Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Caracterização de Vibrio parahaemolyticus isolados de ostras e mexilhões em São Paulo, Brasil

MICROBIOLOGY

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Caracterização de Vibrio parahaemolyticus isolados de ostras e isolados de ostras e mexilhões em São Paulo, Brasil

Martha Virginia Ribeiro Rojas; Maria Helena Matté; Milena Dropa; Miriam Lopes Da Silva; Glavur Rogério Matté;

Public Health Laboratory, School of Public Health/University of São Paulo. Av. Dr. Arnaldo 715, 01246-904 São Paulo, SP, Brazil

^{Correspondence to}

SUMMARY

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the bla_TEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Keywords:Vibrio parahaemolyticus; tlh, tdh and trh Genes; bla_TEM-116

RESUMO

Vibrio parahaemolyticus é uma bactéria marinha, responsável por gastroenterite em humanos. A maioria dos isolados clínicos produzem hemolisina termoestável direta (TDH) e hemolisina TDH-relacionada (TRH) codificadas por genes tdh e trh, respectivamente. Neste estudo, vinte e três V. parahaemolyticus, previamente isolados de ostras e mexilhões foram analisados por PCR utilizando indicadores específicos para o gene 16S rRNA, genes de virulência (tdh, trh e tlh), resistência a diferentes classes de antibióticos, e PFGE. Dezenove isolados foram confirmados por PCR, como V. parahaemolyticus. O gene tlh estava presente em 100% dos isolados, o gene tdh foi identificado em dois (10,5%) dos isolados, enquanto que o gene trh não foi detectado. Cada isolado foi resistente a pelo menos um dos nove antibióticos testados. Além disso, todos os isolados apresentaram resultado positivo para o gene bla_TEM-116. A presença deste gene em V. parahaemolyticus indica a possibilidade de propagação desse gene no ambiente. Cepas atípicas de V. parahaemolyticus foram também detectadas neste estudo.

INTRODUCTION

Vibrio parahaemolyticus is a human pathogenic Gram-negative halophilic bacterium, a natural inhabitant of the marine environment and can be found in crabs, shrimps, fish, oysters, mussels and other seafoods^13,34,41. V. parahaemolyticus infections are associated to the ingestion of contaminated raw or undercooked shellfish, especially bivalve molluscs^10,13,18.

It was first isolated in 1950, during a large outbreak of gastroenteritis that occurred in Japan¹³. Since then this bacterium has been recognized as one of the main agents causing foodborne diseases, in many countries including Asian countries, the United States, France, Mexico, Peru and Chile^{5,14,15,17,20,22,28,41,45}. Moreover, V. parahaemolyticus infections have increased globally^28,33. Studies in Brazil demonstrated the presence of V. parahaemolyticus in environmental samples from a variety of sources as well as in clinical samples^{1,25,26,27,31,34,35,38}. In 2002, according to data of the National Health Foundation (FUNASA), in Ceará, Brazil, an outbreak of gastroenteritis occurred, and Kanagawa-positive strains of V. parahaemolyticus serovar O3:K6 were isolated³¹.

The pathogenicity of this bacterium in humans is associated to the production of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH)³⁰. Many studies have demonstrated that virulent strains of V. parahaemolyticus possess either the gene tdh or trh, or both²¹. The evidence of transmission of these genes through plasmids or insertion elements has already been demonstrated. Another thermolabile haemolysin (TLH) which is encoded by tlh gene is considered as a species-specific marker. This gene has been detected in all clinical and environmental V. parahaemolyticus strains⁴.

Coastal waters and estuarine environments are subjected to waste contamination that could selectively contribute to antimicrobial resistance in bacteria from these environments⁴⁴. Pathogenic strains of V. parahaemolyticus have been reported to be resistant to different classes of antimicrobials such as aminoglycosides, beta-lactams and quinolones^36,46. Thus in this study, antimicrobial susceptibility of V. parahaemolyticus isolated from the environment was evaluated.

V. parahaemolyticus is an important foodborne pathogen, although in São Paulo outbreaks caused by V. parahaemolyticus have not been reported by the Center for Epidemiologic Surveillance (CVE). In addition, consumption of raw fish in Brazilian cities is popular³⁹. Therefore, in this study, V. parahaemolyticus isolated from shellfish were characterized by PCR targeted to 16S rRNA and virulence genes, antibiotic resistance patterns and DNA profiles by PFGE.

MATERIAL AND METHODS

Strains. Twenty-three V. parahaemolyticus strains isolated from oysters and mussels, collected from aquatic environments, fish markets and restaurants in São Paulo, Brazil, between February 1989 and January 1990, were used in this study. Isolates were kept in Luria Bertani broth containing 60% glycerol at -80 °C, in the Public Health Laboratory Culture Collection, School of Public Health /University of São Paulo. The isolates were identified as V. parahaemolyticus by standard biochemical methods as described previously²⁶. To determine the viability, the strains were cultured in thiosulphate citrate bile salts sucrose (TCBS) agar (Difco, France) at 35 °C for 24 hours and screened in Triple Sugar Iron (TSI) agar (Merck, USA) and citochrome oxidase production. A V. parahaemolyticus positive control was kindly provided by Fundação Instituto Oswaldo Cruz (FIOCRUZ - Rio de Janeiro, Brazil). V. harveyi was used as a negative control. DNA extraction was carried out for all samples using the heat-shock technique⁷.

Kanagawa Test. Wagatsuma agar containing 5% human erythrocytes was used to screen all V. parahaemolyticus strains for β-hemolysis by incubation at 35 °C for 24 hours⁴³.

Antimicrobial Susceptibility Test. Antimicrobial susceptibility test was performed using disc-diffusion, Kirby-Bauer method²⁹. The antibiotics used were streptomycin (10 µg), gentamicin (10 µg), chloramphenicol (30 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), cotrimoxazole (25 µg), tetracycline (30 µg), ampicillin (10 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefpodoxime (10 µg) and imipenem (10 µg) (OXOID^®, England).

Polymerase Chain Reaction (PCR). PCR was performed to identify V. parahaemolyticus using 16S rRNA as a targeted gene. Multiplex PCR was established for detection of virulence genes tdh, trh and tlh. In the present study, the majority of V. parahaemolyticus strains were resistant to ampicilin. In addition, bla_TEM genes have recently been detected in many Brazilian environmental bacteria². Therefore, we also investigated the presence of bla_TEM genes in V. parahaemolyticus strains by PCR. All primers used are indicated in Table 1.

Pulsed Field Gel Electrophoresis (PFGE). PFGE technique was performed as previously described⁴⁰. Briefly, DNA was digested with 40U of SfiI restriction enzyme (GE Healthcare, UK). Restriction fragments were separated by pulsed field gel electrophoresis in a Chef Mapper apparatus (Bio-Rad Laboratories, Hercules, CA) in a two-step procedure as follows: Block 1: 6V/cm, 14 °C, for 13h, pulse ramp of 2s to 10s, 120° angle. Block 2: 6V/cm, 14 °C, for six hours, pulse ramp of 20s to 25s, 120° angle. Gels were stained with ethidium bromide. DNA profiles obtained by PFGE were visualized in an Epi Chemi II Darkroom (UVP Bioimaging Systems). PFGE profiles were analyzed using Gel Works 1D Advanced 4.01 and Gel Works 1D Database 1.12 (UVP Bioimaging Systems, Upland, CA).

RESULTS

Identification ofV. parahaemolyticus. Twenty-three strains presumptively identified as V. parahaemolyticus were submitted to biochemical and molecular tests. Four strains (17.4%) were negative for PCR targeted to 16S rRNA. Nineteen strains (82.6%) including five strains with atypical biochemical tests were confirmed as V. parahaemolyticus by PCR. The presence of tlh gene was observed in all 19 V. parahaemolyticus strains. Biochemical tests revealed atypical characteristics in two (8.7%) strains which were sucrose positive. Three strains (13.0%), were negative for ethanol as sole carbon source. Figure 1 summarizes the results of biochemical tests, presence of virulence genes, Kanagawa phenomenon, antimicrobial susceptibility and PFGE profiles of V. parahaemolyticus strains evaluated in this study.

Virulence. The β-hemolysis in Wagatsuma agar (Kanagawa phenomenon) was observed in three strains of V. parahaemolyticus (15.8%). The presence of virulence gene tdh was observed in two (10.5%), but none of the strains were positive for trh gene.

Antibiotics resistance. From 19 strains tested, six were resistant to at least two antimicrobial agents (Fig. 1). All strains were susceptible to imipenem, nalidixic acid and ceftazidime. It is important to highlight that all strains had intermediate results for streptomycin. For bla_TEM genes evaluation, all V. parahaemolyticus isolates presented the expected 972pb fragment, and sequencing of these amplicons demonstrated 100% similarity to bla_TEM-116 (GenBank accession numbers of JF327790 to JF327797 ).

PFGE. The PFGE analysis demonstrated heterogeneity among the V. parahaemolyticus isolates except for isolates FSP1020 and FSP1016 that were very close related, with only one band difference suggesting that they might have originated from the same clone. However, these strains showed differences in antimicrobial susceptibility. Overall, the graphic representation of the Dice's similarity coefficient matrix showed similarity above 80% (Fig. 1).

DISCUSSION

Four strains with typical biochemical reactions were negative for PCR identification of V. parahaemolyticus. On the other hand, FSP367, FSP402, FSP364, FSP1013 and FSP1014, which were strains with atypical phenotypic characteristics (positive reaction for sucrose and negative for ethanol as sole carbon source) were confirmed as Vibrio parahaemolyticus by PCR. It has been demonstrated that phenotypic tests are often unable to discriminate species of vibrios⁹. In this study, tlh gene was a useful marker for confirmation of V. parahaemolyticus and the results of detection of this gene corroborates the results obtained for 16S rRNA gene¹². Therefore, molecular techniques are a useful tool for identification of environmental V. parahaemolyticus strains if atypical V. parahaemolyticus is detected^13,18,42.

In regard to the presence of virulence genes in V. parahaemolyticus, tdh was observed in two (10.5%) strains which corroborates the results of other authors that this gene was absent in the majority of environmental strains of V. parahaemolyticus^3,30. The trh gene was not detected in any tested strains. In the present study it was observed that one strain showed beta-hemolysis on Wagatsuma agar (Kanagawa positive) but it was negative for tdh gene. This phenomenon has been reported in Brazil^1,4,16,24,32 and might be due to deletion or mutation in the tdh gene³³.

PFGE has been used to investigate diversity of V. parahaemolyticus and has been applied to characterize bacterial strains from environment and foodborne outbreaks^37,45. In the present study, genotypic diversity of V. parahaemolyticus strains was achieved by PFGE using SfiI. This methodology is widely used in epidemiological studies for many genus of bacteria⁸ and this is the first application to access the diversity of V. parahaemolyticus strains isolated from Brazilian samples. These results may be kept as a database of environmental strains to be used in investigations in case of outbreaks.

In the present study it was demonstrated that environmental isolates showed low susceptibility to penicillins and aminoglycosides, thus, they may have a low effectiveness in clinical treatment of V. parahaemolyticus.

It has been demonstrated that clinical isolates of V. parahaemolyticus show intermediate susceptibility to gentamicin, resistance to streptomycin and ampicillin, and susceptibility to trimethoprim-sulphamethoxazole²³. In addition, clinical and environmental V. parahaemolyticus strains also show resistance to chloramphenicol, tetracycline and cefotaxime⁴⁶. Results obtained in this study, demonstrated the same resistance profile to streptomycin and ampicillin that was observed in clinical strains, but most of our V. parahaemolyticus environmental isolates were susceptible to chloramphenicol, tetracycline and cefotaxime. Resistance to cotrimoxazole (combination of trimethoprim and sulfamethoxazole) and chloramphenicol was detected in FSP1019 strain.

The acquisition of resistance genes can occur by mobile elements such as plasmids and integrons, which have already been described in V. parahaemolyticus and other Vibrio species⁴².

In the present study, bla_TEM genes were detected in all tested V. parahaemolyticus and sequencing of this gene revealed 100% similarity with bla_TEM-116. The presence of this gene in V. parahaemolyticus isolates from several sites in São Paulo State indicates that bla_TEM-116 genes have been disseminated in the marine environment. In fact, studies demonstrated the presence of this gene in Aeromonas spp. and Klebsiella pneumoniae isolated from environment and clinical samples in the same regions in São Paulo^2,11.

To contribute to a better characterization of the pathogen, further studies on V. parahaemolyticus from its natural environment, seafood or clinical samples are essential. Thus, the molecular methods used in this study may be useful in the monitoring of this microorganism. However, in order for this application to be viable, it is necessary to improve the clinical diagnosis for V. parahaemolyticus infections. Therefore, it is recommended that routine searching for halophilic vibrios and a systematic notification of clinical cases be undertaken in hospital areas located near coastal regions and in places where consumption of seafood is a regular practice in order to identify the prevalence of vibriosis in the population. Consequently, health professionals should be directed, in cases of gastroenteritis associated with seafood consumption, to request specific culture for the isolation and characterization of vibrios, including V. parahaemolyticus. Such measures could provide relevant information to Health Surveillance services.

ACKNOWLEDGEMENTS

Centro Colaborador em Vigilância Sanitária/FSP-CECOVISA/USP-CA no. 06/99-44-ANVSMS, Process # 2001.1.1048.6.9, for support as part of a technology transfer project.

Received: 3 December 2010

Accepted: 20 June 2011

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Publication Dates

• Publication in this collection
  05 Sept 2011
• Date of issue
  Aug 2011

History

• Received
  03 Dec 2010
• Accepted
  20 June 2011