Progress in Microbes and Molecular Biology 1 Original Research Article Determination of antibiotic resistance patterns of Vibrio parahaemolyticus from shrimp and shellfish in Selangor, Malaysia Vengadesh Letchumanan1,2, Nurul-Syakima Ab Mutalib3, Sunny Hei Wong4, Kok-Gan Chan5,6*, Learn- Han Lee1,2,7 1Novel Bacteria and Drug Discovery Research Group, Microbiome and Bioresource Research Strength, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor Darul Ehsan, Malaysia 2Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, PR China. 3UKM Medical Molecular Biology Institute (UMBI), UKM Medical Centre, Universiti Kebangsaan Malaysia, Kuala Lum- pur, Malaysia 4Li Ka Shing Institute of Health Sciences, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong 5Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 6International Genome Centre, Jiangsu University, Zhenjiang 212013, PR China 7Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand Abstract : High consumer demand for seafood has led to the need for large-scale, reliable supply through aquaculture farming. However, bacterial infections - which can spread rapidly among the dense farming area pose a major threat to this industry. The farmers therefore often resort to extensive use of antibiotics, both prophylactically and therapeutically, in order to protect their stocks. The extensive use of antibiotics in aquaculture has been postulated to represent a major contributing factor in the rising incidence of antimicrobial resistant pathogenic bacteria in seafood; which may then lead to the spread of antimicrobial resistant bacteria in the environment as well as posing a significant threat to human health. This study aimed to characterize antibiotic resistance of Vibrio parahaemolyticus from shrimp and shellfish in Selangor, Malaysia. The antibiotic susceptibility of 385 V. parahaemolyticus isolates was investigated against 14 antibiotics followed by plasmid profiling and plasmid curing to determine the antibiotic mediation. A large number of isolates showed resistance to ampicillin (85%), ami- kacin (66.8%), and kanamycin (50.1%). A notable resistance pattern was also observed to the third generation cephalosporins (cefotaxime 55.8% and ceftazidime 34%). Only 338 V. parahaemolyticus isolates had 1-7 different plasmids and could be categorized into 27 patterns based on the number and pattern of plasmid present. Interestingly, there was no correlation be- tween the number of plasmids and antibiotic resistant patterns seen in the isolates. The antibiotic resistance was mediated by both chromosomal and plasmid mediation among the resistant isolates. In summary, our results demonstrate that incidence of pathogenic V. parahaemolyticus in seafood in Selangor remains in relatively assuring levels, however the identification of antibiotic resistance among the isolates does rises a public health concern and warrants for continuous surveillance. Keywords: Consumers; aquaculture; Vibrio parahaemolyticus; antibiotic resistance; Malaysia Received: 22nd January 2019 Accepted: 25th February 2019 Published Online: 15th March 2019 *Correspondence to: Kok-Gan Chan; Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. kokgan@um.edu.my. Citation: Letchumanan V, Ab Mutalib NS, Wong SH, et al. Determination of antibiotic resistance patterns of Vibrio parahae- molyticus from shrimp and shellfish in Selangor, Malaysia. Prog Microbes Mol Biol 2019; 2(1): a0000019. Introduction The global public health is endlessly challenged by the threat of foodborne diseases and the latter restraints the socioeconomic development by affecting the healthcare system, country’s economic, tourism and trade (1,2). Since decades ago, there has been increasing number of food- borne diseases related with consumption of raw or un- dercooked foods in developed and developing countries (3). Among the identified foodborne pathogens are Sal- monella sp. (4,5,6,7), Listeria sp. (8,9) and Vibrio sp. (10,11,12,13) that are often associated with gastroenteritis cases Copyright 2018 by Letchumanan V, et al. and HH Publisher. This work under licensed under the Creative Commons Attribution-Non- Commercial 4.0 International Lisence (CC-BY-NC 4.0) 2 worldwide. Vibrio parahaemolyticus – a member of the Vibriona- ceae family is a Gram-negative, rod-shaped halophilic bacterium that naturally lives in aquatic environments (13,14). V. parahaemolyticus is widely distributed in marine and estuarine environments thus causing gastrointestinal illnesses after being eaten raw or undercooked seafood (15,16). During the 2016, the Centers for Disease Control and Prevention (CDC), United States reported that V. parahaemolyticus was acclaimed as the major foodborne bacterium compared to other Vibrio sp. This pathogen is accounted for nearly 34,664 foodborne cases annually in the United States (17,18). In Malaysia, V. parahaemolyticus is naturally identified in the marine coastal region of Malaysia in all seasons and known to cause foodborne gastroenteritis (19). In the early 1980s, a study revealed the incidence of V. para- haemolyticus in Malaysian shrimp processing industry. It is of interest to note that 21 different serotypes were isolated from Malaysian shrimp, with type 01:K38 and 01:K32 were predominates (20). In addition, the EU coun- tries has rejected the import of frozen black tiger shrimp from Malaysia due the presences of V. parahaemolyticus which subsequently affected the Malaysian economic (21). The virulent V. parahaemolyticus carrying tdh and/ or trh genes was also identified from the frozen shrimps in Malaysia, prompting a possible health risk for people consuming raw shrimp (22). The food safety in Malaysia is further declining due to the rising cases of the detection of antibiotic resistant V. parahaemolyticus strains in seafood and environmental samples. There has been many reported cases of antibi- otic resistant V. parahaemolyticus strains isolated from seafood namely shellfish, fish, and shrimps from Ma- laysia (10,11,19,23,24,25,26). Antibiotics and other chemothera- peutic agents are often incorporated as feed additives or immersion baths in aquaculture farms to control bacte- rial infections (27,28,29). Though antibiotics are effective in controlling bacterial infections, the misuse of antibiotics has caused the occurrence of multidrug resistant bacteria in the environments (25). Henceforth, there is a need for appropriate management and control of the use of antibi- otics in the aquaculture sectors. The increase in bacterial resistance to many clinical an- tibiotics effects many country’s healthcare and food pro- duction sectors (30). In agreement with previous reports and the expected severity of infections, constant inves- tigation on antimicrobial resistance of V. parahaemolyti- cus is needed for epidemiological purpose and guidance in healthcare treatment. For this reason, our study aimed to characterize antibiotic resistance of V. parahaemolyti- cus from shrimp and shellfish in Selangor, Malaysia. Materials and Method Bacterial Strains V. parahaemolyticus isolates from previous study was used for the present study (10,11). A total of 385 V. para- haemolyticus isolates were from shrimp and shellfish – red prawn (Solenocera subnuda), banana prawn (Penaeus indicus), mud crab (Scylla serrate), flower crab (Portunus pelagicus), carpet clam (Paphia texile), hard shell clam (Meretrix meretrix), and mud creeper (Cerithidea ob- tuse) was collected from wetmarket and supermarket in Selangor, Malaysia. All these isolates were confirmed V. parahaemolyticus by toxR-PCR assay and thermostable- related direct haemolysin (trh) gene was detected in the isolates (10,11). Antibiotic Susceptibility Test (AST) The antibiotic susceptibility of V. parahaemolyticus iso- lates was determined using Kirby-Bauer disc diffusion method (31). Fourteen different types of antibiotics discs (Oxoid, UK) was tested: ampicillin (10µg), ampicillin/ sulbactam (30µg), amikacin (30µg), cefotaxime (30µg), ceftazidime (30µg), chloramphenicol (30µg), gentamicin (30µg), imipenem (10µg), kanamycin (30µg), levofloxa- cin (5µg), nalidixic acid (30µg), oxytetracycline (30µg), suphamethox/trimethoprim (25µg), and tetracycline (30µg). E. coli ATCC 25922 with known sensitivity pat- tern was included as a positive control in each test. V. parahaemolyticus isolates was grown in tryptic soy broth (TSB) (HiMedia, India) supplemented with 2% w/v sodium chloride (NaCl) (Vivantis, USA) at 37oC for 18 hours under constant agitation (13). The bacteria cultures are lawn onto Mueller Hilton agar (HiMedia, India) with 2% w/v sodium chloride (NaCl) (Vivantis, USA), placed with antibiotic discs and incubated at 37°C for 18 hours. The zone of inhibition was measured and interpreted fol- lowing the guidelines of Clinical and Laboratory Stan- dards Institute (CLSI) M45-A2 (32). The multiple anti- biotic resistance (MAR) index was calculated based on the ratio isolate’s resistance to the total number of tested antibiotics (33). Plasmid Profiling Plasmid profiling was carried out following the method adapted from previous study with slight modification (27). V. parahaemolyticus cell were grown in tryptic soy broth (TSB) containing 2% w/v sodium chloride and in- cubated at 37oC in a shaker incubator (220rpm) for 18 hours. About 1.5 mL of the culture was transferred into a micro-centrifuge tube followed by centrifugation (10,000 rpm for 2 minutes at 4oC). The supernatant was removed by aspiration leaving the cell pellet as dry as possible. The pellet was resuspended in ice-cold 100ul alkaline lysis solution I (Glucose 50mM; Tris Cl 25mM; EDTA 10Mm) by vigorous vortexing followed by addition of freshly prepared 200uL alkaline lysis solution II (NaOH 2N; SDS 2% w/v). The contents were mixed by vortexing rapidly after which 150ul ice-cold solution III (Potassium acetate 5M: 60ml; Glacial acetic acid 11.5ml; dissolved in 28.5m sterile distilled water) was added to it. The tube was closed and gently vortexed for 10 seconds to disperse solution III through the viscous bacterial lysate. Then the tubes were stored in ice for 5 minutes before being centrifuged at 12,000 rpm for 2 minutes at 4oC. An equal volume of phenol-chloroform (1:1, w/v) was add- ed to the supernatant in a fresh tube, by vortexing. The contents in the micro-centrifuge tube were centrifuged Determination of antibiotic... 3 at 8,000 rpm for 3 minutes at 4oC and the supernatant was transferred into a fresh tube. This was repeated with chloroform: isoamyl-alcohol (24:1, v/v) for removing the phenol. The double stranded DNA was precipitated with 2 volumes of ethanol at room temperature, followed by vor- texing before it was allowed to stand for 5 minutes at room temperature. The aliquot was centrifuged at 12,000 rpm for 12 minutes at 4oC and the supernatant was removed by gentle aspiration. The pellet of double stranded DNA was rinsed with ethanol (1ml, 70% v/v) at 4oC and centrifuged. The supernatant was removed leaving the pellet dry as pos- sible. The pellet was air-dried before it was re-dissolved in 30ul ultrapure water. Electrophoresis was performed using 1% agarose gel. Plasmid Curing The antibiotic resistance mediation of V. parahaemolyti- cus isolate was determined by plasmid curing method us- ing two different intercalating agent, acridine orange (AO) and ethidium bromide (EB) (10,34). The isolates were revived in freshly prepared tryptic soy broth (TSB) supplemented with 0.2 mg/mL of respective curing agent and the tubes were incubated at 37°C for 18 hours under constant agita- tion. The treated culture was subjected to antibiotic sus- ceptibility test as described in section 2.2 to re-examine the antibiotic resistance profiles. The phenotype results were compared with the antibiotic phenotype of non-treated iso- late. The plasmid profiling as described in section 2.3 was performed with the treated culture in order to determine and compared the presences of plasmids before and after treatment. Statistical Analysis The data analysis was performed using IBM SPSS statisti- cal analysis software version 20. Statistical analysis was performed to determine whether there is any significant difference in type of samples and MAR index of resis- tant V. parahaemolyticus isolates. A One-way analysis of variance (ANOVA) followed by suitable post-hoc test (Turkey) was used and p < 0.05 is considered as signifi- cant. Results Antibiotic Resistance of V. parahaemolyticus Strains Fourteen antibiotics belonging to β-lactams, amino- glycosides, carbapenems, quinolones, tetracycline, sul- phonamides, and chloramphenicol were used for the determination of antibiotic susceptibility of V. parahae- molyticus isolates. As shown in Table 1, a large num- ber of isolates showed resistance to ampicillin (85%), amikacin (66.8%), and kanamycin (50.1%). A notable resistance pattern could be observed to the third genera- tion cephalosporins (cefotaxime 55.8% and ceftazidime 34%). In contrast, high susceptibility rate was seen to imipenem (94%), chloramphenicol (92.5%), tetracy- cline (83.1%), ampicillin-sulbactam (81%), levofloxa- cin (76.1%), trimethoprim-sulfamethoxazole (75.8%), nalidixic acid (73.85), and gentamicin (70.6%). A high percentage (68%) of isolates have a significant MAR index more than 0.2. The value of MAR index ranged from 0.00 to 0.79, with the highest MAR index attrib- uted from two isolates respectively (VP152 from super- market banana prawn and SVP129 from supermarket carpet clam) exhibiting resistance profile towards 11/14 antibiotics tested. Letchumanan V et al. Antibiotics No. of resistant isolates (%) No. of intermediate isolates (%) No. of susceptible isolates (%) Ampicillin (10ug) 327 (85) 29 (7.5) 29 (7.5) Ampicillin-sulbactam (30ug) 41 (10.6) 32 (8.3) 312 (81) Cefotaxime (30ug) 215 (55.8) 51 (13.2) 119 (30.9) Ceftazidime (30ug) 131 (34) 98 (25.5) 156 (40.5) Imipenem (10ug) 5 (1.3) 18 (4.7) 362 (94) Amikacin (30ug) 257 (66.8) 90 (23.4) 38 (9.9) Gentamicin (30ug) 28 (7.3) 85 (22.1) 272 (70.6) Kanamycin (30ug) 193 (50.1) 161 (41.8) 31 (8.1) Tetracycline (30ug) 57 (14.8) 8 (2.1) 320 (83.1) Oxytetracycline (30ug) 67 (17.4) 108 (28.1) 210 (54.5) Nalidixic acid (30ug) 38 (9.9) 63 (16.4) 284 (73.8) Levofloxacin (5ug) 31 (8.1) 61 (15.8) 293 (76.1) Trimethoprim-sulfamethoxa- zole (25ug) 18 (4.7) 75 (19.5) 292 (75.8) Chloramphenicol (30ug) 22 (5.7) 7 (1.8) 356 (92.5) Table 1: The percentage of antibiotic resistant V. parahaemolyticus isolates isolated from shrimp and shellfish samples. 4 This study revealed a high percentage of susceptibility to- wards imipenem, however it should be noted that five of the isolates (VP71, SVP90, VP114, VP145, and VP146) exhibited resistance to imipenem. Although the resistance to imipenem is only 1.3% of the total isolates, it still war- rants a concern on the use of antibiotics as Carbapenems are among the beta-lactams that are the last line of anti- biotic used for bacterial treatment (35). These five isolates had MAR index of 0.21 to 0.64, and resistant to more than two different type of antibiotic tested. Imipenem resistance profiles was observed among isolates isolated from both shrimp and shellfish samples, demonstrating that the re- sistance occurred in different seafood samples regardless the habitat of marine organism. The VP114, VP145 and VP146 isolates was isolated from the banana prawn sam- ples whereas VP71 was isolated from the red prawn and SVP90 was isolated from the flower crab sample. Interestingly, the 32 trh-positive V. parahaemolyticus ex- hibited resistance to more than two different type of anti- biotic tested (Table 2). Of the thirty-two isolates, 30 trh- positive isolates were seen resistant to ampicillin. Isolate SVP54 demonstrated resistance to six different antibiotics tested including ampicillin, amikacin, ceftazidime, ce- fotaxime, kanamycin, and levofloxacin. The 32 trh-posi- tive isolates had MAR index of 0.21 to 0.64, with 62.5% (20/32) isolates are resistance to three and more different types of antibiotics tested. The presence of multi-resistant trh-positive isolates in the marine environment may ham- per clinical treatment if one gets infected with these strains. This emphasises the need for frequent monitoring of sea- foods. Based on the One-way ANOVA analysis, there was a sig- nificant effect (p < 0.05) between type of samples and MAR index of V. parahaemolyticus isolates. In line with Tukey’s Post Hoc analysis, there was a significant difference in the mean MAR index (p < 0.05) of V. parahaemolyticus iso- lates between red prawn and all the other type of seafood. There was a significant difference in the mean MAR in- dex between banana prawn with red prawn, p = 0.000 (p < 0.05). The MAR index of swimming crab sample was significantly different with MAR index of red prawn (p = 0.000) and carpet clam (p = 0.041) (p < 0.05). There was no significant difference in the mean MAR index of V. para- haemolyticus isolates between hard shell clam and all the other type of samples, except for red prawn, p = 0.000 (p < 0.05). Figure 1 illustrates the comparison of mean MAR index of V. parahaemolyticus isolates from different type of seafood. Figure 1: Comparison of the mean MAR index of V. parahaemolyticus isolates from different type of seafood. Each bar represents mean MAR index of isolates from type of seafood. The vertical lines associated with the bars represent two times the standard error of the mean. Plasmid Profiles of V. parahaemolyticus Three hundred and eighty-five V. parahaemolyticus isolates were analyzed for the presence of plasmids. Only 338 V. parahaemolyticus isolates have 1-7 differ- ent plasmids (Figure 2) and could be categorized into 27 patterns based on the number and pattern of plas- mid present. The sizes of plasmids ranged from 1.2kb to above 10kb. As shown in Figure 2, from 27 plasmid profiles, the profile that forms the largest group was the plasmid profile 1.3 that consisted of 1 band above 10kb size plasmid. A total of 95 isolates (24.7%) have plasmid profile 1.3. Additionally, in this profile, 22 isolates were from shellfish samples and 73 isolates were from shrimp samples. The isolates grouped in this plasmid profile were identified to be resistant to at least one typed of the antibiotic tested. The isolate VP152 from supermar- ket banana prawn and isolate SVP129 from supermarket carpet clam which exhibited resistance profile towards 11/14 antibiotics tested respectively were grouped un- der plasmid profile 1.3. Isolate VP183 that was resistant towards 5/14 antibiotic tested (AK/AMP/C/OT/TE) and SVP61, a trh-positive isolate resistant towards 4/14 an- tibiotic tested (AMP/CTX/AK/CAZ) respectively har- boured seven plasmids each. Overall, a total of 47/385 isolates (12%) did not express any plasmid profiles. The results demonstrated high discriminatory power of plas- mid profiling conducted in this study. Figure 2: Bar Chart on plasmid profile of 385 V. parahaemolyticus isolates. The Y-axis represents different type of plasmid pattern while the X-axis represents the number of isolates that possess the particular pattern. Determination of antibiotic... 5 Table 2 shows an interesting relationship between the anti- biotic resistance and plasmid profiles of the 32 trh-positive V. parahaemolyticus isolates. 21/32 trh-positive isolate contained 1-7 plasmids, where else another 11 isolates did not exhibit any plasmid profiles. All the trh-positive iso- lates were resistant to at least one type of antibiotic tested in study except isolate VP98 that was not resistant to any antibiotic and did not have any plasmid profile. Isolates Antibiogram MAR Indes SVP54 amp/ak/caz/ctx/k/lev 0.43 SVP55 amp/ak/caz/ctx/k 0.36 SVP56 amp/ak/caz/ctx/k 0.36 SVP70 amp/ak/caz/ctx/k 0.36 VP102 amp/ctx/ak/caz/k 0.36 VP103 amp/ctx/ak/caz/k 0.36 SVP61 amp/ak/caz/ctx 0.29 SVP66 amp/ak/caz/ctx 0.29 SVP69 amp/ak/caz/ctx 0.29 SVP72 amp/ak/caz/ctx 0.29 SVP75 amp/ak/ctx/k 0.29 VP90 amp/ctx/ak/caz 0.29 VP95 amp/ctx/ak/k 0.29 SVP73 ak/ctx/k 0.21 SVP64 amp/ak/ctx 0.21 SVP52 amp/ak/ctx 0.21 VP93 amp/ak/k 0.21 VP101 amp/ak/k 0.21 VP89 amp/ctx/ak 0.21 VP91 amp/ctx/ak 0.21 VP178 amp/ak 0.14 VP99 amp/ctx 0.14 VP175 amp/ctx 0.14 SVP60 AMP 0.07 VP92 amp 0.07 VP96 amp 0.07 VP97 amp 0.07 VP176 amp 0.07 VP177 amp 0.07 VP94 amp 0.07 VP100 amp 0.07 VP98 0.00 Table 2: Antibiotic resistant profile of trh-positive V. para- haemolyticus isolates Ampicillin (AMP), Oxytetracycline (OT), Nalidixic acid (NA), Chloramphenicol (C), Cefotaxime (CTX), Sulfa- methoxazole/Trimethoprim (SXT), Imipenem (IMP), Ami- kacin (AK), Ampicillin/Sulbactam (SAM), Levofloxacin (LEV), Ceftazidime (CAZ), Kanamycin (K), Gentamicin (CN), Tetracycline (TE). Plasmid Curing In this study, two different intercalating agents – acridine orange (AO) and ethidium bromide (EB) were used to de- termine the antibiotic resistance mediation. The plasmid curing revealed that both intercalating agents AO and EB produced same curing profiles of isolate and the re- sults is demonstrated in Figure 3. All 338 V. parahaemolyticus isolates that harbour 1-7 different plasmid ranging of size 1.2kb to above 10kb in size lost their plasmids upon being subjected to cur- ing agents. In Figure 3, it could be observed that 327 V. parahaemolyticus isolates that were resistant towards ampicillin before plasmid curing showed the same phe- notype resistance after plasmid curing. Similar resis- tance pattern could be observed in a group of 57 tet- racycline resistant isolates. The plasmid curing results revealed that 51/57 isolates (89%) were still resistant towards tetracycline. This suggests that the resistance phenotype to ampicillin and tetracycline expressed by the isolates could be chromosomally mediated. All the ampicillin/sulbactam resistant strains lost their plasmid after the curing assay and subsequently were suscep- tible to ampicillin/sulbactam suggesting it was plasmid mediated resistance. The antibiotic resistant patterns of OT/C/CTX/SXT/AK/CAZ/K/CN presented after plas- mid curing had lower number of resistant isolates to- wards respective antibiotic. These results demonstrate that the phenotype resistance observed could be both plasmid and chromosomal mediated. Figure 3: Bar Chart on antibiotic resistance profile of V. parahaemolyticus before and after plasmid curing. The Y-axis represents different type of anti- biotic agents while the X-axis represents the number of resistant isolates to- wards the antibiotic agents. Ampicillin (AMP), Oxytetracycline (OT), Nali- dixic acid (NA), Chloramphenicol (C), Cefotaxime (CTX), Sulfamethoxazole/ Trimethoprim (SXT), Imipenem (IMP), Amikacin (AK), Ampicillin/Sulbactam (SAM), Levofloxacin (LEV), Ceftazidime (CAZ), Kanamycin (K), Gentamicin (CN), Tetracycline (TE). With reference to the 32 trh-positive V. parahaemolyti- Letchumanan V et al. 6 cus isolates (Table 2), the antibiotic resistance profile of the 21 plasmid containing isolates changed after curing while the remaining 10 were unchanged. All 20/21 were ampicil- lin resistant initially, and after curing, the isolates (SVP61, SVP54, SVP75, SVP69, SVP72, VP89, VP90, VP91, VP92, VP93, VP94, VP95, VP99, VP101, VP102, VP102, VP103, VP1175, VP176, VP177, VP178) remained resis- tant to ampicillin and cefotaxime, and became susceptible to the other antibiotics tested. One isolate (SVP73) became susceptible to all antibiotic resistant after plasmid curing, suggesting the resistance phenotype observed was plasmid mediated. This suggests that while antibiotic resistance is mediated by both plasmid and chromosomes in pathogenic V. parahaemolyticus isolates, in plasmid containing strains aside from ampicillin and cefotaxime resistance, most of the remaining resistance phenotypes are plasmid mediated. SVP129 isolate contained one plasmid profile with size more than 10kb and expressed antibiotics resistance to- wards 11/14 antibiotics tested. After plasmid curing, SVP129 isolate lost its plasmid and changed its antibiotic resistance phenotype. SVP129 isolate remained resistant to ampicillin, oxytetracycline, chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim; intermediate resis- tance to amikacin, ceftazidime, cefotaxime and kanamy- cin; and susceptible to gentamycin and ampicillin/sulbac- tam after plasmid curing assay. Discussion In view of many reported cases of antibiotic resistance of V. parahaemolyticus from aquaculture, healthcare person- als and members of the public should be caution in the application of antibiotics in healthcare sectors and aqua- culture sectors. The rising number of antibiotic resistance as well as resistant genes within the V. parahaemolyticus population does causes a global health issue (36-44). Hence, continuous monitoring is required to review the antibiotic resistance patterns and followed by controlling the use of antibiotics in the environments. The study’s susceptibility test placed the 1st generation an- tibiotic – ampicillin at the top of the V. parahaemolyticus resistance scope (85%) (Table 1). This result is in close agreement with previous reports from India, Indonesia, Korea and Malaysia that reported prevalent of ampicillin resistant V. parahaemolyticus strains isolated from seafood samples (10,19,27,45-50). The 1st generation antibiotics including ampicillin has a very low efficacy in treatment of infections due to the misuse of these antibiotics in aquaculture and agriculture which in turn led to a low susceptibility rate (25). These findings signify that ampicillin may longer be an effective antibiotic to treat Vibrio sp. infections. The occur- rence of high ampicillin resistance rate in the environment is still of great concern since the resistance phenotype seen could be chromosomally mediated in the bacteria thus re- quire proper management method to control the resistance phenotype (13). Interestingly, multi-resistance profile was observed among the 32 trh-positive V. parahaemolyticus. These pathogenic isolates were seen resistant to aminoglycosides, 3rd genera- tion cephalosporins, and quinolone. The V. parahaemolyti- cus isolates exhibited high resistance rate towards the 3rd generation cephalosporins – cefotaxime (55.8%) and ceftazidime (34%) in this study. These findings are in line agreement with a study from Terengganu, Malay- sia, who reported ceftazidime and cefuroxime resistant V. parahaemolyticus isolates from shellfish (51). In the neighboring country, Korea, another similar study re- ported high percentage (70%-80%) of V. parahaemolyti- cus isolates from Korean seafood to be resistant to the 3rd generation cephalosporin, cefotaxime and ceftazi- dime (52). In contrast, a study from the US reported low percentage of cefotaxime resistant V. parahaemolyticus isolates isolated from food (53). The inconsistencies in V. parahaemolyticus resistance rate to 3rd generation cephalosporin may be due to difference in sample, geo- graphical variations, or difference in methodology test applied. It is reassuring to note that the isolates in this study are still susceptible to some antibiotics tested including imi- penem (94%) (Table 1). Nevertheless, there were five isolates (SVP90, VP71, VP114, VP145, VP146) exhib- ited resistance towards imipenem. The detection of imi- penem resistant isolates raises concern as carbapenems are the most potent β-lactam antibiotic and is usually administrated in treatment of any serious bacterial in- fections (35). The results are in agreement with previous reports on the isolation of carbapenem-resistant Vibrio sp. from environmental samples. Walsh and colleagues reported carbapenem resistant V. cholerae isolated from drinking water and seepage in New Delhi, India and fur- ther analysis revealed that the carbapenem gene blaNDM-1 was found in the chromosome of V. cholerae isolate (54). The occurrence of carbapenem resistance was also de- tected in a V. cholerae 01 El Tor Ogawa strain isolated from faecal specimen of a 2-year-old child in Puduch- erry, India. Another study reported an increasing trend of carbapenem resistance among V. cholerae 01 or 0139 isolates between 1986 to 2012 in southwest China (55). Recently, Bier and colleagues reported the isolation of four carbapenem resistant V. cholerae from different locations of the German coast line. These four isolates were not only resistant to carbapenem but also exhibited resistance to cefoxitin, aztreonam, and aminopenicillin (56). In addition, there have been reports on the emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) in the United States (US) (57,58). Any infections with carbapenem resistant bacteria may cause higher mortality rates compared to those infections caused by carbapenem-susceptible bacteria. The wide incidence of carbapenem resistant Vibrio sp. is an important emerg- ing threat to public health, thus requires proper manage- ment action to limit the spread of this organism. In this study there was no significant difference between the sampling location and MAR index of V. parahaemo- lyticus isolates. This result demonstrates that the isolates isolated from wetmarket and supermarkets are exposure to antibiotics. Our results came to an agreement with many studies that reported high percentage of V. para- haemolyticus isolated from seafood are resistant to more than one antibiotic tested (28,29,48,59,60). According to the One-way ANOVA analysis results, there was a signifi- cant difference between the groups of sampling location Determination of antibiotic... 7 on the MAR index of V. parahaemolyticus isolates (p < 0.05). The isolates from the supermarket sampling sites had a higher mean MAR index compared to the isolates from wetmarket sampling sites. This situation could be at- tributed by the geographical difference in seafood samples that been sold in supermarket, thus causing a MAR resis- tance profile. In addition, it could be suggested that seafood samples may have originated from similar environmental conditions in terms of antibiotic exposure or cross contam- ination may have occurred during the post-harvest, result- ing in the isolates to have similar MAR index. When compared the antibiotic resistance patterns and plasmid profiles, there was no correlation observed. Even within the isolates with same resistance profiles, the plas- mid profiles were different and a few isolates even did not exhibit any plasmids, which was similar to findings by Laj- nef and colleagues (61). Hence, it could be concluded that the antibiotic resistance is not been influenced by the num- ber of plasmids acquired by the isolates. The exposure of antibiotic in environment causes the bacteria to display a multidrug resistant characteristic. In some strains, the re- sistance observed could be plasmid mediated, and in some are chromosomally coded. Further research could be done to confirm the origin of antibiotic resistance among the iso- lates. In this study, two different plasmid curing agent, acridine orange (AO) and ethidium bromide (EB), both are interca- lating agents. Two different intercalating agents were used because to observed the efficacy of each agent. Intercalat- ing agents such as AO and EB have been successfully used many studies of curing bacterial plasmids (31,37,62-66). The modes of action of intercalating agents are through prefer- ential inhibition of plasmid replication. Both the intercalat- ing agents yield the similar curing profiles for each isolate (Figure 4.8). The present results are closely in agreement with other studies that reported Vibrio sp. isolates lost their plasmids when treated with concentration of 0.2mg/ml acridine or- ange (AO) and the isolates demonstrated changes in their resistance profile (64-67). A Brazilian study reported AO agent was successfully used to cure multi-resistant Vibrio isolates from marine shrimp and concluded the ampicillin resistance strains in study are plasmid mediated (37). In con- trast, another study reported their isolates resistance was chromosomal mediated after AO curing agent treatment (66). Likewise, another study reported the alteration in anti- biotic resistance patterns and loss of plasmid among Vibrio sp. isolates when treated with 0.3mg/mL EB. In that study 79% of the Vibrio sp. isolates loss their plasmid profiles but showed phenotype resistance pattern to amoxicillin, ampicillin, furazolidone and tetracycline after curing as- say, which indicate the resistance may be chromosomally borne (28). Conclusion In conclusion, the current study provides an overview on the seafood contamination levels in Selangor, Malay- sia and the distribution of V. parahaemolyticus in shrimp and shellfish samples. The shrimp and shellfish samples analyzed were contaminated with V. parahaemolyticus regardless their sampling locations. There was no cor- relation observed between the antibiotic resistance and plasmid profiles. Yet, the antibiotic resistance mediation was studied via the plasmid curing assay. In some iso- lates, the resistance was plasmid mediated, while others were chromosomally borne resistance. The information derived from this curing assay is useful for public health personnel to understand better on the antibiotic resis- tance of V. parahaemolyticus in shrimp and shellfish from Selangor, Malaysia. The plasmid curing assay is fast, cost saving, provides fundamental knowledge, and may influence effective antibiotic management policies in the aquaculture industry. With this knowledge, the aquaculture farmers may alternate the antibiotics in their aquaculture fields from time to time in order to allow withdrawal of antibiotic resistance among the bacteria (34). In summary, the antibiotic resistance presented by V. parahaemolyticus isolates could be due to the exces- sive use of antibiotic in aquaculture to control bacterial infection and huge production loses (68,69). In addition, antimicrobial resistance is also caused by exposure of antibiotics via agriculture runoff, wastewater treatment plants, and thru mobile genetic elements or horizon- tal gene transfers among bacteria (70). There have been prevalent cases of multiple resistance reported among environmental pathogens such as Salmonella sp. (71,72), V. vulnificus (73), Listeria monocytogenes, Escherichia coli and V. parahaemolyticus (74). Hence, the present results would provide a baseline information on the severity of resistance among V. parahaemolyticus in shrimp and shellfish in Selangor Malaysia, then may allow manage- ment personal to overcome this problem with proper management strategies. 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