phoPQ carrying Salmonella in bile of cattle

Abstract

The objectives of this study were to determine the prevalence of Salmonella in bile of cattle by comparing immunomagnetic separation (IMS) based cultivation and conventional cultivation techniques, to verify the isolates by the detection of oriC gene, to determine phoPQ gene by PCR, to identify the isolates by serotyping and to find out the antibiotic resistance profiles using disc diffusion method. A total of 188 cattle bile samples were collected from two slaughterhouses near Ankara between MaySeptember 2007. In three (1.6 %) of the samples Salmonella spp. were detected by IMS based cultivation technique and two (1.1 %) of the samples with conventional cultivation technique. It was shown that IMS based cultivation technique is more sensitive for the detection of Salmonella in bile of cattle. From the each contaminated samples three colonies were picked and coded (A, B and C). Isolates were verified by detection of oriC gene and phoPQ gene was detected using PCR. According to serotyping; two of them (A and B) were found to be S. Dublin and one (C) S. Bredeney. Disc diffusion method indicated that, S. Bredeney was resistant to ampicillin, cephalothin, tetracycline, nalidixic acid and sulphamethoxazole. All S. Dublin and S. Bredeney isolates were intermediately resistant to streptomycin, also S. Dublin to sulphamethoxazole and S. Bredeney to amoxicillin/clavulanic acid and cephazolin. In conclusion bile can be a site of Salmonella in cattle and all the isolates carried phoPQ gene that may play a significant role in the survival of Salmonella spp. in bile

Keywords

• Antibiotic resistance
• bile
• cattle
• IMS
• PCR
• PhoPQ
• Salmonella
• serotype

Sığır safrasında phoPQ taşıyan Salmonell

Abstract

Bu çalışmada, sığır safra örneklerinde Salmonella varlığının klasik kültür ve IMS (immuno-manyetik separasyon) yöntemleriyle tespit edilmesi, elde edilen izolatların PCR yöntemiyle oriC geni ile doğrulanması, phoPQ geninin belirlenmesi, izolatların serotiplendirilmesi ve antibiyotik direnç profillerinin belirlenmesi amaçlanmıştır. Bu amaçla 2007 yılı Mayıs-Eylül ayları arasında Ankara bölgesinde yer alan iki mezbahadan 188 sığır safra örneği toplanmıştır. Örneklerden 3 tanesi (% 1.6) IMS ve 2 tanesi (% 1.1) klasik kültür yöntemiyle Salmonella spp. olarak tespit edilmiştir. Sonuçlar ile sığır safrasında Salmonella’nın belirlenmesinde IMS bazlı kültür tekniğinin klasik kültür tekniğine gore daha duyarlı olduğu görülmektedir. Salmonella tespit edilen örneklerden 3’er koloni alınarak A, B ve C şeklinde kodlanmıştır. İzolatlar PCR yöntemiyle oriC ve phoPQ genleri yönünden analiz edilmiştir. Serotiplendirme işlemi sonucunda elde edilen izolatların ikisinin (A ve B) S. Dublin, birinin (C) S. Bredeney olduğu tespit edilmiştir. Disk difüzyon yöntemiyle S. Bredeney izolatının ampisilin, sefalotin, tetrasiklin, nalidiksik asit ve sülfametaksazole karşı dirençli olduğu tespit edilmiştir. Bütün izolatların (her iki S. Dublin ve S. Bredeney) streptomisine karşı, S. Dublin’in sülfametaksazole ve S. Bredeney’in amoksisilin/klavulanik asite karşı orta düzeyde dirençli olduğu belirlenmiştir. Sonuç olarak, sığır safralarının Salmonella yönünden önemli bir kaynak olabileceği ve izolatların taşıdığı phoPQ geninin Salmonella türlerinin safra koşullarında canlı kalmasında etkin rol oyanayabileceği belirlenmiştir

Keywords

• Antibiyotik direnç
• IMS
• PCR
• phoPQ
• safra
• Salmonella
• serotip
• sığır

References

1. Anon (1991): Dynabeads anti-Salmonella: For rapid selective enrichment of Salmonella. Dynal, A.S. Oslo, Norway, 1-2 (Manufacturer information).
2. Anon (2002): Microbiology of foods and animal feeding staff-horizontal method for the detection of Salmonella spp. International Standart (ISO, 6579).
3. Anon (2006): Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement. M100-S16 Vol.26 No.3.
4. Begley M, Gahan CGM, Hill C (2005): The interaction between bacteria and bile. FEMS Microbiol Rev, 29, 625- 651.
5. Buchwald DS, Blaser MJ (1984): A review of human salmonellosis. II. Duration of excretion following infection with nontyphi Salmonella. Rev Infect Dis, 6, 345-356.
6. Chandra M, Singh BR, Shankar H, Agarwal M, Agarwal RK, Sharma G, Babu N (2006): Study on prevalance of Salmonella infection in goats. Small Rum Res, 65, 24-30.
7. Cormican M, Delappe N, O’Hare C, Doran G, Morris D, Feeney G, Fanning S, Daly M, Fitzgerald M, Moorre J (2002): Salmonella enterica serotype Bredeney: Antimicrobial susceptibility and molecular diversity of isolates from Ireland and Northern Ireland. Appl Environ Microbiol, 68(1), 181-186.
8. Davis MA, Hancock DD, Besser TE, Daniels JB, Baker KNK, Call DR (2007): Antimicrobial resistance in Salmonella enterica serovar Dublin isolates from beef and dairy sources. Vet Microbiol, 119, 221-230.

9. Deng SX, Cheng AC, Wang MS, Cao P, Yan B, Yin NC, Cao SY, Zhang ZH (2008): Quantitative studies of the regular distribution pattern for Salmonella Enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction. World J Gastroenterol, 14(5), 782-789.
10. Dias FS, dos Santos LF, Franco RM, Nascimento ER (2010): Antimicrobial resistance in Salmonella spp. isolated from cattle gallbladder slaughtered in the South of the state of Rio de Janeiro. R Bras Ci Vet, 17 (3/4), 104- 107.
11. Duffy G, Cloak OM, O'Sullivan MG, Guillet A, Sheridan JJ, Blair IS, McDowell DA (1999): The incidence and antibiotic resistance profiles of Salmonella spp. on Irish retail meat products. Food Microbiol, 16, 623–631.
12. Erol I, Hildebrandt G, Kleer J, Yurtyeri A (1999): Kopplung von Immunomagnetischer separation und Polymerase-kettenreaktion zum Schnellachweis von Salmonellen in Hackfleisch und Geflügelinnereien. Berl Münch Tieraerztl Wschr, 112, 100-103.
13. Erol I, Hildebrandt G, Goncuoglu M, Ormanci FSB, Yurtyeri A, Kleer J, Kuplulu O (2009): Incidence and serotype distribution of Salmonella in spices retailed in Turkey. Fleischwirtschaft International, 24(6), 50-52.
14. Ertas HB, Ozbey A, Kilic A, Muz A (2003): Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction. J Vet Med B, 50, 294-297.
15. Fluit AC, Widjojoatmodjo MN, Box ATA, Torensma R, Verhoef J (1993): Rapid detection of Salmonella in poultry with the magnetic Immuno-Polymerase Chain Reaction assay. Appl Environ Microbiol, 59(5), 1342- 1346.
16. Gay JM, Rice DH, Steiger JH (1994): Prevalence of faecal Salmonella shedding by cull dairy cattle marketed in Washington State. J Food Prot, 57, 195-197.
17. Goncuoglu M, Erol I, Ayaz ND, Ormanci FSB, Kaspar CW (2010): Isolation and genomic characterization of Escherichia coli O157:H7 in bile of cattle. Ann Microbiol, 60, 293-297.
18. Gunn JS, Hohmann EL, Miller SI (1996): Transcriptional regulation of Salmonella virulence: a PhoQ periplasmic domain mutation results in increased net phosphotransfer to PhoP. J Bacteriol, 178, 6369–6373.
19. Gunn JS (2000): Mechanisms of bacterial resistance and response to bile. Microbes Infect, 2, 907–913.
20. Guo L, Lim K, Gunn JS, Bainbridge B, Darveau R, Hackett M, Miller SI (1997): Regulation of lipid A modifications by Salmonella Typhimurium virulence genes phoP-phoQ. Science, 276, 250–253.
21. Helms M, Vastrup P, Gerner-Smidt P, Mİlbak K (2003): Short and long term mortality associated with foodborne bacterial gastrointestinal infections: registry based study. Br Med J, 326, 357-361.
22. Jeong KC, Kang MY, Heimke C, Shere JA, Erol I, Kaspar CW (2007): Isolation of Escherichia coli O157:H7 from the gall bladder of inoculated and naturally-infected cattle. Vet Microbiol, 119, 339-345.
23. Lai CW, Chan RCY, Cheng AFB, Sung JY, Leung JWC (1992): Common bile duct stones: a cause of chronic salmonellosis. Am J Gastroenterol, 87, 1198–1199.
24. Lomborg SR, Agerholm JS, Jensen AL, Nielsen LR (2007): Effects of experimental immunosuppression in cattle with persistently high antibody levels to Salmonella Dublin lipopolysaccharide O-antigens. BMC Vet Res, 3, 17.
25. Popoff MY (2001): Antigenic formulas of the Salmonella serovars. WHO Colaborating Centre for Reference and Research on Salmonella, Institute Pasteur, France.
26. Soto SM, Guerra B, Del Cerro A, González-Hevia MA, Mendoza MC (2001): Outbreaks and sporadic cases of Salmonella serovar Panama studied by DNA 37. Widjojoadmodjo MN, Fluit AC, Torensma R, Keller fingerprinting and antimicrobial resistance. Int J Food Microbiol, 71, 35–43.
27. Stoffregen WC, Pohlenz JFL, Dean-Nystrom EA (2004): Escherichia coli O157:H7 in the gallbladders of experimentally infected calves. J Vet Diagn Investig, 16, 79-83.
28. Tennant SM, Diallo S, Levy H, Livio S, Sow SO, Tapia M, Fields PI, Mikoleit M, Tamboura B, Kotloff KL, Nataro JP, Galen JE, Levine MM (2010): Identification by PCR of non-typhoidal Salmonella enterica serovars associated with invasive infections among febrile patients in Mali. PLoS Negl Trop Dis, 4(3), e621.
29. Thanassi DGL, Cheng W, Nikaido H (1997): Active efflux of bile salts by Escherichia coli. J Bacteriol, 179, 2512–2518.
30. Tsolis RM, Young GM, Solnick JV, Baumler AJ (2008): From bench to bedside: Stealth of enteroinvasive pathogens. Nat Rev Microbiol, 6(12), 883−892.
31. Vaishnavi C, Singh S, Kochhar R, Bhasin D, Singh G, Singh K (2005): Prevalence of Salmonella enterica serovar Typhi in bile and stool of patients with biliary diseases and those requiring biliary drainage for other purposes. Jpn J Infect Dis, 58, 363-365.
32. Van Velkinburgh JC, Gunn JS (1999): PhoP-PhoQ regulated loci are required for enhanced bile resistance in Salmonella spp. Infect Immun, 67, 1614–1622.
33. Walker RA, Lindsay E, Woodward MJ, Ward LR, Threlfall EJ (2001): Variation in clonality and antibiotic- resistance genes among multiresistant Salmonella enterica serotype Typhimurium phage type U302 (MR U302) from humans, animals and foods. Microb Drug Resist, 7, 13–21.
34. Way JS, Josephson KL, Pillai SD, Abbaszadagan M, Gesba CP, Pepper IL (1993): Specific detection of Salmonella spp. by multiplex polymerase chain reaction. Appl Environ Microbiol, 59, 1473–1479.
35. Wegner HC, Aarestrup FM, Gerner-Smidt P, Bager F (1999): Transfer of antibiotic resistant bacteria from animals to man. Acta Vet Scand Suppl, 92, 51-57.