JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1997, p. 2778–2781 0095-1137/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 35, No. 11
Phenotypic and Genotypic Characterization of Vagococcus fluvialis, Including Strains Isolated from Human Sources ´ CIA M. TEIXEIRA,1,2* MARIA DA GLO ´ RIA S. CARVALHO,1,2 VA ˆ NIA LU ´ CIA C. MERQUIOR,1,3 LU ARNOLD G. STEIGERWALT,2 DON J. BRENNER,2 AND RICHARD R. FACKLAM2 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941,1 and Faculdade de Cieˆncias Me´dicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro 20551,3 Brazil, and Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303332 Received 11 April 1997/Returned for modification 2 June 1997/Accepted 28 July 1997
This study presents phenotypic and genotypic data for seven isolates of Vagococcus fluvialis, including four strains recovered from human clinical sources, one strain isolated from an environmental source, and two strains isolated from pigs. On the basis of phenotypic characteristics, most isolates were initially classified as “unidentified enterococci,” because they resembled atypical arginine-negative enterococcal species. All seven strains as well as the type strain of V. fluvialis reacted with the AccuProbe Enterococcus genetic probe. The seven isolates had virtually indistinguishable whole-cell protein profiles that were similar to that of the V. fluvialis type strain and distinct from those of Enterococcus and Lactococcus species. DNA-DNA reassociation experiments confirmed that the strains were V. fluvialis. They were 71% or more related to the V. fluvialis type strain under optimum and stringent conditions, with 2.5% or less divergence within related sequences. All strains were susceptible to ampicillin, cefotaxime, trimethoprim-sulfamethoxazole, and vancomycin and were resistant to clindamycin, lomefloxacin, and ofloxacin. Strain-to-strain variation was observed in relation to susceptibilities to 18 other antimicrobial agents. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI. Distinctive PFGE patterns were generated, suggesting the nonclonal nature of V. fluvialis strains. Although the number of strains was small, this report provides molecular characterization of V. fluvialis and the first evidence of a possible connection of this species with human infections. The genus Vagococcus was proposed in 1989 (2) to accommodate the motile cocci resembling lactococci, which were once referred to as motile “lactic” streptococci and were shown to be distinct from all known lactococci (9, 10). It was demonstrated in 16S rRNA sequencing studies that such strains formed a distinct line of descent within the lactic acid bacteria and represented a new species, which was named Vagococcus fluvialis (2). A subsequent molecular taxonomic investigation resulted in the description of a second species, named Vagococcus salmoninarum (15). Although distinct, the genus Vagococcus had a close phylogenetic relationship with the genera Enterococcus and Lactococcus, and some species are difficult to differentiate solely on the basis of phenotypic characteristics. On the other hand, except for a few reports of the isolation of V. salmoninarum from diseased salmonid fish (11) and of V. fluvialis from domestic animals (8), the significance, if any, of the vagococci as human pathogens remained unknown. The putative involvement of the vagococci as a cause of infections in human beings has possibly been hindered by difficulties in their precise identification, because they may have been misidentified or overlooked in clinical laboratories. This report describes the phenotypic and genotypic characteristics of isolates of V. fluvialis, including strains recovered from human clinical sources. (This study was presented in part at the 96th Annual Meeting of the American Society for Microbiology, New Orleans, La., 19 to 23 May 1996.)
MATERIALS AND METHODS Bacterial strains. Nine Vagococcus strains were studied, including seven isolates of V. fluvialis. Among those, four strains were recovered from human sources (two, strains 2117-82 and 2296-95, from blood cultures; one, strain 2143-93, from peritoneal fluid; and one, strain 1981-94, from a wound), one strain (strain 546-90) was isolated from an environmental source (well water), and two strains (strains 2063-92 and 2064-92) were isolated from pigs (unknown clinical sources). The type strains of V. fluvialis (ATCC 49515 and NCFB 2497) and V. salmoninarum (NCFB 2777) were included for comparative purposes. Representative strains of species of enterococci that were physiologically most similar to the clinical isolates were also included. Characterization of strains. The strains were tested for their phenotypic characteristics by conventional physiological tests as described previously (3, 4). Serogrouping was performed by the Lancefield hot-acid extraction procedure and by capillary precipitation tests with group D antiserum prepared at the Centers for Disease Control and Prevention. Strains were also tested for reactivity by the AccuProbe Enterococcus culture confirmation test (Gen-Probe, Inc., San Diego, Calif.) as directed by the manufacturer. Analysis of whole-cell protein profiles by SDS-PAGE. Strains were grown on brain heart infusion sheep blood agar plates for 24 h at 37°C. Preparation of extracts and analysis of whole-cell protein profiles by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described by Merquior et al. (6). Protein profiles were compared by using the correlation coefficients to form a similarity matrix and were clustered by the unweighted pair group method with averages by using the Whole Band Analyzer software of the Bio Image Electrophoresis Analyzer System (Bio Image/Millipore Corp., Ann Arbor, Mich.). Antimicrobial susceptibility testing. MICs were determined by the broth microdilution assay, by using the Pasco MIC gram-positive panel (Difco Laboratories, Detroit, Mich.) following the manufacturer’s instructions. Cefaclor and meropenem were tested by the Sensititre panel (Radiometer America Inc., West Lake, United Kingdom). DNA reassociation studies. Each strain was grown in 2 liters of Todd-Hewitt broth at 37°C for 18 to 20 h with gentle shaking. Harvesting and lysis of the bacterial cells were performed according to the recommendations of Teixeira et al. (12). The procedures used to extract and purify the DNA and for determination of DNA relatedness by the hydroxyapatite hybridization method were essentially those recommended by Brenner et al. (1). The DNAs were labeled enzymatically with [32P]dCTP by using a nick translation reagent kit (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.). DNA hybridization experiments were performed at 55°C for optimal DNA reassociation. DNA reassociation was also tested at the stringent temperature of 70°C.
* Corresponding author. Mailing address: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS Bloco I Cidade Universitaria, 21941 Rio de Janeiro RJ, Brazil. Phone: 55 21 260 4193. Fax: 55 21 560 8344. E-mail:
[email protected]. 2778
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TABLE 1. Phenotypic characteristics of the Vagococcus strains included in this studya V. fluvialis Characteristic
V. salmoninarum
ATCC 49515T
2117-82
546-90
2063-92
2064-92
2143-93
1981-94
2296-95
Growth under the following conditions: in 6.5% NaCl at 10°C at 45°C
1 1 2
1 1 2
1 1 1
1 1 1
2 1 2
2 1 2
1b 1b 2
2 1 2
1 2 2
Pyruvate utilization
1
1
2
2
1b
1
1
2
2
Tellurite tolerance
2
2
1
2
1
1
1
2
2
Motility
2
1
1
1
1
1
1
1
1
Voges-Proskauer test
2
2
2
2
1
1
1
2
2
Acid production from the following: Glycerol Mannitol Raffinose Sorbitol Sucrose
2 2 1 2 1
1 1 2 1 1
1 1 2 1 1b
1 1 2 1 1
1b 1b 2 1 2
1 1 2 1 1
1 1 2 1 1b
1b 1b 2 1 1b
2 1 2 1 2
Serogroup
Db
Db
2
2
Db
Db
Db
2
2
a
All strains were positive for pyrrolidonyl arylamidase activity, leucine aminopeptidase activity, and hydrolysis of esculin in the presence of bile; were susceptible to vancomycin and nonpigmented; produced acid from maltose, ribose, and trehalose; reacted with the AccuProbe Enterococcus genetic probe; did not produce gas in Lactobacillus Mann-Rogosa-Sharp broth; did not hydrolyze arginine; and did not produce acid from arabinose, inulin, lactose, melibiose, or sorbose. b Delayed or weak reaction.
Analysis of chromosomal DNA restriction patterns by PFGE. Genomic DNA was prepared in agarose plugs by a procedure based on that recommended by Murray et al. (7), with some modifications. Briefly, the bacteria were grown in 5 ml of Todd-Hewitt broth at 37°C for 5 h. Bacterial cells from 1-ml aliquots were harvested and suspended in 0.5 ml of PIV buffer (1.0 M NaCl, 10 mM Tris-HCl [pH 7.6]). This suspension was mixed with an equal volume of 2.0% low-meltingtemperature agarose (NuSieve GTG Agarose; FMC BioProducts, Rockland, Maine) in PIV buffer at 58°C, and then the mixture was distributed into plug molds (Bio-Rad Laboratories, Richmond, Calif.). For lysis, the plugs were placed in 4 ml of fresh lysis solution (6 mM Tris hydrochloride [pH 7.6], 1.0 NaCl, 100 mM EDTA [pH 7.5], 0.5% Brij 58, 0.2% sodium deoxycholate, 0.5% sodium lauroyl sarcosine, 1 mg of lysozyme per ml, 5 U of mutanolysin per ml). After overnight incubation at 37°C with gentle shaking, this solution was replaced with 4 ml of ESP (0.5 M EDTA [pH 8.0], 1% sodium lauroyl sarcosine, 0.1 mg of proteinase K per ml), followed by overnight incubation at 50°C with gentle shaking. The plugs were stored in ES (0.5 M EDTA [pH 8.0], 1% sodium lauroyl sarcosine) at 4°C until use. Prior to digestion with restriction enzyme, the plugs were washed four times (twice for 1 h each time and twice for 2 h each time) with TE (10 mM Tris-HCl [pH 7.6], 0.1 mM EDTA) at 37°C, followed by overnight incubation at 25°C with specific restriction enzyme buffer. The DNA in the plugs was restricted with SmaI according to the manufacturer’s instructions (Boehringer Mannheim Corporation, Indianapolis, Ind.). The fragments were resolved by pulsed-field gel electrophoresis (PFGE) in 1.2% agarose gels (Pulsed Field Certified Agarose; Bio-Rad) in 0.53 Tris-borate-EDTA buffer by using a CHEF-DR III system (Bio-Rad). The following parameters were used: running time, 19 h; temperature, 14°C; voltage gradient, 6 V/cm; initial pulse time, 5 s; final pulse time, 40 s; included angle, 120°C. The gels were stained with ethidium bromide and photographed under UV light.
RESULTS AND DISCUSSION The physiologic characteristics of the strains studied are presented in Table 1. All V. fluvialis strains were positive for pyrrolidonyl arylamidase activity, leucine aminopeptidase activity, and hydrolysis of esculin in the presence of bile. Growth at 10°C, at 45°C, and in broth containing 6.5% NaCl was variable. The strains were susceptible to vancomycin, were motile and nonpigmented, and were negative for production of gas and for arginine and hippurate hydrolysis. Variable results were obtained for pyruvate utilization, for tellurite tolerance, and by Voges-Proskauer tests. All strains produced acid from
maltose, D-mannitol, ribose, D-sorbitol, and trehalose and failed to form acid from L-arabinose, inulin, melibiose, raffinose, and sorbose. Weak or trace reactions with anti-group D sera were observed for four strains. Although vagococci are phylogenetically distinct, the difficulty in distinguishing these microorganisms from other lactic acid bacteria by phenotypic criteria is widely recognized (4, 8–10, 17). On the basis of phenotypic characteristics, most isolates were initially classified as “unidentified enterococci,” because they resembled atypical arginine-negative enterococcal species. Results of motility and arginine hydrolysis tests allowed for the differentiation of the isolates from the lactococci, which usually give negative and positive reactions, respectively (4, 13). All Vagococcus strains tested reacted positively with the AccuProbe Enterococcus genetic probe. Therefore, the enterococcal probe can be used as a tool to separate the vagococci from the lactococci, which test negative with the enterococcal probe (4, 13). The differentiation of vagococcal strains from the enterococci remains problematic. Our data indicate that production of acid from L-arabinose and raffinose may be useful, since all V. fluvialis strains tested were negative and the motile Enterococcus species, E. gallinarum and E. casseliflavus, are positive (5, 14). The arginine test may also be a clue for such differentiation because most strains belonging to these enterococcal species are positive (5, 14). To clarify the identification of the strains, analysis of wholecell protein profiles was carried out by SDS-PAGE. The profiles were compared with those obtained for the type strains of both Vagococcus species and for representative strains of enterococcal and lactococcal species. The isolates had virtually indistinguishable protein profiles that were similar to that of the V. fluvialis type strain (Fig. 1) and distinct from those of V. salmoninarum and enterococcal and lactococcal species. The results were confirmed by DNA-DNA reassociation experiments (Table 2). All of the isolates were shown to have species-
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FIG. 1. (A) SDS-PAGE patterns of whole-cell protein extracts of Vagococcus strains. Lanes 1 and 9, molecular mass markers; lane 2, V. salmoninarum NCFB 2777T; lane 3, V. fluvialis (ATCC 49515T, NCFB 2497); lane 4, strain 2117-82; lane 5, strain 546-90; lane 6, strain 2063-92; lane 7, strain 2064-92; lane 8, strain 2143-93. The numbers on the left indicate the positions of molecular mass markers (in kilodaltons). (B) Dendrogram resulting from a computer-assisted analysis of the protein profiles in panel A. The scale represents average percentages of similarity.
level relatedness to the type strain of V. fluvialis and a low level of relatedness to the V. salmoninarum and the enterococcal species tested. All of the isolates conformed to the three main species-level DNA-DNA relatedness criteria (16); they had relatedness levels ranging from 78 to 91% under optimal conditions, divergence percentages ranging from 1.0 to 2.5% within related sequences, and relatedness levels ranging from
71 to 87% under stringent conditions. Analysis of electrophoretic whole-cell protein profiles, in conjunction with physiologic tests and tests with the enterococcal genetic probe, was shown to be reliable and relatively simple method for the identification of V. fluvialis, as confirmed by DNA hybridization experiments. Results of MIC determinations (Table 3) indicated that all strains were susceptible to ampicillin, cefotaxime, trimethoprimsulfamethoxazole, and vancomycin, whereas all strains were re-
TABLE 2. Levels of DNA relatedness of Vagococcus strains and some phenotypically related enterococcal species
a
Source of unlabeled DNA
Vagococcus fluvialis ATCC 49515T (SS-1339) 2117-82 546-90 2063-92 2064-92 2143-93 1981-94 2296-95 Vagococcus salmoninarum NCFB 2777T (SS-1340)
Labeled DNA from Vagococcus fluvialis ATCC 49515T RBRb at 55°Cb
100 82 88 83 84 78 91 85
5 5
Enterococcus casseliflavus ATCC 25788T (SS-1229) 1319-94
5 5
Enterococcus faecalis ATCC 19433T (SS-1273)
MIC (mg/ml) %Dc
RBR at 70°C Antimicrobial agent
0.0 2.0 1.5 1.5 1.0 2.5 2.0 2.5
100 77 82 83 87 77 71 72
7
Enterococcus gallinarum ATCC 49573T (SS-1228) 447-94
TABLE 3. Antimicrobial susceptibilities of Vagococcus strains
8
a ATCC, American Type Culture Collection; SS, standard strain from the culture collection of the Centers for Disease Control and Prevention; NCFB, National Collection of Food Bacteria. b RBR, relative binding ratio. c %D, percent divergence. Calculated to the nearest 0.5%.
Ampicillin Ampicillin-sulbactam Cefaclor Cefazolin Cefixime Cefotaxime Ceftriaxone Cefuroxime Chloramphenicol Ciprofloxacin Clarithromycin Clindamycin Erythromycin Gentamicin Meropenem Lomefloxacin Ofloxacin Oxacillin Penicillin Piperacillin-tazobactam Rifampin Tetracycline Tobramycin Trimethoprim-sulfamethoxazole Vancomycin
V. fluvialis V. salmoninarum NCFB 2777T
0.25 #2/1 8 16 .2 16 8 16 #4 0.5 #0.5 1 #5 1 2 4 2 .6 0.25 8/4 #1 #2 1 #0.5/9.5 #1
ATCC 49515T
0.25 #2/1 16 8 .2 #4 #4 #4 #4 .2 1 .2 1 1 0.5 .4 .4 2 0.25 4/4 #1 .8 2 #0.5/9.5 #1
Clinical isolates
0.25–1 #2/1 8–16 8–16 .2 #4–8 #4–16 #4–16 #4–.16 1–.2 #0.5–.4 .2 #0.5–.4 1–6 0.5–8 .4 .4 1–6 0.25–1 4/4–8/4 #1–2 #2–.8 2–.8 #0.5/9.5 #1–2
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Estudos e Projetos (FINEP), Fundac¸˜ao de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Ministe´rio da Cie ˆncia e Tecnologia (MCT/PRONEX) of Brazil. We thank Bala Swaminathan and Susan Hunter of the Centers for Disease Control and Prevention for providing facilities for computerassisted analysis and Carlos Ausberto B. de Souza of the Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, for technical assistance. REFERENCES
FIG. 2. PFGE of chromosomal DNA of Vagococcus strains after digestion with SmaI. Lane 1, molecular size markers (in kilobases; bacteriophage lambda DNA concatemers ranging from 48.5 to 1,018.5 kb); lane 2, V. salmoninarum NCFB 2777T; lane 3, V. fluvialis ATCC 49515T (NCFB 2497); lane 4, strain 2117-82; lane 5, strain 546-90; lane 6, strain 2063-92; lane 7, strain 2064-92; lane 8, strain 2143-93; lane 9, strain 1981-94; lane 10, strain 2296-95.
sistant to clindamycin, lomefloxacin, and ofloxacin. Strain-tostrain variation was observed in relation to the susceptibilities to the other 18 antimicrobial agents tested. The distinctive PFGE patterns generated after digestion of chromosomal DNA with SmaI (Fig. 2) indicated the potential ability of this typing technique to discriminate between Vagococcus isolates and suggested strain-to-strain variability within V. fluvialis. The two strains isolated from pigs (strain 2063-92 [Fig. 2, lane 6] and strain 2064-92 [Fig. 2, lane 7]) had fragment patterns more closely related to each other than to those of the other strains tested. These two strains were also more closely related when protein profiles were compared (Fig. 1B). Although the numbers of isolates tested are still low, the present report provides extensive information on the characterization of V. fluvialis and may be the first evidence of the possible connection of this species as a cause of infections in human beings. Very little clinical information was submitted with the cultures identified in our laboratory. One strain (strain 2143-93) was isolated from the peritoneal fluid of a nephrology patient, and another (strain 1981-94) was isolated from an infected bite wound in a person who was bitten by a lamb. The other two isolates from humans (strains 2117-82 and 2296-95) were recovered from blood cultures, and no additional information on the clinical condition or associated illness was available. After the present work was completed, two additional strains were received for identification. One was isolated from a finger wound of a patient in Canada, and the other was recovered from the cerebrospinal fluid of a patient with meningitis in Argentina. The strains had phenotypic characteristics typical of V. fluvialis. As more attention and accurate procedures are incorporated into the identification schemes to properly detect and characterize vagococcal strains in the clinical setting, more information will become available. ACKNOWLEDGMENTS This study was supported in part by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´gico (CNPq), Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Financiadora de
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