http://orcid.org/0000-0001-5580-1997
Figures
Abstract
Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n = 54) and environmental samples (n = 38) in Huzhou were analyzed by serological typing, virulence gene detection, antibiotic resistance testing, and pulsed-field gel electrophoresis (PFGE) for molecular typing. O3:K6 was the main serotype and tlh+tdh+trh- was the most frequently detected virulence genotype in clinical strains. O2:Kut was the main serotype and tlh+tdh-trh- was the most frequently detected virulence genotype in environmental strains. Antibiotic resistance testing indicated that the isolates were highly resistant to ampicillin (90.76%), followed by gentamicin and tetracycline. Following the restriction enzyme NotI digestion, the 91 strains yielded 81 PFGE patterns, and 16 clones had similarity values of > 85.00%, indicating a high level of diversity. Finally, there may be cross-contamination between freshwater and seawater products, so it is necessary to strengthen supervision of food processing.
Citation: Yan W, Ji L, Xu D, Chen L, Wu X (2020) Molecular characterization of clinical and environmental Vibrio parahaemolyticus isolates in Huzhou, China. PLoS ONE 15(10): e0240143. https://doi.org/10.1371/journal.pone.0240143
Editor: Yung-Fu Chang, Cornell University, UNITED STATES
Received: July 28, 2020; Accepted: September 20, 2020; Published: October 2, 2020
Copyright: © 2020 Yan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Vibrio parahaemolyticus is a Gram-negative haemolyticus widely found in seawater, seafloor sediments, and aquatic products such as fish, shrimp, and shellfish near the coast. It is one of the main pathogens causing infectious diarrhea in coastal areas [1–3] and can cause acute gastroenteritis and primary sepsis. In 1950, V. parahaemolyticus was discovered in a food poisoning incident in Japan, in which at least 272 people were sickened and 20 died due to ingestion of small sardines that were partially dried [4]. In recent years, rising demand has led more seafood to be transported to inland areas, contributing to an increase in V. parahaemolyticus cases each year [5].
Huzhou is located in inland China, and many residents eat raw or undercooked aquatic products (Fish et al.). Therefore, V. parahaemolyticus has become the most important pathogen causing infectious diarrhea in Huzhou [6]. Despite the high risk of V. parahaemolyticus, information on its prevalence or molecular epidemiology is not readily available.
We investigated V. parahaemolyticus strains collected from clinical samples and environmental samples in Huzhou in terms of their serotypes, virulence genes, genotypic traits, antimicrobial resistance, and molecular characteristics. The findings will provide vital information to government agencies for performing risk assessments of V. parahaemolyticus infection.
Materials and methods
Bacterial isolates
A total of 92 strains of V. parahaemolyticus was isolated from clinical (stool samples) and environmental (water surface samples) collected at food markets during foodborne disease surveillance in 2019 from several regions of Huzhou. The 92 strains of V. parahaemolyticus included 54 isolates from stool samples and 38 from environmental samples. The 92 isolates were stored at −80°C in trypticase soy broth (TSB) containing 20% glycerol. The quality control strain for pulsed-field gel electrophoresis (PFGE)—Brendan Lupussalmonellae (H9812) [7]—was from the Zhejiang Center for Disease Control and Prevention, China.
Human ethics approval was not requested because no human subjects were involved.
Serotyping
Serological analysis of the lipopolysaccharide (O) and capsular (K) antigens of the V. parahaemolyticus isolates was performed by agglutination tests using a commercial V. parahaemolyticus Antiserum Test Kit (Denka Seiken, Tokyo, Japan). Pure cultures of bacterial strains on 3% NaCl TSA plate were subjected to glass-slide agglutination to detect K antigen. Freshly cultured colonies were ground into sterilized 3% NaCl to produce a bacterial suspension, which was inactivated at 121°C for 1 h and centrifuged at 4000 r/min for 10 min. The supernatant was discarded, and the bacterial suspension was precipitated and resuspended in sterilized 3% NaCl. The bacterial suspension was subjected to O-antigen analysis; normal saline was used as the control.
Analysis of virulence genes
DNA was extracted by boiling. A ring of fresh colony was ground in 200 μL of sterilized water. The bacterial suspension was bathed at 100°C for 10 min and centrifuged at 10000 r/min for 5 min. The supernatant contained strain DNA was collected and stored at −80°C until use.
Virulence genes of the V. parahaemolyticus isolates were analyzed by fluorescence PCR using a commercial V. parahaemolyticus Triple Nucleic Acid Testing Kit (Biogen Biological Co., Ltd., Shenzhen, China), in accordance with the manufacturer’s instructions.
Antimicrobial susceptibility testing
The antimicrobial susceptibility of the 92 V. parahaemolyticus isolates was tested using the broth microdilution method. The 10 antibacterial agents were ampicillin (AMP), tetracycline (TET), chloramphenicol (CHL), cotrimoxazole (SXT), ceftazine (CAZ), gentamicin (GEN), imipenem (IMI), ciprofloxacin (CIP), amikacin (AMI), and levofloxacin (LEV). Antimicrobial susceptibility was interpreted as sensitive, intermediate, or resistant, with reference to the Interpretation Criteria for Other non-Enterobacteriaceae in the Clinical and Laboratory Standards Institute (CLSI) Drug Sensitivity Test Guidelines. Escherichia coli ATCC 25922 was used as a control. The results were analyzed using the CLSI breakpoints.
PFGE
PFGE analysis of the V. parahaemolyticus isolates was performed according to the PFGE Standard Operating Method of Vibrio parahaemolyticus [8]. First, chromosomal DNA was digested using the restriction enzyme NotI (TaKaRa, Japan). The digestion fragments were subjected to PFGE in a 1% Seakem Gold Agarose gel (Lonza Company, Swiss) in 0.5% Tris-boric acid-ethylenediaminetetraacetic acid (EDTA) buffer using a CHEF Mapper XA system (Bio-Rad Laboratories, Richmond, CA, USA). The electrophoresis conditions were as follows: fragment size 78 to 396 kb, electrophoresis time 19 h, and pulse time 10 to 35 s. The XbaI-digested DNA from Salmonella enterica serotype Braenderup strain H9812 was used as a standard. The PFGE patterns were analyzed using Chinese Pathogen Identification Net. Clustering was performed using the unweighted pair group method and the Dice correlation coefficient with a position tolerance of 1.5%. Clusters were defined on the basis of a 90% similarity cutoff [9].
Results
Serotyping
Serological analysis of the 54 V. parahaemolyticus isolates from clinical samples revealed six serovars with O3:K6 (35 strains, 64.81%), followed in order of frequency by O4:Kut (10 strains, 18.52%). Serological analysis of the 38 V. parahaemolyticus isolates from environmental samples revealed nine serovars with O2:Kut (eight strains, 21.05%), followed in order of frequency by O5:Kut (seven strains, 18.42%) (Table 1).
Analysis of virulence genes
All of the 92 isolates were positive for tlh. Among 54 isolates from clinical samples, 50 isolates had tdh and only 1 isolate had trh. Six isolates from environmental samples had tdh, and all of the 38 isolates from environmental samples were negative for trh (Table 2).
Antimicrobial resistance
The majority of the V. parahaemolyticus strains was resistant to ampicillin (90.76%), compared to 4.35% and 2.17% for gentamicin and tetracycline, respectively. All of the isolates were sensitive to the other seven antibiotics. Additionally, 100% of the isolates were sensitive to chloramphenicol, ceftazine, ciprofloxacin, and levofloxacin (Table 3).
PFGE and cluster analysis
Genotyping of the 92 V. parahaemolyticus isolates showed that one was non-typable due to DNA degradation during endonuclease digestion. PFGE analysis of the remaining 91(53 from clinical samples and 38 from environmental samples) isolates yielded 81 distinguishable patterns. Among them, 45 isolates were clustered in 16 patterns with a more than 85% similarity threshold, which contained isolates belonging to serotypes O3:K6 and O4:Kut. The similarity threshold for the other 46 isolates was less than 85.00%. The cluster map showed that the similarity of clinical isolates was higher than that of environmental isolates. Also, the maximum similarity between the clinical and environmental strains was 60.00% (Fig 1).
Strain identification number, seromarkers, and sample source.
Discussion
V. parahaemolyticus is an important pathogen causing food poisoning and infectious diarrhea. Food poisoning caused by V. parahaemolyticus has surpassed Salmonella and ranks first among bacterial causes of food poisoning [10–12]. The incidence of food-borne diseases caused by V. parahaemolyticus is related to local seafood farming [13]. Huzhou is known as the land of fish and rice and has a well-developed aquaculture industry. The consumption of aquatic products is increasing, and the food safety issue caused by V. parahaemolyticus is increasingly important.
Thermolabile hemolysin (TLH) is an important virulence factor of V. parahaemolyticus [14], together with heat-resistant direct hemolysin (TDH) and heat-resistant related hemolysin (TRH). Clinical, environmental, and food isolates carry the TLH gene, which is species-specific but not a virulence factor [15]. TDH mediates the Kanagawa phenomenon, a determinant of the virulence of V. parahaemolyticus. TRH and TDH are potentially lethal enterotoxins. In this study, all 92 V. parahaemolyticus isolates had tlh, which was consistent with their biochemical characteristics. Of the clinical isolates, 95.80% were TDH+ and TRH−, suggesting strong pathogenicity, while 84.21% of the environmental isolates were TDH− and TRH−, suggesting relatively weak virulence, as reported by Li Xue [16]. Although some strains do not harbor tdh and trh, they can cause severe diarrhea [17], so it is necessary to strengthen the monitoring of V. parahaemolyticus virulence genes.
V. parahaemolyticus can be classified into 13 thermally stable O-antigen groups (O1–O13) and 71 thermally unstable K-antigen groups (K1–K71). In addition, some strains cannot be serologically classified, among which O3 predominates at present [18]. Here, 92 V. parahaemolyticus isolates from clinical and environmental samples were classified into 11 serotypes. The serotypes of clinical and food isolates were inconsistent. The serotypes of clinical isolates were principally O3:K6 and O4:Kut. The serotypes of environmental isolates were diverse but mainly O2, similar to the report by Xiuying [19].
The problem of bacterial resistance has become more serious with the widespread use of antibiotics. V. parahaemolyticus was sensitive to most of the tested drugs, but the resistance rate to AMP was 90.76%, and some strains were resistant to tetracycline and gentamicin. These results are different from those in other regions [20–22], possibly due to use of different antibiotics in different regions. Monitoring drug resistance in V. parahaemolyticus can guide clinical decision making. As the aquaculture industry expands in Huzhou, the increasing use of antibiotics may promote drug resistance in V. parahaemolyticus. Therefore, monitoring V. parahaemolyticus drug resistance is critical.
PFGE is the gold standard for bacterial typing and can analyze the relationship between strains at the molecular level and trace their origins. We found polymorphisms, low genetic similarity, and no obvious epidemiological relationship between the clinical and environmental strains. There were 16 clones with a similarity of more than 85.00%, among which 12 were from clinical samples and 4 from environmental samples. The cluster map showed that the similarity of clinical strains was greater than that of environmental strains. Also, the maximum similarity between the clinical and environmental strains was 60.00%, such as S19094, S19218, S19080, and S19070, suggesting no genotypic homology between the environmental and clinical strains. Notably, the similarity between S19008 (seawater sample) and S19009 (freshwater sample) was 86.67%. The samples were taken from non-adjacent stalls in the same farmers’ market, suggesting the possibility of cross-contamination between freshwater and seawater during transportation or sale. Therefore, the appropriate departments need to strengthen supervision, provide early warning, and reduce the incidence of foodborne diseases caused by V. parahaemolyticus.
Conclusions
We analyzed V. parahaemolyticus isolates from clinical and environmental samples in Huzhou. A variety of serotypes was detected. Although the V. parahaemolyticus strains were sensitive to most common antimicrobial agents, attention should be paid to the potential emergence of resistant strains and the management of antimicrobials needs to be strengthened. In addition, there may be cross-contamination between freshwater and seawater products, so it is necessary to strengthen supervision of food processing. This information will be useful for the control and treatment of foodborne illnesses caused by V. parahaemolyticus in Huzhou.
Acknowledgments
We thank the outpatients, nurses, and clinicians of the participating hospitals for their cooperation.
The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/2FzyzL
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