Identification of Gram-Positive Coccal and Coccobacillary Vancomycin-Resistant Bacteria

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1989, p Vol. 27, No /89/ $02.00/0 Identification of Gram-Positive Coccal and Coccobacillary Vancomycin-Resistant Bacteria R. FACKLAM,1* D. HOLLIS,' AND M. D. COLLINS2 Respiratory Diseases Branch, Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Department of Microbiology, Institute of Food Research, Agricultural Food Research Council, Reading Laboratory, Shinfield, Reading RG2 9AT, United Kingdom2 Received Il October 1988/Accepted 30 December 1988 A total of 84 of 150 vancomycin-resistant (defined as no inhibition of bacterial growth around a 30-,ug vancomycin disk placed on 5% sheep blood-trypticase soy agar [BBL Microbiology Systems, Cockeysville, Md.]) bacteria were definitively identified by determining the phenotypic criteria. The identity of representatives was also confirmed by DNA-DNA hybridizations. The following strains were identified: 1 Enterococcus faecium, 18 Leuconostoc mesenteroides, 15 Leuconostoc citreum, 9 Leuconostoc pseudomesenteroides, 2 Leuconostoc lactis, 20 Pediococcus acidilactici, 5 Pediococcus pentosaceus, and 14 Lactobacillus confusus. The remaining vancomycin-resistant strains were identified as probable Leuconostoc (6 strains), probable Pediococcus (1 strain), and probable lactobacilli (28 strains). A total of 32 strains of gram-positive coccobacillary bacteria remained unidentified. Tests used for the phenotypic identification of strains to the genus level included a Gram stain of bacteria grown in thioglycolate broth, gas production in Lactobacillus Mann, Rogosa, and Sharpe broth, hydrolysis of pyrrolidonyl-jg-naphthylamide, bile-esculin reaction, demonstration of streptococcal group D antigen, and growth at 10 and 45 C and in 6.5% NaCl broth. Strains were identified to the species level by hydrolysis of esculin, reactions in litmus milk, slime production on 5% sucrose agar, acidification of maltose, melibiose, and raffinose broths, deamination of arginine, and growth at 42 C and in 6.5% NaCl broth. Vancomycin is used for treating infection caused by gram-positive cocci (16, 17). Although resistance to vancomycin among species of Streptococcus has been reported (27), it has also been disputed (C. Thornsberry and R. R. Facklam, Antimicrob. Newsl. 1:63-64, 1984). We screened more than 500 strains of Aerococcus, Enterococcus, and Streptococcus for susceptibility to the 30-pug vancomycin disk and found that this test could be used to help identify non-beta-hemolytic strains of the three aforementioned genera (8). No vancomycin-resistant aerococci, enterococci, or streptococci were identified with a zone-no zone criterion used for the screening test (8). Recently (17), we reported vancomycin resistance in an Enterococcus species, but the level of resistance for this species (MIC, 16,ug/ml) was much lower than that reported for the Leuconostoc species (MIC, >1,000 ptg/ml) (20). Strains of Enterococcus faecalis and Enterococcus faecium have been isolated in epidemics with vancomycin MICs of >16,ug/ml (28). Colman and Efstratiou (5) reported three vancomycin-resistant gram-positive cocci (human isolates) which were similar to pediococci. The level of vancomycin resistance was not reported for these strains. Other reports indicate that some species of Leuconostoc have been isolated from human diseases (2, 6, 15, 16, 19, 23, 24). These reports include evidence that the vancomycinresistant streptococci are members of the Leuconostoc genus, although no definitive DNA studies with human isolates and type strains of either Leuconostoc or Pediococcus species have yet been reported. Seven genera of gram-positive cocci are listed as facultatively anaerobic bacteria in Bergey's Manual of Systematic Bacteriology (25). Two of these genera possess cytochrome enzymes (Staphylococcus and Stomatococcus), while the remainder of the genera do not. This investigation included * Corresponding author. 724 only cytochrome-negative bacteria as measured by a negative catalase reaction. For identification of catalase-negative gram-positive cocci, it has been suggested that clinical microbiologists use methods to identify only species of the Aerococcus and Streptococcus genera because bacteria that belong to the Gemella, Leuconostoc, and Pediococcus genera are not considered pathogenic for humans (25) or are poorly defined and refractory to identification (9). Recently, the genus Streptococcus was split into three genera: Enterococcus, Lactococcus, and Streptococcus (for a review, see reference 26). Although most Lactococcus spp. are not considered pathogenic for humans, we have recently isolated Lactococcus garviae from human sources (unpublished data). The clinical microbiologist is therefore confronted with the problem of identifying seven different genera of catalase-negative, facultatively anaerobic bacteria: Aerococcus, Enterococcus, Gemella, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus. In this report, we outline the basic procedures necessary to place the gram-positive cocci into the seven genera. We will also describe the phenotypic characteristics and laboratory methods used to identify the genus and species of 84 vancomycin-resistant strains we have identified from human infections. MATERIALS AND METHODS Strains. The following type strains were used in this study: Leuconostoc mesenteroides ATCC 8293, Leuconostoc dextranicum ATCC 19255, Leuconostoc cremoris ATCC 19254, Leuconostoc lactis ATCC 19256, Leuconostoc oenos ATCC 23279, Leuconostoc paramesenteroides ATCC 33313, Pediococcus acidilactici ATCC 33314, Pediococcus pentosaceus ATCC 33316, Pediococcus damnosus ATCC 29358, Pediooccus halophilus ATCC 33315, Pediococcus dextrinicus ATCC 33087, Pediococcus parvulus ATCC 19371, Gemella

2 VOL. 27, 1989 VANCOMYCIN-RESISTANT COCCOID BACTERIA 725 TABLE 1. Reactions in tests used to aid in differentiating gram-positive cocci and coccobacillary genera of bacteria (human isolates) % Positive (no. of strains) Test Enterococci Streptococci Lactococci Aerococci Gemella Pediococci Leuconostoc Lactobacillus (200) (645)" (16) (42) (15) (26) (50) (42) Gas from glucose < Vancomycin resistance < Reaction to streptococcal group D antiserum Bile-esculin reaction PRYase Growth: In 6.5% NaCi broth At 45 C At 10 C 85 < " All streptococcal strains tested were viridans group strains. Only 44 of these strains were tested for gas production. haemolysans ATCC 10379, and Aerococcus viridans ATCC Type strains for the species of Enterococcus, Lactococcus, and Streptococcus species were retrieved from the culture collection of the Centers for Disease Control Streptococcus Laboratory (11), and type strains for the DNA homology studies were obtained from the National Collection of Food Bacteria, Reading, United Kingdom, and Deutsche Sammlung von Mikroorganismen, Braunschweig, Federal Republic of Germany. Strains of vancomycin-resistant clinical isolates from infected persons were sent to the Centers for Disease Control Streptococcus or Special Bacteriology Laboratories by state and city public health laboratories in the United States. Most of the bacteria were recent clinical isolates, but some strains had been received as early as Inocula for tests performed in the Streptococcus Laboratory were prepared by selecting a single colony and inoculating 5 ml of Todd-Hewitt broth. The Todd-Hewitt broth was incubated overnight at 35 C and then was used to inoculate all routine tests for the identification of aerococci, enterococci, lactococci, and streptococci. Preparation and interpretation of media and tests have been previously described (9, 11). Bacteria were tested for vancomycin resistance as previously described (8). Briefly, a nonstandardized heavy inoculum was spread over one-half of a Trypticase soy agar-5% sheep blood plate (BBL Microbiology Systems, Cockeysville, Md.) with a wire loop or cotton swab to achieve confluent growth. A 30-,ug vancomycin disk was placed in the center of the inoculated plate, and the plate was incubated in a candle extinction jar for 16 to 18 h at 35 C. If any zone of inhibition was observed, the strain was considered susceptible. Strains that exhibited growth up to the disk were considered resistant. Production of gas from glucose was determined by inoculating Lactobacillus Mann, Rogosa, and Sharpe (MRS) broth (Difco Laboratories, Detroit, Mich.) (7). The inoculated tube was overlaid with melted petrolatum and incubated for up to 7 days at 35 C. Gas production was recorded as positive when the petrolatum plug was completely separated from the broth in the tube. Pyrrolidonylarylamidase (PRYase) activity was determined by the 4-h broth test (Carr-Scarborough) (11). The Coblenz modification of the Voges-Proskauer test was used to measure the production of acetyl methyl carbinol (11). Inocula for tests performed in the Special Bacteriology Laboratory were prepared by selecting single colonies of bacteria and inoculating 5 ml of heart infusion broth and heart infusion agar slant. After incubation at 35 C, heart infusion broth and heart infusion agar were used in all tests performed for identifying the facultatively anaerobic bacteria. Preparation and interpretation of media and tests have been previously described (3). Preparation of inocula and interpretation of test results for the Rapid Strep system (Analytab Products, Plainview, N.Y.) have been previously described (11). Gram stains were prepared for growth obtained in thioglycolate broth (1 to 2 days at 35 C). Strains of pediococci for DNA studies were grown in yeast-glucose-phosphate broth (13), whereas Leuconostoc and Lactobacillus strains were grown in MRS broth to the late exponential phase at 25 C. DNA-DNA hybridizations were performed under stringent conditions (10 C below the melting temperature) by the membrane filter method described by Garvie (13). RESULTS Gram stains of the vancomycin-resistant strains were prepared in both Centers for Disease Control laboratories. Two of us (R.F. and D.H.) achieved a consensus by sharing slides and observations. Gram stain results alone could not be used to identify any of the eight genera listed in Table 1. However, they were useful in certain situations (see Discussion). Gas production in MRS broth was consistently observed with type strains and clinical isolates of Leuconostoc species. One strain of E. faecalis and approximately half of the Lactobacillus species examined produced gas in MRS broth (Table 1). All the type strains and clinical isolates of the Leuconostoc and Pediococcus genera, with the exception of the type strains of L. oenos and P. halophilus, were resistant to vancomycin. Neither of the latter strains grew on a Trypticase soy agar-5% sheep blood plate. P. halophilis ATCC was susceptible to vancomycin on glucose-yeast-naci agar (13). L. oenos ATCC grew only in MRS broth and on tomato juice agar. Vancomycin resistance in the latter was not determined. The majority (90%) of Lactobacillus strains in our study were also vancomycin resistant. With the exception of one strain of E. faecium, all other Enterococcus, Streptococcus, Lactococcus, Aerococcus, and Gemella strains were susceptible by our screening procedure (Table 1). Lancefield extracts of the type strains of L. mesenteroides, L. paramesenteroides, P. acidilactici, P. parvulus, and P. pentosaceus reacted with streptococcal group D antisera. Among the clinical isolates, 80% of the enterococci, 95% of the pediococci, 35% of the leuconostocs, and 25% of the lactobacilli reacted with serogroup D antiserum.

3 726 FACKLAM ET AL. J. CLIN. MICROBIOL. TABLE 2. DNA-DNA homologies of vancomycin-resistant Leuconostoc strains, Lactobacillus confusus, and Pediococcus strains No. of Range of homology between type strain DNA and test strain DNA (%) strains tested L. citreum L. lactis L. mesenteroides L. pseudomesenteroides L. confusus P. acidilactici P. pentosaceus NT NT NT NT NT NT 2 NTI NT NT NT NT NT S51 NT NT NT 10 il NT NT NT 8 NT NT NT NT NT NT NT NT NT NT aleuconostoc and Lactobacillus strains were also tested for homology with type strains of L. cremoris, L. dextranicum, and L. paramesenteroides. Pediococcus strains were tested for homology with P. damnosus, P. dextrinicus, P. halophilus, P. parvulus, P. urinaeequi, and Aerococcus viridans. Percent homologies were less than 50% in al instances. " NT, Not tested. All the type strains of the Pediococcus species except P. damnosus gave positive bile-esculin reactions. Among the Leuconostoc type strains, only L. mesenteroides gave a positive bile-esculin reaction. The majority of vancomycinresistant clinical isolates were also positive for bile-esculin reactions (Table 1). None of the type strains or clinical isolates from the Pediococcus or Leuconostoc genus gave positive PRYase reactions. Only three Lactobacillus strains were PRYase positive. All the Enterococcus and Aerococcus spp. and the majority of Lactococcus and Gemella spp. were also PRYase positive. Tolerance tests and growth in 6.5% NaCI (salt) broth and at 10 and 45 C were useful in differentiating enterococci from streptococci (salt broth), aerococci from enterococci (10 and 45 C), and aerococci from gemellas (salt broth). These tests were of little value in differentiating or recognizing the genus of vancomycin-resistant strains. A summary of the results of the DNA homology studies with 50 vancomycin-resistant strains is presented in Table 2. Four Leuconostoc sp., two Pediococcus sp., and Lactobacillus confusus strains were identified. Two of the Leuconostoc spp. are recently proposed new species (J. A. E. Farrow, R. R. Facklam, and M. D. Collins, submitted for publication). Four of the vancomycin-resistant strains failed to form sufficient homology to be identified as any known Leuconostoc species, although homologies of 10 to 51% indicate a strong relationship. Details of the experiments and interpretation of data are given in the above-mentioned report submitted for publication. The physiologic characteristics of the Leuconostoc spp. and Lactobacillus confusus strains determined by conventional tests in the Special Bacteriology (30 strains) and Streptococcus (58 strains) Laboratories as well as by the API Rapid Strep system tests (38 strains) are listed in Table 3. There is a reasonable overall agreement of test results between the two conventional test systems. The base for the carbohydrate acidifications is different in the two laboratories, so minor differences can be expected. Overall, a 91% agreement was observed among 16 tests performed in both laboratories. The range of agreement was 74% (mannitol and lactose acidification) to 100% (melibiose and sucrose acidification and deamination of arginine). By using the results from the DNA hybridization (Table 2) and physiologic tests (Table 3), we constructed Table 4. Table 4 lists the physiologic characteristics that can be used to identify the Leuconostoc spp. and Lactobacillus confusus strains isolated from human infections. The identification of L. citreum, L. lactis, L. mesenteroides, and Lactobacillus confusus is clear; however, the identification of L. pseudomesenteroides is difficult. Because both strains of L. pseudomesenteroides confirmed by DNA homologies failed to grow in 6.5% NaCI broth, we used this test to differentiate L. mesenteroides from L. pseudomesenteroides. Using the criteria listed in Table 4, 15 strains of L. citreum, 2 strains of L. lactis, 18 strains of L. mesenteroides, 9 strains of L. pseudomesenteroides, and 14 strains of Lactobacillus confusus were identified. Four strains could not be placed into any Leuconostoc species because the DNA homologies were inconclusive. Two strains were omitted from this analysis because of major discrepancies between DNA homologies and phenotypic characteristics. None of the DNA homology or physiologic test experiments were repeated. The results with the API 20 Strep system indicated that L. citreum, L. lactis, L. mesenteroides, and Lactobacillus confusus could be identified by hydrolysis of esculin, deamination of arginine, acidification of raffinose, and alphagalactosidase activity. L. pseudomesenteroides could not be differentiated from L. mesenteroides. Appreciable differences between both conventional tests and the acidification of mannitol and Voges-Proskauer tests in the API 20 Strep system were apparent. Difference in substrates is the probable reason. The physiologic characteristics of the Pediococcus spp. determined by conventional tests in the Special Bacteriology Laboratory (8 strains), Streptococcus Laboratory (26 strains), and API Strep system (15 strains) are listed in Table 5. Overall correlation between the two conventional test systems was high (about 90%). The range of correlation was broader with these strains (0 to 80%) than with the leuconostocs. Too few strains were tested in the Special Bacteriology Laboratory, however, to draw a final conclusion. The DNA homology data confirmed that these human strains were identical to nonhuman strains of P. acidilactici and P. pentosaceus. The two species are differentiated by acidification of maltose broth: P. pentosaceus acidifies maltose, while P. acidilactici does not. Of the 26 strains, 20 were identified as P. acidilactici and 5 were identified as P. pentosaceus; 1 strain, although phenotypically P. acidilactici, failed to join the group by DNA homology. The same differences between conventional Voges- Proskauer test results and the API Voges-Proskauer test results observed with the leuconostocs were also observed with the pediococci. The two Pediococcus species, P. acidilactici and P. pentosaceus, may be differentiated by the arabinose reaction in the API system (Table 5). Too few strains have been tested in the API system, however, to suggest that this reaction is reliable.

4 VOL. 27, 1989 VANCOMYCIN-RESISTANT COCCOID BACTERIA 127 TABLE 3. Reactions of Leuconostoc species and Lactobacillus confusus in conventional and API Strep system testsa % Positive' Test L. citreum L. lactis L. mesenieroides L. pseudomesenteroides L. confusus Acid formation in: Adonitol 0 -' Arabinose Q Cellobiose Dulcitol D-Galactose Lactose Maltose Mannitol Melibiose Raffinose Ribose Salicin Trehalose Xylose Deamination of arginine Hydrolysis of esculin Growth at 42 or 45oCd il Methyl red Voges-Proskauer Litmus milk: Acid Clot Slime on 5% sucrose agar Acid butt and slant in TSI i0o Growth in 6.5% NaCl O x-galactosidase activity O [-Galactosidase activity a All strains formed acid in fructose, glucose, mannose, and sucrose broths. Ail strains grew on tomatojuice agar, in heart infusion broth containing 2 and 4% NaCI, and at 25 and 35 C. All strains were catalase, gelatinase, nitrate, oxidase, and urease negative. None of the cultures hydrolyzed starch or hippurate. None of the cultures grew on SS agar or MacConkey agar, produced indole or H2S in TSI agar, or tolerated 0.04% tellurite and 0.25% tetrazolium. None of the cultures decarboxylated lysine or ornithine or utilized pyruvate. None of the strains were motile Or produced extracellular polysaccharide in 5% sucrose broth. None of the strains produced acid from dextrin, erythritol, glycerol, glycogen, inositol, inulin, melezitose, rhamnose, sorbitol, sorbose, or starch. None of the strains were alkaline phosphatase and P-glucuronidase negative. All the leuconostocs and four of six strains of Lactobacillus confusus were leucine aminopeptidase negative. b Results are grouped in subheads according to test: 1, Special Bacteriology Laboratory; 2, Streptococcus Laboratory; 3, API 20 Strep. C-, Not tested. d Growth at 42 C in the Special Bacteriology Laboratory test and at 45'C in the Streptococcus Laboratory test. DISCUSSION gram-positive cocci. The greatest difficulties arise in differ- Table 6 lists the most useful laboratory tests that can be entiating the Enterococcus, Streptococcus, Lactococcus, used in the clinical microbiology laboratory to identify the Leuconostoc, and Lactobacillus species. Cellular arrangegram-positive cocci and coccobacillary bacteria. Gram ment is basically too similar to differentiate the species. stains are very useful as adjuncts in identifying genera. Cellular morphology (cocci versus rods) is useful in some Cellular arrangement in clusters and tetrads were consis- instances. The Leuconostoc species we observed were more tently observed with species of Aerococcus, Gemella, and coccobacillary than coccal in shape. The Lactobacillus Pediococcus but were not observed with species of the other species do not form definite cocci, but coccobacillary forms TABLE 4. Test Differential characteristics of Leuconostoc species and Lactobacillus confusus by conventional microbiologic tests Reaction of': L. citreum L. lactis L. mesenteroides L. pseudomesenteroides L. confusus Esculin + (100) - (0) + (100) + (100) + (100) Litmus milk (acid and clot) - (0) + (100) - (10) + (67) - (0) Slime on 5% sucrose agar + (93) - (0) + (90) + (75) + (100) Acid formation in: Raffinose - (0) + (50) + (100) + (67) - (0) Melibiose - (0) + (100) + (100) + (100) - (10) Growth in 6.5% NaCI broth V (50)b - (O) + (89) - (0) V (57)b Growth at 42 C - (O) - (O) - (0) - (0) + (100) Deamination of arginine - (0) - (0) - (0) - (0) + (100) b'v, Variable. a The number in parentheses is the percent positive.

5 728 FACKLAM ET AL. J. CLIN. MICROBIOL. TABLE 5. Reactions of Pediococcus species in conventional and API Strep system tests" % Positive' Test Pediococcus acidilactici Pediococ cus pentosaceus Unknown Pediococcus sp Acidification of: Arabinose Glycerol 80 0 '` Lactose 60 0 il Maltose Rhamnose Ribose Salicin Sucrose Trehalose Xylose Hydrolysis of hippurate Growth: At 45 C On MacConkey agar In 4.0% NaCI In 6.5% NaCI Voges-Proskauer ax-galactosidase activity " All strains formed acid in cellobiose, D-galactose, glucose, fructose, and mannose broths. Ali strains hydrolyzed esculin and deaminated arginine. All strains grew at 25, 35, and 42'C. AIl strains formed acid on both the slant and butt of triple sugar iron agar and were leucine aminopeptidase positive. Reactions in litmus milk were limited to very weak acid or no reaction. None of the strains formed acid in adonitol, dextrin, dulcitol, erythritol, glycogen, inositol, inulin, mannitol, rtelibiose, melezitose, raffinose, sorbitol, or sorbose. All strains failed to hydrolyze starch or urea, and ail were nonmotile and catalase, oxidase, and nitrate negative. None of the cultures grew on SS agar, produced indole or HS in TSI agar, or tolerated 0.04% tellurite and 0.25% tetrazolium. None of the cultures decarboxylated lysine or ornithine or utilized pyruvate. None of the cultures produced extracellular polysaccharide on 5% sucrose agar or broth. All cultures were at-galactosidase, P-glucuronidase, and alkaline phosphatase negative. Results are grouped in subheads according to test: 1, Special Bacteriology Laboratory; 2, Streptococcus Laboratory; 3, API 20 Strep. -, Not tested. were often observed. Because the physiologic characteristics of some strains of Leuconostoc and Lactobacillus species are so similar, Gram strains were sometimes the only distinguishing characteristic. We feel that growth in thioglycolate broth gave the most consistent Gram stain results. If cultures grew poorly or not at all in thioglycolate broth, we used growth in MRS broth for Gram stains. To identify the Leuconostoc bacteria, microbiologists must determine gas production from glucose. Lactobacillus MRS broth is commercially available and has performed well for us. As with ail other tests in this system (Table 6), gas production in MRS broth alone does not identify the leuconostocs, and many Lactobacillus species also produce gas in this broth. To assist in identifying the Leuconostoc genus, TABLE 6. Tests useful in differentiating gram-positive cocci and coccobacillary genera of bacteria (human clinical isolates) Reaction of': Test Enterococci Streptococci Lactococci Aerococci Gemella Pediococci Leuconostoc Lactobacillus Morphology' Cocci Coccobacilli Rods Arrangement Chains Pairs Clusters Gas from glucose V Vancomycin S S S S S R R RS Streptococcal group antigen D A-V N - - D - -'C Bile-esculin + V V V - + V PRYase + Vd Vd + + Leucine aminopeptidase V Growth: In 6.5% NaCl + - V + - V V V At 45 C + V -' V At 10 C +' -C V a Abbreviations: V, variable reaction; S, susceptibility to vancomycin (presence of a zone of inhibition); R, resistance to vancomycin (absence of a zone of inhibition). h Determined after incubation for 1 to 2 days in thioglycolate broth. There was an occasional exception. d Positive for Streptococcus pyogenes and Lactococcus garviae; negative for all other species.

6 VOL. 27, 1989 deamination of arginine should also be determined since none of the Leuconostoc species deaminate arginine. AIthough we do not claim that the tests listed in Table 6 will identify all lactobacilli, those that are most often confused with Streptococcus and Leuconostoc species can be identified (12). It is also likely that all the Leuconostoc bacteria encountered from clinical material will be resistant to vancomycin. This level of resistance is much higher than that of vancomycin-resistant enterococci (>2,000 versus 16 to 32 jxg/ml) (17, 20). Clinical microbiologists can take advantage of this fact. With the vancomycin screening test, al but one each of the Leuconostoc and Pediococcus species are resistant to vancomycin. Although P. halophilus was judged susceptible to vancomycin, we tested the culture under unusual conditions, and there is a question of the correct taxonomic status of this species (29). Vancomycin resistance is intrinsic in Leuconostoc species and could not be transferred to streptococci or enterococci (20). It is likely that vancomycin resistance will emerge within the Enterococcus genus; strains of vancomycin-resistant E. faecalis and E. faecium for which MICs are very high have been reported (18, 28). Vancomycin resistance in E. faecium was plasmid related and could be transferred to Streptococcus sanguis but not other enterococcal species (20). The PRYase test is very useful in this scheme. None of the Leuconostoc or Pediococcus strains were positive with the Carr-Scarborough 4-h test (11) or the PRYase test in the API 20 Strep system. All of the Enterococcus and Aerococcus species included in this study were PRYase positive. Thus, vancomycin-resistant Enterococcus species can be differentiated from the Leuconostoc and Pediococcus species by the PRYase test. In addition to the PRYase test in the API 20 gallery, the leucine aminopeptidase test helps to differentiate the gram-positive cocci. All the leuconostocs were negative by the leucine aminopeptidase test, while all the pediococci were positive. Because an appreciable number of vancomycin-resistant strains were bile-esculin reaction positive and did not grow in 6.5% NaCi broth, the strains were presumptively identified as group D streptococci (Streptococcus bovis or Streptococcus equinus), not enterococci. Lancefield extracts of most of the Pediococcus and some of the Leuconostoc and Lactobacillus species reacted with group D antiserum from the Centers for Disease Control. Thus, considerable confusion arose over the proper identity of the vancomycinresistant strains. Most of the Pediococcus strains had been previously identified as S. equinus. The type strain of S. equinus is susceptible to vancomycin, however, and does not deaminate arginine. We have not yet confirmed an S. equinus strain that has been isolated from an infected human. Interpreting the value of our tolerance tests is difficult. Species within each genera listed in Table 6 vary in their capacity to grow in broth containing 6.5% NaCI and at 10 and 45 C. This capacity to grow at these temperatures and salt concentrations is dependent upon base medium, type of incubation (water bath or air incubator), temperature (for NaCi tolerance), and inoculum (unpublished data). The tolerance tests we used in the Streptococcus Laboratory were not devised to identify or differentiate Pediococcus, Leuconostoc, or Lactobacillus species. Some of these strains grew very poorly, if at all, at the optimum temperature for growth in heart infusion broth. Thus, interpretations of these tests were not valid in some instances. Experiments are under way that will determine which of the variables, VANCOMYCIN-RESISTANT COCCOID BACTERIA 729 including base medium, will optimize the reliability of these tests. Recognition of the species within each genus listed in Table 6 was not addressed in this study with the exception of the vancomycin-resistant strains. First of all, the microbiologist must recognize the fact that some enterococcal species may be vancomycin resistant. The PRYase reaction will help place these bacteria in the proper genus, and species identification can be accomplished by several methods (4, 8, 9). Some of the isolates of Leuconostoc strains we examined had physiologic characteristics consistent with L. mesenteroides (18 strains) and L. lactis (2 strains). About half of the human isolates we examined were members of two recently described species, L. citreum (15 strains) and L. pseudomesenteroides (9 strains). Of the 50 strains identified as leuconostocs, 44 (88%) were placed into one of the four species by the tests listed in Table 4. Pigmentation (a yellow citruslike growth in MRS broth) may be useful as an aid in identifying L. citreum. The identification of L. pseudomesenteroides as described in Table 4 is tenuous. The failure of cultures to grow in a tolerance test not specifically designed for this species must be interpreted cautiously. Perhaps future experiments will find more reliable tests or procedures to identify this species. Differentiation of the two Pediococc us species was achieved by the acidification of maltose. P. acidilactici does not acidify maltose broth, while P. pentosaceus does. Comprehensive reviews of the Pediococcus genus are available (1, 22). Because Lactobacillus confusus has unique phenotypic characteristics and is often confused with L. mesenteroides (12), we included the identification in this study. As with L. mesenteroides, Lactobacillus confusus produces gas in MRS broth, is vancomycin resistant, and produces extracellular polysaccharide; however, Lactobacillus confusus deaminates arginine, while L. mesenteroides does not. Some of our unidentified vancomycin-resistant lactobacilli (more than 30 strains) resemble Lactobacillus oris (10). Studies are under way to confirm this observation. The majority of strains studied were blood culture isolates (60 strains). Endocarditis was given as a clinical diagnosis in three patients (two with L. citreum and one with L. pseudomesenteroides). Sepsis, pneumonia, and bacteremia were the diagnoses given for other patients with positive blood cultures of all other species of vancomycin-resistant bacteria. L. citreurm, L. mesenteroides, and Lactobacillus confuses were isolated from the cerebrospinal fluid of patients with meningitis (one case each). Leuconostoc, Pediococcuts, and Lacbtobacilluis strains were also isolated from urine and wound cultures. The sources and clinical diagnoses of patients from whom the vancomycin-resistant strains were isolated are similar to sources and clinical diagnoses of patients with enterococcal, streptococcal or both types of infections. Implementation of the tests and procedures described in this study will assist in the microbiological identification of vancomycin-resistant bacteria found in human infections. LITERATURE CITED 1. Bergan, T., R. Solberg, and O. Solberg Fatty acid and carbohydrate cell composition in pediococci and aerococci, and identification of related species, p In T. Bergan (ed.), Methods in microbiology. Academic Press, Inc., New York. 2. Buu Hoi, A., C. Branger, and F. J. Acar Vancomycinresistant streptococci or Leuconostoc sp. Antimicrob. Agents Chemother. 28: Clark, W. A., D. G. Hollis, R. E. Weaver, and P. 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