Vancomycin-Resistant Gram-Positive Bacteria Isolated from Human Sources

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1 JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1988, p /88/ $02.00/0 Copyright 1988, American Society for Microbiology Vol. 26, No. 10 Vancomycin-Resistant Gram-Positive Bacteria Isolated from Human Sources KATHRYN L. RUOFF,l.2* DANIEL R. KURITZKES,3'4 JOHN S. WOLFSON,3'4 AND MARY JANE FERRARO"4 Francis Blake Bacteriology Laboratories' and Infectious Disease Unit, Medical Services,3 The Massachusetts General Hospital, Boston, Massachusetts 02114, and Department of Microbiology and Molecular Genetics2 and Department of Medicine,4 Harvard Medical School, Boston, Massachusetts Received 28 March 1988/Accepted 14 July 1988 Recent reports of infections with vancomycin-resistant gram-positive bacteria prompted us to study vancomycin-resistant isolates from human sources to characterize the types of bacteria displaying this phenotype. Thirty-six vancomycin-resistant gram-positive isolates, 14 from clinical specimens and 22 from stool samples, were identified. These isolates were tentatively identified as Lactobacillus spp. (25 strains), Leuconostoc spp. (6 strains), and Pediococcus spp. (3 strains) on the basis of morphology and physiological tests. Two isolates of indeterminate morphology could not be unambiguously assigned to a genus. Four isolates of vancomycin-resistant lactobacilli from normally sterile body sites were considered to be clinically significant. Vancomycin-resistant gram-positive bacteria may represent an emerging class of nosocomial pathogens. Better methods for distinguishing the various genera in the clinical microbiology laboratory are needed. Despite decades of vancomycin use, few reports of clinically significant vancomycin resistance among isolates of gram-positive bacteria have appeared in the literature. Recently, isolates of vancomycin-resistant coagulase-negative staphylococci (17) and enterococci (19) have been described. Vancomycin resistance has been reported in clinical isolates of Leuconostoc and Pediococcus, genera of "lactic acid bacteria" that resemble streptococci morphologically (3, 5, 6, 9, 15). Although vancomycin resistance has been reported in a strain of viridans group streptococcus (18), this isolate most likely represents a case of a lactobacillus mistaken for a streptococcus (C. Thornsberry and R. Facklam, Antimicrob. Newsl. 1:63-64, 1984). These reports emphasize the resemblance of certain lactic acid bacteria to streptococci and the difficulties in correctly identifying vancomycinresistant gram-positive organisms. We examined 36 isolates of vancomycin-resistant grampositive bacteria recovered from specimens submitted to the clinical bacteriology laboratory to study their morphological and physiological characteristics and to assess their clinical significance. MATERIALS AND METHODS Bacterial strains and cultivation media. Isolates were routinely cultured on plates containing brucella agar supplemented with 5% horse blood (GIBCO Diagnostics, Madison, Wis.). Cultures were incubated at 35 C in an atmosphere containing 5% C02. Long-term storage of organisms was accomplished by freezing suspensions of the bacteria in horse blood. Stock strains used in these studies were obtained from the American Type Culture Collection (ATCC), Rockville, Md., and included Leuconostoc mesenteroides ATCC 8293, Leuconostoc dextranicum ATCC 19255, Leuconostoc lactis ATCC 19256, Leuconostoc paramesenteroides ATCC 33313, Lactobacillus casei subsp. rhamnosus ATCC 7469, Lactobacillus fermentum ATCC 9338, Lactobacillus leichmannii ATCC 4797, Lactobacillus plantarum ATCC 8014, Pediococcus acidilactici ATCC 25742, and Pediococcus pentosaceus ATCC A clinical isolate of * Corresponding author Aerococcus sp. from The Massachusetts General Hospital Bacteriology Laboratory culture collection was also used in some studies. Isolation of vancomycin-resistant strains from stool specimens. Portions of approximately 100 stool specimens submitted to the laboratory for culturing of enteric pathogens were streaked onto plates containing Thayer-Martin medium (GIBCO). After overnight incubation at 35 C in the presence of air containing 5% CO2, colonies were picked, restreaked on brucella agar containing 5% horse blood, and checked for purity. Vancomycin susceptibility tests. All isolates were tested for vancomycin susceptibility with 30-,ug disks (Organon Teknika, Irving, Tex.) by the disk diffusion method (12) with certain modifications. Inocula were grown in lactobacilli MRS broth (Difco Laboratories, Detroit, Mich.) and streaked onto blood-supplemented Mueller-Hinton agar plates (GIBCO). Plates were incubated at 35 C in increased (5%) C02. All isolates were tested for their ability to grow in MRS broth containing 256,ug of vancomycin (Sigma Chemical Co., St. Louis, Mo.) per ml according to National Committee for Clinical Laboratory Standards methodology (13). Bacteriological studies. The ability of the isolates to produce gas from glucose was tested by growth at 35 C in MRS broth overlaid with petrolatum. Cellular morphology was determined from Gram stains of cells grown on horse blood agar plates, in thioglycolate broth (GIBCO), and in MRS broth sealed with petrolatum. Cells for the assay of catalase production, tested by hydrogen peroxide decomposition, were grown on horse blood agar medium. Catalase-positive isolates were subcultured on brucella agar slants (GIBCO) and retested for catalase production. All isolates with positive catalase reactions on blood agar or on both blood agar and brucella agar were tested by the method of Wong (21) for the production of porphobilinogen and porphyrin from 8- aminolevulinic acid. Stock strains of Micrococcus luteus and Enterococcus faecalis were used, respectively, as positive and negative controls. All strains were tested for polysaccharide production by observation of growth on heart infusion agar (Difco) containing 5% sucrose. Cultures on solid

2 VOL. 26, 1988 TABLE 1. Characteristics and sources of 36 vancomycinresistant gram-positive isolates No. of No. of isolates from: Geu To.tof Cellular isolates Genus.no. of morphology producing Clinical Stool isolates gas from speci- speciglucose mens mens Lactobacillus 25 Rods Leuconostoc 6 Cocci Pediococcus 3 Cocci in tetrads Indeterminatea 2 Indeterminate a Cells were neither definitely rod shaped nor coccoid. media were incubated at 35 C in the presence of 5% C02. Cultures in liquid media were incubated at 35 C in an ambient atmosphere. Heterofermentative strains and those identified as pediococci were tested with the API Rapid Strep system according to the manufacturer's recommendations (Analytab Products, Plainview, N. Y.). Pediococci were also tested for their ability to produce acid from maltose in Hugh-Leifson oxidation-fermentation and cystine tryptic agar media (GIBCO). Growth in air containing 5% C02 versus that in an anaerobic (GasPak, BBL Microbiology Systems, Cockeysville, Md.) environment was observed by incubating horse blood agar media inoculated with the Pediococcus and Aerococcus strains in both atmospheres. Interaction of isolates with vancomycin. Selected strains were tested for their ability to alter the antimicrobial action of vancomycin in a bioassay described by Orberg and Sandine (14). Staphylococcus aureus ATCC served as the vancomycin-susceptible indicator organism. RESULTS Characteristics of isolates. All isolates and stock strains were determined to be resistant to vancomycin by disk diffusion testing, with the exceptions of Lactobacillus leichmannii and the Aerococcus isolate, which showed inhibition zones of 29 and 21 mm, respectively. All strains found vancomycin resistant by disk testing were also able to grow in MRS broth containing 256 zug of vancomycin per ml. All of the vancomycin-resistant strains studied were grampositive bacteria which displayed definite rodlike, coccoid, or indeterminate (questionable short rods or coccobacilli) cell morphology. Of the 36 isolates, 8 were able to decompose hydrogen peroxide when grown on blood agar; 5 of these also produced positive reactions with hydrogen peroxide when grown on a medium (brucella agar) devoid of blood. All eight peroxide-decomposing strains produced negative results for porphobilinogen and porphyrin synthesis when tested by the method of Wong (21). Some basic characteristics of the 36 isolates are summarized in Table 1. Twenty-three of the isolates failed to produce gas from glucose when grown in MRS broth overlaid with petrolatum. Twenty of these displayed rodlike morphology in Gramstained smears and were tentatively identified as lactobacilli. Some strains had coccoid cells, usually when grown on blood agar, but rod-shaped cells were observed in Gram stains of broth cultures. The remaining three strains that failed to produce gas from glucose formed coccoid cells arranged in tetrads and pairs. Two of these strains produced positive catalase reactions but were negative in tests for porphyrin and porphobilinogen. The isolates grew well on blood agar plates incubated Genus VANCOMYCIN-RESISTANT BACTERIA 2065 TABLE 2. Characteristics of heterofermentative vancomycin-resistant isolates No. of isolates positive for: Total no. of isolates Arginine Leucine Polysaccharide hydrolysis arylamidase production Lactobacillus Leuconostoc Indeterminate a Cells were neither definitely rod shaped nor coccoid. anaerobically, distinguishing them from the Aerococcus (a genus of porphyrin-negative tetrad-forming cocci) strain tested, which grew very poorly when incubated anaerobically. The Aerococcus strain also showed an inhibition zone of 21 mm with a 30-p.g vancomycin disk. The three vancomycin-resistant strains showed colony and cellular morphology similar to that displayed by the P. acidilactici and P. pentosaceus ATCC strains. These homofermentative species, isolated from vegetable material and dairy products, grow equally well under aerobic and anaerobic conditions and may be catalase positive, although cytochromes are absent. P. acidilactici and P. pentosaceus display similar physiological characteristics, with the exception of acid production from maltose, which is characteristic of P. pentosaceus but not of P. acidilactici (8). The two ATCC strains and two of the three Pediococcus isolates had identical API Rapid Strep profiles ( ), with positive reactions in the following tests: Voges-Proskauer, esculin hydrolysis,,- galactosidase, leucine arylamidase, arginine hydrolysis, and acid production from ribose, arabinose, and raffinose. The biochemical profile of the remaining isolate was identical, except for a positive reaction with lactose. The P. pentosaceus ATCC strain and two of the three Pediococcus isolates produced acid from maltose in oxidation-fermentation and cystine tryptic agar media, while the P. acidilactici ATCC strain and the remaining Pediococcus isolate were inactive on maltose. Thus, our three vancomycin-resistant isolates that formed coccoid cells arranged in tetrads appeared to be either P. acidilactici or P. pentosaceus. Of the 13 isolates capable of gas production from glucose, 11 were tentatively identified as lactobacilli or leuconostocs on the basis of cellular morphology. The remaining two isolates showed indeterminate morphology, with cells resembling either very short rods or coccobacilli, and thus, on the basis of morphology, could not be placed in either genus. Reactions of the heterofermentative isolates on API Rapid Strep strips were variable, except for the reactions noted in Table 2. Four of the five rod-shaped bacterial strains, tentatively identified as lactobacilli, hydrolyzed arginine and produced leucine arylamidase. The remaining strain with rod-shaped cells was exceedingly unreactive and produced a positive reaction on the API strip only in the Voges-Proskauer test. The six isolates with definite coccoid morphology were all negative in the arginine hydrolysis and leucine arylamidase tests and were tentatively identified as Leuconostoc strains. Some of these strains formed polysaccharides when grown on a sucrose-containing medium, a characteristic of Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc mesenteroides subsp. dextranicum (7). The remaining two heterofermentative isolates showed neither clearly rod-shaped nor coccoid morphology in stains of cells from any growth medium and thus, on the basis of morphology, are referred to here as "indeterminate" with

3 2066 RUOFF ET AL. J. CLIN. MICROBIOL. TABLE 3. Clinical features of patients with vancomycin-resistant gram-positive organisms Patient Age Sexa Genus Site Coisolates Clinical presentation Outcome (yr) 1 45 F Lactobacillus Hepatic abscess Klebsiella pneumoniae, Citro- Multiple pyogenic liver ab- Deathb bacterfreundii, and Entero- scesses and polymicrobial coccus sp. sepsis 2 63 M Lactobacillus Intra-abdominal abscess Escherichia coli, Klebsiella Peridiverticular abscess Recovery pneumoniae, Staphylococcus epidermidis, Bacteroides vulgatus, and mixed anaerobes 3 41 M Lactobacillus Blood culture Intra-abdominal abscess and Recovery idiopathic necrotizing pancreatitis 4 94 M Indeterminatec Blood culture Enterobacter cloacae and 18% Body surface area third- Deathb Staphylococcus epidermidis degree burns and sepsis 5 56 F Leuconostoc Gastrostomy site Clostridium difficile colitis Recovery 6 75 M Leuconostoc Gastrostomy site Staphylococcus epidermidis Respiratory failure after re- Recovery and Enterococcus sp. placement of stenotic prosthetic mitral valve 7 69 F Lactobacillus Gastrostomy site Klebsiella pneumoniae, Ente- Chronic obstructive pulmo- Deathb rococcus sp., Clostridium nary disease and respiratory perfringens, and Citro- failure bacter freundii 8 83 M Leuconostoc Tracheostomy site Proteus mirabilis Supraglottitis (epiglottitis) and Recovery laryngeal cancer 9 37 F Indeterminatec Jackson-Pratt drain Lactobacillus Sp.d, Candida Renal/pancreatic allograft and Deathb sp., and Bacteroides dista- chronic draining urinary fissonis tula M Lactobacillus Enterocutaneous fistula Staphylococcus aureus Chronic enterocutaneous fis- Recovery tula following total pancreatectomy a F, Female; M, male. b Not related to infection with a vancomycin-resistant organism. C Cells were neither definitely rod shaped nor coccoid. d Morphologically distinct from the vancomycin-resistant indeterminate isolate. respect to genus affiliation. These isolates resembled the Leuconostoc strains in their arginine hydrolysis and leucine arylamidase reactions and ability to produce polysaccharide (Table 2). The possibility that resistant strains were able to destroy or inactivate vancomycin was explored with ATCC strains of Lactobacillus plantarum, P. acidilactici, and P. pentosaceus, the three stool isolates of pediococci, and five clinical isolates (two strains of lactobacilli, two strains of leuconostocs, and one strain of questionable identity). Inhibition zones of 17 mm were noted against S. aureus with disks seeded with uninoculated broth containing 1,000,ug of vancomycin per ml and with spent vancomycin-supplemented broth from 10 of the 11 isolates tested. Spent broth from the remaining isolate tested produced inhibition zones of 16 mm against the test organism. S. aureus was not inhibited by spent broth from vancomycin-free cultures of the 11 strains. Clinical correlation. The charts of 10 patients from whom vancomycin-resistant gram-positive organisms were isolated were available for review (Table 3). In three patients the vancomycin-resistant organism was identified as a Leuconostoc sp.; in five patients the vancomycin-resistant organism was identified as a Lactobacillus sp. In two patients the isolates could not be assigned unambiguously to either genus. In four patients, isolates were recovered from normally sterile body sites (patients 1 to 4, Table 3). In two cases (patients 1 and 2), the isolates were considered probably clinically significant. In two other cases (patients 3 and 4), the isolates were considered possibly significant, but specific findings precluded the assignment of a pathogenic role to the vancomycin-resistant organisms. Six patients (patients 5 to 10, Table 3) had vancomycinresistant gram-positive organisms recovered from cultures of wounds or drains. None of these isolates was associated with fever, leukocytosis, or cellulitis at the site of the wound. Therefore, these isolates were considered clinically insignificant and appear to represent colonization of those sites.

4 VOL. 26, 1988 DISCUSSION The accurate identification of vancomycin-resistant members of the lactic acid bacteria is often difficult. Thornsberry and Facklam (Antimicrob. Newsl. 1:63-64, 1984) noted that since vancomycin resistance is usually not found in streptococci, resistant isolates showing cellular and colony morphology suggestive of that of the viridans group streptococci are probably lactobacilli. Subsequent reports have shown that leuconostocs and pediococci may also be mistaken for vancomycin-resistant streptococci. Thornsberry and Facklam stressed the importance of observing Gram-stained growth from broth cultures for accurate determination of cell morphology. We agree with these authors that broth-grown cells provide the most reliable information on cellular morphology. Gram stains made with growth from agar plates may be misleading, since lactobacilli often form coccoid cells when grown on solid media. In addition to homofermentative lactobacilli, we also recovered three vancomycin-resistant strains of non-gasproducing bacteria which appeared to be members of the genus Pediococcus. Two additional Pediococcus strains obtained from the ATCC were also vancomycin resistant. Pediococci, usually isolated from vegetable material, may be confused with aerococci or even micrococci, owing to their cellular morphology (gram-positive cocci in tetrads) and the positive catalase reactions of some strains. Pediococci, unlike micrococci, do not synthesize cytochromes and may be differentiated from both aerococci and micrococci by their ability to grow luxuriantly under anaerobic conditions (8). Additionally, 24 Aerococcus strains examined by Swenson and co-workers were found to be vancomycin susceptible (J. M. Swenson, G. S. Bosley, R. R. Facklam, and C. Thornsberry, Program Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 786, 1987). Whereas pediococci have been recovered from feces (1), as our three isolates were, to our knowledge there is only one report describing the isolation of pediococci from clinical specimens (5). However, pediococci, like leuconostocs before the first report of their clinical significance (3), may simply be unrecognized when encountered in clinical laboratories. Previous reports have stressed gas production from glucose as an important identifying feature of leuconostocs. While this characteristic will separate leuconostocs from streptococci and homofermentative lactobacilli, it is of no use for differentiation between leuconostocs and heterofermentative lactobacilli. These two groups of organisms may have very similar morphology, as discussed by Garvie (7) and as observed with our two isolates of indeterminate genus affiliation. Nucleic acid homology studies are needed to clarify the taxonomic divisions among these closely related bacteria. The data in Table 2 suggest that arginine hydrolysis and leucine arylamidase reactions may be useful for distinguishing leuconostocs and heterofermentative lactobacilli, but lactobacilli may be arginine hydrolysis negative (7, 11). We also noted that the ATCC strain of Leuconostoc paramesenteroides produced a consistently positive leucine arylamidase reaction in our experiments. Colman and Ball (4), using the API 20 Strep kit, reported that three of four Leuconostoc strains they examined were leucine arylamidase positive. VANCOMYCIN-RESISTANT BACTERIA 2067 Bridge and Sneath (2) reported that six leuconostoc strains hydrolyzed gelatin, a characteristic not found among the lactobacilli (11). This trait could be an easily determined characteristic for Leuconostoc-Lactobacillus differentiation. However, we observed negative reactions for gelatin hydrolysis with all ATCC and other Leuconostoc strains tested on nutrient gelatin medium and in thioglycolate broths containing either 5 or 2% gelatin after incubation at 35 C for 14 days. Isenberg and co-workers (10) have presented data on a limited number of strains which suggest that enzymatic profiles may aid in the differentiation of Leuconostoc and Lactobacillus species. The lack of reliable differential tests for leuconostocs and heterofermentative lactobacilli at the present time forces workers in clinical laboratories to rely on morphology for identification, but as mentioned previously, morphological determinations may be inconclusive. We did not recover any vancomycin-resistant staphylococci or enterococci. Reports of resistance in these organisms are rare (17, 19), and it has been suggested that resistant isolates arise in response to long-term vancomycin treatment of their hosts. In contrast, vancomycin resistance in lactobacilli, leuconostocs, and pediococci appears to be a natural phenomenon. All of the vancomycin-resistant isolates studied were able to grow in high concentrations of the drug, as has been reported previously for members of the genera Leuconostoc (3, 6, 9, 14, 15) and Lactobacillus (20). The Leuconostoc isolates examined by others (10, 14) and the Leuconostoc, Lactobacillus, and Pediococcus strains tested here failed to inactivate vancomycin when tested in a bioassay, suggesting that these organisms form cell walls whose synthesis is unaffected by the antibiotic. Eight patients with clinically significant isolates of Leuconostoc spp. have been reported (3, 6, 8a, 9, 15). All but one of these patients had an underlying medical condition, such as prior surgery (15), intravenous catheter (9, 15), or steroid therapy (15), that predisposed them to infection. The only previous report of Leuconostoc spp. causing infection in a normal host was that of a 16-year-old girl with purulent meningitis (6). In four of the patients reported here the isolation of vancomycin-resistant gram-positive organisms was thought to have probable (patients 1 and 2) or possible (patients 3 and 4) clinical significance. The common occurrence of coinfecting organisms in these patients may reflect the severity of their underlying disease (as in patient 4) or the site of isolation (as in patients 1 and 2). Patients 1, 3, and 4 had multisystem diseases which required prolonged hospitalization and numerous courses of antibiotics. By contrast, patient 2 was a previously healthy man whose only predisposing medical problem was diverticulosis. Leuconostocs and vancomycin-resistant lactobacilli may therefore represent a newly recognized group of nosocomial pathogens. Although five of our patients had been treated with vancomycin prior to the isolation of the organisms (patients 4, 5, 6, 7, and 10), four patients had not (patients 1, 2, 8, and 9). The antibiotic exposure of patient 3 could not be established. The absence of a suitably matched control group of patients from whom vancomycin-resistant organisms were not recovered prevents us from correlating vancomycin usage with the emergence of these organisms. Of our 10 isolates, 6 came from sites contiguous with the gastrointestinal tract. A previous report (15) described the isolation of Leuconostoc spp. from the gastrostomy site of a patient who also had leuconostoc infection of a Broviak catheter. Leuconostoc spp. are normally found in association with vegetables or milk and dairy products (7). Thus, the common finding of these organisms in the alimentary tract is not surprising. Although it may seem that we are now seeing increasing numbers of vancomycin-resistant gram-positive clinical iso-

5 2068 RUOFF ET AL. lates because of increased vancomycin usage, larger numbers of immunocompromised patients, or simply increased awareness, these organisms have probably been isolated but incorrectly identified in the past. A previous report from our laboratory described 24 unidentified viridans group streptococci among a group of 500 clinical viridans group isolates collected in 1982 (16). Nine of the unidentified strains were arginine hydrolysis negative and were recently reexamined. Two were found to be vancomycin resistant (we did not routinely test gram-positive isolates for vancomycin susceptibility when these strains were originally collected). One resistant isolate was identified as a Leuconostoc strain, and the other appeared to be a homofermentative lactobacillus. Our observations along with those of other workers suggest that routine vancomycin susceptibility testing of gram-positive isolates is necessary to reveal the resistant strains which are undoubtedly encountered in clinical specimens. Clinical microbiologists are in need of simple methods for further characterizing these vancomycin-resistant bacteria. ACKNOWLEDGMENTS We thank Lisa Genewicz and Mimma Mogavero for technical assistance and Denise Kettell for exceptional secretarial support. LITERATURE CITED 1. Atterbery, H. R., V. L. Sutter, and S. M. Finegold Normal human intestinal flora, p In A. Balows, R. M. DeHaan, V. R. Dowell, Jr., and L. B. Guze (ed.), Anaerobic bacteria: role in disease. Charles C Thomas, Publisher, Springfield, Bridge, P. D., and P. H. A. Sneath Numerical taxonomy of Streptococcus. J. Gen. Microbiol. 129: Buu-Hoi, A., C. Branger, and J. F. Acar Vancomycinresistant streptococci or Leuconostoc sp. Antimicrob. Agents Chemother. 28: Colman, G., and L. C. Bail Identification of streptococci in a medical laboratory. J. Apple. Bacteriol. 57: Colman, G., and A. Efstratiou Vancomycin-resistant leuconostocs, lactobacilli and now pediococci. J. Hosp. Infect. 10: Coovadia, Y. M., Z. Solwa, and J. van den Ende Meningitis caused by vancomycin-resistant Leuconostoc sp. J. Clin. Microbiol. 25: Garvie, E. I Genus Leuconostoc van Tieghem 1878, 198AL emend mut. char. Hucker and Pederson 1930, 66AL, p In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. J. CLIN. MICROBIOL. 8. Garvie, E. I Genus Pediococcus Claussen 1903, 68AL, p In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 8a.Hardy, S., K. L. Ruoff, E. A. Catlin and J. I. Santos Catheter-associated infection with a vancomycin-resistant gram-positive coccus of the Leuconostoc sp. Pediatr. Infect. Dis. J. 7: Horowitz, H. W., S. Handwerger, K. G. van Horn, and G. P. Wormser Leuconostoc, an emerging vancomycin-resistant pathogen. Lancet ii: Isenberg, H. D., E. M. Vellozzi, J. Shapiro, and L. G. Rubin Clinical laboratory challenges in the recognition of Leuconostoc spp. J. Clin. Microbiol. 26: Kandler, O., and N. Weiss Genus Lactobacillus Beijerinck 1901, 212AL p In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams and Wilkins Co., Baltimore. 12. National Committee for Clinical Laboratory Standards Performance standards for antimicrobial disk susceptibility tests. Approved standard M2-A3. National Committee for Clinical Laboratory Standards, Villanova, Pa. 13. National Committee for Clinical Laboratory Standards Approved standard M7-A. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 14. Orberg, P. K., and W. E. Sandine Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp. Apple. Environ. Microbiol. 48: Rubin, L. G., E. Vellozzi, J. Shapiro, and H. D. Isenberg Infection with vancomycin-resistant "streptococci" due to Leuconostoc species. J. Infect. Dis. 157: Ruoff, K. L., J. A. Fishman, S. B. Calderwood, and L. J. Kunz Distribution and incidence of viridans streptococcal species in routine clinical specimens. Am. J. Clin. Pathol. 80: Schwalbe, R. S., J. T. Stapleton, and P. H. Gilligan Emergence of vancomycin resistance in coagulase-negative staphylococci. N. Engl. J. Med. 316: Shlaes, D. M., J. Marino, and M. R. jacobs Infection caused by vancomycin-resistant Streptococcus sanguis II. Antimicrob. Agents Chemother. 25: Uttley, A. H. C., C. H. Collins, J. Naidoo, and R. C. George Vancomycin-resistant enterococci. Lancet i: Watanakunakorn, C The antibacterial action of vancomycin. Rev. Infect. Dis. 3:S210-S Wong, J. D Porphyrin test as an alternative to benzidine test for detecting cytochromes in catalase-negative gram-positive cocci. J. Clin. Microbiol. 25: