Environmental & anthropogenic modulators of multi-drug resistant Vibrios in the food chain: Diarrheal disease perspective Suvajit Saha, Madhumanti Halder, Subham Mookerjee & Anup Palit ICMR- National Institute of Cholera and Enteric Diseases (ICMR-NICED) P- 33, Scheme-XM, CIT Road, Beliaghata, Kolkata-700 010, India Email: anup.palit@gmail.com Abstract Background: The advent of antibiotic resistance in pathogenic enterobacteria is a pressing global health crisis. Rationale: Diarrhoea-menace and its etiological agents is the major public-health concern in Bengal-delta. Controlling outbreaks induced by pathogenic Vibrios and associated health consequences always remain a challenge for healthcare executives. Disseminating multi-drug resistant Vibrios into community aggravates the diseases paradigm manifold and no clear knowledge is available about the reason of accelerated rates of increases in drug susceptibility. Strategy: Various environmental sources (riverine, potable-water, aquaculture-ponds, raw-seafood & restaurant-cooked seafood) were targeted to isolate Vibrios and subjected to PCR for species identification & molecular characterization and disk-diffusion methods for antibiotic-assay. Plasmids were analyzed by kit-based methods to detect the mode of antibiotic resistant gene (ARGs) acquisition. Results: The present study elucidates multiple environmental/anthropogenic factors triggering increasing rate of drug-resistant Vibrio species in the food-chain and also facilitate acquisition of ARGs. Though a considerable load of resistant vibrio strains has been detected from all the sources, prevalence of resistant strains against highly effective drugs viz. carbapenem, quinolone, 2nd/3rdgeneration cephalosporin and amino-glycosides were remarkably high in aquaculture ponds and seafood items. Additionally high frequencies of ARGs were also detected from these sources, which signifies uncontrolled use of antibiotics in aquaculture farms and poor infrastructure in food preservation processes is responsible. We also observed the presence of ‘sxt’ element increases ARGs (strB, tetA, sul2, bla-DHA, floR) acquisition manifold in environmental Vibrios and the resistant strains are able to tolerate high-flame cooking temperature and pressure. Thus, systemic surveillance is strongly advocated.
Introduction Multi-drug resistant zoonotic enteropathogens present on food pose a serious public health threat globally1. Seafood is a widely perishable delicacy in India, which is the second largest aquaculture producer in the world2. Vibrios are one of the prime etiological agents of acute diarrheal diseases, which utilizes water and various seafood organisms as transmission vehicle3. Increasing drug-resistant strains amongst pathogenic Vibrios in Indo-Gangetic plain becoming serious challenges for health practitioners and the environmental cause of resistance acquisition is hitherto unexplored. We aimed to investigate diversified sources of possible Vibrio transmission routs to reveal modulators stimulating the rate of increase of antibiotic resistant pathogens in this century old diarrhoea endemic foci4.
Fig: Riverine water sampling
Fig: Study Foci
A significant phase of Vibrio transmission where uncontrolled uses of antibiotics and lack of proper handling causes drastic changes in drug-resistant profile amongst the pathogens
Fig: Antibiotic assay
Fig: Antibiotic susceptibility profile of Vibrios X-axis represent percentage of positive isolates
Findings A considerable load of multi-drug resistant enteropathogenic Vibrios were detected in the potable/drinking water and seafood sources. It has been observed that seafood borne Vibrios are more vulnerable to acquire antibiotic resistant genes (ARGs) in comparison to water borne progenies. Surprisingly, the drug resistant strains were also found to tolerate boiling temperature and high pressure of cooking procedure. Uncontrolled usages of antibiotics in culture ponds were found to pose selective pressure to amplify the prevalence of drug resistant strains in pathogenic Vibrio pools, which facilitates transmission of antibiotic resistance character through zoonotic spillover. Resistance against all of the commonly used antibiotics along with ARGs have been increased significantly (p<0.05) amongst the Vibrio strains isolated from raw and cooked seafood, which signifies the role of chitinous microenvironment in acquisition of ARGs. Noticeable increase of ‘sxt’ element amongst highly resistant pathogenic pool signifies that ‘sxt’ play a crucial role in acquisition of antibiotic resistance amongst environmental Vibrios by means of horizontal gene transfer. No significant variation in plasmid numbers have been found in between the highly resistant and non-resistant bacterial strains.
Abbreviations P: Penicillin derivatives; Q: Quinolones; CE: Cephalosporins; TE: Tetracyclins: AM: Aminoglycosides; CA: Carbapenem; CH: Chloramphenicol; SU: Sulfonamides ARGs: Antibiotic Resistance Genes
Reference 1. Verraes, Claire, et al. "Antimicrobial resistance in the food chain: a review." International journal of environmental research and public health 10.7 (2013): 2643-2669. 2. FAO (Food and Agriculture Organization of the United Nations). 2014. The state of world fisheries and aquaculture: 345 opportunities and challenges. Rome. [accessed 2018 Dec 16]. http://www.fao.org/3/a-i3720e.pdf. 3. Batabyal, Prasenjit, et al. "High prevalence of toxin producing enteropathogenic Vibrios among estuarine crab in Ganges delta of West Bengal, India." Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 26 (2014): 359. 4. Halder, Madhumanti, et al. "Environmental Vibrio cholerae non O1/non O139 from the Gangetic delta: a diarrhoeal disease purview." International Journal of Environmental Health Research 27.4 (2017): 241-251.
Acknowledgement The study was funded by Ministry of Earth Sciences, Govt. od India.