Elsevier

Marine Pollution Bulletin

Volume 101, Issue 2, 30 December 2015, Pages 526-532
Marine Pollution Bulletin

Detection and characterization of integrative and conjugative elements (ICEs)-positive Vibrio cholerae isolates from aquacultured shrimp and the environment in Shanghai, China

https://doi.org/10.1016/j.marpolbul.2015.10.062Get rights and content

Highlights

  • We detected the prevalence and resistance of V. cholerae in Shanghai, China.

  • We reported the occurrence of ICEs-positive V. cholerae environmental isolates.

  • The presence of ICEs did not affect resistance to antibiotics or heavy metals.

  • ICEs are not responsible for resistance to antibiotics or heavy metals in V. cholera.

Abstract

Increasing industrialization and use of antimicrobial agents in aquaculture production, have led to heavy metals and multidrug resistant (MDR) pathogens becoming serious problems. These resistances are conferred in two ways: intrinsic and transfer via conjugation, or transformation by the major transmission mediators. Integrative and conjugative elements (ICEs) are one of the major mediators; however, few studies on ICEs of environmental origin have been reported in Asia. Herein, we determined the prevalence, antimicrobial susceptibility, heavy metal resistance and genotypes of 126 strains of Vibrio cholerae isolated from aquatic products and the environment in Shanghai, China. 92.3% of isolates were ICEs-positive from aquaculture water and 89.3% of isolates from shrimp showed MDR. Tracing the V. cholerae genotypes, showed no significant relevance of genotype among the antimicrobial resistance strains bearing the ICEs or not. Thus, in aquaculture, ICEs are not the major transmission mediators of resistance to antibiotics or heavy metals.

Introduction

Vibrio cholerae is regarded as the etiological agent of the most severe, watery, life-threatening diarrheal disease, causing cholera epidemics, and is particularly prevalent in developing countries with poor sanitary conditions (Rajpara et al., 2015, Zhang and Gou, 2014). An estimated 3–5 million cases and over 100,000 deaths occur each year around the world (http://www.cdc.gov/cholera/general/). Cholera toxin (CT) encoded by ctxA and ctxB, is the major virulence determinant of V. cholera. In addition, the toxR gene of V. cholerae encodes a transmembrane, DNA-binding protein that positively controls the transcription of the cholera toxin genes, and those encoding the TCP pili and other proteins important in cholera pathogenesis (Boyd et al., 2000, Parsot and Mekalanos, 1990). Unlike clinical strains, most of the environmental strains do not produce CT, but might acquire CT genes under conditions similar to those of the aquatic environment (Faruque and Nair, 2002, Goel et al., 2007); therefore, non-pathogenic V. cholerae strains are likely to convert into epidemic strains if they acquire virulence genes, which is a threat to public health. However, multidrug-resistant V. cholerae strains that have been reported to cause outbreaks of cholera have become a significant threat to public health and indicate a high incidence and dissemination of antimicrobial resistance (AMR) in bacteria derived from aquatic environments (Chitanand et al., 2010, Teophilo, 2004). Aquaculture ponds with rising pollution of heavy metals and antibiotics have been proved as hotbeds of superbugs. Moreover, a previous study indicated that critical concentrations of heavy metals accumulated can trigger co-selection of antibiotic resistance (Seiler and Berendonk, 2012), which suggests that occurrence of heavy metal and antibiotic resistance has potential relevance. In fact, drug-resistance mechanisms revealed not only the resistance determinants, but also the genetic elements that contribute to the emergence of multidrug resistance (MDR) (Li and Nikaido, 2009, Sjölund-Karlsson et al., 2011). Mutation and acquisition of resistance genes on mobile genetic elements are one way of developing AMR in Vibrio spp. (Frost et al., 2005, Kitaoka et al., 2011).

Integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements (MGEs) that allow bacteria to acquire complex new traits through horizontal gene transfer (HGT). ICEs are found in Gram-positive and Gram-negative bacteria, and are key driving forces in bacterial evolution (Burrus et al., 2002, Wozniak and Waldor, 2010). ICEs are recognized for their role in bacterial genome plasticity, and encode a wide variety of genetic information, including resistance to antibiotics and heavy metals (Davies et al., 2009), and the capacity to degrade aromatic compounds (Ravatn et al., 1998), which may be beneficial under certain environmental conditions. (Bordeleau et al., 2010, Ceccarelli et al., 2013, Wozniak et al., 2009). To date, MGEs with ICE-like properties have been described in several species of Gammaproteobacteria, mainly Vibrios. On the Indian subcontinent in late 1992, a major cholera epidemic was caused by a novel non-O1 serogroup of V. cholerae named V. cholerae O139, was found to harbor an SXT element, which confers resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin, and has ICE-like properties (Group et al., 1868, Waldor et al., 1996).

To date, few studies on ICEs of environmental origin have been reported in Asia. MGEs play a crucial role in the genomic plasticity and fitness of V. cholerae; therefore, in this study, we investigated the prevalence, antimicrobial susceptibility, heavy metal resistance and genotypes of V. cholerae isolated from one of the major shrimp production regions in China, to better understand the dynamic of their dissemination and their correlation with resistance to drugs. Our data produced a well-characterized collection of strains that could be used as controls for several molecular-epidemiological approaches to antimicrobial resistance and virulence.

Section snippets

Bacterial isolation

Shrimp samples were confirmed whether to be free of V. cholerae using culture methods by Zhang et al. (Zhang et al., 2015), in accordance with the Bacteriological Analytical Manual standard method (Kaysner and DePaola, 2004) with some modifications. After collection, live shrimps were immediately cooled in iceboxes and transported to our laboratory and homogenized within 2 h. Meanwhile, water samples were collected using 10 L sterile plastic bottles, and immediately transferred on ice to the

Prevalence of virulence associated-genes and ICEs conserved genes

126 V. cholerae strains were isolated and identified from the aquatic environment: 84 were isolated from water and 42 were isolated from shrimps. As shown in Table 2, the toxR gene was detected in 99.2% of samples, representing a substantial proportion of environmental samples. Meanwhile, the rtxA gene was detected in 42.06% of samples. All the environmental isolates were negative for ctxAB, tcpA, sto, ace and zot. In addition, 91 of the isolates were positive for conserved ICEs genes,

Discussion

The microbial status of seafood is closely related to environmental conditions and the microbiological quality of the water (Feldhusen, 2000). Previous studies disclosed that V. cholerae is a very diverse species and is an opportunistic pathogen in aquatic environments that is highly successful in adapting to changing environmental conditions (Song et al., 2013, Thompson et al., 2003). Since 2002, China has become the world's largest producer of aquatic products (People's Republic of China,

Acknowledgments

This work was supported by grants from the Shanghai Key Laboratory of Meteorology and Health (No. QXJK201207), the Shanghai Municipal Education Commission (No. B-9500-10-0004), and the National Natural Science Foundation of China (No. 31271830).

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    These authors contributed equally to this work.

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