Environmental Science and Pollution Research

, Volume 25, Issue 7, pp 6228–6239 | Cite as

Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India

  • Onkar A. Naik
  • Ravindranath Shashidhar
  • Devashish Rath
  • Jayant R. Bandekar
  • Archana RathEmail author
Research Article


Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFU g−1. Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54–0.77. The fish microflora showed high prevalence of AR genes like bla TEM, Class I integron, tetA, aph(3′)-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX-M, dfr1, tetA, bla TEM, and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.


Metagenomic analysis Food-borne pathogens Multiple antibiotic resistance Antibiotic-resistant genes Integrons R-plasmid Horizontal gene transfer Marine fish 


Funding information

The present study was supported by the financial grant from the Department of Atomic Energy-Board of Research in Nuclear Sciences (DAE-BRNS), Sanction No. 2012/37B/50/BRNS.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11356_2017_945_MOESM1_ESM.pdf (287 kb)
ESM 1 (PDF 287 kb)
11356_2017_945_MOESM2_ESM.xlsx (19 kb)
ESM 2 (XLSX 18 kb)


  1. Aguilo-Ferretjans MM, Bosch R, Martin-Cardona C, Lalucat J, Nogales B (2008) Phylogenetic analysis of the composition of bacterial communities in human-exploited coastal environments from Mallorca Island (Spain). Syst Appl Microbiol 31(3):231–240. CrossRefGoogle Scholar
  2. Bharucha N (2017) Mumbai dumps 2,100mn litres of human waste in sea daily. The Times Of India. Accessed15 May 2017, 12:18 IST
  3. Carlson SA, Bolton LF, Briggs CE et al (1999) Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR. Mol Cell Probe 13:213–222Google Scholar
  4. Chandrasekaran S, Venkatesh B, Lalithakumari D (1998) Transfer and expression of a multiple antibiotic resistance plasmid in marine bacteria. Curr Microbiol 37(5):347–351. CrossRefGoogle Scholar
  5. Clinical and Laboratory Standards Institute (2008) Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Approved Standard, 3rd edn. CLSI document M31-A3. Clinical and Laboratory Standards Institute, WayneGoogle Scholar
  6. Costa D, Vinue L, Poeta P et al (2009) Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers. Vet Microbiol 138(3–4):339–344. CrossRefGoogle Scholar
  7. Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74(3):417–433. CrossRefGoogle Scholar
  8. DeSantis TZ, Hugenholtz P, Keller K, Brodie EL, Larsen N, Piceno YM, Phan R, Andersen GL (2006a) NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucleic Acids Res 34:W394–W399. CrossRefGoogle Scholar
  9. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006b) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72(7):5069–5072. CrossRefGoogle Scholar
  10. European Committee on Antimicrobial Susceptibility Testing (2017) Breakpoint tables for interpretation of MICs and zone diameters, version 5.0.
  11. Food and Agriculture Organization (FAO) of the United Nations (2016) The State of World Fisheries and Aquaculture 2016. Contributing to food security and nutrition for all. Rome. 200 pp. Available from:
  12. Gevers D, Hurys G, Swings J (2003) In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria. FEMS Microbiol Lett 225(1):125–130. CrossRefGoogle Scholar
  13. Gilbert JA, Steele JA, Caporaso JG et al (2012) Defining seasonal marine microbial community dynamics. ISME J 6:298–308CrossRefGoogle Scholar
  14. Han JE, Mohney LL, Tang KFJ, Pantoja CR, Lightner DV (2015) Plasmid mediated tetracycline resistance of Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (AHPND) in shrimps. Aquaculture Rep 2:17–21CrossRefGoogle Scholar
  15. He Y, Jin L, Sun F, Hu Q, Chen L (2016) Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China. Environ Sci Pollut Res 23(15):15033–15040. CrossRefGoogle Scholar
  16. Jeyasanta KI, Aiyamperumal V, Patterson J (2012) Prevalence of antibiotic resistant Escherichia coli in sea foods of Tuticorin coast, Southeastern India. Adv Biol Res 6(2):70–77Google Scholar
  17. Kelly KM, Chistoserdov AY (2001) Phylogenetic analysis of the succession of bacterial communities in the Great South Bay (Long Island). FEMS Microbiol Ecol 35(1):85–95. CrossRefGoogle Scholar
  18. Kotlarska E, Łuczkiewicz A, Pisowacka M, Burzyński A (2015) Antibiotic resistance and prevalence of class 1 and 2 integrons in Escherichia coli isolated from two wastewater treatment plants, and their receiving waters (Gulf of Gdansk, Baltic Sea, Poland). Environ Sci Pollut Res 22(3):2018–2030. CrossRefGoogle Scholar
  19. Kulkarni P (2005) The Marine Seafood Export Supply Chain in India: Current State and Influence of Import Requirements. Report by Consumer Unity and Trust Society (CUTS), International Institute for Sustainable Development, ManitobaGoogle Scholar
  20. Lalzampuia H, Dutta TK, Warjri I, Chandra R (2013) PCR-based detection of extended-spectrum β-lactamases (bla CTX-M-1 and bla TEM) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae isolated from pigs in North Eastern India (Mizoram). Indian J Microbiol 53(3):291–296. CrossRefGoogle Scholar
  21. Lozupone CA, Stombaugh J, Gonzalez A, Ackermann G, Wendel D, Vazquez-Baeza Y, Jansson JK, Gordon JI, Knight R (2013) Meta-analyses of studies of the human microbiota. Genome Res 23(10):1704–1714. CrossRefGoogle Scholar
  22. Maloo A, Fulke AB, Mulani N, Sukumaran S, Ram A (2017) Pathogenic multiple antimicrobial resistant Escherichia coli serotypes in recreational waters of Mumbai, India: a potential public health risk. Environ Sci Pollut Res 24(12):11504–11517. CrossRefGoogle Scholar
  23. Meervenne EV, Coillie EV, Kerckhof F-M, Devlieghere F, Herman L, De Gelder LSP, Top EM, Boon N (2012) Strain-specific transfer of antibiotic resistance from an environmental plasmid to foodborne pathogens. J BioMed Biotechnol Article ID 834598, 2012:1–8Google Scholar
  24. Modak A, Kumar V, Rao KVB (2014) Microbial quality assessment of broiler chicken meat and evaluation of antibiotic susceptibility profile of isolates from retail outlets of Vellore, Tamilnadu, India. Res J Pharm Biol Chem Sci 5:1195–1202Google Scholar
  25. Nagar V, Shashidhar R, Bandekar JR (2011) Prevalence, characterization, and antimicrobial resistance of Aeromonas strains from various retail food products in Mumbai, India. J Food Sci 76(7):M486–M492. CrossRefGoogle Scholar
  26. Nagar V, Shashidhar R, Bandekar JR (2013) Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis. World J Microbiol Biotechnol 29(4):745–752. CrossRefGoogle Scholar
  27. Ng KH, Samuel L, Kathleen MM, Leong SS, Felecia C (2014) Distribution and prevalence of chloramphenicol-resistance gene in Escherichia coli isolated from aquaculture and other environment. Int Food Res J 21:1321–1325Google Scholar
  28. Nordmann P, Poirel L (2005) Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother 56(3):463–469. CrossRefGoogle Scholar
  29. Nossa CW, Oberdorf WE, Yang L, Aas JA, Paster BJ, Desantis TZ, Brodie EL, Malamud D, Poles MA, Pei Z (2010) Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World J Gastroenterol 16(33):4135–4144. CrossRefGoogle Scholar
  30. Novotny L, Dvorska L, Lorencova A, Beran V, Pavlik I (2004) Fish: a potential source of bacterial pathogens for human beings. Vet Med –Czech 49:343–358CrossRefGoogle Scholar
  31. O’Hara CM, Brenner FW, Miller JM (2000) Classification, identification, and clinical significance of Proteus, Providencia and Morganella. Clin Microbiol Rev 13(4):534–546. CrossRefGoogle Scholar
  32. Pitout JDD, Church DL (2004) Emerging gram-negative enteric infections. Clin Lab Med 24(3):605–626. CrossRefGoogle Scholar
  33. Rajini RDB, Mahadevan A (1992) Plasmid mediated metal and antibiotic resistance in marine Pseudomonas. Biometals 5(2):73–80CrossRefGoogle Scholar
  34. Rit K, Saha R (2012) Multidrug-resistant Acinetobacter infection and their susceptibility patterns in a tertiary care hospital. Niger Med J 53:126–128CrossRefGoogle Scholar
  35. Ryu SH, Park SG, Choi SM, Hwang YO, Ham HJ, Kim SU, Lee YK, Kim MS, Park GY, Kim KS, Chae YZ (2012) Antimicrobial resistance and resistance genes in Escherichia coli strains isolated from commercial fish and seafood. Int J Food Microbiol 152(1-2):14–18. CrossRefGoogle Scholar
  36. Schmidt AS, Bruun MS, Larsen JL, Dalsgaard I (2001) Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas. J Antimicrob Chemother 47(6):735–743. CrossRefGoogle Scholar
  37. Schwarz S, Kehrenberg C, Doublet B, Cloeckaert A (2004) Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol Rev 28(5):519–542. CrossRefGoogle Scholar
  38. Sergeant MJ, Constantinidou C, Cogan TA, Bedford MR, Penn CW, Pallen MJ (2014) Extensive microbial and functional diversity within the chicken cecal microbiome. PLoS One 9(3):e91941. CrossRefGoogle Scholar
  39. Serrano HP (2005) Responsible use of antibiotics in aquaculture. FAO Fisheries Technical Paper No. 469, 97 pagesGoogle Scholar
  40. Smriga S, Sandin SA, Azam F (2010) Abundance, diversity, and activity of microbial assemblages associated with coral reef fish guts and faeces. FEMS Microbiol Ecol 73(1):31–42. Google Scholar
  41. Sullam KE, Essinger SD, Lozupone CA et al (2012) Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol Ecol 21(13):3363–3378.
  42. Sung JY, Oh JE (2014) Distribution and characterization of integrons in Enterobacteriaceae isolates from chickens in Korea. J Microbiol Biotechnol 24(7):1008–1013. CrossRefGoogle Scholar
  43. Sung JY, Koo SH, Kwon KC (2014) Epidemiological characterizations of class 1 integrons from multidrug-resistant Acinetobacter isolates in Daejeon, Korea. Ann Lab Med 34(4):293–299. CrossRefGoogle Scholar
  44. Verner-Jeffreys DW, Welch TJ, Schwarz T, Pond MJ, Woodward MJ, Haig SJ, Rimmer GSE, Roberts E, Morrison V, Baker-Austin C (2009) High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water. PLoS One 4(12):e8388. CrossRefGoogle Scholar
  45. Visnuvinayagam S, Joseph TC, Murugadas V, Chakrabarti R, Lalitha KV (2014) Status on methicillin resistant and multiple drug resistant Staphylococcus aureus in fishes of Cochin and Mumbai coast, India. J Environ Biol 36:571–575Google Scholar
  46. Zaniani FR, Meshkat Z, Nasab MN et al (2012) The prevalence of TEM and SHV genes among extended-spectrum beta-lactamases producing Escherichia coli and Klebsiella pneumoniae. Iran J Basic Med Sci 15(1):654–660Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Onkar A. Naik
    • 1
  • Ravindranath Shashidhar
    • 2
  • Devashish Rath
    • 3
  • Jayant R. Bandekar
    • 2
  • Archana Rath
    • 1
    Email author
  1. 1.Department of BiotechnologyUniversity of MumbaiMumbaiIndia
  2. 2.Food Technology DivisionBhabha Atomic Research CentreMumbaiIndia
  3. 3.Molecular Biology DivisionBhabha Atomic Research CentreMumbaiIndia

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