Şükrü KIRKAN, Elif Oya ORAL, Uğur PARIN, Hafize Tuğba YÜKSEL DOLGUN

Detection of Rhodococcus equi by PCR from foals and determination of antimicrobial susceptibility

Abstract: The scope of this research was determination of Rhodococcus equi isolates and antimicrobial susceptibilities in nasal swab specimen taken from animals between 0 and 6 months of age period. R. equi was identified from 10 isolates (11.1%) by automatic identification device. The verification of isolates were conducted by 16S ribosomal RNA PCR. VapA gene presence was detected in two (20%) isolates and multidrug resistance was identified against 11 antimicrobials. The antimicrobial resistance to macrolides and rifampin is arising worldwide since the excessive usage of the chemotherapeutics in various breeding farms just by evaluating monitoring outcomes. These results exhibit the presence of resistant strains among equine populations. The development of fast, economical and highly specific diagnostic technique against the threat of R. equi is essensial for better understanding of gene expressions related to resistance and development of effective immunization.Key words: Rhodococcus equi, foal pneumonia, PCR, vapA, antibacterial resistance

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1. Cohen ND, Chaffin MK, Martens RJ. How to prevent and control pneumonia caused by Rhodococcus equi at affected farms. In: Proceedings of the Annual Convention of the AAEP; Orlando, FL, USA; 2002. pp. 295-299.
2. Ainsworth DM. The impact of foal pneumonia on future racing performance. In: Proceedings Dubai International Equine Symposium; Dubai, UEA; 1997. pp. 241-243.
3. Vazquez-Boland JA. Focus Article: recent advances in Rhodococcus equi. AHT/BEVA/DEFRA Equine Quarterly Disease Surveillance Report 2010; 6 (4): 13-17.
4. Giguère S, Prescott JF. Clinical manifestations, diagnosis, treatment and prevention of Rhodococcus equi infections in foals. Veterinary Microbiology 1997; 56: 313-334. doi: 0.1016/ S0378-1135(97)00099-0
5. Prescott JF. Rhodococcus equi: an animal and human pathogen. Clinical Microbiology Reviews 1991; 4: 20-34. doi: 10.1128/ cmr.4.1.20
6. Takai S, Ohbushi S, Koike K, Tsubaki S, Oishi H et al. Prevalence of virulent Rhodococcus equi in isolates from soil and feces of horses from horse breeding farms with and without endemic infections. Journal of Clinical Microbiology 1991; 29: 2887- 2889. doi: 10.1128/JCM.29.12.2887-2889.1991
7. Bargen KV, Haas A. Molecular and infection biology of the horse pathogen Rhodococcus equi. FEMS Microbiology Reviews 2009; 33 (5): 870-891. doi: 10.1111/j.1574-6976.2009.00181.x
8. Vazquez-Boland JA, Prescott JF, Meijer WG, Leadon DP, Hines SA. Havemeyer workshop report: Rhodococcus equi comes of age. Equine Veterinary Journal 2009; 41: 93-95. doi: 10.2746/042516409X383564
9. Takai S. Restriction fragment length polymorphism of virulence plasmids in Rhodococcus. Journal of Clinical Microbiology 1999; 37 (10): 3417-3420.
10. Takai S, Hines SA, Sekizaki T, Nicholson VM, Alperin DA et al. DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103. Infection and Immunology 2000; 68 (12): 6840-6847. doi: 10.1128/ iai.68.12.6840-6847.2000
11. Letek M, Ocampo-Sosa AA, Sanders M, Fogarty U, Buckley T et al. Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids. Journal of Bacteriology 2008; 190 (17): 5797-5805. doi: 10.1128/JB.00468-08
12. Valero-Rello A, Hapeshi A, Anastasi E, Alvarez S, Scortti M et al. An invertron-like linear plasmid mediates intracellular survival and virulence in bovine isolates of Rhodococcus equi. Infection and Immunology 2015; 83 (7): 2725-2737. doi: 10.1128/IAI.00376-15
13. Ren J, Prescott JF. Analysis of virulence plasmid gene expression of intra-macrophage and in vitro grown Rhodococcus equi ATCC33701. Veterinary Microbiology 2003; 94: 167-182. doi: 10.1016/s0378-1135(03)00099-3
14. Byrne GA, Russell DA, Chen X, Meijer WG. Transcriptional regulation of the virR operon of the intracellular pathogen Rhodococcusequi. Journal of Bacteriology 2007; 189: 5082- 5089. doi: 10.1128/JB.00431-07
15. Ocampo-Sosa AA, Lewis DA, Navas J, Quigley F, Callejo R et al. Molecular epidemiology of Rhodococcus equi based on traA, vapA, and vapB virulence plasmid markers. Journal Infectious Diseases 2007; 196 (5): 763-769. doi: 10.1086/519688
16. Mir IA, Kumar B, Taku A, Bhardwaj RK, Bhat MA et al. Prevalence and antibiogram study of Rhodococcus equi in equines of Jammu and Kashmir, India. Journal Equine Science 2015; 26 (1) 21-24. doi: 10.1294/jes.26.21
17. Al-Graibawi MAA, Mohammed QA. Molecular Detection of vapA and vapB genes in Rhodococcus equi isolated from human and horses by multiplex PCR in Baghdad. Journal of Animal Health and Production 2016; 4 (1): 9-14. doi: 10.14737/journal. jahp/2016/4.1.9.14
18. Clinical and Laboratory Standards Institute (CLSI) National Committee for Clinical Laboratory Standards (M100- S15). Performance Standards for Antimicrobial Susceptibility Testing, Vol. 15th Informational Supplement. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2005.
19. Riesenberg A, Feßler AT, Erol E, Prenger-Berninghoff E, Stamm I et al. MICs of 32 antimicrobial agents for Rhodococcus equi isolates of animal origin. Journal Antimicrobial Chemotherapy 2014; 69 (4): 1045-1049. doi: 10.1093/jac/dkt460
20. Takai S, Ikeda T, Sasaki Y, Watanabe Y, Ozawa T et al. Identification of virulent Rhodococcusequi by amplification of gene coding 15- to 17- kilodalton antigens. Journal Clinical Microbiology 1995; 33: 1624-1627.
21. Cohen ND, Chaffin MK, Vandenplas ML, Edwards RF, Nevill M et al. Study of serum amyloid A concentrations as a means of achieving early diagnosis of Rhodococcus equi pneumonia. Equine Veterinary Journal 2005; 37: 212-216. doi: 10.2746/0425164054530704
22. Chaffin MK, Cohen ND, Martens RJ, Edwards RF, Nevill M. Foal-related risk factors associated with development of Rhodococcus equi pneumonia on farms with endemic infection. Journal of American Veterinary Medical Association 2003; 223: 1791-1799. doi: 10.2460/javma.2003.223.1791
23. Chaffin MK, Cohen ND, Martens RJ. Evaluation of equine breeding farm characteristics as risk factors for development of Rhodococcus equi pneumonia in foals. Journal of American Veterinary Medical Association 2003; 222: 467-475. doi: 10.2460/javma.2003.222.467
24. Hondalus MK, Mosser DM. Survival and replication of Rhodococcus equi in macrophages. Infection and Immunology 1994; 62: 4167-4175.
25. Lavoie JP, Fiset L, Laverty S. Review of 40 cases of lung abscesses in foals and adult horses. Equine Veterinary Journal 1994; 26: 348-352. doi: 10.1111/j.2042-3306.1994.tb04401.x
26. Falcon J, Smith BP, O’Brien TR. Clinical and radiological findings in Corynebacterium equi pneumonia of foals. Journal of American Veterinary Medicals Association 1985; 186: 593- 599.
27. Sellon DC, Besser TE, Vivrette SL. Comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of Rhodococcus equi pneumonia in foals. Journal Clinical Microbiology 2001; 39: 1289. doi: 10.1128/JCM.39.4.1289- 1293.2001
28. Hashikura S, Higuchi T, Taharaguchi S, Orita Y, Nanao Y et al. Evaluation of nasotracheal aspiration as a diagnostic tool for Rhodococcus equi pneumonia in foals. Equine Veterinary Journal 2000; 32 (6): 560-564. doi: 10.2746/042516400777584587
29. Woolcock JB, Farmer AMT, Mutimer MD. Selective Medium for Corynebacterium equi isolation. Journal Clinical Microbiology 1979; 9: 640-642.
30. Takai S, Fukunaga N, Ochiai S, Sakai T, Sasaki Y et al. Isolation of virulent and intermediately virulent Rhodococcus equi from soil and sand on parks and yards in Japan. Journal Medicine Science 1996; 58: 669-672. doi: 10.1292/jvms.58.669
31. Soedarmanto I, Oliveria R, Lämmler CH, Dürrling H. Identification and epidemiological relationship of Rhodococcus equi isolated from cases of Iymphadenitisin cattle. Zentralblatt für Bakteriologie 1997; 286: 457-467 (in German). doi: 10.1016/S0934-8840(97)80047-3
32. Fuhrmann C, Lämmler CH. Characterization of Rhodococcus equi isolates from horse and man. Berliner und Münchner Tierärztliche Wochenschrift 1997; 110: 54-59 (in German).
33. Vivrette SL, Sellon DC, Gibson DS. Clinical application of a polymerase chain reaction assay in the diagnosis of pneumonia caused by Rhodococcus equi in a horse. Journal of American Veterinary Medicine Association 2000; 217 (9): 1348-1350.
34. Takai S, Imai Y, Fukunaga N, Uchida Y, Kamisawa K et al. Identification of virulence associated antigens and plasmids in Rhodococcus equi from patients with AIDS. Journal of Infectious Diseases 1995; 172: 1306-1311. doi: 10.1093/ infdis/172.5.1306
35. Tan C, Prescott JF, Patterson MC, Nicholson VM. Molecular characterization of a lipid modified virulence associated protein of Rhodococcus equi and its potential in protective immunity. Canadian Journal of Veterinary Research 1995; 59: 51-59.
36. Monego F, Maboni F, Krewer C. Molecular characterization of Rhodococcus equi from horse-breeding farms by means of multiplex PCR for the vap gene family. Current Microbiology 2009; 58: 399. doi: 10.1007/s00284-009-9370-6
37. Takai S. Epidemiology of Rhodococcus equi infections: a review. Veterinary Microbiology 1997; 56: 167-176. doi: 10.1016/ S0378-1135(97)00085-0
38. Wada R, Kamada M, Anzai T, Nakanishi A, Kanemaru T et al. Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge. Veterinary Microbiology 1997; 16 (56): 301-312. doi: 10.1016/S0378-1135(97)00098-9
39. Cohen ND, O’Conor MS, Chaffin MK. Farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals. Journal of American Veterinary Medicals Association 2005; 226: 404-413. doi: 10.2460/javma.2005.226.404
40. Anastasi E, MacArthur I, Scortti M, Alvarez S, Giguère S et al. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biology and Evolution 2016; 23 (8): 3140-3148. doi: 10.1093/gbe/evw222
41. Erganiş O, Hadimli HH, Sayın Z, Sakmanoğlu A, Pınarkara Y et al. The effectiveness of anti-R. equi hyperimmune plasma against R. equi challenge in thoroughbred Arabian foals of mares vaccinated with R. equi vaccine. The Scientific World Journal 2014; 2014: 1-10. doi: 10.1155/2014/480732
42. Erganiş O, Hadimli HH, Sayın Z, Sakmanoğlu A, Pınarkara Y et al. Efficacy of experimental inactivated and live Rhodococcus equi vaccines for thoroughbred Arabian mares in mice. Turkish Journal of Veterinary and Animal Science 2015; 39: 295-301. doi: 10.3906/vet-1410-44
43. Tel OY, Keskin O, Erdenliğ Gürbilek S. Isolation of virulent Rhodococcus equi from Arabian horses in Şanlıurfa, Turkey. Turkish Journal of Veterinary and Animal Sciences 2016; 40: 417-420. doi: 10.3906/vet-1505-25
44. Özgür NY, İkiz S, Bağcıgil AF, Carioğlu B, Akay Ö et al. Studies on early diagnosis of Rhodococcus equi Infection by ELISA in foals from racehorse breeding studfarms in the Marmara Region. Turkish Journal of Veterinary and Animal Sciences 2002; 26: 1427-1434.
45. Nordmann P, Ronco E. In vitro antimicrobial susceptibility of Rhodococcus equi. Journal Antimicrobial Chemotherapy 1992; 29: 383-393. doi: 10.1093/jac/29.4.383
46. Asoh N, Watanabe H, Fines-Guyon M, Watanabe K, Oishi K et al. Emergence of rifampin-resistant Rhodococcus equi with several types of mutations in the rpoB gene among AIDS patients in northern Thailand. Journal of Clinical Microbiology 2003; 41 (6): 2337-2340. doi: 10.1128/JCM.41.6.2337-2340.2003
47. Giguère S, Lee E, Williams E, Cohen ND, Chaffin MK et al. Determination of the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in Rhodococcus equi isolates and treatment outcome in foals infected with antimicrobial-resistant isolates of R equi. Journal of American Veterinary Medicine Association 2010; 237: 74-81. doi: 10.2460/ javma.237.1.74
48. Chaffin K, Cohen ND, Martens RJ, O’Connor M, Benstein LR. Chemoprophylactic effects of gallium maltolate against Rhodococcus equi pneumonia among foals at endemic equine breeding farms. Proceedings of the 55th annual meeting of the American Association of Equine Practitioners 2009; 55: 36-37.
49. Coleman M, Kuskie K, Liu M, Chaffin K, Libal M et al. In vitro antimicrobial activity of gallium maltolate against virulent Rhodococcus equi. Veterinary Microbiology 2010; 146: 175- 178. doi: 10.1016/j.vetmic.2010.05.027
50. Burton AJ, Giguère S, Sturgill TL, Berghaus LJ, Slovis NM et al. Macrolide and rifampin resistant Rhodococcus equi on horse breeding farm, Kentucky, USA. Emerging Infectious Diseases 2013; 19: 282-285. doi: 10.3201/eid1902.121210
51. Riesenberg A, Feßler A, Frömke C, Kadlec K, Klarmann D et al. Harmonization of antimicrobial susceptibility testing by broth microdilution for Rhodococcus equi of animal origin. Journal of Antimicrobial Chemotherapy 2013; 68 (9): 2173-2175. doi: 10.1093/jac/dkt134
52. Carlson K, Kuskie K, Chaffin K, Libal M, Giguère S et al. Antimicrobial activity of tulathromycin and other antimicrobials against virulent Rhodococcus equi in vitro. Veterinary Therapeutics: Research in Applied Veterinary Medicine 2010; 11: E1-E9.
53. Giguère S, Lee EA, Guldbech KM, Berghaus LJ. In vitro synergy, pharmacodynamics, and post antibiotic effect of 11 antimicrobial agents against Rhodococcus equi. Veterinary Microbiology 2012; 160: 207-213. doi: 10.1016/j. vetmic.2012.05.03