Microbes and Infectious Diseases Antibiotic sensitivity and plasmid assessment of ampicillin-resistant Vibrio cholerae from seafoods in coastal areas of Ondo State

Background: Vibrio cholerae is an epitome of non-invasive organisms, which only affects the small intestine via the liberation of enterotoxin. The plasmid profile and beta lactamase production in ampicillin-resistant Vibrio species isolated from seafoods collected from coastal area of Ondo State, Nigeria were investigated. Methods: One hundred and six (106) samples of seafoods were collected in February, 2020 and January, 2021. The isolation and identification of Vibrio species, antibiotic sensitivity pattern, plasmid analysis and beta-lactamase detection activity were conducted via pour plate, Kirby-Bauer disc diffusion, alkaline lysis, neutralization, electrophoresis methods, use of 10 % sodium dodecyl sulphate (SDS) and double disk synergistic test (DDST) respectively. Results: Pathogenic bacteria such as Bacillus subtilis , Enterobacter cloacae , Enterococcus faecalis , Escherichia coli, Proteus vulgaris , Pseudomonas aeruginosa , Salmonella enterica , Staphylococcus aureus , and Vibrio cholerae were isolated from 10 crabs, 54 fishes, and 42 shrimps. Findings also revealed that 66% of Vibrio cholerae that showed multiple antibiotic resistance tested positive to beta lactamase production test. The result of plasmid analysis indicated that they were more susceptible to antibiotics after plasmid curing of the microorganisms. Conclusion: The resistance of Vibrio cholerae in this investigation was found to be plasmid-mediated consequent to its loss after curing. The detection of multidrug resistance, ESBLs-producing Vibrio organisms, suggests risks public health. The risk of seafood-borne infections can be reduced by decrease in pollution levels, ensuring appropriate cooking temperatures, and prevention of cross-contamination during harvesting, handling and processing.


Introduction
The presence of pathogenic bacteria in the marine environment raises concerns of human on food safety due to their potential in causing disease outbreaks depending on the environmental conditions [1]. The genus Vibrio comprises about 100 species which are autochthonous inhabitants of aquatic environments and play vital roles in sustaining the aquatic milieu [2]. Vibrio species are Gram-negative bacteria, some are halophilic, from the family Vibrionaceae. They are widely distributed in fresh, estuarine, marine and coastal surroundings based on their species and can be isolated from sea foods [3], where they predominantly persist in culturable or nonculturable state, and are considered to exhibit both fermentative and respiratory metabolisms [4].
Several investigations have also shown the prevalence of Vibrio species in surface water throughout the world, and their prevalence in the environment is influenced by season, location, and the analytical methods employed [4]. They are usually found in a free swimming state, with its motility conferred by a single polar flagellum affixed to inert and animate surfaces including zooplankton, fish, shell-fish or any suspended matter under water [5,6].
Vibrio species cause diarrhoea including cholera infection. Vibrio cholerae serogroup O1 is responsible for the majority of cholera cases globally [7]. In rare cases, Vibrio parahaemolyticus causes wound and ear infections or septicemia that may be life-threatening to individuals with pre-existing medical conditions [8]. The pathogenic Vibrio species of health relevance which are generally transmitted through water and seafood include Vibrio cholerae, Vibrio fluvial, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio tubiashi [9]. Vibrio cholerae is an example of a non-invasive organism, which only affects the small intestine via the release of enterotoxin and is the etiological agent of cholera, whereas Vibrio parahaemolyticus and Vibrio vulnificus are considered as intrusive microorganisms largely affecting the colon [2]. The aim of this study is to identify and detect extended spectrum beta-lactamases in ampicillin resistant Vibrio species from Seafoods in Coastal Areas of Ondo State and determine the plasmid profile of the isolates.

Description of the study area
Erin-Amah is a town in Aheri kingdom of Ilaje in Ondo State. The coastal areas are largely found in Ilaje Local Government Area. Ilaje is situated between longitude 6º 20` and 6º00 North and latitude 4º45` and 5º45` East. It shares boundary with Ese-Odo Local Government of Ondo State in the North, the Atlantic Ocean in the South, Ogun State in the West and Delta State in the East. The local government has about 80 KM long shoreline, thereby giving Ondo State one of the largest coastlines in Nigeria [10].

Collection of samples
One hundred and six (106) samples of seafood which comprised ten (10)

Preparation of culture media
All culture media were prepared according to the manufacturers specifications. Dehydrated Thiosulfate Citrate Bile Salt Sucrose agar (16.84 g) (Hi-Media, Mumbai, India), Tryptose Soya Agar (8 g) (Hi-Media, Mumbai, India) Mueller-Hinton agar (7.6 g) (Hi-Media, Mumbai, India) were separately dissolved in 200 ml of distilled water in a conical flask. Complete dissolution was achieved on a hot plate at 60 °C for 5 min. Thereafter, the mixture was autoclaved at 121°C for 15 min at 15psi [11].

Detection and identification of Vibrio species
Seafood samples were investigated for the presence of Vibrio species. The procedure of Oramadike and Ogunbamiwo, [12] was adopted for the isolation. Alkaline peptone water (APW) at pH 8.4 and Alkaline Peptone Salt Broth (APSB) (Hi-Media, Mumbai, India) i.e. (APW supplemented with 3% NaCl) at pH 8.4 were used for the enrichment of the samples. A 25 g portion of each sample (crab, fish and shrimp) was blended separately with sterile blender, 25 g of sediment was mixed with 225 ml of sterile Alkaline peptone water and Alkaline Peptone Salt Broth (APSB) each (i.e. 1:10 dilution).
The alkaline peptone water homogenate was incubated at 37 °C for 6 h. Thereafter, 1 ml of the broth culture showing positive growth (turbidity) was transferred to a sterile Petri dish, then pour plated with sterilized cooled Thiosulphate Citrate Bile Salt Sucrose Agar -TCBS Agar (Hi-Media, Mumbai, India) to obtain discrete colonies for each of the different samples. The plates were incubated for 18 -24 h at 37 °C, after which the discrete colonies were sub-cultured to obtain pure isolates of Vibrio species on dried Tryptose Soya agar (TSA) (Hi-Media, India) supplemented with 3% NaCl and incubated overnight for further identification of the isolates. Typical colonies of Vibrio species are either golden yellow (V cholera, V. alginolyticus, V. fluvialis, V. vulnificus) or green (V. parahaemolyticus), on TCBS.

Characterization and identification of bacterial isolates
All the bacterial isolates were further confirmed using microscopic examination, staining techniques and standard biochemical tests as described by Tripathi and Sapra, [13].

Standardization of bacterial inoculum
A loop full of test bacterial isolates was inoculated on nutrient broth and incubated for 24 h. 0.2 ml from the 24 h broth culture of the bacteria was dispensed into 20 ml sterile nutrient broth and incubated for 3 to 5 h to standardize the culture to 0.5 McFarland standards (10 6 CFU/ml) before use. One percent (1%) of Sulphuric acid (H2SO4) was prepared by adding 1ml of concentrated Sulphuric acid to 99/ml of distilled water. This solution was mixed properly. Also, 1% solution of barium (BaCl2) chloride was prepared by dissolving 0.5g of dehydrated barium chloride (BaCl2H2O) in 50/ml of distilled water. About 0.6/ml of barium chloride solution was added to 99.4/ml of Sulphuric acid solution and it was then mixed together to make the McFarland standard. The bacterial suspension turbidity was made sure to be equivalent to the turbidity of McFarland standard. The solution was transferred into capped tube of the same type used for both the control and the test inoculums. The solution was kept at a temperature of 4 o C until it was ready for use [14,15].

Antibiotic sensitivity test
Vibrio isolates were tested for sensitivity to conventional antibiotics (CELTECH, Belgium). Using the disk diffusion method according to guidelines set by the Clinical Laboratory Standards Institute [16]. The test was to determine the susceptibility and resistance patterns of the organisms to the antimicrobial drugs.

Detection of extended spectrum beta-lactamase producing ampicillin resistance Vibrio isolates using Double Disc Synergy Test (DDST)
All the ampicillin-resistant Vibrio isolates were selected for detection of extended spectrum betalactamase using double disc synergy test [17]. A disc of amoxicillin clavulanic acid (30 μg) + ceftazidime (30 μg) + amoxicillin clavulanic acid (30 μg) was placed at appropriate distance of 20mm apart on a Mueller Hinton agar plate inoculated with a bacterial suspension of 0.5 McFarland turbidity standards, and then incubated for 18-24 h at 37 °C. An increase in the inhibition zone diameter of > 5mm for a combination disc versus ceftazidime or cefotaxime disc alone was confirmed as ESBLs production.

Extraction of plasmid DNA
Ampicillin-resistant Vibrio isolates were revived in Tryptose Soya agar and subsequently, they were respectively grown for 18 -24 h in Luria-Bertani (LB) broth at 37ºC with aeration using an orbital shaker and plasmid DNA was extracted from lyzed isolates using the Zyppy™ Plasmid Miniprep Kit, (Inqaba Biotech., South Africa) following alkaline lysis method. A 600 µl of Vibrio culture grown in Luria-Bertani broth medium were transferred to a 1.5 ml micro centrifuge tube containing the culture for 30s at 14,000 rpm. The supernatant was discarded, 100 µl of 7X lysis Buffer was added and mixed together by inverting the solution in the tube for 4-6 times.
Thereafter, the solution was centrifuged at 14000 rpm for 2-4 minutes. However, 900 µl of the supernatant was transferred into a provided Zymo-Spin™ IIN column, placed into a collection tube and centrifuged at the proceeding measure (14000 rpm) for 15 seconds. The flow-through in the column was discarded and the column was placed back into the same collection tube. Thereafter, 200 µl of Endo-Wash buffer was added to the column and centrifuged for 30 seconds after which 400 µl of Zyppy™ wash buffer was added to the column and centrifuged for 1 minute. The column was transferred into a clean 1.5 ml micro centrifuge tube, 30 µl of Zyppy™ elution buffer was added to the column matrix and left alone for one minute at room temperature. Hence, the constituent was centrifuged to obtain the plasmid DNA. The column was then removed after the centrifugation and the micro-centrifuge tubes containing the plasmid DNA was stored at -20°C until used [18].

Plasmid profiling
Plasmid profiling was done starting with the preparation of agarose gel. 1g of agarose powder was measured into a sterile conical flask and mixed with 100ml of Tris-acetate-ethylene diaamine tetra-acetic acid (TAE). The solution was heated for homogenization for about 1-3minutes. The agarose solution was allowed to cool for 5 minutes and 10µl EZ vision™ DNA dye was added in order to visualize the DNA under ultraviolet (UV) light. The solution was poured in a gel tray and allowed to solidify at room temperature for about 20-30 minutes [18].
Once the gel was ready i.e. solidified, the purified plasmid DNA was loaded and run on the agarose gel by the addition of loading buffer. Now, the gel was placed into the gel box (electrophoresis unit) and filled with 1xTAE until the gel is covered. The molecular weight ladder was loaded carefully to the first lane of the gel, purified plasmid DNA is loaded into the remaining wells of the gel. The current was supplied to the tank at about 90V for 1 hour, the resulting bands were observed under UV trans-illuminator and compared with100bp and 1kb ladder.

Plasmid curing
Plasmid curing was performed by inoculating 100ml of culture grown in Tryptose Soya broth containing 10% of Sodium Dodecyl Sulfate (SDS). After Inoculation, the cultures were incubated at 37 °C for 24 hrs with constant shaking with the aid of water bath. After 24hrs incubations, the extraction of plasmid DNA procedure as explained earlier on, was used to purify the plasmid [14].

Post-curing sensitivity test
Freshly prepared Tryptose Soya broths supplemented with 3% NaCl were again prepared and then inoculated with the plasmid-cured tested cultures and incubated at 37 °C for 24 hours. After incubation, the standardized inocula of these bacteria were swabbed into the Mueller Hinton Agar (MHA) (Hi-Media, Mumbai, India) plates and incubated at 37 °C for 18hrs as a confirmatory antibacterial assay. Then Antibiotic sensitivity was carried out again according to Kirby-Bauer disc diffusion method, as previously described by Kebede et al. [19] using same convention antibiotics earlier described. Results were examined and compared with the formal for successful examination.

Statistical analysis
All experiments were carried out in triplicate, and data obtained were subjected to one way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) version 20 and treatment means were separated using Duncan`s New Multiple Range Test (DNMRT) at p ≤ 0.05 level of significance.

Antibiotic sensitivity patterns of vibrio species isolates from seafood samples
The results of the antibiotics sensitivity pattern of the three Vibrio cholerae isolates are presented in table (2). The zones of inhibition ranges from 0.00±0.58 to 17.00±1.58 with ofloxacin being the most effective on the Vibrio cholerae; while ampicillin, ceftriaxone sulbactam, nalidixic acid augmentin, cefotaxime, imipenem, and cefexime were least effective with no zone of inhibition.

Occurrence of extended spectrum beta lactamases in isolated Vibrio species
Two of the three tested ampicillin resistant Vibrio cholerae showed beta-lactamases production with an increase in the inhibition zone diameter of > 5mm towards amoxicillin-clavulanate disc from the two extended spectrum beta-lactamase disc of ceftazidime and cefotaxime.

Plasmid profile and curing of ampicillin resistant Vibrio species
As shown in plate (1A), isolated plasmids from the Vibrio cholerae isolates indicated the presence of plasmids of high molecular weight in all samples. Plasmids of molecular weight ranging from about 3.8Kbp to 9Kbp were isolated from Vibrio cholerae sample 1 and 2 while plasmids of molecular weight of about 9Kbp were isolated from samples 3. Additionally, samples 1 indicated the presence of another plasmid of molecular weight at about 700bp while sample 2 indicated the presence of two additional plasmids of molecular weight of about 580bp and 700bp similar to that of sample 1. However, plasmid curing was successful in the lower molecular weight plasmids possessed by sample 1 and 2 represented by 4 and 5 on plate (1B), however, the high 9kbp plasmid were not cured. this was illustrated by the absence of plasmid lower than 9Kbp in samples 1 and 2 cured. Plasmid curing for samples 3 represented by 6 on plate (1B) was successful as no plasmid was found after curing.

Post-curing antibiotics sensitivity pattern of ampicillin resistant Vibrio isolates
The results of the post-curing antibiotics sensitivity pattern of the three Vibrio cholerae isolates are presented in table (4). The zones of inhibition ranges from 0.00±0.00 to 24.00±2.08 with ofloxacin being the most effective on the Vibrio cholerae; followed by entamycin 23.33±3.33, levofloxacin, ciprofloxacin, ceftriaxone-sulbactam and cefexime 21.00±2.08, cefuroxime and augmentin were 16.67.±8.82 and 14.00±7.02 respectively, while ampicillin, nalidixic acid, cefotaxime, and imipenem, were least effective with no zone of inhibition.

Discussion
Vibrio cholerae implicated in this study is a significant diarrhoeic pathogen which when ingested causes cholera and gastroenteritis. Vibrio cholerae isolated from crabs corroborates with the investigations of Sivasubramanian et al. [20], and Soundarapandian and Sowmiya [21]. Also, Ashiru et al. [22] report supported the findings of this research as they isolated Vibrio species including the V. cholerae from the intestine of crabs. Okonkwo et al. [23] did not isolate any Vibrio species from crab samples in their findings; a result which supported absolutely with the result gotten from this findings. Vibrio cholerae reported by Jahan et al. [24] supported the findings from this study as two V. cholerae were identified from fish samples. No Vibrio species was isolated from shrimp samples in their findings, an outcome which was parallel to Okonkwo et al. [23].
Two of the three Vibrio tested positive for the production of extended spectrum betalactamases which were identified as Vibrio cholerae were positive and this correlated with the findings of Canellas et al. [25] who reported that four strains of tested Vibrio cholerae were producers of extended-spectrum beta-lactamases and their antimicrobial susceptibility testing confirmed resistance to aminoglycosides, betalactams (including carbapenems and thirdgeneration cephalosporins), fluoroquinolones, sulfonamides, and tetracyclines. The report also correlates with the findings of Ceccarelli et al. [26] whose finding was positive for detection of Vibrio beta-lactamases producers among screened isolates.
The electrophoresis band pattern of plasmid profiling for uncured and cured three (3) Ampicillin resistant Vibrio isolates indicated that 100% of the isolates harbored plasmid with molecular weight as high as 9 Kbp. During curing process, Vibrio isolates 1 and 2 lost plasmids of molecular weights 580 bp and 700 bp but retained 9 Kbp size, and remained resistant to ampicillin, nalidixic acid, cefotaxime and imipenem. However, Vibrio isolate 3 was successfully cured but also remained resistant to ampicillin, nalidixic acid, cefotaxime and imipenem. The 3 isolates were moderately resistant to Cefuroxime, and Augmentin. Isolate 2 was moderately resistant to ciprofloxacin and ceftriaxone, while isolate 1 was moderately resistant ciprofloxacin, and Isolate 3 to ceftriaxone. the results suggested that the resistance to antibiotics in isolated Vibrio species might be due to chromosomal and plasmid-borne as the isolates shows sensitivity to other antibiotics. The results correlated with the reports from Amalina et al. [27] and Garben et al. [7]. Vibrio cholerae isolates in this study showed high susceptibility to Ofloxacin, and high levels of resistance to Ampicillin, and other antibiotics similar to findings of Das et al. [28] and Garbern et al. [7].
Antimicrobial drug resistance in Vibrio species can develop through mutation or through acquisition of resistance genes transferred between bacteria [6]. Thus, plasmid is one of the mediators that play an important role in spreading of resistance genes since it consists most of the genetic determinants of antibiotic resistance. Acquisition of plasmids by the microorganisms in collected in Ondo State Seaside is possible as the site is about 35min to the urban centre of densely industrialized Lekki Zone in Lagos, Nigeria. The phenomenon which has been supported by several studies on the effect of released effluents, industrial and household waste and feacal contaminants into the water body. This study also shows correlation between plasmid and antibiotic resistance among Vibrio species which has been reported by Amalina et al. [27]. The post-curing antibiotic susceptibility profiles obtained in current study indicate clearly that ofloxacin, levofloxacin, gentamycin, cefexime, and ciprofloxacin are effective against tested Vibrio cholerae. Emergence of high resistance Vibrio strains against Ampicillin and many other WHO recommended antibiotics was related with the extensive used of the antibiotics and could influence the disease management in aquaculture system (Food and Agriculture Organization) [FAO], [29]. Thus, ampicillin is ineffective for treatment of Vibrio infection.
Vibrio cholerae in the seafoods from coastal area of Ondo State which might be released into this environment from human activities such as release of effluents which results in the modification of the genetic makeup of the pathogens and in-turn acquaintance of resistance genes and plasmids which aid antimicrobial resistance; and faecal contamination of the water body by tourist and those who visit the beach for recreation activities.

Conclusion
Pathogenic organisms such as Vibrio cholerae are recognized as pathogenic microorganisms of seafood-borne illnesses among which are diarrhoeic agents. Marine environment harbor varieties of seafood and pathogenic microorganisms which tolerates the salty environment. They cause diseases in aquatic life and also in the consumers of these sea foods.
Seawater an environment where Vibrio species are autochthonous which leads to unsafe seafood. This findings indicate the presence of antimicrobial-resistant and potentially pathogenic However, foodborne pathogen are consumed via consumption of uncooked or insufficiently heatcooked seafood or the toxins produced by these pathogens. The detection of multidrug resistance, ESBLs-producing Vibrio organisms, suggests risks to public health. The risk of seafood-borne infections can be reduced by decrease in pollution levels, ensuring appropriate cooking temperatures, and prevention of crosscontamination during harvesting, handling and processing. By doing so, exposure to aquatic environments and consumption of seafood can be limited to minimize the risks of infection. Findings from the study has shown that sea foods from coastal area of Ondo State consequent to the presence of Vibrio cholerae; antibiotic sensitivity patterns of the Vibrio species from the sea foods and plasmid as the basis of the antibiotic resistance shown by the Vibrio cholerae.