Vol. 26, No. 10JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1988, p. 2064-20680095-1137/88/102064-05$02.00/0Copyright © 1988, American Society for Microbiology

Vancomycin-Resistant Gram-Positive BacteriaIsolated 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 GeneralHospital, Boston, Massachusetts 02114, and Department of Microbiology and Molecular Genetics2 and

Department of Medicine,4 Harvard Medical School, Boston, Massachusetts 02115

Received 28 March 1988/Accepted 14 July 1988

Recent reports of infections with vancomycin-resistant gram-positive bacteria prompted us to studyvancomycin-resistant isolates from human sources to characterize the types of bacteria displaying thisphenotype. Thirty-six vancomycin-resistant gram-positive isolates, 14 from clinical specimens and 22 fromstool 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 physiologicaltests. Two isolates of indeterminate morphology could not be unambiguously assigned to a genus. Four isolatesof 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. Bettermethods for distinguishing the various genera in the clinical microbiology laboratory are needed.

Despite decades of vancomycin use, few reports of clini-cally significant vancomycin resistance among isolates ofgram-positive bacteria have appeared in the literature. Re-cently, isolates of vancomycin-resistant coagulase-negativestaphylococci (17) and enterococci (19) have been described.Vancomycin resistance has been reported in clinical isolatesof Leuconostoc and Pediococcus, genera of "lactic acidbacteria" that resemble streptococci morphologically (3, 5,6, 9, 15). Although vancomycin resistance has been reportedin a strain of viridans group streptococcus (18), this isolatemost likely represents a case of a lactobacillus mistaken fora streptococcus (C. Thornsberry and R. Facklam, Antimi-crob. Newsl. 1:63-64, 1984). These reports emphasize theresemblance of certain lactic acid bacteria to streptococciand the difficulties in correctly identifying vancomycin-resistant gram-positive organisms.We examined 36 isolates of vancomycin-resistant gram-

positive bacteria recovered from specimens submitted to theclinical bacteriology laboratory to study their morphologicaland physiological characteristics and to assess their clinicalsignificance.

MATERIALS AND METHODSBacterial strains and cultivation media. Isolates were rou-

tinely cultured on plates containing brucella agar supple-mented with 5% horse blood (GIBCO Diagnostics, Madison,Wis.). Cultures were incubated at 35°C in an atmospherecontaining 5% C02. Long-term storage of organisms wasaccomplished by freezing suspensions of the bacteria inhorse blood. Stock strains used in these studies were ob-tained from the American Type Culture Collection (ATCC),Rockville, Md., and included Leuconostoc mesenteroidesATCC 8293, Leuconostoc dextranicum ATCC 19255, Leu-conostoc lactis ATCC 19256, Leuconostoc paramesente-roides ATCC 33313, Lactobacillus casei subsp. rhamnosusATCC 7469, Lactobacillus fermentum ATCC 9338, Lacto-bacillus leichmannii ATCC 4797, Lactobacillus plantarumATCC 8014, Pediococcus acidilactici ATCC 25742, andPediococcus pentosaceus ATCC 33316. A clinical isolate of

* Corresponding author.

Aerococcus sp. from The Massachusetts General HospitalBacteriology Laboratory culture collection was also used insome studies.

Isolation of vancomycin-resistant strains from stool speci-mens. Portions of approximately 100 stool specimens sub-mitted to the laboratory for culturing of enteric pathogenswere streaked onto plates containing Thayer-Martin medium(GIBCO). After overnight incubation at 35°C in the presenceof air containing 5% CO2, colonies were picked, restreakedon brucella agar containing 5% horse blood, and checked forpurity.Vancomycin susceptibility tests. All isolates were tested for

vancomycin susceptibility with 30-,ug disks (OrganonTeknika, Irving, Tex.) by the disk diffusion method (12) withcertain modifications. Inocula were grown in lactobacilliMRS broth (Difco Laboratories, Detroit, Mich.) andstreaked onto blood-supplemented Mueller-Hinton agarplates (GIBCO). Plates were incubated at 35°C in increased(5%) C02. All isolates were tested for their ability to grow inMRS broth containing 256 ,ug of vancomycin (Sigma Chem-ical Co., St. Louis, Mo.) per ml according to NationalCommittee for Clinical Laboratory Standards methodology(13).

Bacteriological studies. The ability of the isolates to pro-duce gas from glucose was tested by growth at 35°C in MRSbroth overlaid with petrolatum. Cellular morphology wasdetermined from Gram stains of cells grown on horse bloodagar plates, in thioglycolate broth (GIBCO), and in MRSbroth sealed with petrolatum. Cells for the assay of catalaseproduction, tested by hydrogen peroxide decomposition,were grown on horse blood agar medium. Catalase-positiveisolates were subcultured on brucella agar slants (GIBCO)and retested for catalase production. All isolates with posi-tive catalase reactions on blood agar or on both blood agarand brucella agar were tested by the method ofWong (21) forthe production of porphobilinogen and porphyrin from 8-aminolevulinic acid. Stock strains of Micrococcus luteus andEnterococcus faecalis were used, respectively, as positiveand negative controls. All strains were tested for polysac-charide production by observation of growth on heart infu-sion agar (Difco) containing 5% sucrose. Cultures on solid

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TABLE 1. Characteristics and sources of 36 vancomycin-resistant gram-positive isolates

No. of No. of isolates from:

Geu To.tof Cellular isolatesGenus .no. of morphology producing Clinical Stoolisolates gas from speci- speci-

glucose mens mens

Lactobacillus 25 Rods 5 7 18Leuconostoc 6 Cocci 6 5 1Pediococcus 3 Cocci in tetrads 0 0 3Indeterminatea 2 Indeterminate 2 2 0

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 anambient atmosphere.

Heterofermentative strains and those identified as pedio-cocci were tested with the API Rapid Strep system accord-ing to the manufacturer's recommendations (Analytab Prod-ucts, Plainview, N. Y.). Pediococci were also tested for theirability to produce acid from maltose in Hugh-Leifson oxida-tion-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 agarmedia inoculated with the Pediococcus and Aerococcusstrains in both atmospheres.

Interaction of isolates with vancomycin. Selected strainswere tested for their ability to alter the antimicrobial actionof vancomycin in a bioassay described by Orberg andSandine (14). Staphylococcus aureus ATCC 29213 served asthe vancomycin-susceptible indicator organism.

RESULTS

Characteristics of isolates. All isolates and stock strainswere determined to be resistant to vancomycin by diskdiffusion testing, with the exceptions of Lactobacillus leich-mannii and the Aerococcus isolate, which showed inhibitionzones of 29 and 21 mm, respectively. All strains foundvancomycin resistant by disk testing were also able to growin MRS broth containing 256 zug of vancomycin per ml.

All of the vancomycin-resistant strains studied were gram-positive bacteria which displayed definite rodlike, coccoid,or indeterminate (questionable short rods or coccobacilli)cell morphology. Of the 36 isolates, 8 were able to decom-pose hydrogen peroxide when grown on blood agar; 5 ofthese also produced positive reactions with hydrogen perox-ide when grown on a medium (brucella agar) devoid ofblood. All eight peroxide-decomposing strains producednegative results for porphobilinogen and porphyrin synthesiswhen tested by the method of Wong (21). Some basiccharacteristics 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 Gram-stained smears and were tentatively identified as lactobacilli.Some strains had coccoid cells, usually when grown onblood agar, but rod-shaped cells were observed in Gramstains 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 butwere negative in tests for porphyrin and porphobilinogen.The isolates grew well on blood agar plates incubated

TABLE 2. Characteristics of heterofermentativevancomycin-resistant isolates

No. of isolates positive for:Genus Total no.

of isolates Arginine Leucine Polysaccharidehydrolysis arylamidase production

Lactobacillus 5 4 4 0Leuconostoc 6 0 0 3Indeterminate 2 0 0 2

a Cells were neither definitely rod shaped nor coccoid.

anaerobically, distinguishing them from the Aerococcus (agenus of porphyrin-negative tetrad-forming cocci) straintested, which grew very poorly when incubated anaerobi-cally. The Aerococcus strain also showed an inhibition zoneof 21 mm with a 30-p.g vancomycin disk. The three vanco-mycin-resistant strains showed colony and cellular morphol-ogy similar to that displayed by the P. acidilactici and P.pentosaceus ATCC strains. These homofermentative spe-cies, isolated from vegetable material and dairy products,grow equally well under aerobic and anaerobic conditionsand may be catalase positive, although cytochromes areabsent. P. acidilactici and P. pentosaceus display similarphysiological characteristics, with the exception of acidproduction from maltose, which is characteristic of P. pen-tosaceus but not of P. acidilactici (8). The two ATCC strainsand two of the three Pediococcus isolates had identical APIRapid Strep profiles (5057010), with positive reactions in thefollowing tests: Voges-Proskauer, esculin hydrolysis, ,-galactosidase, leucine arylamidase, arginine hydrolysis, andacid production from ribose, arabinose, and raffinose. Thebiochemical profile of the remaining isolate was identical,except for a positive reaction with lactose. The P. pentosa-ceus ATCC strain and two of the three Pediococcus isolatesproduced acid from maltose in oxidation-fermentation andcystine tryptic agar media, while the P. acidilactici ATCCstrain and the remaining Pediococcus isolate were inactiveon maltose. Thus, our three vancomycin-resistant isolatesthat formed coccoid cells arranged in tetrads appeared to beeither P. acidilactici or P. pentosaceus.Of the 13 isolates capable of gas production from glucose,

11 were tentatively identified as lactobacilli or leuconostocson the basis of cellular morphology. The remaining twoisolates showed indeterminate morphology, with cells re-sembling either very short rods or coccobacilli, and thus, onthe basis of morphology, could not be placed in either genus.

Reactions of the heterofermentative isolates on API RapidStrep strips were variable, except for the reactions noted inTable 2. Four of the five rod-shaped bacterial strains,tentatively identified as lactobacilli, hydrolyzed arginine andproduced leucine arylamidase. The remaining strain withrod-shaped cells was exceedingly unreactive and produced apositive reaction on the API strip only in the Voges-Pros-kauer test. The six isolates with definite coccoid morphologywere all negative in the arginine hydrolysis and leucinearylamidase tests and were tentatively identified as Leuco-nostoc strains. Some of these strains formed polysaccha-rides when grown on a sucrose-containing medium, a char-acteristic of Leuconostoc mesenteroides subsp. mesen-teroides and Leuconostoc mesenteroides subsp. dextrani-cum (7).The remaining two heterofermentative isolates showed

neither clearly rod-shaped nor coccoid morphology in stainsof cells from any growth medium and thus, on the basis ofmorphology, are referred to here as "indeterminate" with

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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- Deathbbacterfreundii, and Entero- scesses and polymicrobialcoccus sp. sepsis

2 63 M Lactobacillus Intra-abdominal abscess Escherichia coli, Klebsiella Peridiverticular abscess Recoverypneumoniae, Staphylo-coccus epidermidis, Bacte-roides vulgatus, and mixedanaerobes

3 41 M Lactobacillus Blood culture Intra-abdominal abscess and Recoveryidiopathic necrotizing pan-creatitis

4 94 M Indeterminatec Blood culture Enterobacter cloacae and 18% Body surface area third- DeathbStaphylococcus 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- Recoveryand Enterococcus sp. placement of stenotic pros-

thetic mitral valve

7 69 F Lactobacillus Gastrostomy site Klebsiella pneumoniae, Ente- Chronic obstructive pulmo- Deathbrococcus sp., Clostridium nary disease and respiratoryperfringens, and Citro- failurebacter freundii

8 83 M Leuconostoc Tracheostomy site Proteus mirabilis Supraglottitis (epiglottitis) and Recoverylaryngeal cancer

9 37 F Indeterminatec Jackson-Pratt drain Lactobacillus Sp.d, Candida Renal/pancreatic allograft and Deathbsp., and Bacteroides dista- chronic draining urinary fis-sonis tula

10 46 M Lactobacillus Enterocutaneous fistula Staphylococcus aureus Chronic enterocutaneous fis- Recoverytula following total pancre-atectomy

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 theLeuconostoc strains in their arginine hydrolysis and leucinearylamidase reactions and ability to produce polysaccharide(Table 2).The possibility that resistant strains were able to destroy

or inactivate vancomycin was explored with ATCC strainsof Lactobacillus plantarum, P. acidilactici, and P. pentosa-ceus, the three stool isolates of pediococci, and five clinicalisolates (two strains of lactobacilli, two strains of leuco-nostocs, and one strain of questionable identity). Inhibitionzones of 17 mm were noted against S. aureus with disksseeded with uninoculated broth containing 1,000 ,ug ofvancomycin per ml and with spent vancomycin-supple-mented broth from 10 of the 11 isolates tested. Spent brothfrom the remaining isolate tested produced inhibition zonesof 16 mm against the test organism. S. aureus was notinhibited by spent broth from vancomycin-free cultures ofthe 11 strains.

Clinical correlation. The charts of 10 patients from whomvancomycin-resistant gram-positive organisms were isolated

were available for review (Table 3). In three patients thevancomycin-resistant organism was identified as a Leuco-nostoc sp.; in five patients the vancomycin-resistant organ-ism was identified as a Lactobacillus sp. In two patients theisolates could not be assigned unambiguously to eithergenus.

In four patients, isolates were recovered from normallysterile body sites (patients 1 to 4, Table 3). In two cases(patients 1 and 2), the isolates were considered probablyclinically significant. In two other cases (patients 3 and 4),the isolates were considered possibly significant, but specificfindings precluded the assignment of a pathogenic role to thevancomycin-resistant organisms.

Six patients (patients 5 to 10, Table 3) had vancomycin-resistant gram-positive organisms recovered from cultures ofwounds or drains. None of these isolates was associatedwith fever, leukocytosis, or cellulitis at the site of thewound. Therefore, these isolates were considered clinicallyinsignificant and appear to represent colonization of thosesites.

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DISCUSSION

The accurate identification of vancomycin-resistant mem-bers of the lactic acid bacteria is often difficult. Thornsberryand Facklam (Antimicrob. Newsl. 1:63-64, 1984) noted thatsince vancomycin resistance is usually not found in strepto-cocci, resistant isolates showing cellular and colony mor-

phology suggestive of that of the viridans group streptococciare probably lactobacilli. Subsequent reports have shownthat leuconostocs and pediococci may also be mistaken forvancomycin-resistant streptococci. Thornsberry and Fack-lam stressed the importance of observing Gram-stainedgrowth from broth cultures for accurate determination of cellmorphology. We agree with these authors that broth-growncells provide the most reliable information on cellular mor-

phology. Gram stains made with growth from agar platesmay be misleading, since lactobacilli often form coccoidcells when grown on solid media.

In addition to homofermentative lactobacilli, we alsorecovered three vancomycin-resistant strains of non-gas-

producing bacteria which appeared to be members of thegenus Pediococcus. Two additional Pediococcus strainsobtained from the ATCC were also vancomycin resistant.Pediococci, usually isolated from vegetable material, may beconfused with aerococci or even micrococci, owing to theircellular morphology (gram-positive cocci in tetrads) and thepositive catalase reactions of some strains. Pediococci,unlike micrococci, do not synthesize cytochromes and may

be differentiated from both aerococci and micrococci bytheir ability to grow luxuriantly under anaerobic conditions(8). Additionally, 24 Aerococcus strains examined by Swen-son and co-workers were found to be vancomycin suscepti-ble (J. M. Swenson, G. S. Bosley, R. R. Facklam, and C.Thornsberry, Program Abstr. 27th Intersci. Conf. Antimi-crob. Agents Chemother., abstr. no. 786, 1987). Whereaspediococci have been recovered from feces (1), as our threeisolates were, to our knowledge there is only one reportdescribing the isolation of pediococci from clinical speci-mens (5). However, pediococci, like leuconostocs before thefirst report of their clinical significance (3), may simply beunrecognized when encountered in clinical laboratories.

Previous reports have stressed gas production from glu-cose as an important identifying feature of leuconostocs.While this characteristic will separate leuconostocs fromstreptococci and homofermentative lactobacilli, it is of no

use for differentiation between leuconostocs and heterofer-mentative 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 toclarify the taxonomic divisions among these closely relatedbacteria.The data in Table 2 suggest that arginine hydrolysis and

leucine arylamidase reactions may be useful for distinguish-ing leuconostocs and heterofermentative lactobacilli, butlactobacilli may be arginine hydrolysis negative (7, 11). Wealso noted that the ATCC strain of Leuconostoc paramesen-

teroides produced a consistently positive leucine arylami-dase reaction in our experiments. Colman and Ball (4), usingthe API 20 Strep kit, reported that three of four Leuconostocstrains they examined were leucine arylamidase positive.Bridge and Sneath (2) reported that six leuconostoc strainshydrolyzed gelatin, a characteristic not found among thelactobacilli (11). This trait could be an easily determinedcharacteristic for Leuconostoc-Lactobacillus differentiation.However, we observed negative reactions for gelatin hydrol-

ysis with all ATCC and other Leuconostoc strains tested onnutrient gelatin medium and in thioglycolate broths contain-ing either 5 or 2% gelatin after incubation at 35°C for 14 days.Isenberg and co-workers (10) have presented data on alimited number of strains which suggest that enzymaticprofiles may aid in the differentiation of Leuconostoc andLactobacillus species. The lack of reliable differential testsfor leuconostocs and heterofermentative lactobacilli at thepresent time forces workers in clinical laboratories to rely onmorphology for identification, but as mentioned previously,morphological determinations may be inconclusive.We did not recover any vancomycin-resistant staphylo-

cocci or enterococci. Reports of resistance in these organ-isms are rare (17, 19), and it has been suggested that resistantisolates arise in response to long-term vancomycin treatmentof their hosts. In contrast, vancomycin resistance in lacto-bacilli, leuconostocs, and pediococci appears to be a naturalphenomenon.

All of the vancomycin-resistant isolates studied were ableto grow in high concentrations of the drug, as has beenreported previously for members of the genera Leuconostoc(3, 6, 9, 14, 15) and Lactobacillus (20). The Leuconostocisolates examined by others (10, 14) and the Leuconostoc,Lactobacillus, and Pediococcus strains tested here failed toinactivate vancomycin when tested in a bioassay, suggestingthat these organisms form cell walls whose synthesis isunaffected by the antibiotic.

Eight patients with clinically significant isolates of Leuco-nostoc spp. have been reported (3, 6, 8a, 9, 15). All but oneof these patients had an underlying medical condition, suchas prior surgery (15), intravenous catheter (9, 15), or steroidtherapy (15), that predisposed them to infection. The onlyprevious report of Leuconostoc spp. causing infection in anormal host was that of a 16-year-old girl with purulentmeningitis (6).

In four of the patients reported here the isolation ofvancomycin-resistant gram-positive organisms was thoughtto have probable (patients 1 and 2) or possible (patients 3 and4) clinical significance. The common occurrence of coinfect-ing organisms in these patients may reflect the severity oftheir underlying disease (as in patient 4) or the site ofisolation (as in patients 1 and 2). Patients 1, 3, and 4 hadmultisystem diseases which required prolonged hospitaliza-tion and numerous courses of antibiotics. By contrast,patient 2 was a previously healthy man whose only predis-posing medical problem was diverticulosis. Leuconostocsand vancomycin-resistant lactobacilli may therefore repre-sent a newly recognized group of nosocomial pathogens.Although five of our patients had been treated with van-

comycin 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 patientsfrom whom vancomycin-resistant organisms were not recov-ered prevents us from correlating vancomycin usage with theemergence of these organisms.Of our 10 isolates, 6 came from sites contiguous with the

gastrointestinal tract. A previous report (15) described theisolation of Leuconostoc spp. from the gastrostomy site of apatient who also had leuconostoc infection of a Broviakcatheter. Leuconostoc spp. are normally found in associa-tion with vegetables or milk and dairy products (7). Thus, thecommon finding of these organisms in the alimentary tract isnot surprising.

Although it may seem that we are now seeing increasingnumbers of vancomycin-resistant gram-positive clinical iso-

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lates because of increased vancomycin usage, larger num-

bers of immunocompromised patients, or simply increasedawareness, these organisms have probably been isolated butincorrectly identified in the past. A previous report from our

laboratory described 24 unidentified viridans group strepto-cocci among a group of 500 clinical viridans group isolatescollected 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 notroutinely test gram-positive isolates for vancomycin suscep-

tibility when these strains were originally collected). Oneresistant isolate was identified as a Leuconostoc strain, andthe other appeared to be a homofermentative lactobacillus.Our observations along with those of other workers suggestthat routine vancomycin susceptibility testing of gram-posi-tive isolates is necessary to reveal the resistant strains whichare undoubtedly encountered in clinical specimens. Clinicalmicrobiologists are in need of simple methods for furthercharacterizing these vancomycin-resistant bacteria.

ACKNOWLEDGMENTSWe thank Lisa Genewicz and Mimma Mogavero for technical

assistance and Denise Kettell for exceptional secretarial support.

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2. Bridge, P. D., and P. H. A. Sneath. 1983. Numerical taxonomyof Streptococcus. J. Gen. Microbiol. 129:565-597.

3. Buu-Hoi, A., C. Branger, and J. F. Acar. 1985. Vancomycin-resistant streptococci or Leuconostoc sp. Antimicrob. AgentsChemother. 28:458-460.

4. Colman, G., and L. C. Bail. 1984. Identification of streptococciin a medical laboratory. J. Apple. Bacteriol. 57:1-14.

5. Colman, G., and A. Efstratiou. 1987. Vancomycin-resistantleuconostocs, lactobacilli and now pediococci. J. Hosp. Infect.10:1-3.

6. Coovadia, Y. M., Z. Solwa, and J. van den Ende. 1987. Menin-gitis caused by vancomycin-resistant Leuconostoc sp. J. Clin.Microbiol. 25:1784-1785.

7. Garvie, E. I. 1986. Genus Leuconostoc van Tieghem 1878,198AL emend mut. char. Hucker and Pederson 1930, 66AL, p.1071-1075. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, andJ. G. Holt (ed.), Bergey's manual of systematic bacteriology,vol. 2. The Williams & Wilkins Co., Baltimore.

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8a.Hardy, S., K. L. Ruoff, E. A. Catlin and J. I. Santos. 1988.Catheter-associated infection with a vancomycin-resistantgram-positive coccus of the Leuconostoc sp. Pediatr. Infect.Dis. J. 7:519-520.

9. Horowitz, H. W., S. Handwerger, K. G. van Horn, and G. P.Wormser. 1987. Leuconostoc, an emerging vancomycin-resis-tant pathogen. Lancet ii:1329-1330.

10. Isenberg, H. D., E. M. Vellozzi, J. Shapiro, and L. G. Rubin.1988. Clinical laboratory challenges in the recognition of Leu-conostoc spp. J. Clin. Microbiol. 26:479-483.

11. Kandler, O., and N. Weiss. 1986. Genus Lactobacillus Beije-rinck 1901, 212AL p. 1209-1234. In P. H. A. Sneath, N. S. Mair,M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual ofsystematic bacteriology, vol. 2. The Williams and Wilkins Co.,Baltimore.

12. National Committee for Clinical Laboratory Standards. 1984.Performance standards for antimicrobial disk susceptibilitytests. Approved standard M2-A3. National Committee for Clin-ical Laboratory Standards, Villanova, Pa.

13. National Committee for Clinical Laboratory Standards. 1985.Approved standard M7-A. Methods for dilution antimicrobialsusceptibility tests for bacteria that grow aerobically. NationalCommittee for Clinical Laboratory Standards, Villanova, Pa.

14. Orberg, P. K., and W. E. Sandine. 1984. Common occurrence ofplasmid DNA and vancomycin resistance in Leuconostoc spp.Apple. Environ. Microbiol. 48:1129-1133.

15. Rubin, L. G., E. Vellozzi, J. Shapiro, and H. D. Isenberg. 1987.Infection with vancomycin-resistant "streptococci" due to Leu-conostoc species. J. Infect. Dis. 157:216.

16. Ruoff, K. L., J. A. Fishman, S. B. Calderwood, and L. J. Kunz.1983. Distribution and incidence of viridans streptococcal spe-cies in routine clinical specimens. Am. J. Clin. Pathol. 80:854-858.

17. Schwalbe, R. S., J. T. Stapleton, and P. H. Gilligan. 1987.Emergence of vancomycin resistance in coagulase-negativestaphylococci. N. Engl. J. Med. 316:927-931.

18. Shlaes, D. M., J. Marino, and M. R. jacobs. 1984. Infectioncaused by vancomycin-resistant Streptococcus sanguis II. An-timicrob. Agents Chemother. 25:527-528.

19. Uttley, A. H. C., C. H. Collins, J. Naidoo, and R. C. George.1988. Vancomycin-resistant enterococci. Lancet i:57-58.

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21. Wong, J. D. 1987. Porphyrin test as an alternative to benzidinetest for detecting cytochromes in catalase-negative gram-posi-tive cocci. J. Clin. Microbiol. 25:2006-2007.

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