of -glucuronidase. Pyroglutamic acid arylamidase and glucose fermentation tests are useful for differentiation of A. prevotii and A. tetradius, but only a limited number of strains were available for testing (Table 2; Fig. 1). Glucose fermentation can be used as a key test to distinguish the saccharolytic from the asaccharolytic GPAC strains. Among the asaccharolytic strains, P. micros and Finegoldia magna can be readily distinguished from the rest by being positive for pyroglutamic acid arylamidase. A combination of colonial and microscopic morphology and proteolytic enzyme profiles can be used to differentiate between P. micros and F. magna. An anaerobic coccus with a milky halo around the colonies and small cells (diameter, 0.6 m) can be presumptively identified as P. micros, and it can be differentiated from F. magna by enzymatic tests: P. micros is positive for proline arylamidase, phenylalanine arylamidase, and tyrosine arylamidase, in contrast to F. magna, which is either negative or weakly positive (Table 2; Fig. 1). The asaccharolytic Peptoniphilus species can be differentiated by their enzyme profiles. Peptoniphilus ivorii can be easily distinguished from the others by being negative for arginine arylamidase and histidine arylamidase but positive for proline arylamidase. Peptoniphilus lacrimalis and two newly recognized groups of bacteria (groups I and II) can be differentiated from P. asaccharolyticus and P. harei by being serine arylamidase positive. Two newly recognized groups of bacteria (groups I and II) produce an enzyme profile similar to that of P. lacrimalis; however, organisms in group I can be differentiated from P. lacrimalis and strains of group II by producing glutamyl glutamic acid arylamidase but not alanine arylamidase. Organisms of group II can be distinguished from P. lacrimalis by being alkaline phosphatase positive. Strains of P. asaccharolyticus produce an enzyme profile very similar to that of strains of P. harei. The results we obtained from three P. asaccharolyticus strains we tested indicated that they can be readily distinguished from P. harei by being alkaline phosphatase positive (Table 2; Fig. 1). Among the saccharolytic GPAC strains, Anaerococcus vaginalis and A. lactolyticus can be differentiated from Anaerococcus octavius and Anaerococcus hydrogenalis by producing arginine arylamidase and leucine arylamidase, while A. octavius and A. hydrogenalis do not, and they can be readily differentiated from each other by features such as urease and -galactosidase (Table 2). Again, we recognized a group of saccharolytic GPAC strains (group III) that has a proteolytic enzyme profile very similar to that of A. lactolyticus, but they can be readily differentiated from A. lactolyticus by being urease negative. A. hydrogenalis can be differentiated from A. octavius by producing -glucosidase and indole but not proline arylamidase (Table 2; Fig. 1). Metabolic end products of glucose metabolism. Gram-positive anaerobic cocci are classified into the following five groups on the basis of fatty acid end products of metabolism

3 514 SONG ET AL. J. CLIN. MICROBIOL. TABLE 2. Key differential characteristics of gram-positive anaerobic cocci Organism Result of the following test: Production of the following saccharolytic or proteolytic enzyme d : GLC a SPS b Glucose c Urease AlkP -GAL -GLU -GUR AlaA ArgA TyrA ProA HisA PheA PyrA GGA SerA P. asaccharolyticus A, b R v P. harei A, b R v /w v Group I A, b R /w Group II A, b R P. lacrimalis A, b R v v /w A. hydrogenalis B, a R w w A. prevotii B, a R v v v /w /w /w /w A. tetradius B, a R v w /w A. lactolyticus B, a R /w v Group III B, a R v /w v A. vaginalis B, a R v v v v A. octavius B, a, c R P. ivorii IV R P. anaerobius A, IC S P. stomatis A, IC S F. magna A R v /w /w /w /w /w P. micros A R /w /w a For end products from glucose fermentation. Capital letters indicate major components; lowercase letters indicate minor components. A (a), acetate; B (b), butyrate; c, n-caproic acid; IV, isovalerate; IC, isocaproate. b R, either there is no zone of inhibition or the zone of inhibition is 2 mm; S, zone of inhibition is 12 mm. c Glucose fermentation tests were performed using prereduced, anaerobically sterilized peptone-yeast-glucose broth tubes (Anaerobe Systems, Morgan Hill, CA). ApHof 5.5 in the PYG tubes was interpreted as positive fermentation ( ) andaphof 5.9 as negative fermentation ( ). d All the enzymatic tests were done using rapid ID 32A systems (API biomérieux, Marcy l Etoile, France), according to the manufacturer s instructions. AlkP, alkaline phosphatase; -GAL, -galactosidase; -GLU, -glucosidase; -GUR, -glucuronidase; AlaA, alanine AMD; ArgA, arginine AMD; HisA, histidine AMD; TyrA, tyrosine AMD; ProA, proline AMD; PheA, phenylalanine AMD; PyrA, pyroglutamyl AMD, GGA, glutamyl glutamic acid AMD; SerA, serine AMD., 90% negative; w, weakly positive;, 90% positive; v, variable. analyzed by GLC (see Table 2). (i) The acetate (A) group, containing F. magna and P. micros, produces only acetic acid. (ii) The butyrate-acetate (Ba) group produces butyric acid as its major terminal volatile fatty acid (VFA) and acetic acid as a second acid; this group contains all of the species in the genus Anaerococcus. (iii) The acetate-butyrate (Ab) group produces acetic acid as its major terminal VFA and butyric acid as the second acid; this group contains all of the species in the genus Peptoniphilus except for P. ivorii. (iv) The caproate (C) group produces large quantities of longer-chain VFAs. The most important species in this group is P. anaerobius, the only species of gram-positive anaerobic cocci to produce a major terminal peak of isocaproic acid. (v) The isovaleric acid (IV) group contains P. ivorii the only species of gram-positive anaerobic cocci that produces a major terminal peak of isovaleric acid. GLC is also useful for identifying the rarely isolated Peptococcus niger and P. octavius, which produce n-caproic acid. DISCUSSION GPAC are a major part of the normal human flora and are frequently recovered from human clinical material; they constituted 24 to 31% of all isolates in four surveys of anaerobic pathogens (1, 3, 6, 11). However, studies of the significance of isolates of GPAC have been hindered by inadequate classification and the lack of a valid identification scheme. For example, P. asaccharolyticus is a gram-positive anaerobic coccal species frequently isolated from human clinical specimens, but strains that are identified phenotypically are genetically diverse. To complicate matters further, the type strain of P. asaccharolyticus is highly atypical in its whole-cell composition (8) and some biochemical properties (7). Our previous study (10) based on 16S rrna gene sequence analysis indicated that most of the strains that were identified phenotypically as P. asaccharolyticus shared only a low sequence similarity ( 90%) with the corresponding sequence of the type strain of P. asaccharolyticus. To eliminate the possibility of incorrect collection strains or culture contamination, cultures of the type strains of P. asaccharolyticus (ATCC T and CCUG 9988 T ) held in different collections were sequenced and the original data were confirmed. Members of this group of bacteria which were identified phenotypically as P. asaccharolyticus isolates shared high sequence similarity ( 99%) with another P. asaccharolyticus strain, ATCC 29743, and a blood isolate, strain (GenBank accession no. AY ), and the type strain most closely related phylogenetically was that of P. harei (approximately 98.6% sequence similarity). Although there is no an accepted cutoff value of 16S rrna sequence similarity for species definition, it is apparent from the results of studies of numerous diverse taxa that the majority of recognized species that have been examined to date differ in their 16S rrna sequences from related species of the same genus in at least 1% of the sequence positions, and typically more. In this study, we identified this group of so-called P. asaccharolyticus isolates as P. harei because of their relatively high sequence similarity (98.6%) and very similar phenotypic characteristics with P. harei. Further study involving DNA-DNA hybridization is being carried out to determine whether this group of bacteria merits classification as a novel species or a subspecies of P. harei. Previous studies (5) indicated that strains of P. asaccharolyticus produce an enzyme profile very similar to that of strains

4 VOL. 45, 2007 IDENTIFICATION OF GPAC BY A BIOCHEMICAL SCHEME 515 FIG. 1. Flow chart with key characteristics for identification and differentiation of gram-positive anaerobic cocci. 1, the SPS test was done using an SPS disk (Anaerobe Systems, Morgan Hill, CA). All gram-positive anaerobic cocci are resistant to SPS except for P. anaerobius and P. stomatis, which show zones of inhibition of 12 mm around an SPS disk. P. micros also exhibits a zone of inhibition with SPS; however, the zone is usually 12 mm in diameter. R, there is no zone or the zone of inhibition is 12 mm; S, the zone of inhibition is 12 mm. 2, all the enzymatic tests were done using rapid ID 32A systems (API; biomérieux, Marcy l Etoile, France) according to the manufacturer s instructions. -GLU, -glucosidase; -GAL, -galactosidase; -GUR, -glucuronidase; ArgA, arginine AMD; GGA, glutamyl glutamic acid AMD; ProA, proline AMD; PyrA, pyroglutamyl AMD; SerA, serine AMD; AlkP, alkaline phosphatase. 3, glucose fermentation tests were performed using a tube with PRAS peptone-yeast-glucose broth (Anaerobe Systems, Morgan Hill, CA). A ph of 5.5 in the peptone-yeast-glucose tubes was interpreted as positive fermentation and a ph of 5.9 as negative fermentation. of P. harei, but they can be easily differentiated by their clearly different cell and colony morphology. However, in this study, based on the solid identification obtained from 16S rrna gene sequencing, we found that there are two types of colonies in P. harei isolates that have 99.5% sequence similarity with the type strain of P. harei. One type is the same as that described previously for P. harei strains: colonies of 5-day cultures on enriched blood agar are approximately 1 mm in diameter, entire, flat, and translucent. However, the other type is the same as that described previously for P. asaccharolyticus strains: colonies are convex, circular, entire, opaque, and 2 to 3 mm in diameter, with a distinctive lemon-yellow tinge and a central peak. Therefore, we concluded that colony morphology is not a good criterion for differentiation between P. harei and P. asaccharolyticus. Our data indicated that the alkaline phosphatase test might be useful to distinguish between the two species; however, more strains of P. asaccharolyticus need to be tested. Another problem involves the designations A. prevotii and A. tetradius, which are still often used as a loose description for all strains of indole-negative, butyrate-producing GPAC. They were reported as common species of gram-positive anaerobic cocci in human clinical material in early surveys (1, 3, 11). Again, our study based on 16S rrna gene sequencing indicates that a large percentage of organisms identified as A. prevotii and A. tetradius are actually strains of A. vaginalis. Based on our data and those of Murdoch s group (7, 8, 9, 10), it is likely that strictly defined strains of A. prevotii and A. tetradius are only occasionally recovered from most clinical specimens. It has been shown that the GPAC species, as supported by the molecular data, can be discriminated by routine phenotypic methods. However, to our knowledge, not much effort has been made to devise a phenotypic scheme for identification of GPAC that would be effective and relatively rapid. Murdoch summarized the differential characteristics of the GPAC, including the newly described species, based on VFA profiles, carbohydrate fermentation reactions, and the enzyme profiles obtained with the rapid ID 32A commercial kit (5). GLC is very useful for classifying GPAC into groups. Our data indicate that the GPAC strains we tested can be divided into five groups based on the major end products of metabolism, and the GLC results correlate very closely with those of biochemical tests. Although GLC analysis of VFA is useful for grouping GPAC, many laboratories do not have ready access to GLC equipment. Further identification has relied mainly on the fermen-

5 516 SONG ET AL. J. CLIN. MICROBIOL. tation of carbohydrates and other standard bacteriological tests. However, relatively few species of GPAC produce acid from carbohydrates; many strains have been characterized on the basis of negative reactions. Most identification schemes have used indole production as a key test for the differentiation of butyrate-producing GPAC. For example, until recently, all butyrate-producing GPAC that formed indole were identified as P. asaccharolyticus; thus, indole-negative strains of P. asaccharolyticus presented a problem. It has long been accepted that most species of GPAC are strongly proteolytic and use the products of protein or peptide decomposition as a major energy source. In this study, we attempted to apply mainly saccharolytic and proteolytic enzyme tests selectively; to this end, we set up an accurate identification tree (flow chart) based on solid identification obtained from 16S rrna gene sequencing, rather than doing all the phenotypic tests on all isolates. We were able to identify the key phenotypic tests for relatively rapid identification of GPAC, and our results show good agreement with the data summarized by Murdoch (5). However, for those species for which we had insufficient numbers of strains to test (P. asaccharolyticus, P. lacrimalis, P. ivorii, A. hydrogenalis, A. lactolyticus, A. octavius, and A. tetradius), the distinctions cannot be generalized. There is strong evidence that related species of clinical importance still await formal description. In this study, we found three groups of GPAC (groups I, II, and III) that are phylogenetically and phenotypically distinct from the established GPAC species; they merit separate-species status. We are proposing groups I, II, and III as Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov., respectively (unpublished data). Systematic description of new bacterial species recovered from patients may contribute to the description of emerging infections. Therefore, efforts should be made to report novel species, even if only very few strains have been isolated. In conclusion, we developed a short biochemical scheme, based on the solid identification we obtained from sequencing, to provide clinical laboratories with an inexpensive and simple alternative for identification of GPAC. ACKNOWLEDGMENT This work has been carried out, in part, with financial support from Veterans Administration Merit Review funds. REFERENCES 1. Brook, I Recovery of anaerobic bacteria from clinical specimens in 12 years at two military hospitals. J. Clin. Microbiol. 26: Finegold, S. M Anaerobic bacteria in human disease. Academic Press, New York, NY. 3. Holland, J. W., E. O. Hill, and W. A. Altemeier Numbers and types of anaerobic bacteria isolated from clinical specimens since J. Clin. Microbiol. 5: Jousimies-Somer, H. R., P. Summanen, D. M. Citron, E. J. Baron, H. M. Wexler, and S. M. Finegold Wadsworth anaerobic bacteriology manual, 6th ed. Star Publishing Co., Belmont, CA. 5. Murdoch, D. A Gram-positive anaerobic cocci. Clin. Microbiol. Rev. 11: Murdoch, D. A., I. J. Mitchelmore, and S. Tabaqchali The clinical importance of gram-positive anaerobic cocci isolated at St Bartholomew s Hospital, London, in J. Med. Microbiol. 41: Murdoch, D. A., and J. Mitchelmore The laboratory identification of gram-positive anaerobic cocci. J. Med. Microbiol. 34: Murdoch, D. A., and J. T. Magee A numerical taxonomic study of the Gram-positive anaerobic cocci. J. Med. Microbiol. 43: Murdoch, D. A., and M. Kelly Pepstostreptococcus prevotii or Peptostreptococcus vaginalis? Identification and clinical importance of a new species of Peptostreptococcus. Anaerobe 3: Song, Y., C. Liu, M. McTeague, and S. M. Finegold S ribosomal DNA sequence-based analysis of clinically significant gram-positive anaerobic cocci. J. Clin. Microbiol. 41: Wren, M. W. D., A. W. F. Baldwin, C. P. Eldon, and P. J. Sanderson The anaerobic culture of clinical specimens: a 14-month study. J. Med. Microbiol. 10:49 61.