Molecular Detection and Antibiotic Resistance of Vibrio cholerae , Vibrio parahaemolyticus , and Vibrio alginolyticus from Shrimp ( Penaeus monodon ) and Shrimp Environments in Bangladesh

Some Vibrio species can cause food-borne diseases in humans, including cholera, vomiting, septicemia


Introduction
Te aquaculture sector of Bangladesh has become a major food production sector, contributing to national economies and supporting rural and farming families.Total shrimp and prawn production in 2017-18 was 2.54 × 10 5 metric tons, as reported by the Deputy Director (Shrimp), Department of Fisheries, Dhaka, and District Fisheries Ofces [1].Aquaculture, fsheries, and related processing are important for food security in Bangladesh, contributing nearly 60% of animal proteins.In addition, they make a substantial contribution to gross domestic product (GDP) and export earnings.Tis sector accounts for 3.57% of the national GDP and about a quarter of the agricultural GDP of Bangladesh (25.30%), making it vitally important to the rural economy and employment [1].Globally, Bangladesh ranks 3rd in inland open water capture production and 5th in aquaculture production [2].In terms of the value of exports, shrimp like the black tiger (Penaeus monodon) and freshwater scampi (Macrobrachium rosenbergii) bring in the most foreign currency, indicating the economic importance of shrimp in our country.
Consumption of contaminated fresh-raw shellfsh, shrimp, and other sea foods has been linked to numerous cases of food-borne infection in humans [3,4].Shrimp contamination can occur due to improper and unhygienic conditions during cultivation, processing, preservation, and storage [5,6].Consequently, shrimp may be tainted with diferent Vibrio species that not only contribute to food spoilage but also to the spread of cholera and other foodborne diseases [7,8].Vibrio spp.are Gram-negative halophilic bacteria with a curved rod shape and a single fagellum that are typically found in aquatic environments.Moreover, Vibrio spp.are widespread in marine environments, including sea fsh, mollusks, and crustaceans [9][10][11].Twelve of the 30 Vibrio spp.are considered human pathogens; among these, V. parahaemolyticus, V. cholerae, and V. vulnifcus are the most frequently reported pathogens of zoonotic signifcance [10,12,13].Tey can develop a wide range of human diseases, such as cholera, wound infection, bloody diarrhea, vomiting, abdominal pain, fever, and nausea [10,14].V. cholerae is the causal agent of cholera and has the potential to be transmitted to humans through contact with fsh, seafood, and water [15].V. parahaemolyticus causes gastroenteritis and food poisoning associated with the consumption of sea fsh and foods [16,17].V. vulnifcus, also known as the fesh-eating bacteria, is the leading cause of primary septicemia in people who consume raw or undercooked seafood [18].V. alginolyticus is associated with wound infection through the exposure of cuts or abrasions of the skin to contaminated seawater [5].
Te rise of antimicrobial resistance (AMR) poses a serious risk to human and animal health, food production, and economic growth around the globe [19].It is predicted that AMR will result in hundreds of millions of deaths, massive economic shortfalls, and a sharp decline in livestock production if it is not encapsulated by the year 2050 [20,21].Overuse of antibiotics in both animals and humans creates a selective pressure that leads to the evolution of AMR [22,23].Antibiotic-resistant bacteria can be spread throughout the environment due to the careless use of antibiotics and a general lack of knowledge among the public [24].Consequently, antibiotic-resistant Vibrio spp.could be transferred from marine products (e.g., shrimp) to their environments (e.g., water and mud) and vice versa.
Te shrimp export market in Bangladesh is at risk because of the possible deterioration in the quality of processed shrimp products brought on by improper handling and the subsequent spread of food-borne illnesses [25].Shrimp contaminated with Vibrio spp.are of lower quality, leading to their rejection at export.So, if these pathogenic microorganisms can be detected early in the shrimp industry, it will increase the likelihood that high-quality shrimp will be produced, which in turn will increase the number of export markets, bring in more foreign currency, and ensure the safety of shrimp for human consumption.Several studies on shrimp and shrimp products have been conducted so far in Bangladesh, but most of them focused solely on identifying Vibrio spp. at the genus level and quantifying their numbers [26][27][28][29][30][31][32][33][34][35].However, as far as we know, very limited studies on detecting Vibrio spp.from shrimp and shrimp environments have been conducted in Bangladesh regarding species identifcation based on molecular approaches.Considering the current situation, the present study was undertaken with the following objectives: (1) to detect V. cholerae, V. parahaemolyticus, V. vulnifcus, and V. alginolyticus from shrimp and shrimp environments (water and mud) of the Khulna region of Bangladesh by molecular approach and (2) to determine antibiotic resistance patterns of isolated Vibrio species.

Ethical Statement.
No ethical statement was required during the sample collection.Two expert veterinarians and one microbiologist were involved in the sample collection.Moreover, consent to collect samples was obtained verbally from the farm owners or authorities.1).Tese areas were selected based on the high number of shrimp farms and the high demand for shrimp and shrimp farming.

Sampling
A total of 150 samples containing shrimp, water, and sediments were collected randomly from each pond.Casting nets were used to collect the shrimp, and in order to avoid crosscontamination, each shrimp sample was placed in an individual sterile plastic zipper bag.Falcon tubes were used to collect water and sediment samples from the ponds where shrimp were harvested.All the samples were thereafter transferred to the Cary-Blair transport medium and transported to the laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, as soon as possible for further processing and analysis.If one of three samples taken from a certain pond tested positive for Vibrio spp., that pond was labeled as contaminated with the bacteria.

Sample Processing.
A loop of mud or sediment samples was directly inoculated into BHI broth containing 2% NaCl (2% BHI broth).Water samples were centrifuged at 3000 rpm for 1 min, and then the sediments were inoculated into 2% BHI broth.Te brain, leg, muscle, and intestine from each shrimp sample were blended and taken for enrichment in 2% BHI broth.After that, the broths were incubated overnight at 37 °C for bacterial enrichment.

Isolation of Vibrio spp.
A loop full of surface growth was streaked on a preset thiosulfate-citrate-bile salt-sucrose (TCBS) agar plate and incubated at 37 °C for 18-24 h, as previously described [36].After incubation, bacterial colonies were tentatively identifed as Vibrio spp.based on their morphology (shape, size, and color).A single, clearly defned colony (yellow or green in color) was then streaked onto freshly prepared TCBS and blood agar plates for further purifcation.Isolates thought to be colonies of Vibrio spp.were Gram-stained to determine their morphology.As previously mentioned by Bergey et al. [37], other biochemical assays, including catalase and oxidase tests, were also conducted.

Molecular Detection of Vibrio spp. Final confrmation of
Vibrio spp. was done by a polymerase chain reaction (PCR) test targeting the genus-specifc groEL gene (Table 1).Genomic DNA was extracted by the rapid boiling method following the procedures described by Talukder et al. [41].In brief, 1 mL of overnight bacterial broth culture was centrifuged at 5,000 rpm for 3 min at 4 °C.After centrifugation, the supernatant was discarded, and the pellet was resuspended in 500 μL of sterile distilled water.Again, the sample was centrifuged at 5,000 rpm for 3 min, and the supernatant was discarded.Te pellet was resuspended in 200 μL of sterile distilled water.Te mixture was then boiled for 10 min and immediately cooled at −20 °C for 10 min.Subsequently, the sample was centrifuged at 10,000 rpm for 10 min at 4 °C.Te supernatant containing the genomic DNA was transferred into a sterile Eppendorf tube and stored at −20 °C until used.
A 25 μL of PCR mixture comprising 12.5 μL of 2X master mix (Promega, Termo Fisher Scientifc, MA, USA), 5 μL of genomic DNA, one μL of each of the designated forward and reverse primers, and 5.5 μL of nuclease-free water were used for amplifcation.Tereafter, 5 μL of the PCR product was loaded onto a 1.5% agarose gel and run in 1X TAE bufer for 25 min at 100 V. Ethidium bromide was used for staining, and PCR products were visualized under an ultraviolet transilluminator (Biometra, Aquaculture Research Germany).For molecular weight DNA markers, 1 kb and 100 bp DNA ladders (Promega, Termo Fisher Scientifc, MA, USA) were used.
2.6.Molecular Detection of V. cholerae, V. parahaemolyticus, V. Vulnifcus, and V. alginolyticus.All the Vibrio isolates were subjected to a simplex PCR for the detection of V. cholerae, V. parahaemolyticus, V. vulnifcus, and V. alginolyticus molecularly.Te primer sequence, annealing temperature, and their amplicon size are documented in Table 1.

MDR and MAR Profles of Vibrio spp. Twenty-seven
Vibrio isolates (52.9%, 95% CI: 39.5%-65.9%)were MDR in nature.Isolates have a MAR index that is greater than 0.2 in a substantial number of isolates (27/51).Te MAR index had a range of 0.0 to 1.0.One isolate showed resistance to all 13 used antibiotics (under eight antimicrobial classes).Also, there were 14 diferent resistant patterns observed among Vibrio isolates (Table 4).

Discussion
Vibrio species are marine and estuarine bacteria that pose a serious hazard to human health due to their role as primary microbial agents in food-borne infections.Consumption of seafood contaminated by antimicrobial-resistant Vibrio species has the potential to have serious consequences for humans.Tis study reported the molecular detection of antimicrobial-resistant Vibrio spp. in shrimp and shrimprearing environments in Bangladesh by a molecular approach.
In this study, 34% (51/150) of the shrimp and shrimp environmental samples were contaminated with Vibrio spp., of which 24.7%, 15.3%, and 4% of the samples were positive for V. cholera, V. parahaemolyticus, and V. alginolyticus, respectively.All the samples were negative for V. vulnifcus.Te prevalence of shrimp contamination found in the present study is comparable with the previous study [26], in which the prevalence of Vibrio spp. was 44%.Another study conducted in the southwest coastal area of Bangladesh on 216 shrimp and shrimp environmental samples found a 60.2% prevalence of Vibrio spp.Conversely, a similar study carried out in southern Bangladesh found that only 27% of shrimp samples were contaminated with V. parahaemolyticus [35], whereas shrimp and water samples from Gher farms were less frequently contaminated with Vibrio spp.Shrimp and water samples from Gher were found to be less contaminated and even absent of Vibrio spp. in comparison to the market samples in a study of the Kaliganj area in Satkhira district, which hold most of the shrimp farms in Bangladesh [47].To the best of our knowledge, this is the frst study in Bangladesh that has used a molecular approach to detect all three species of Vibrio-V.parahaemolyticus, V. alginolyticus, and V. cholerae from shrimp and shrimp environments in the same study.International studies make interesting comparisons (similar, higher, and lower) with the present data [5,[48][49][50][51][52].Te diferences between these fndings and our fndings might be due to variations in the geographical distributions, environments, sample types and sizes, test methodologies, and others.
Temperature and salinity are the two most important factors afecting Vibrio distribution worldwide [53].Te concentration of marine Vibrio spp. is directly linked to water temperature.A study conducted in an island in Brazil revealed Vibrio spp.contamination in oysters was higher in warm water [54].As water surface temperatures and salinity infuence concentrations of most Vibrio spp., infections usually arise during the summer and fall when surface waters are comparatively warm [53].Tus, the lower prevalence of Vibrio spp.found in the present study might be explained by the fact that the sampling was done in the winter.However, the variation among the studies might be due to the environmental conditions, nutrient concentration, the salinity of the water, and sample collection time.
Based on the present results, the prevalence of Vibrio spp.contamination in shrimp and the shrimp culture environment, together with the identifcation of zoonotic species (V.parahaemolyticus, V. alginolyticus, and V. cholerae), indicates a signifcant risk to public health.It is  Values with diferent superscripts difer signifcantly (p < 0.05) within the variable under assessment, CI � confdence interval, P number and percentage of positive isolates, Q 95% confdence interval, and NC � not computed.
Aquaculture Research feasible that other toxicogenic strains could have been present, but identifcation of these was beyond the scope of the present study.Nonetheless, future studies should focus on attempting to identify additional pathogenic species, as well as considering the efects of season and water temperature and correlating the pH and salinity of the sampling source water for the best clarifcation of the factors that promote the multiplication of this organism in the shrimp industry.A routine surveillance program could be carried out to monitor the pathogenic bacteria, which would, in turn, allow for an efective management program to be implemented to control the risk of spreading diseases.In such programs, applications of conventional and molecular methods are strongly recommended to confrm and diferentiate the pathogenic strains of Vibrio spp.
Antimicrobial resistance is becoming an increasingly serious threat to public health [55,56].Antibiotic resistance among Vibrio species is a problem that afects not only human health on a worldwide scale but also poses a possible obstacle to the process of treating infectious diseases that afect aquatic animals.Although chromosomal mutations can be involved in AMR in Vibrio species, the greatest contributors to Vibrio drug resistance are the frequent acquisition of extrachromosomal mobile genetic elements (e.g., replicating plasmids and integrating conjugative elements) and/or insertion sequences from closely related or distantly related bacterial species [57].In this study, a higher percentage of Vibrio isolates (92.2%) were resistant to ampicillin, which shows agreement with the previous studies in Bangladesh [34,35].Resistance to penicillin may have resulted from the widespread use of antibiotics in aquaculture and the impact of leftover antibiotics in aquaculture systems.As a result, infections caused by Vibrio spp.are difcult to treat using penicillin-based antibiotics.Also, Vibrio isolates showed a higher percentage of resistance to cefotaxime (29.4%) and ceftazidime (27.5%), which is analogous to other studies reported in diferent countries [58][59][60].In the present study, Vibrio isolates were highly resistant to nalidixic acid, ciprofoxacin, levofoxacin, amikacin, gentamicin, and tetracyclines; however, a low level of resistance was exhibited to imipenem, meropenem, chloramphenicol, and trimethoprimsulfamethoxazole.In line with our fndings, Heenatigala and Fernando [61] reported that Vibrio isolates showed a very high level of resistance to antibiotics that are commonly used in aquaculture, including gentamycin, kanamycin, oxytetracycline, chloramphenicol, and trimethoprim.Te presence of resistance to these antibiotics reveals a serious threat by limiting the treatment options because antibiotics under the classes of cephalosporins, aminoglycosides, fuoroquinolones, and folate pathway inhibitors are recommended to treat Vibrio spp.infections [60].
In bivariate analysis, we found a very high signifcant positive correlation between the resistance patterns of imipenem and meropenem, cefotaxime and ceftazidime, ciprofoxacin and levofoxacin, nalidixic acid and ciprofoxacin, nalidixic acid and levofoxacin, gentamicin and amikacin, imipenem and chloramphenicol, meropenem and chloramphenicol, amikacin and ceftazidime, ciprofoxacin and gentamicin, amikacin and cefotaxime, tetracycline and amikacin, and levofoxacin and gentamicin.Since they belong to the same class of antibiotics, it is not surprising to fnd a high degree of similarity between most of the antibiotics.Te random administration of antibiotics to aquaculture systems is a possible cause of the other signifcant associations.Te signifcance of these fndings is tied to the fact that Vibrio isolates have shown signs of developing resistance against various types of antimicrobials [62].
Humans are vulnerable to infections produced by MDR microorganisms [63].Pathogens pose a signifcant threat to healthcare systems everywhere because MDR strains are spreading due to AMR [64].In this study, 52.9% of the Vibrio isolates were phenotypically MDR in nature.It has become a common practice to use MAR analysis to discern bacteria isolated from settings where conventional antimicrobials are safe and efective for human treatment.Bacterial MAR is typically linked to the presence of plasmids that carry single or multiple resistance genes [65].Bacterial strains with MAR indices above 0.2 were likely exposed to many antibiotics or isolated from tainted environments, while those with indices below 0.2 were less likely to have been treated with antibiotics [45,66].More than half (52.9%) of the Vibrio isolates in this investigation had a MAR index value larger than 0.2, suggesting that these samples came from a high-risk contamination source where several antibiotics were utilized.Previously, Lee et al. [67] reported that 70% of the Vibrio isolates showed a MAR index of more than 0.2, which is more than in our present study.Tere may be a correlation between the heavy use of antibiotics in the aquaculture industry to combat bacterial infections and the high prevalence of multiple antibiotic-resistant isolates observed in this study.Antimicrobial resistance in aquatic bacteria is a result of environmental contamination in coastal and estuarine  8 Aquaculture Research waters, most notably from wastewater treatment plants and agricultural runof, which may carry a variety of antimicrobials and heavy metals [35].

Conclusion
Te fndings observed in this study embrace the comprehensive reports about the occurrence and antimicrobial resistance profles of Vibrio species, especially V. cholerae, V. parahaemolyticus, and V. alginolyticus isolates sourced from shrimp and shrimp environments.Our study suggests that shrimp and shrimp environments are contaminated with antimicrobial-resistant Vibrio species, indicating the requirement for extensive surveillance with a large number of farms and samples across the country.Hence, it is recommended that the shrimp culture industry should be aware of the risks of antimicrobial-resistant Vibrio contamination and take appropriate precautions during the culture, harvest, and processing of shrimp.Monitoring the antibiotic resistance of Vibrio species on a continuous basis is essential for determining the most efective treatment for patients with gastroenteritis and ensuring the safety of seafood.Moreover, it is necessary to do additional research in order to determine the risk factors that could result in disease outbreaks in shrimp farms with Vibrio species.

Table 1 :
List of primers used for specifc detection of target Vibrio species.

Table 4 :
Multidrug resistance and multiple antibiotic resistance index of Vibrio isolates sourced from shrimp environments.