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Journal of Clinical Microbiology, October 2004, p. 4879-4881, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4879-4881.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multilocus Variable-Number Tandem-Repeat Polymorphism among Brazilian Enterococcus faecalis Strains

Ricardo Titze-de-Almeida,^1* Rob J. L. Willems,^2,3 Janetta Top,^2,3 Isabela Pereira Rodrigues,⁴ Renato Fonseca Ferreira II,¹ Hélène Boelens,⁵ Maria Christina C. Brandileone,⁶ Rosemeire C. Zanella,⁶ Maria Sueli Soares Felipe,⁷ and Alex van Belkum⁵

Molecular Microbiology and Biotechnology,¹ Infection Control Sector, Universitarian Hospital of Brasília,⁴ Molecular Biology Laboratory, Institute of Biological Sciences, University of Brasília, Brasília,⁷ Bacteriology Division, Adolfo Lutz Institute, São Paulo, Brazil,⁶ Research Laboratory of Infectious Diseases, National Institute of Public Health and the Environment, Bilthoven,² Erasmus MC, Department of Medical Microbiology & Infectious Diseases, Rotterdam,⁵ Division of Acute Internal Medicine and Infectious Diseases, Department of Internal Medicine, University Medical Center Utrecht, Utrecht, The Netherlands³

Received 29 March 2004/ Returned for modification 30 June 2004/ Accepted 6 July 2004

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Multilocus variable-number tandem-repeat analysis (MLVA) for seven genomic loci was developed for Enterococcus faecalis. MLVA and pulsed-field gel electrophoresis (PFGE) resulted in 37 and 31 genotypes among 83 strains, respectively. Both typing schemes were highly concordant (90.4%). MLVA is an excellent alternative to PFGE.

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Enterococci are opportunistic pathogens associated with cardiovascular, urinary tract, and wound infections (5). Especially when these bacteria have acquired resistance to multiple antibiotics, they are considered prominent agents of nosocomial infection (2). Enterococci resistant to glycopeptides (vancomycin-resistant enterococci [VRE]) are a striking example of a nosocomial pathogen for which therapeutic options are limited. In order to investigate the bacterial epidemiology of both VRE and vancomycin-susceptible enterococci (VSE), several typing methods have been used. Pulsed-field gel electrophoresis (PFGE) is currently considered the "gold standard" method for epidemiological investigation of hospital outbreaks (4, 7). However, the use of PFGE in peripheral and small hospital laboratories, especially those in developing countries, is limited because of high complexity and costs. Less-complex and cheaper alternatives are usually based on DNA amplification. Some of these methods aim at assessing allelic polymorphism among variable-number tandem repeats (VNTRs) (11-13). We have set out to define genome heterogeneity for Brazilian VRE and VSE by PFGE and to develop an alternative multilocus VNTR analysis (MLVA) system.

A heterogeneous set of 83 Enterococcus faecalis isolates, 12 VRE and 71 VSE, was studied. The VRE were obtained from rectal swabs (n = 6), a wound lesion (n = 1), tracheal secretions (n = 2), catheters (n = 2), and a urine specimen (n = 1) from patients involved in the first Brazilian VRE outbreak, which occurred in the Santa Marcelina Hospital in São Paulo (1998). The VSE were isolated from rectal swabs from patients hospitalized in intensive care units of the Universitarian Hospital of Brasília (n = 49), the Santa Luzia Hospital (n = 9) and the Regional Hospital of Taguatinga (n = 13) during a 3-year period (2000 to 2002). Standard biochemical tests and (vanA) PCRs were performed for identification (3). PFGE was carried out as described before, and isolates were classified according to published criteria (9, 14).

The MLVA was based on analysis of the number of repeat units present at seven regions of the E. faecalis genome. The ace (B region) and esp (A and C regions) sequences were described earlier (6, 8). The four new repeats described here have been named efa2, efa3, efa5, and efa6. They were identified in the E. faecalis genome (The Institute for Genomic Research; http://www.tigr.org/) by a specific algorithm (http://cbsu.tc.cornell.edu/index.htm). The number of repeat units was calculated on the basis of the sizes of the amplified sequences. MLVA types were determined by the number of repeat units in the seven different targets. MLVA genogroups represent isolates that shared at least six of seven alleles. PCR was performed after automated DNA extraction (Magna Pure kit; Roche Pharmaceuticals, Basel, Switzerland). For each PCR, 100 ng of DNA was suspended in 50 µl of a mixture containing a locus-specific pair of oligonucleotide primers (25.0 pmol per primer; Table 1), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 0.2 mM deoxynucleotide triphosphates, and 1.2 U of Taq DNA polymerase (SuperTaq; SphaeroQ, Leiden, The Netherlands). The PCR was carried out by initial denaturation at 94°C for 4 min, 15 cycles of "touchdown" amplification-denaturation steps at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 2 min. The annealing temperature for the first cycle was 70°C and decreased 1°C at each cycle during the next 15 cycles. PCR products were sized on 1% agarose gels (Hispanagar; SphaeroQ).

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TABLE 1. Oligodeoxynucleotide primers

Differences in repeat numbers resulted in various alleles for the seven loci. The number of different alleles for each VNTR locus varied strongly (Table 2). It is notable that three of the seven loci in 50% or more of the strains studied comprise 0 or 1 repeat copies. This is a striking finding, probably related to the fact that we focused on repeats with large unit lengths. The combination of alleles for the seven loci resulted in an MLVA profile, and each profile represents a specific MLVA type. Thirty-seven different MLVA types could be discriminated (Table 3), and these were classified into MLVA groups, in which isolates share at least six of the seven alleles (Table 4). MLVA identified four major genogroups among VSE isolates (I to IV) and one major genogroup (V) among VRE (Table 4).

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TABLE 2. Diversity in MLVA allele scores as established by the number of repeats present in given loci

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TABLE 3. MLVA types and corresponding repeat profiles

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TABLE 4. MLVA and PFGE clusters as identified for VRE and VSE

The isolates were also typed by PFGE in order to assess the discriminatory power of the MLVA. PFGE discriminated 31 different genotypes, identifying four main genogroups composed of VSE, named D, J, M, and P, and one primarily composed of VRE, named X (Table 4). The rate of concordance between MLVA and PFGE results for the five main clusters of isolates was 90.4%, varying between 71.4 and 100%. For instance, MLVA clustered in genogroup V the seven VRE classified as PFGE genotype X and another isolate considered possibly related to them (9). Isolates of PFGE types J and M were discriminated in MLVA types 6, 9, 11, 28, and 34. These results indicated that MLVA can be superior to PFGE, discriminating isolates with similar or identical PFGE banding patterns (data not shown). Overall, 26 isolates were identified as unique genotypes by MLVA and 22 by PFGE. Of these, 18 were typed as unique by both methods. This leads us to the conclusion that there is adequate congruence between the data generated with the two technologies. The Simpson index of diversity for the current MLVA scheme is 0.933 (95% confidence interval, 0.905 to 0.961).

An MLVA typing scheme for E. faecalis was based upon seven targets obtained from known genes and new ones selected from the genome sequence available at The Institute for Genomic Research database. The targets showed sufficient variability for the identification of 37 different MLVA types in a relatively small group of independent E. faecalis isolates from Brazil. This is comparable to the 31 types identified by PFGE. MLVA-based clustering of isolates included in the present study (85.7% allele similarity) was close to the values described earlier for PFGE (86%) and analysis of randomly amplified polymorphic DNA (75%) (14). MLVA detected five main clusters of genetically related isolates from four Brazilian hospitals recovered during different periods. Persistence of enterococcal isolates sharing the same PFGE genotype and their intra- and interhospital spreading have also been described in previous investigations (1, 10). In conclusion, MLVA accomplishes most of the desired typing features, including sufficient discriminatory power, speed, ease of use, and simple result interpretation.

 
We acknowledge the cooperation of the staffs of the Universitarian Hospital of Brasília, Santa Marcelina Hospital, Santa Luzia Hospital, Molecular Biology Laboratory—University of Brasília, Public Health Bacteriology Laboratory of Federal District, Integrated Microbiology Laboratory—University of Brasília, and Adolfo Lutz Institute.

R. Titze-de-Almeida was supported by a grant from the CAPES Foundation, Brazil.

 
* Corresponding author. Mailing address: Microbiologia Molecular & Biotecnología. Faculdade de Agronomia e, Medicina Veterinária, Campus Universitário Darcy Ribeiro, C. Postal 04508, CEP 70910-970, Brasilia-DF, Brazil. Phone: (55) 61 3071162. Fax: (55) 61 2736593. E-mail: titze{at}unb.br.

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Journal of Clinical Microbiology, October 2004, p. 4879-4881, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4879-4881.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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