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Multilocus sequence typing of multidrug-resistant Salmonella strains circulating in poultry farms of Bangladesh

Abstract

Salmonella is one of the most important foodborne zoonotic pathogens, and becoming multidrug-resistant (MDR), which represents a serious public health concern worldwide. This study aimed to identify the circulating MDR strains of Salmonella through cutting edge molecular techniques including gene specific PCR, RAPD-PCR, ribosomal gene sequencing, and multilocus sequence types (MLST) in the poultry industry of Bangladesh. Two hundred Salmonella isolates were retrieved from 154 samples comprising droppings (n = 60), cloacal swabs (n = 60), feeds (n = 14), feeding water (n = 14), and handler’s swab (n = 6) from 14 commercial layer farms of Bangladesh. The isolates were confirmed as Salmonella through invA gene specific PCR, and further genotyping was done by RAPD-PCR, and 16S rRNA sequencing. The isolates were distributed into 18 different genotypes according to RAPD typing. The phylogenetic analysis identified three diverging phylogroups such as S. enterica Litchfield, S. enterica Enteritidis and S. enterica Kentucky with 11, 8, and 6 strains, respectively. The in vitro antibiogram profiling the Salmonella isolates through disc diffusion method using 13 commercially available antibiotics revealed highest resistance against doxycycline (91.5%) followed by tetracycline and ampicillin (86.0%, in each), and 72.0% isolates as MDR, being resistant to ≥ 5 antibiotics. The MLST typing was carried out based on the PCR amplification of seven housekeeping genes (aroC, hisD, hemD, purE, secA, thrA, and dnaN). MLST typing also revealed three sequence types (STs) such as ST11, ST198, and ST214 in these isolates, and eBURST analysis showed ST11 as the founder genotype. The three STs were highly resistant to tetracyclines and quinolone group of antibiotics, and all of the isolates harboring S. enterica Litchfield showed the highest resistance. Circulating common MLSTs with MDR properties in different farms confirmed the possibility of a common route of intra-farm transmission. We report for the first time of the association serovar Litchfield (ST11) in avian salmonellosis with MDR properties which is an urgent public health concern in Bangladesh.

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References

  1. 1.

    Chattaway MA, Langridge GC, Wain J (2021) Salmonella nomenclature in the genomic era: a time for change. Sci Rep 11:7494

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Sedeik ME, Nahed A, Awad AM, Elfeky SM, Abd El-Hack ME, Hussein EO, Alowaimer AN, Swelum AA (2019) Isolation, conventional and molecular characterization of Salmonella spp. from newly hatched broiler chicks. AMB Express 9(1):136

    PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Soultose N, Koidis P, Madden RH (2003) Prevalence of Listeria and Salmonellae in retail chicken in Northern Ireland. Letter in Applied Microbiology 37(5):421–423

    Article  Google Scholar 

  4. 4.

    Helms M, Vastrup P, Gerner-Smidt P, Mølbak K (2002) Excess mortality associated with antimicrobial drug-resistant Salmonella Typhimurium. Emerg Infect Dis 8(5):490

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Li R, Lai J, Wang Y, Liu S, Li Y, Liu K, Wu C (2013) Prevalence and characterization of Salmonella species isolated from pigs, ducks and chickens in Sichuan Province. China International Journal of Food Microbiology 163(1):14–18

    PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Nair VT, D., Venkitanarayanan, K. and Kollanoor Johny, A. (2018) Antibiotic-resistant Salmonella in the food supply and the potential role of antibiotic alternatives for control. Foods 7(10):167

    CAS  Article  Google Scholar 

  7. 7.

    Baratto CM, Gelinski JMLN, Bernardi AZ, Marafon A, Braun F (2012) Potential use of molecular-typing methods for the identification and characterization of Salmonella enterica serotypes isolated in the poultry production chain. Braz J Poultry Sci 14(3):173–179

    Article  Google Scholar 

  8. 8.

    Kotetishvili M, Stine OC, Kreger A, Morris JJG, Sulakvelidze A (2002) Multilocus sequence typing for characterization of clinical and environmental Salmonella strains. J Clin Microbiol 40(5):1626–1635

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Achtman M, Wain J, Weill FX, Nair S, Zhou Z, Sangal V, Dougan G (2012) Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog 8(6):e1002776

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Prabakaran R (2003) Good practices in planning and management of integrated commercial poultry production in South Asia Food and Agricultural Organization of the United Nations. Rome 159, 71–82 

  11. 11.

    Antunes P, Mourão J, Campos J, Peixe L (2016) Salmonellosis: the role of poultry meat. Clin Microbiol Infect 22(2):110–121

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Foley SL, Johnson TJ, Ricke SC, Nayak R, Danzeisen J (2013) Salmonella pathogenicity and host adaptation in chicken-associated serovars. Microbiol Mol Biol Rev 77(4):582–607

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 13.

    Mendonça EP, de Melo RT, Nalevaiko PC, Monteiro GP, Fonseca BB, Galvão NN, Giombelli A, Rossi DA (2019) Spread of the serotypes and antimicrobial resistance in strains of Salmonella spp. isolated from broiler. Braz J Microbiol: [publication of the Brazilian Society for Microbiology] 50(2):515–522. https://doi.org/10.1007/s42770-019-00054-w

    CAS  Article  Google Scholar 

  14. 14.

    Barua H, Biswas PK, Talukder KA, Olsen KE, Christensen JP (2014) Poultry as a possible source of non-typhoidal Salmonella enterica serovars in humans in Bangladesh. Vet Microbiol 168(2–4):372–380

    PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Le Hello S, Hendriksen RS, Doublet B, Fisher I, Nielsen EM, Whichard JM et al (2011) International spread of an epidemic population of Salmonella enterica serotype Kentucky ST198 resistant to ciprofloxacin. J Infect Dis 204(5):675–684

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  16. 16.

    Andrews WH Jacobson A Hammack (2014) BAM Chapter 5: Salmonella, p.5.01–05.020. In: Food and Drug Administration (ed) Bacteriological analytical manual, 8th ed. AOAC International, Gaithersburg

  17. 17.

    Buchanan RE, Gibbons NE (1949) Bergey’s manual of determinative bacteriology. Williams & Wilkins

  18. 18.

    Guibourdenche M, Roggentin P, Mikoleit M, Fields PI, Bockemühl J, Grimont PA et al (2010) Supplement 2003–2007 (No. 47) to the White-Kauffmann-Le Minor scheme . Res Microbiol 161(1):26–29

    PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Saha O, Hoque MN, Islam OK, Rahaman M, Sultana M, Hossain MA (2020) Multidrug-Resistant avian pathogenic Escherichia coli strains and association of their virulence genes in Bangladesh. Microorganisms 8(8):1135

    CAS  PubMed Central  Article  Google Scholar 

  20. 20.

    Hoque MN, Istiaq A, Clement RA, Sultana M, Crandall KA, Siddiki AZ, Hossain MA (2019) Metagenomic deep sequencing reveals association of microbiome signature with functional biases in bovine mastitis. Sci Rep 9(1):1–14

    Google Scholar 

  21. 21.

    Nandi SP, Sultana M, Hossain MA (2013) Prevalence and characterization of multidrug-resistant zoonotic Enterobacterspp. in poultry of Bangladesh. Foodborne Pathog Dis 10(5):420–427

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galan JE, Ginocchio C, Curtiss R, Gyles CL (1992) Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 6(4):271–279

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Hejazi A, Keane CT, Falkiner FR (1997) The use of RAPD-PCR as a typing method for Serratia marcescens. J Med Microbiol 46(11):913–919

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    CLSI (2020) Performance Standards for Antimicrobial Susceptibility Testing. 30th Ed CLSI Supplement M100. Wayne, PA: Clinical and laboratory Standards Institute 2020 (https://clsi.org/media/3481/m100ed30_sample.pdf)

  26. 26.

    Hoque MN, Das ZC, Rahman ANMA, Haider MG, Islam MA (2018) Molecular characterization of Staphylococcus aureus strains in bovine mastitis milk in Bangladesh. Int J Vet Sci Med 6(1):53–60

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Turner KM, Hanage WP, Fraser C, Connor TR, Spratt BG (2007) Assessing the reliability of eBURST using simulated populations with known ancestry. BMC Microbiol 7(1):1–14

    Article  CAS  Google Scholar 

  28. 28.

    Francisco AP, Bugalho M, Ramirez M, Carriço JA (2009) Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach. BMC Bioinformatics 10(1):152

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  29. 29.

    Huson DH, Bryant D (2005) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23(2):254–267

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  30. 30.

    Hoque MN, Istiaq A, Clement RA, Gibson KM, Saha O, Islam OK, Hossain MA (2020) Insights into the resistome of bovine clinical mastitis microbiome, a key factor in disease complication. Front Microbiol 11:860

    PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Magiorakos AP, Srinivasan A, Carey RB, Carmeliy F, M.E., Giske, C.G., and Paterson, D.L. (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18(3):268–281

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    World Organization for Animal Health (O.I.E.) (2015) World Animal Health Information System. Available from: <Available from: http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Diseasetimelines >. Accessed: Jan. 15, 2015

  33. 33.

    Threlfall EJ, Wain J, Peters T, Lane C, de Pinna E, Little CL, Davies RH (2014) Egg-borne infections of humans with Salmonella: not only an S Enteritidis Problem. Worlds Poult Sci J 70(1):15–26

    Article  Google Scholar 

  34. 34.

    Borges KA, Furian TQ, Souza SND, Salle CTP, Moraes HLDS, Nascimento VPD (2019) Antimicrobial resistance and molecular characterization of Salmonella enterica serotypes isolated from poultry sources in Brazil. Braz J Poult Sci 21(1):1–6

    Article  Google Scholar 

  35. 35.

    Suresh T, Hatha AAM, Sreenivasan D, Sangeetha N, Lashamanaperumalsamy P (2006) Prevalence and antimicrobial resistance of Salmonella enteritidis and other Salmonellas in the eggs and egg-storing trays from retails markets of Coimbatore. South India Food Microbiol 23(3):294–299

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Petrovic R, Chai LC, Usha MR, Lee HY, Fatimah AB, Farinazleen MG, Son R (2011) Characterization of Salmonella enterica isolated from street food and clinical samples in Malaysia. Asian Food J 14(3):161–173

    Google Scholar 

  37. 37.

    Alam SB, Mahmud M, Akter R, Hasan M, Sobur A, Nazir KHM, Noreddin A, Rahman T, El Zowalaty ME, Rahman M (2020) Molecular detection of multidrug resistant Salmonella Species isolated from broiler farm in Bangladesh. Pathogens 9(3):201

    CAS  PubMed Central  Article  Google Scholar 

  38. 38.

    Islam MM, Islam MN, Sharifuzzaman FM, Rahman MA, Sharifuzzaman JU, Sarker EH, Shahiduzzaman M, Mostofa M, Sharifuzzaman MM (2014) Isolation and identification of Escherichia coli and Salmonella from poultry litter and feed. Int J Nat Soc Sci 1(1):1–7

    Google Scholar 

  39. 39.

    Shanmugasamy M, Velayutham T, Rajeswar J (2011) Inv A gene specific PCR for detection of Salmonella from broilers. Vet World 4(12):562

    Article  Google Scholar 

  40. 40.

    Henton MM, Eagar HA, Swan GE, Van Vuuren M (2011) Antibiotic management and resistance in livestock production. SAMJ: South Afr Med J 101(8):583–586

    CAS  Google Scholar 

  41. 41.

    Parvej MS, Nazir KNH, Rahman MB, Jahan M, Khan MFR, Rahman M (2016) Prevalence and characterization of multi-drug resistant Salmonella Enterica serovar Gallinarum biovar Pullorum and Gallinarum from chicken. Veterinary World 9(1):65

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Aditya A (2015) Drug resistant Salmonella in broiler chicken sold at local market in Bangladesh and its public health significance. Afr J Biotech 14(43):2995–3000

    CAS  Article  Google Scholar 

  43. 43.

    Samanta I, Joardar SN, Das PK, Sar TK, Bandyopadhyay S, Dutta TK, Sarkar U (2014) Prevalence and antibiotic resistance profiles of Salmonella serotypes isolated from backyard poultry flocks in West Bengal. India Journal of Applied Poultry Research 23(3):536–545

    CAS  Article  Google Scholar 

  44. 44.

    Patel SR, Bharti S, Pratap CB, Nath G (2017) Drug Resistance Pattern in the Recent Isolates of Salmonella Typhi with Special Reference to Cephalosporins and Azithromycin in the Gangetic Plain. J Clin Diagn Res 11:DM01–DM03

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Hooda Y Sajib MS Rahman H Luby SP Bondy-Denomy J Santosham M, ... Saha S (2019) Molecular mechanism of azithromycin resistance among typhoidal Salmonella strains in Bangladesh identified through passive pediatric surveillance. PLoS Neglected Tropical Diseases, 13(11), e0007868

  46. 46.

    Sabat AJ, Budimir A, Nashev D, Sá-Leão R, van Dijl JM, Laurent F et al (2013) Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveill 18(4):20380

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Campioni F, Pitondo-Silva A, Bergamini AM, Falcão JP (2015) Comparison of four molecular methods to type Salmonella Enteritidis strains. APMIS 123(5):422–426

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48.

    Torpdahl M, Skov MN, Sandvang D, Baggesen DL (2005) Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism. J Microbiol Methods 63(2):173–184

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Howe K, Salehi S, Hartford Bailey R, Brooks JP, Wills R, Lawrence ML, Karsi A (2017) Supplemental invasion of Salmonella from the perspective of Salmonella enterica serovars Kentucky and Typhimurium. BMC Microbiol 17(1):88

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. 50.

    Xu Q, Yin Y, Liu D, Zhang J, Wu Q, Tian P, Shi X, Wang D (2018) Prevalence and characterization of Salmonella serovars isolated from farm products in Shanhai. Food Control 85:269–275

    Article  Google Scholar 

  51. 51.

    Noda T, Murakami K, Asai T, Etoh Y, Ishihara T, Kuroki T et al (2011) Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004. Acta Vet Scand 53(1):38

    PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Doublet B, Praud K, Bertrand S, Collard JM, Weill FX, Cloeckaert A (2008) Novel insertion sequence- and transposon-mediated genetic rearrangements in genomic island SGI1 of Salmonella enterica serovar Kentucky. Antimicrob Agents Chemother 52(10):3745–3754

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Sultana M, Bilkis R, Diba F, Hossain MA (2014) Predominance of Multidrug Resistant Zoonotic Salmonella Enteritidis Genotypes in Poultry of Bangladesh. J Poult Sci 51(4):424–434

    CAS  Article  Google Scholar 

  54. 54.

    Orji MU, Onuigbo HC, Mbata TI (2005) Isolation of Salmonella from poultry droppings and other environmental sources in Awka. Nigeria International Journal of Infectious Diseases 9(2):86–89

    PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    Hoque MN, Istiaq A, Rahman MS, Islam MR, Anwar A, Siddiki AZ, Sultana M, Crandall KA, Hossain MA (2020) Microbiome dynamics and genomic determinants of bovine mastitis. Genomics 112(6):5188–5203

    CAS  PubMed  Article  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to acknowledge Bangladesh Academy of Science–United States Department of Agriculture (BAS–USDA) (Grant no: BAS -USDA PALS DU LSc-34) for supporting the project and a PhD student. We would like to further acknowledge University Grants Commission (UGC), Ministry of Science and Technology, Bangladesh, for supporting reagents and equipment.

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KFS and OS carried out the studies (sampling, sequencing, molecular, and data analysis) and participated in drafting the manuscript. MNH visualized figures, interpreted data and results, critically reviewed and edited the manuscript. MS and MAH developed the hypothesis, supervised the whole work, and helped to prepare and critically revise the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Munawar Sultana or M. Anwar Hossain.

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Ethical approval was granted from the Ethics Committee of the Faculty of Biological Sciences, University of Dhaka, Bangladesh who has approved the procedure under the Reference 71/Biol.Scs./2018-2019.

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The authors declare that they have no competing interests.

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Sultana, K.F., Saha, O., Hoque, M.N. et al. Multilocus sequence typing of multidrug-resistant Salmonella strains circulating in poultry farms of Bangladesh. Braz J Microbiol (2021). https://doi.org/10.1007/s42770-021-00577-1

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Keywords

  • Salmonella
  • MLST
  • MDR
  • Poultry farms
  • Bangladesh
  • Khandokar Fahmida Sultana
  • OtunSaha
  • And M. Nazmul Hoque contributed equally