Determination of In Vitro Antibiotic Susceptibility of Campylobacter species Isolated from Stool Samples and Detection of Macrolide Resistance by Genotypic Methods

Öz

Most gastroenteritis caused by Campylobacter are self-limiting. However, antimicrobial treatment (e.g., macrolides, fluoroquinolones) is necessary in severe or prolonged cases. The objective of this study was to identify Campylobacter species isolated from stool samples, to determine in vitro antibiotic susceptibility and detect macrolide resistance by genotypic methods. We analyzed a total of 65 strains that were isolated from stool samples between August 2014 and August 2019. Antibiotic susceptibilities of the isolates were detected by disk diffusion method and the results were evaluated according to the European Committee of Antimicrobial Susceptibility Testing (EUCAST)guidelines. Macrolide resistance was investigated by mismatch amplification mutation assay- polymerase chain reaction (MAMA-PCR). Of the isolates, 60 (92.3%) were identified as Campylobacter jejuni, five (7.7%) were Campylobacter coli. The rates of resistance of the isolates were 76.92% for ciprofloxacin and 16.92 % for erythromycin. In 11 macrolide resistant isolates, 23S rRNA, ERY2074 and/or ERY2075 mutations were detected. Isolated ERY2025 and ERY2024 mutations were observed in five and three isolates, respectively. The high presence of resistance to macrolides and quinolones, which are used to treat campylobacteriosis in humans, highlights the need for proper antimicrobial surveillance and control measures.

Anahtar Kelimeler

• Campylobacter species
• erythromycin
• ciprofloxacin
• in vitro resistance
• MAMA-PCR

Dışkı örneklerinden izole edilen Campylobacter türlerinin in vitro antibiyotik duyarlılıklarının belirlenmesi ve makrolid direncinin genotipik yöntemlerle saptanması.

Öz

Campylobacter enfeksiyonu genellikle kendi kendini sınırlar, ancak ciddi veya uzun süreli vakalarda antimikrobiyal tedavi (örn. makrolidler, florokinolonlar) gereklidir. Bu çalışmanın amacı dışkı örneklerinden izole edilen Campylobacter türlerinde, in vitro antibiyotik duyarlılığı saptamak ve makrolid direncini genotipik yöntemlerle tespit etmektir. Ağustos 2014 ile Ağustos 2019 arasında dışkı örneklerinden elde edilen toplam 65 Campylobacter izolatı analiz edildi. İzolatların antibiyotik duyarlılıkları gradiyent test yöntemiyle tespit edildi. Sonuçlar Avrupa Antimikrobiyal Duyarlılık Testi Komitesi (European Committee of Antimicrobial Susceptibility Testing - EUCAST) kılavuzuna göre değerlendirildi. Makrolid direnci uyumsuzluk amplifikasyon mutasyon testi (mismatch amplification mutation assay) polimeraz zincir reaksiyonu (MAMA-PZR)ile araştırıldı. Altmış (%92.3) izolat C.jejuni ve beş (%7.7) izolat C. coli olarak tanımlandı. İzolatların direnç oranları siprofloksasine %76.92, eritromisine ise %16.92 olarak tespit edildi. Makrolid dirençli 11 izolatta, 23S rRNA, ERY2074 ve/veya ERY2075 mutasyonu tespit edildi. Sadece 23S rRNA, ERY2025 ve ERY2024 mutasyonu sırasıyla beş ve üç izolatta gözlendi. İnsanlarda kampilobakteriyozu tedavi etmek için kullanılan makrolidler ve kinolonlara karşı yüksek direnç oranları, uygun antimikrobiyal gözetim ve kontrol önlemlerine olan ihtiyacı vurgulamaktadır.

Anahtar Kelimeler

• Campylobacter türleri
• eritromisin
• siprofloksasin
• in vitro direnç
• MAMA-PZR

Kaynakça

1. 1. Aarestrup FM, Engberg J. Antimicrobial resistance of thermophilic Campylobacter. Vet. Res. 2001;32(3-4):311-21. https://doi.org/10.1051/vetres:2001127
2. 2. Alonso R, Mateo E, Churruca E, et al. MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains. J Microbiol Methods. 2005;63(1):99-103. https://doi.org/10.1016/j.mimet.2005.03.013
3. 3. Blaser M, Engberg J. Clinical Aspects of Campylobacter jejuni and Campylobacter coli infections, “Nachamkin I, Szymanski CM (eds). Campylobacter, 3.baskı” kitabında s.99-121, ASM Press, Washington (2008). https://doi.org/10.1128/9781555815554.ch6
4. 4. Coker AO, Isokpehi RD, Thomas BN, Amisu KO, Obi CL. Human campylobacteriosis in developing countries. Emerg Infect Dis. 2002;8(3):237-43. https://doi.org/10.3201/eid0803.010233
5. 5. Eryıldız C, Sakru N, Kuyucuklu G. Investigation of antimicrobial susceptibilities and resistance genes of Campylobacter isolates from patients in Edirne, Turkey. Iran J Public Health. 2022;51(3):569-77. https://doi.org/10.18502/ijph.v51i3.8933
6. 6. Fitzgerald C, Whichard J, Nachamkin I. Diagnosis and antimicrobial susceptibility of Campylobacter species. “Nachamkin I, Szymanski CM, Blaser MJ (eds). Campylobacter, 3.baskı” kitabında s.227-243, ASM Press, Washington (2008) https://doi.org/10.1128/9781555815554.ch12
7. 7. Fliegelman RM, Petrak RM, Goodman LJ, Segreti J, Trenholme GM, Kaplan RL. Comparative in vitro activities of twelve antimicrobial agents against Campylobacter species. Antimicrob Agents Chemother. 1985;27(3):429-30. https://doi.org/10.1128/aac.27.3.429
8. 8. Gao F, Tu L, Chen M, et al. Erythromycin resistance of clinical Campylobacter jejuni and Campylobacter coli in Shanghai, China. Front Microbiol. 2023;14:1-12. https://doi.org/10.3389/fmicb.2023.114558

9. 9. García-Fernández A, Dionisi AM, Arena S, et al. Human campylobacteriosis in Italy: emergence of multi-drug Resistance to ciprofloxacin, tetracycline, and erythromycin. Front Microbiol. 2018;9(1906):1-8. https://doi.org/10.3389/fmicb.2018.01906
10. 10. Gupta A, Nelson JM, Barrett TJ, et al. Antimicrobial resistance among Campylobacter strains, United States, 1997-2001. Emerg Infect Dis. 2004;10(6):1102-9. https://doi.org/10.3201%2Feid1006.030635
11. 11. Hızlısoy H, Kılıç H. Broiler karkaslarından izole edilen Campylobacter jejuni izolatlarının makrolid, kinolon ve tetrasiklin grubu antibiyotiklere karşı direnç durumu. Erciyes Üniv. Vet. Fak. Derg. 2015;12(2):81-92. https://dergipark.org.tr/en/pub/ercivet/issue/25187/281578
12. 12. Johnson TJ, Shank JM, Johnson JG. Current and Potential Treatments for Reducing Campylobacter Colonization in Animal Hosts and Disease in Humans. Front Microbiol. 2017;8:487. https://doi.org/10.3389/fmicb.2017.00487
13. 13. Kaakoush NO, Castano-Rodriguez N, Mitchell NM, Man SM. Global Epidemiology of Campylobacter Infection, Clin Microbiol Rev. 2015;28(3):687-720. https://doi.org/10.1128/cmr.00006-15
14. 14. Kayman T, Abay S, Hızlısoy H. Campylobacter Türlerinin Fenotipik Yöntemler ve Multipleks Polimeraz Zincir Reaksiyonu ile Tanımlanması ve Antibiyotik Duyarlılıkları. Mikrobiyol Bul 2013;47(2):230-9. https://doi.org/10.5578/mb.4532
15. 15. Kurincic M, Botteldoorn N, Herman L, Smole Mozina S. Mechanisms of erythromycin resistance of Campylobacter spp. isolated from food, animals and humans. Int J Food Microbiol. 2007;120(1-2):186-90. https://doi.org/10.1016/j.ijfoodmicro.2007.03.012
16. 16. Lindmark H, Harbom B, Thebo L, et al. Genetic characterization and antibiotic resistance of Campylobacter jejuni isolated from meats, water, and humans in Sweden. J Clin Microbiol. 2004; 42(2):700-6. https://doi.org/10.1128/jcm.42.2.700-706.2004
17. 17. Luangtongkum T, Jeon B, Han J, et al. Antibiotic resistance in Campylobacter: emergence, transmission and persistence. Future Microbiol. 2009;4(2):189-200. https://doi.org/10.2217/17460913.4.2.189
18. 18. Otsuka Y, Hagiya H, Takahashi M, et al. Clinical characteristics of Campylobacter bacteremia: a multicenter retrospective study. Sci Rep. 2023;13:647-53. https://doi.org/10.1038/s41598-022-27330-4
19. 19. Paton JH, Reeves DS. Fluoroquinolone antibiotics: Microbiology, pharmacokinetics and clinical use. Drugs. 1988; 36(2):193-228. https://doi.org/10.2165/00003495-198836020-00004
20. 20. Pfister P, Jenni S, Poehlsgaard J, et al. The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23SrRNA positions 2058 and 2059. J Mol Biol. 2004;342(5):1569-81. https://doi.org/10.1016/j.jmb.2004.07.095
21. 21. Rivera-Mendoza D, Martínez-Flores I, Santamaría R, Lozano L, Bustamante V. Genomic analysis reveals the genetic determinants associated with antibiotic resistance in the zoonotic pathogen Campylobacter spp. distributed globally. Front Microbiol. 2020;11:513070. https://doi.org/10.3389/fmicb.2020.513070
22. 22. Sheppard SK, Dallas JF, MacRae M, et al. Campylobacter genotypes from food animals, environmental sources and clinical disease in Scotland 2005/6. Int J Food Microbiol. 2009;134(1-2):96-103. https://doi.org/10.1016/j.ijfoodmicro.2009.02.010
23. 23. Skarp CPA, Hanninen ML, Rautelin HIK. Campylobacteriosis: the role of poultry meat. Clin Microbiol Infect. 2016;22(2):103-9. https://doi.org/10.1016/j.cmi.2015.11.019
24. 24. Wang S, Huang F, Wu C, Tang K. Clinical significance of erythromycin-resistant Campylobacter jejuni in children. J Microbiol Immunol Infect. 2011;44(1):63-6. https://doi.org/10.1016/j.jmii.2011.01.012
25. 25. Wieczorek K, Osek J. Antimicrobial Resistance Mechanisms among Campylobacter. Biomed Res Int. 2013;340605:1-12. https://doi.org/10.1155/2013/340605
26. 26. Yan M, Sahin O, Lin J, Zhang Q. Role of the Cme ABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure. J Antimicrob Chemother. 2006;58(6):1154-9. https://doi.org/10.1093/jac/dkl412
27. 27. Zirnstein G, Li Y, Swaminathan B, Angulo F. Ciprofloxacin resistance in Campylobacter jejuni isolates: Detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol. 1999;37(10):3276-80. https://doi.org/10.1128/jcm.37.10.3276-3280.1999