Amjad Islam AQIB, Abdullah Saghir AHMAD, Shahid Hussain FAROOQI, Aftab Ahmad ANJUM, Muhammad IJAZ, Aneela Zameer DURRANI, Kashif HUSSAIN

Molecular Typing of Clostridium perfringens Toxins (α, β, ε, ι) and Type ‘A’ Multidrug Resistance Profile in Diarrheic Goats in Pakistan

Clostridium perfringens (C. perfringens) hayvanlarda enterotoksemi olarak adlandırılan hastalığına neden olur. Bu bakteri hayvanların gastrointestinal sisteminin normal florasında yer alır ve uygun şartlar oluştuğunda fırsat bularak koloni sayısını artırarak ve aynı zamanda toksinlerini salarak zararlı hale gelir. Bu çalışma Pakistan'da ishalli keçilerde Clostridium perfringens'in (?, ?, ?, ?) moleküler tiplendirilmesini ve tip 'A' çoklu ilaç direnç profilinin belirlenmesini amaçlamaktadır. İshalli dışkı örnekleri (n=192) keçilerden toplandı ve örneklerin %80.73'ü (155/192) kültürde üreme ve PCR analizleri ile C. perfringens pozitif olarak belirlendi. Pozitif örneklerin %33.55'inde (52/155) artmış C. perfringens miktarları (>107 CFU/g) tespit edildi. Pozitif örneklerin %66.45'inde (103/155) bakteri miktarları normal aralıklarda (104-107 CFU/g) gözlemlendi. Moleküler tespit amacıyla PCR kullanılarak C. perfringens'in cpa (?), cpb (?), etx (?) ve iap (?) toksin genleri hedef alındı. Pozitif örnekler arasında izolatların %78.06'sı tip 'A', %5.16'sı tip 'B', %3.23'ü tip 'C' ve %13.55'i tip 'D' C. perfringens olarak belirlendi. İzolatların hiçbiri iap toksin geni (tip 'E') için pozitif değildi. C. perfringens tip 'A'ya in-vitro antibiyotik sensitivite testi uygulandı. Bakterilerin penisilin, siprofloksasin ve seftriaksona karşı duyarlı oldukları, basitrasin, amoksisilin ve ampilisine karşı ise en az duyarlı oldukları tespi edildi. Bu çalışma, Pakistan'da C. perfringens tip 'A'nın keçiler arasında oldukça yaygın olduğunu ve klinik enterotoksemi vakalarında penisilin, siprofloksasin ve seftriaksonun etkili bir şekilde kullanılabileceğini göstermiştir

Pakistan’da İshalli Keçilerde Clostridium perfringens Toksinlerinin (α, β, ε, ι) Moleküler Tiplendirilmesi ve Tip ‘A’ Çoklu İlaç Direnç Profili

Clostridium perfringens (C. perfringens) causes disease, generally, named as enterotoxemia in the animals. This bacterium is a normal inhabitant in gastro-intestinal tract (GIT) of animals and become harmful by increasing its colony counts as well as toxin liberation whenever gets an opportunity of favorable conditions. This study focused molecular typing of C. perfringens (α, β, ε, ι toxins) and type ‘A’ multidrug resistance profile in diarrheic goats in Pakistan. Diarrheic fecal samples (n=192) were collected from goats and 80.73% (155/192) of the samples were found positive for C. perfringens on the basis of culture growth and PCR. Elevated C. perfringens counts (>107 CFU/g) were recorded in 33.55% (52/155) of positive samples, while, 66.45% (103/155) of the positive sample appeared in normal range of bacterial counts (104-107 CFU/g). Molecular detection was carried out by targeting specific toxin genes i.e. cpa (α), cpb (β), etx (ε) and iap (ι) of C. perfringens using PCR. Among the positive samples, 78.06% of the isolates were found as type ‘A’, 5.16% isolates were type ‘B’, 3.23% isolates were type ‘C’ while, 13.55% of the positive samples were type ‘D’ C. perfringens. None of the isolates was found positive for iap toxin gene (type ‘E’). C. perfringens type ‘A’ was subjected to in-vitro antibiotic sensitivity test. Penicillin, ciprofloxacin and ceftriaxone were found sensitive while bacitracin, amoxicillin and ampicillin were found least sensitive antibiotics. This study concludes that C. perfringens type ‘A’ is highly prevalent among goats in Pakistan and clinical cases of enterotoxemia can be effectively dealt with penicillin, ciprofloxacin and ceftriaxone antibiotics

PDF

___

1. Li J, Adams V, Bannam TL, Miyamoto K, Garcia JP, Uzal FA, Rood JI, McClane BA: Toxin plasmids of Clostridium perfringens. Microbiol Mol Bio. Rev, 77 (2): 208-233, 2013. DOI: 10.1128/MMBR.00062-12
2. Ezzeldeen NA, Ammar AM, Shalaby B, Haririr ME, Omar WS: Rapid detection of Clostridium perfringens in seafood. Adv Environ Biol, 10 (4): 174-182, 2016.
3. Petit L, Gibert M, Popoff MR: Clostridium perfringens: Toxinotype and genotype. Trends Microbiol, 7, 104-110, 1999. DOI: 10.1016/S0966- 842X(98)01430-9
4. Songer JG: Clostridial enteric disease of domestic animals. Clin Microbiol Rev, 9 (2): 216-234, 1996.
5. Garmory HS, Chanter N, French NP, Bueschel D, Songer JG, Titball RW: Occurrence of Clostridium perfringens beta2-toxin amongst animals, determined using genotyping and subtyping PCR assays. Epidemiol Infect, 124 (1): 61-67, 2000.
6. Rood JI. Virulence genes of C. perfringens. Ann Rev Microbiol, 50, 333– 360, 1998. DOI: 10.1146/ANNUREV.MICRO.52.1.333
7. Verherstraeten S, Goossens E, Valgaeren B, Pardon B, Timbermont L, Haesebrouck F, Ducatelle R, Deprez P, Wade KR, Tweten R, Van Immerseel F: Perfringolysin O: The underrated Clostridium perfringens toxin?. Toxins, 7 (5): 1702-1721, 2015. DOI: 10.3390/toxins7051702
8. Biswas R, Deepika K, Sistla S, Sistla SC, Amaranathan A: Clostridium perfringens-A bacterial pathogen gaining recognition in necrotizing pancreatitis. Anaerobe, 47, 111-114, 2017. DOI: 10.1016/j.anaerobe. 2017.05.006
9. Tammy Dray: Clostridium perfringens type A and β2 toxin associated with enterotoxemia in a 5-week-old goat. Can Vet J, 45, 251-253, 2004. 10. Miyakawa MF, Uzal FA: The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep. Vet Res Commun, 27 (3): 231-241, 2003
11. Meer RR, Songer JG: Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am J Vet Res, 58, 702-705, 1997.
12. Hedberg M, Nord CE: ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria. Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe. Clin Microbiol Infect, 9, 475-488, 2003. DOI: 10.1046/j.1469-0691.2003.00674.x
13. Lõivukene K, Naaber P: Antibiotic susceptibility of clinically relevant anaerobes in Estonia from 1999 to 2001. Anaerobe, 9, 57-61, 2003. DOI: 10.1016/S1075-9964(03)00041-6
14. Van Asten AJ, van der Wiel CW, Nikolaou G, Houwers DJ, Gröne A: A multiplex PCR for toxin typing of Clostridium perfringens isolates. Vet Microbiol, 12, 136 (3-4): 411-412, 2009. DOI: 10.1016/j.vetmic.2008.11.024
15. Uzal FA: Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. Anaerobe, 10, 135-143, 2004. DOI: 10.1016/j.anaerobe. 2003.08.005
16. Kumar NV, Sreenivasulu D, Reddy YN: Prevalence of Clostridium perfringens toxin genotypes in enterotoxemia suspected sheep flocks of Andhra Pradesh. Vet World, 7 (12): 1132-1136, 2014. DOI: 10.14202/ vetworld.2014.1132-1136
17. Vaikosen ES, Ikhatua UJ: Detection of high level of enterotoxin of Clostridum perfingens types C and D in small ruminants in Nigeria. Small Ruminant Res, 58 (3): 287-290, 2005. DOI: 10.1016/j.smallrumres. 2004.11.004
18. Gokce HI, Genç O, Sozmen M, Gökçe G: Determination of Clostridium perfringens toxin-types in sheep with suspected enterotoxemia in Kars province, Turkey. Turk J Vet Anim Sci, 31 (5): 355-360, 2007.
19. Ahsani MR, Bafti MS, Esmailizadeh AK, Mohammadabadi MR: Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep. Small Ruminant Res, 95 (1): 65-69, 2011. DOI: 10.1016/j.smallrumres.2010.09.001
20. Albini S, Brodard I, Jaussi A, Wollschlaeger N, Frey J, Miserez R, Abril C: Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates. Vet Microbiol, 127, 179-185, 2008. DOI: 10.1016/j.vetmic.2007.07.024
21. Aras Z, Hadimli HH: Detection and molecular typing of Clostridium perfringens isolates from beef, chicken and turkey meats. Anaerobe, 32, 15-17, 2015. DOI: 10.1016/j.anaerobe.2014.11.004
22. Hashimoto A, Tsuchioka H, Higashi K, Ota N, Harada H: Distribution of enterotoxin gene-positive Clostridium perfringens spores among human and livestock samples and its potential as a human fecal source tracking indicator. J Water Environ Technol, 14 (6): 447-454, 2016. DOI: 10.2965/jwet.16-022
23. Gkiourtzidis K, Frey J, Bourtzi-Hatzopoulou E, Iliadis N, Sarris K: PCR detection and prevalence of α-, β-, β2-, ε-, ι-and enterotoxin genes in Clostridium perfringens isolated from lambs with clostridial dysentery. Vet Microbiol, 82 (1): 39-43, 2001. DOI: 10.1016/S0378-1135(01)00327-3
24. Mignaqui AC, Marcellino RB, Ronco T, Pappalardo JS, Nonnemann B, Pedersen K, Robles CA: Isolation and molecular characterization of Clostridium perfringens from healthy Merino lambs in Patagonia region, Argentina. Anaerobe, 43, 35-38, 2017. DOI: 10.1016/j.anaerobe.2016.10.015
25. Novak A, Rubic Z, Dogas V, Goic-Barisic I, Radic M, Tonkic M: Antimicrobial susceptibility of clinically isolated anaerobic bacteria in a University Hospital Centre Split, Croatia in 2013. Anaerobe, 31, 31-36, 2015. DOI: 10.1016/j.anaerobe.2014.10.010
26. Khan M, Nazir J, Anjum AA, Ahmad MD, Nawaz MA, Shabbir MZ: Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat. J Food Sci Technol, 52 (8): 5323-5328, 2015. DOI: 10.1007/s13197-014-1584-3
27. Dever, Laura A, Terence S, Dermody: Mechanisms of bacterial resistance to antibiotics. Arch Intern Med, 151 (5): 886-895, 1991. DOI: 10.1001/archinte.1991.00400050040010