Faruk PEHLIVANOGLU, Dilek OZTURK, Hulya TURUTOGLU

Carriage of Plasmidic AmpC Beta-Lactamase Producing Escherichia coli in Cattle and Sheep and Characterisation of the Isolates in Terms of Antibiogram Profiles, Phylogeny and Virulence

AmpC type beta-lactamase enzyme production by Escherichia coli confers resistance to penicillin and cephalosporins including oxyiminocephalosporin, cephamycin and aztreonam (variably). Screening of AmpC beta-lactamase determinants in both commensal and pathogenic E. coli isolates in livestock is important to reveal the resistance status of the bacteria. Therefore, we aimed to investigate the AmpC beta-lactamase producing E. coli isolates in cattle and sheep populations in Burdur, Turkey. The fecal samples were collected from 250 Holstein cows, older than 12 months of age and apparently healthy, and from 225 sheep from different breeds, older than 6 months of age and apparently healthy. After selective isolation and identification of the agent in coliform/E. coli selective medium supplemented with cefotaxime (2 µg/mL) or ceftazidim (2 µg/mL), the cefoxitin resistant E. coli isolates were determined by agar disc diffusion test (ADDT). Then, pAmpC beta-lactamase genes were determined by multiplex polimerase chain reaction (PCR) as gold standart test for pAmpC beta-lactamase producing E. coli. Finally, the isolates were characterized by PCR for phylogeny and Enterohemorrhagic/Shiga toxin producing E. coli (EHEC/STEC) related virulence genes. Totally 17 (6.8%) cattle fecal samples were found positive for pAmpC beta-lactamase producing E. coli. None of the sheep fecal samples yielded culture positive results for the bacteria of interest. Among the E. coli isolates, only CIT (origin, Citrobacter freundii) family pAmpC gene was found. The predominant phylogenetic group was found as group A and only eae gene was detected in only one E. coli isolate. Multidrug resistance (MDR) was observed in 7 (41.2%) isolates. Consequently, the present study revealed that pAmpC beta-lactamase producing E. coli, with MDR and low phylogenetic group diversity, exists in cattle population, but not in sheep population.

Sığır ve Koyunlarda Plasmidik AmpC Beta Laktamaz Üreten Escherichia coli Taşıyıcılığı ve İzolatların Antibiyogram Profilleri, Filogenetik ve Virulans Yönünden Karakterizasyonu

Escherichia coli tarafından AmpC beta laktamaz enzim üretimi penisilinlere, oksiimino sefalosporinler dahil tüm sefalosporinlere, sefamisinlere ve değişken olmakla birlikte aztreonama direnç sağlar. Çiftlik hayvanlarında komensal ve patojenik E. coli izolatlarında AmpC beta laktamazların taranması bakterilerdeki antibiyotik dirençliliğinin durumunu göstermesi açısından önemlidir. Bu nedenle, Burdur ilindeki sığır ve koyun popülasyonunda AmpC beta laktamaz üreten E. coli yaygınlığını ortaya çıkarmayı amaçladık. Bu çalışmada, 12 aylık yaştan daha büyük ve sağlıklı görünümdeki 250 Holştayn ırkı sığır ve 6 aylıktan daha büyük ve sağlıklı görünümdeki 225 değişik ırktan koyundan dışkı örneği toplandı. Sefotaksim (2 µg/ mL) veya seftazidim (2 µg/mL) ilave edilmiş Koliform/E. coli besi yerinde selektif izolasyon ve identifikasyon gerçekleştirildikten sonra, agar disk difüzyon testi (ADDT) ile sefoksitine dirençli E. coli izolatları belirlendi. Takiben pAmpC beta laktamaz genleri, pAmpC beta laktamaz üreten E. coli belirlenmesi için altın standart test olan multipleks polimeraz zincir reaksiyonu (PZR) ile belirlendi. Son olarak, izolatlar PZR ile filogenetik ve enterohemorajik/Siga toksin üreten E. coli (EHEC/STEC) virulans genleri açısından karakterize edildi. Toplam 17 (%6.8) sığır dışkı örneği pAmpC beta laktamaz üreten E. coli yönünden pozitif bulunurken koyun örneklerinin tümü negatif bulundu. Izolatlarda sadece CIT ailesi pAmpC geni tespit edildi. İzolatlarda en yaygın filogenetik grubun grup A olduğu ve sadece 1 izolatta eae virulans geninin olduğu tespit edildi. Çoklu antibiyotik dirençliliği 7 (%41.2) izolatta tespit edildi. Sonuç olarak, bu çalışma pAmpC beta laktamaz üreten E. coli’nin koyunlarda bulunmadığı ve sığırlarda çoklu antibiyotik dirençliliğine sahip ve az sayıda filogenetik çeşitlilikte var olduğu belirlendi.

PDF

___

1. Frere JM: Beta-lactamases and bacterial resistance to antibiotics. Mol Microbiol, 16, 385-395, 1995. DOI: 10.1111/j.1365-2958.1995.tb02404.x

2. Seiffert SN, Hilty M, Perreten V, Endimiani A: Extended-spectrum cephalosporin-resistant gram-negative organisms in livestock: An emerging problem for human health? Drug Resist Updat, 16, 22-45, 2013. DOI: 10.1016/j.drup.2012.12.001

3. Bush K, Jacoby GA: Updated functional classification of betalactamases. Antimicrob Agents Chemother 54 (3): 969-976, 2010. DOI: 10.1128/AAC.01009-09

4. Vardanyan R, Hruby V: Antibiotics. In, Sysnthesis of Best Seller Drugs. 573-643, Elsevier B.V., 2016.

5. Bush K, Jacoby GA, Medeiros AA: Functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother, 39 (6): 1211-1233, 1995. DOI: 10.1128/AAC.39.6.1211

6. Ambler RP: The structure of β-lactamases. Philos Trans R Soc Lond B Biol Sci, 289 (1036): 321-331, 1980. DOI: 10.1098/rstb.1980.0049

7. Jacoby GA: AmpC β-lactamases. Clin Microbiol Rev, 22, 161-182, 2009. DOI: 10.1128/CMR.00036-08

8. Pérez-Pérez FJ, Hanson ND: Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol, 40, 2153-2162, 2002. DOI: 10.1128/JCM.40.6.2153-2162.2002

9. Lerner A, Matthias T, Aminov R: Potential effects of horizontal gene exchange in the human gut. Front Immunol, 8:1630, 2017. DOI: 10.3389/ f immu.2017.01630

10. Bugarel M, Martin A, Fach P, Beutin L:Virulence gene profiling of enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains: A basis for molecular risk assessment of typical and atypical EPEC strains. BMC Microbiol, 11:142, 2011. DOI: 10.1186/1471-2180-11-142

11. Venegas-Vargas C, Henderson S, Khare A, Mosci RE, Lehnert JD, Singh P, Ouellette LM, Norby B, Funk JA, Rust S, Bartlett PC, Grooms D, Manning SD: Factors associated with Shiga toxin-producing Escherichia coli shedding by dairy and beef cattle. Appl Environ Microbiol, 82, 5049-5056, 2016. DOI: 10.1128/AEM.00829-16

12. Asai T, Masani K, Sato C, Hiki M, Usui U, Baba K, Ozawa M, Harada K, Aoki H, Sawada T: Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan. Acta Vet Scand, 53:52, 2011. DOI: 10.1186/1751-0147-53-52

13. Hille K, Fischer J, Falgenhauer L, Sharp H, Brenner GM, Kadlec K, Friese A, Schwarz S, Imirzalioglu C, Kietzmann M, Von Münchhausen C, Kreienbrock L: On the occurence of extended-spectrum-and AmpCbeta-lactamase-producing Escherichia coli in livestock: Results of selected European studies. Berl Munch Tierarztl Wochenschr, 127 (9-10): 403-411, 2014.

14. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control): The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2017. EFSA J, 17 (2):e05598, 2019. DOI: 10.2903/j.efsa.2019.5598

15. Tewari R, Mitra S, Ganaie F, Das S, Chakraborty A, Venugopal N, Shome R, Rahman H, Shome BR: Dissemination and characterisation of Escherichia coli producing extended-spectrum β-lactamases, AmpC β-lactamases and metallo-β-lactamases from livestock and poultry in Northeastern India: A molecular surveillance approach. J Glob Antimicrob Resist, 17, 209-215, 2019. DOI: 10.1016/j.jgar.2018.12.025

16. Pehlivanoglu F: Existence of plasmid mediated AmpC betalactamase-producing Escherichia coli isolates in healthy laying hens. Van Vet J, 28 (2): 63-67, 2017.

17. Aslantaş Ö, Elmacıoğlu S, Yılmaz EŞ: Prevalence and characterization of ESBL-and AmpC-producing Escherichia coli from cattle. Kafkas Univ Vet Fak Derg, 23, 63-67, 2017. DOI: 10.9775/kvfd.2016.15832

18. Gumus B, Celik B, Kahraman BB, Siğirci BD, Ak S: Determination of extended spectrum beta-lactamase (ESBL) and AmpC beta-lactamase producing Escherchia coli prevalence in faecal samples of healthy dogs and cats. Revue Med Vet, 168, 46-52, 2017

19. Aslantas O, Yilmaz ES: Prevalence and molecular characterization of extended-spectrum beta-lactamase (ESBL) and plasmidic AmpC betalactamase (pAmpC) producing Escherichia coli in dogs. J Vet Med Sci, 79, 1024-1030, 2017. DOI: 10.1292/jvms.16-0432

20. Thrusfield M: Surveys. In, Veterinary Epidemiology. 3rd ed., 228-242, Blackwell Publishing, Ames, Iowa, 2007.

21. Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G: The Enterobactericeae. In, Koneman EW (Ed): Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed., 211-302, Lippincott Williams and Wilkins, Philadelphia, 2006.

22. Wang G, Clark CG, Rodgers FG: Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 shiga toxin family by multiplex PCR. J Clin Microbiol, 40, 3613-3619, 2002. DOI: 10.1128/ JCM.40.10.3613-3619.2002

23. Pehlivanoglu F, Turutoglu H, Ozturk D: CTX-M-15-type extendedspectrum beta-lactamase-producing Escherichia coli as causative agent of bovine mastitis. Foodborne Pathog Dis, 13 (9): 477-482, 2016. DOI: 10.1089/fpd.2015.2114

24. CLSI (Clinical and Laboratory Standards Institute): Performance standards for antimicrobial susceptibility testing. 26th Informational Supplement, CLSI Document M100-S26, Pennsylvania, USA, 2016.

25. Polsfuss S, Bloemberg GV, Giger J, Meyer V, Böttger EC, Hombach M: Practical approach for reliable detection of AmpC beta-lactamase producing Enterobactericeae. J Clin Microbiol, 49, 2798-2803, 2011. DOI: 10.1128/JCM.00404-11

26. Clermont O, Bonacorsi S, Bingen E: Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol, 66, 4555-4558, 2000. DOI: 10.1128/aem.66.10.4555-4558.2000

27. Bai J, Paddock ZD, Shi X, Li S, An B, Nagaraja TG: Application of a multiplex PCR to detect the seven major shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Pathog Dis, 9, 541-548, 2012. DOI: 10.1089/fpd.2011.1082

28. Osek J: Development of a multiplex PCR approach for the identification of shiga toxin-producing Escherichia coli strains and their major virulence factor genes. J Appl Microbiol, 95, 1217-1225, 2003. DOI: 10.1046/j.1365-2672.2003.02091.x

29. Posse B, Zutter LD, Heyndrickx M, Herman L: Metabolic and genetic profiling of clinical O157 and non-O157 shiga-toxin-producing Escherichia coli. Res Microbiol, 158, 591-599, 2007. DOI: 10.1016/j.resmic.2007.06.001

30. Paton AW, Paton JC: Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA and saa. J Clin Microbiol, 40, 271-274, 2002. DOI: 10.1128/JCM.40.1.271-274.2002

31. CLSI (Clinical and Laboratory Standards Institute): Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, Approved standard, 3rd Ed., CLSI document M31-A3, Pennsylvania, USA, 2010.

32. CLSI (Clinical and Laboratory Standards Institute): Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, 2nd informational supplement, CLSI document VET01-S2, Pennsylvania, USA, 2013.

33. Higgins J, Hohn C, Hornor S, Frana M, Denver M, Joerger R: Genotyping of Escherichia coli from environmental and animal samples. J Microbiol Methods, 70, 227-235, 2007. DOI: 10.1016/j.mimet. 2007.04.009

34. Escobar-Páramo P, Le Menac’h A, Le Gall T, Amorin C, Gouriou S, Picard B, Skurnik D, Denamur E: Identification of forces shaping the commensal Escherichia coli genetic structure by comparing animal and human isolates. Environ Microbiol, 8, 1975-1984, 2006. DOI: 10.1111/ j.1462-2920.2006.01077.x

35. Pehlivanlar Onen S, Aslantas O, Yılmaz ES, Kurekci C: Prevalence of β-lactamase -producing Escherichia coli from retail meat in Turkey. J Food Sci, 80 (9): M2023-M2029, 2015. DOI: 10.1111/1750-3841.12984

36. Ozpinar H, Tekiner IH, Sarici B, Cakmak B, Gokalp F, Ozadam A: Phenotypic characterization of ESBL- and AmpC- type beta- lactamases in Enterobacteriaceae from chicken meat and dairy products. Ankara Univ Vet Fak Derg, 64 (4): 267-272, 2017.

37. Ozdikmenli Tepeli S, Demirel Zorba NN: Frequency of extendedspectrum β-lactamase (ESBL)- and AmpC β-lactamase-producing Enterobacteriaceae in a cheese production process. J Dairy Sci, 101, 29062914, 2018. DOI: 10.3168/jds.2017-13878

38. Smith JL, Fratamico PM, Gunther NW: Extraintestinal pathogenic Escherichia coli. Foodborne Pathog Dis, 4 (2): 134-163, 2007. DOI: 10.1089/ fpd.2007.0087

39. Chakraborty A, Saralaya V, Adhikari P, Shenoy S, Baliga S, Hegde A: Characterization of Escherichia coli phylogenetic groups associated with extraintestinal infections in South Indian population. Ann Med Health Sci Res, 5 (4): 241-246, 2015.

40. McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB: A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci USA, 92 (5): 1664-1668, 1995. DOI: 10.1073/ pnas.92.5.1664

41. Voets GM, Fluit AC, Scharringa J, Schapendonk C, Van Den Munckhof T, Leverstein-Van Hall MA, Stuart JC: Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands. Int J Food Microbiol, 167, 359-362, 2013. DOI: 10.1016/j.ijfoodmicro.2013.10.001

42. Huijbers PMC, Graat EAM, Haenen APJ, Van Santen MG, Van Essen-Zandbergen A, Mevius DJ, Van Duijkeren E, Van Hoek AHAM: Extended-spectrum and AmpC β-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: Prevalence, risk factors and molecular characteristics. J Antimicrob Chemother, 69 (10): 2669-2675, 2014. DOI: 10.1093/jac/dku178

43. Ljungquist O, Ljungquist D, Myrenås M, Ryden C, Finn M, Bengtsson B: Evidence of household transfer of ESBL-/pAmpCproducing Enterobacteriaceae between humans and dogs - A pilot study. Infect Ecol Epidemiol, 6:31514, 2016. DOI: 10.3402/iee.v6.31514