Prevalence and diversity of extended-spectrum-β-lactamase-producing Enterobacteriaceae from marine beach waters

doi:10.1016/j.marpolbul.2014.11.021

Marine Pollution Bulletin

Volume 90, Issues 1–2, 15 January 2015, Pages 60-67

https://doi.org/10.1016/j.marpolbul.2014.11.021 Get rights and content

Highlights

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We analyzed 1351 Enterobacteriaceae isolates from public beach waters in Croatia.
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Approximately 35% of the strains were multidrug resistant.
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ESBLs were detected in 4.2% of the isolated Enterobacteriaceae.
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E. coli was predominant ESBL producer, followed by K. pneumoniae and E. cloacae.
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The bla_TEM-1+SHV-12 was the predominant ESBL genotype, followed by bla_CTX-M-15.

Abstract

A total of 1351 Enterobacteriaceae isolates from 144 seawater samples were collected over a four-year period from three public beaches in the eastern Adriatic Sea in Croatia. Approximately 35% of the strains were multidrug-resistant. Bla_ESBL genes were detected in 4.2% of the isolated Enterobacteriaceae, the main species of which were Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae. Bla_TEM-1+SHV-12 was the most dominant genotype, followed by bla_CTX-M-15. Raoultella terrigena and E. intermedius simultaneously harboured bla_TEM-1, bla_SHV-11/12 and bla_CTX-M-15. Isolate fingerprinting revealed that marine E. coli isolates were clonally related to CTX-M-producing strains from a regional university hospital. These results indicate that marine beach waters are reservoirs of ESBL-producing Enterobacteriaceae and thus constitute a public health problem with further potential to act as mediators in gene flow between marine coastal areas and clinical settings.

Introduction

In the past decade, the evolving threat of antimicrobial resistance has imposed a global problem from the medical, ecological and economical points of view (WHO, 2012). The detection and prompt identification of antibiotic-resistance traits in bacteria persisting in a specific environment have been defined as key steps in the evaluation of the potential of a certain environment to represent a reservoir of antibiotic resistance. In this regard, the aquatic ecosystems highly impacted by human activities have been recognized as hot-spots that significantly contribute to the dissemination of resistant strains and associated genes and even to the proliferation of novel resistance mechanisms and pathogens (Girlich et al., 2011, Tacăo et al., 2012).

In Gram-negative bacteria, resistance to β-lactam antibiotics is mostly related to the production of β-lactamases (Bush and Jacoby, 2010), of which the number of extended-spectrum β-lactamases (ESBLs) has been markedly increased during the last two decades (Cantón et al., 2012). ESBLs confer resistance to all penicillins, narrow and extended cephalosporins, and monobactams. Since first reported in 1983 (Knothe et al., 1983), the prevalence of infections and the list of ESBL-producing bacteria have been constantly increasing. ESBLs have been reported in a variety of Gram-negative species, most often in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii (Poirel et al., 2012). TEM, SHV and CTX-M are the most frequently reported ESBL families, followed by GES, PER, and VEB types. Currently, CTX-Ms. are the most prevalent ESBLs in Enterobacteriaceae worldwide, and Escherichia coli is the predominant species among ESBL-producing enterobacteria (Cantón et al., 2012, Poirel et al., 2012). Furthermore, the spread of ESBL-encoding genes among bacterial pathogens is of particular concern because these are mostly located on plasmids that confer resistance to other classes of antimicrobial agents, resulting in very few treatment options.

The analysis of aquatic environments has shown that ESBL-producing Enterobacteriaceae are abundantly found in wastewater (Ojer-Usoz et al., 2014, Reinthaler et al., 2010), polluted freshwater (Lu et al., 2010, Tacăo et al., 2012) and estuaries (Henriques et al., 2006, Machado et al., 2009). To the best of our knowledge, ESBL-producing Enterobacteriaceae from coastal marine waters were investigated only once, in a study of the heavily polluted Jiaozhou Bay in China (Wang et al., 2008), but bla genes in these isolates have not been identified by sequencing.

Taking into account the panoply of infections in which enterobacteria are implicated, the present study focused for the first time on an urban coastal marine environment comprising three public beaches in the area of Kaštela Bay in Croatia. Kaštela Bay, which is located on the eastern coast of the Adriatic Sea, is a semi-enclosed bay with a surface area of 61 km² and an average depth of 23 m. The narrow coastal strip is a highly developed area with a population of approximately 350,000 inhabitants. Due to an inadequate sewerage system, urban and industrial wastewaters are partially released into the bay. Despite pollution, the coastal beach waters are extensively used for bathing and other recreational activities by the local population and tourists. This study provides the first investigation of the molecular diversity of genes encoding ESBLs in Enterobacteriaceae from coastal marine waters designated for recreational use in Croatia. We investigated the occurrence and antibiotic resistance patterns of Enterobacteriaceae from seawater and determined the frequency and molecular diversity of ESBL-related traits. In addition, enterobacterial repetitive intergenic consensus (ERIC) PCR analysis and pulsed-field gel electrophoresis (PFGE) were used to determine the population structure and genetic relatedness between the ESBL-producing isolates recovered in this study and the strains isolated from a regional university hospital.

Section snippets

Study area and sample collection

Sampling was performed in the period from September 2009 to September 2013 at three public beaches located in Kaštela Bay: Kaštel Sućurac (S1; 43°32′43′′N, 16°25′38′′E), Kaštel Gomilica (S2; 43°32′50′′N, 16°23′50′′E) and Kaštel Stari (S3; 43°33′03′′N, 16°27′07′′E) (Fig. 1). One sample from each site was collected every month, resulting in a total of 144 samples analysed in this study. Seawater was collected 1 m from the coast and approximately 20 cm below the surface using sterile bottles. All of

Water quality

Based on the analysis of the microbiological parameters (see Fig. S1 in the Supplemental Material) collected over the four-year sampling period and according to the quality classification of marine water used for bathing in Croatia, the surface waters collected from all three studied locations were designated as polluted. The water samples from all of the sites at all of the samplings contained high to exceptionally high levels of faecal coliforms (E. coli) and faecal enterococci, exceeding the

Discussion

The implications of polluted natural environments in the promotion of antibiotic resistance have been emphasized in many studies (Tacăo et al., 2012, Wright, 2010, Zhang et al., 2009). As common nosocomial and community-acquired opportunistic pathogens as well as natural commensals of human and animals, Enterobacteriaceae have been pointed to play a significant role in the ongoing mobilization of resistance genes from environmental microbes to other species and eventually to human pathogens,

Conclusions

This study constitutes the first report of the occurrence of ESBL-producing Enterobacteriaceae in public beach waters from the eastern Adriatic Sea, providing insights into the prevalence, bacterial species affiliation and molecular epidemiology of ESBLs. Herein, we identified enterobacterial species carrying clinically important ESBL determinants that have not been previously isolated from natural environments. Our data highlight the public health problem arising from the presence of

Acknowledgments

The authors thank I. Gracin (Split University Hospital, Croatia) for providing the clinical strains. This work was supported by the Ministry of Science, Education and Sports, Croatia, in the form of grants to M. Skočibušić and J. Puizina (177-0000000-3182 and177-1191196-0829).

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Prevalence and diversity of extended-spectrum-β-lactamase-producing Enterobacteriaceae from marine beach waters

Highlights

Abstract

Introduction

Section snippets

Study area and sample collection

Water quality

Discussion

Conclusions

Acknowledgments

Diagn. Microbiol. Infect. Dis.

Int. J. Food Microbiol.

High dissemination of extended-spectrum β-lactamase-producing Enterobacteriaceae in effluents from wastewater treatment plants

Clin. Microbiol. Infect.16

Infect. Genet. Evol.

Water Res.

Curr. Opin. Microbiol.

Updated functional classification of β-lactamases

Antimicrob. Agents Chemother.

CTX-M enzymes: origin and diffusion

Front. Microbiol.

Performance Standard for Antimicrobial Susceptibility Testing—Approved Standard M100–S22

CTX-M-15-producing Escherichia coli clone B2-O25b-ST131 and Klebsiella spp. isolates in municipal wastewater treatment plant effluents

J. Antimicrob. Chemother.

Diversity of clavulanic acid-inhibited extended- spectrum β-lactamases in Aeromonas spp. from the Seine River, Paris, France

Antimicrob. Agents Chemother.

Incidence and antimicrobial susceptibility of Escherichia coli and Klebsiella pneumoniae and extended-spectrum ß-lactamases in community- and hospital-associated intraabdominal infections in Europe: results of the 2008 Study for Monitoring Antimicrobial Resistance Trends (SMART)

Antimicrob. Agents Chemother.

Analysing diversity among β-lactamase encoding genes in aquatic environments

FEMS Microbiol. Ecol.

Characterization of extended-spectrum β- lactamases in Enterobacteriaceae causing nosocomial infections in a Zagreb University hospital

J. Chemother.