PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

PCR-RFLP do 16S DNA ribossomal para confirmar a identificação de Enterococcus gallinarum e Enterococcus casseliflavus isolados de amostras clínicas e alimentares

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PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

PCR-RFLP do 16S DNA ribossomal para confirmar a identificação de Enterococcus gallinarum e Enterococcus casseliflavus isolados de amostras clínicas e alimentares

Aline Weber Medeiros^I; Pedro d'Azevedo^II; Rebeca Inhoque Pereira^III; Ana Paula Cassenego^I; Sueli Van Der Sand^III; Jeverson Frazzon^IV; Ana Paula Guedes Frazzon^III

^IGraduate Program of Agricultural and Environmental Microbiology, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

^IIDepartment of Basic Health Sciences, Microbiology Department, Federal Health Sciences University of Porto Alegre, Porto Alegre, RS, Brazil

^IIIMicrobiology Department, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

^IVScience and Food Technology Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

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ABSTRACT

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP.

METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus.

RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species.

CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

Key-words: PCR-RFLP of 16S rDNA. Enterococcus gallinarum. Enterococcus casseliflavus.

RESUMO

INTRODUÇÃO: O objetivo deste estudo foi confirmar a identificação de amostras clínicas e alimentos de Enterococcus gallinarum e Enterococcus casseliflavus por PCR-RFLP.

MÉTODOS: Cinquenta e duas cepas identificadas por exames bioquímicos convencionais foram submetidos a amplificação por PCR e digestão com HinfI. Apenas 20 (38,5%) das 52 amostras apresentaram um padrão de DNA esperado E. gallinarum e E. casseliflavus.

RESULTADOS: Analise dos resultados deste estudo demonstraram que, algumas vezes E. gallinarum e E. casseliflavus são erroneamente identificados e confirmaram a potencial aplicação da análise do 16S rDNA para identificação exata destas espécies.

CONCLUSÕES: A correta identificação é importante a fim de distinguir entre resistência intrínseca e adquirida à vancomicina.

Palavras-chaves: PCR-RFLP de 16S rDNA. Enterococcus gallinarum. Enterococcus casseliflavus.

INTRODUCTION

Enterococci are opportunistic pathogens and well known as the principal microorganisms associated with the development of infections, especially in immunosuppressed patients. Furthermore, strains have been recognized as emerging human pathogens mostly associated with nosocomial infections¹. The emergence of enterococci in nosocomial infections has grown in parallel with the rise in strains resistant to a large number of antimicrobial drugs used in the treatment of human infections. Enterococcus gallinarum and Enterococcus casseliflavus exhibit low-level intrinsic resistance to vancomycin, conferred by the vanC-1 gene². Commercial kits for species identification of Enterococcus are unable to distinguish E. gallinarum and E. casseliflavus from other enterococci³. Rapid and reliable differentiation of these species in patients infected with vancomycin resistant enterococci (VRE) is essential for an infection control program. The aim of this work was to confirm the identification of E. gallinarum and E. casseliflavus using the PCR-restriction fragment length polymorphism (PCR-RFLP) technique.

METHODS

In the current study, E. gallinarum (n=32) and E. casseliflavus (n=20) isolated from clinical samples and food identified by conventional biochemical were analyzed. Two references strains E. gallinarum (PAD 262) and E. casseliflavus (PAD 71) were obtained from the culture collection at the laboratory of microbiology of the Federal University of Health Sciences (Universidade Federal de Ciências da Saúde) of Porto Alegre and used as controls (Table 1). Extraction of total DNA from cells followed the method described by Riboldi et al⁴ The amplifications were performed with a thermal cycler (Eppendorf Mastercycler Personal). The primers 16Sent-F (5'-CTGACGCTGAGGCTCGAAAGCG-3') and 16Sent-R (5'-TGTGACGGGCGGTGTGTACAAGGGGG-3') corresponded to nucleotide sequences of 16SrDNA of the genus Enterococcus. The PCR product of 661 bp amplified was submitted to digestion with the restriction enzyme HinfI (Jena Bioscience GmbH, Germany), according to the manufacturer's instructions. The DNA fragments obtained were resolved by electrophoresis on 2% agarose gel stained by ethidium bromide.

RESULTS AND DISCUSSION

The PCR-RFLP results from reference strains of E. gallinarum and E. casseliflavus showed two distinguishable DNA fragments of 589bp and 72bp (Figure 1). PCR-RFLP from the 52 strains tested demonstrated that 47% (15/32) of E. gallinarum and 25% (5/20) showed the expected PCR-RFLP patterns (Figure 1). Two PCR-RFLP positive E. gallinarum and one E. casseliflavus were analyzed by the SDS-PAGE method and confirmed the results obtained. On the other hand, 53% (17/32) of E. gallinarum and 75% (15/20) of E. casseliflavus strains showed three DNA fragments of 504, 85 and 72bp (Figure 1). These strains were resubmitted to a new set of biochemical tests and reclassified as: E. faecium, E. faecalis and Enterococcus sp. The 16S rDNA gene has been useful for the identification of Enterococcus genus and species^5,6. All 16S rDNA sequences deposited in GenBank of the NCBI of E. gallinanum and E.casseliflavus have a conserved thymidine (T) at position 1248, while other species of enterococci predominantly present a cytosine (C) or T at the equivalent position. A single conserved base substitution in this position in E. gallinarum and E. casseliflavus eliminates the restriction endonuclease site for HinfI. The present results demonstrate that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis to accurately identify these species. Correct identification is very important to discriminate between natural and VRE strains.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FINANCIAL SUPPORT

The authors grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico, the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior and the Fundação de Amparo a Pesquisa do Rio Grande do Sul for their financial support.

Received in 16/11/2009

Accepted in 13/01/2010

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• 6. Patel R, Piper KE, Rouse MS, Steckelberg JM, Uhl JR, Kohner P, et al. Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates. J Clin Microbiol 1998; 36: 3399-3407

Publication Dates

• Publication in this collection
  17 Mar 2010
• Date of issue
  Feb 2010

History

• Accepted
  13 Jan 2010
• Received
  16 Nov 2009