Üriner Sistem Enfeksiyonlu Bireylerden İzole Edilen Escherichia coli ve Klebsiella pneumoniae Suşlarında CTX-M, TEM ve SHV Tipi Genişlemiş Spektrumlu Beta-Laktamaz Aktivitesinin Otomatize Sistem ve Moleküler Yöntemlerle Araştırılması

Yıl 2021, Cilt: 11 Sayı: 2, 307 - 317, 01.08.2021

Sibel Deniz Fatih Büyük Kenan Murat

Öz

Amaç: Bu çalışmada, Kars Harakani Devlet Hastanesi’ne idrar yolları yakınması ile başvuran bireylere ait idrar örneklerinden izole edilen Escherichia coli ve Klebsiella pneumoniae suşları arasında GSBL pozitifliğinin otomatize identifikasyon sistemi ve moleküler yöntemlerle araştırılması amaçlandı.
Materyal ve Metot: Çalışmaya, Nisan-Ağustos 2019 tarihleri arasında örneklenen 5000 idrar örneği dâhil edildi. Örnekler, Mac Conkey ve %7 koyun kanlı agarda kültüre edildikten sonra elde edilen izolatların identifikasyonu BD Phoenix™ 100 ID paneli, antibiyotik duyarlılıkları ve GSBL aktiviteleri ise AST paneli ile belirlendi. İzolatların CTX-M, TEM ve SHV genleri PCR ile analiz edildi.
Bulgular: İdrar örneklerinden %19,5 oranında aerobik bakteriyel etken kültür pozitifliği elde edildi ve bunların 120 (%13,33)’sinin GSBL aktivitesi olduğu saptandı. GSBL pozitif izolatların 102 (%85)’si E. coli ve 14 (%11,67)’ü K. pneumoniae olarak identifiye edildi. GSBL pozitifliği toplamda idrar örneklerinde %2,4, E. coli ve K. pneumoniae için ise sırasıyla %2,04 ve %0,28 olarak belirlendi. İzolatların, beta laktam grubu antibiyotiklere toplam direnci %57,46 belirlenirken, en yaygın dirençlilik sefalosporinlere (%92,53) ve en yaygın duyarlılık ise karbapenemlere (%47,19) karşı saptandı. PCR ile GSBL pozitif izolatların %90,52’sinde en az bir veya birden fazla direnç geni saptandı ve en yaygın saptanan direnç geni CTX-M idi. GSBL pozitifliği ile yaş grupları arasında istatistiksel olarak anlamlı bir ilişki saptanamazken, hastaların cinsiyetleri arasında %21,9 oranında pozitif yönde bir ilişki saptandı.
Sonuç: GSBL kaynaklı enfeksiyonların insidansının azaltılmasında risk faktörlerinin ortadan kaldırılmasının yanı sıra enfeksiyöz etkenlerin identifikasyonu, ilgili GSBL profillerinin çıkarılması ve bu modellerine göre geliştirilecek doğru antibiyotik tercihi oldukça önemlidir. Sınırlı bir lokasyonda, Kars yöresinde, yapılan bu sistematik çalışmanın bu yönde fayda sağlayacağı umulmaktadır.

Anahtar Kelimeler

insan, idrar, GSBL, kültür, Phoenix, PCR, E. coli, K. pneumoniae

Investigation of CTX-M, TEM and SHV Type Extended Spectrum Beta-Lactamase Activity with Automated System and Molecular Methods in Escherichia coli and Klebsiella pneumoniae Strains Isolated from Individuals with Urinary Track Infections

Öz

Aim: This study aimed to investigate ESBL positivity among E. coli and K. pneumoniae strains isolated from urine samples belonging to individuals who applied to Kars Harakani State Hospital with urinary tract complaints by automated identification system and molecular methods.
Material and Method: 5.000 urine samples obtained between April and August 2019 were included. The samples were cultured on Mac Conkey and 7% sheep blood agar, identification was done by ID panel of BD Phoenix ™ 100 and antibiotic susceptibility and ESBL activities were determined by AST panel. CTX-M, TEM and SHV genes were analyzed by PCR.
Results: 19.5% aerobic bacteria culture positivity was obtained from the urine samples and 120 (13.33%) of them were found to have ESBL activity. 102 (85%) of the isolates were identified as E. coli and 14 (11.67%) as K. pneumoniae. The ESBL positivity was obtained 2.4% for total urine samples, and 2.04% and 0.28% for E. coli and K. pneumoniae, respectively.
While the total resistance to beta-lactam antibiotics was 57.46%, the most common resistance was observed against cephalosporins (92.53%) and the most common sensitivity (47.19%) was to carbapenems. At least one or more resistance genes were detected in 90.52% of ESBL positive isolates by PCR and CTX-M was the most common resistance gene. While there was no statistically significant relationship between ESBL positivity and age groups, however, a positive relationship was found between ESBL and gender of the patients at a rate of 21.9%.
Conclusion: In addition to eliminating risk factors in reducing the incidence of ESBL-related infections, identification of microorganism, disclosure of relevant ESBL profiles and the right antibiotic preference to be developed according to these emerged models are important. It is hoped that this systematic study carried out in a limited location in Kars region of Turkey will benefit in this context.

Anahtar Kelimeler

Human, Urine, ESBL, Culture, Phoenix, PCR, E. coli, K. pneumoniae

Kaynakça

• 1. Ambler RP. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci 1980;289:321-31.
• 2. Falagas ME, Karageorgopoulos DE. Extended-spectrum beta-lactamase-producing organisms. J Hosp Infect 2009;73:345-54.
• 3. Calbo E, Garau J. The changing epidemiology of hospital outbreaks due to ESBL-producing Klebsiella pneumoniae:The CTX-M-15 type consolidation. Future Microbiol 2015;10:1063-75.
• 4. Coque TM, Baquero F, Canton R. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill 2008;13:19044.
• 5. Kadanalı A. Üriner sistem enfeksiyonları. Eurasian J Med 2006;38:119-23.
• 6. Bektaş A, Güdücüoğlu H, Gürsoy NC, Berktaş M, Gültepe BS, Parlak M, ve ark. Genişlemiş spektrumlu beta-laktamaz (GSBL) üreten Escherichia coli ve Klebsiella pneumoniae suşlarının GSBL genlerinin araştırılması. FLORA 2018;23:116-23.
• 7. Gholipour A, Soleimani N, Shokri D, Mobasherizadeh S, Kardi M, Baradaran A. Phenotypic and molecular characterization of extended-spectrum β-lactamase produced by Escherichia coli, and Klebsiella pneumoniae isolates in an educational hospital. Jundishapur J Microbiol 2014;7:e11758.
• 8. Lim YK, Lee MK, Kim TH. Management of extended-spectrum beta-lactamase-positive Gram-negative bacterial urologic infections. Urogenit Tract Infect 2015;10:84-91.
• 9. Rodriguez-Bano J, Gutierrez-Gutierrez B, Machuca I, Pascual A. Treatment of infections caused by extended-spectrum-betalactamase-, AmpC-, and carbapenemase producing Enterobacteriaceae. Clin Microbiol Rev 2018;31:e00079-17.
• 10. Arda M. Temel Mikrobiyoloji. 2011. Medisan Yayınevi, 4. Baskı, Ankara.
• 11. Marmur J. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 1961;3:208-18.
• 12. Edelstein M, Pimkin M, Palagin I, Edelstein I, Stratchounski L. Prevalence and molecular epidemiology of CTX-M extended-spectrumlactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob Chemother Agent 2003;47:3724-32.
• 13. Hosoglu S, Gundes S, Kolaylı F, Karadenizli A, Demirdağ K, Günaydın M, et al. Extended spectrum β-lactamases in ceftazidime resistant Escherichia coli and Klebsiella pneumonia isolates in Turkish hospitals. Indian J Med Microb 2007;25:346-50.
• 14. Arlet G, Rouveau M, Philippon A. Substitution of alanine for aspartate at position 179 in the SHV-6 extended-spectrum -lactamase. FEMS Microbiol Lett 1997;152:163-7.
• 15. Arıcıgil Çilan Ç. Sosyal Bilimlerde Kategorik Verilerle İlişki Analizi. 2013. Pegem Akademi, 2. Baskı, Ankara.
• 16. Karagöz Y. İlişki katsayıları ile öğrenci başarısını etkileyen faktörlerin belirlenmesi. Elektronik Sosyal Bilimler Dergisi 2010;9:425-46.
• 17. United Nations. Provisional Guidelines on Standard International Age Classifications. 1982. United Nations Publication, Sales no. E.82.XVII.5. New York:United Nations.
• 18. Stamm WE, Norrby SR. Urinary tract infections:Disease panorama and challenges. J Infect Dis 2001;183:S1-4.
• 19. Amin M, Mehdinejad M, Pourdangchi Z. Study of bacteria isolated from urinary tract infections and determination of their susceptibility to antibiotics. Jundishapur J Microbiol 2009;2:118-23.
• 20. Begum SA, Afreen S, Ahamed F, Chowdhury A, Jobayer M, Samsuzzaman SM. Etiology of UTI and frequency of ESBL producing bacteria isolated from patients of Dhaka Medical College Hospital with their antimicrobial susceptibility pattern. J Dhaka Med Coll 2016;25:26-31.
• 21. Mert D, Çeken S, Ertek M. İdrar yolu enfeksiyonlarında kültürden izole edilen bakteriler ve antibiyotik duyarlılıkları. Turk Hij Den Biyol Derg 2020;77:25-32.
• 22. Mollick S, Dasgupta T, Hasnain J, Ahmed M. Isolation and characterization of pathogens responsible for urinary tract infection in Bangladesh and determination of their antibiotic susceptibility pattern. J Appl Pharm Sci 2016;6:72-6.
• 23. Yilmaz Y, Tazegun ZT, Aydin E, Dulger M. Bacterial uropathogens causing urinary tract infection and their resistance patterns among children in Turkey. Iran Red Crescent Med J 2016;18:e26610.
• 24. Carroll KC, Weinstein MP. Manual and Automated Systems for Detection and Identification of Microorganisms, pp:192-217. In:Murray PR (ed), Manual of Clinical Microbiology. 2007, 9th ed. ASM Press, Washington, DC.
• 25. Marucco AP, Minervini P, Snitman GV, Sorge A, Guelfand LI, Moral LL, et al. Comparison of the identification results of Candida species obtained by BD PhoenixTM and Maldi-TOF (Bruker Microflex LT Biotyper 3.1). Rev Argent Microbiol 2018;50:337-40.
• 26. Snyder JW, Munier GK, Johnson CL. Direct comparison of the BD Phoenix System with the MicroScan WalkAway System for identification and antimicrobial susceptibility testing of Enterobacteriaceae and nonfermentative Gram-negative organisms. J Clin Microbiol 2008;46:2327-33.
• 27. Swati M, Balasubramanian U. Comparison of BD Phoenix Automated System with conventional methods for identification & susceptibility testing of common bacteria. Int J Sci Res 2017;6:252-5.
• 28. Sanguinetti M, Posteraro B, Spanu T, Ciccaglione D, Romano L, Fiori B, et al. Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method. J Clin Microbiol 2003;41:1463-8.
• 29. Leverstein-van HMA, Fluit AC, Paauw A, Box ATA, Brisse S, Verhoef J. Evaluation of the Etest ESBL and the BD Phoenix, VITEK 1, and VITEK 2 automated instruments for detection of extended-spectrum β-lactamases in multiresistant Escherichia coli and Klebsiella spp. J Clin Microbiol 2002;40:3703-11.
• 30. Wiegand I, Geiss HK, Mack D, Stürenburg E, Seifert H. Detection of extended-spectrum beta-lactamases among Enterobacteriaceae by use of semiautomated microbiology systems and manual detection procedures. J Clin Microbiol 2007;45:1167‐74.
• 31. Doi Y, Park YS, Rivera JI, Adams-Haduch JM, Hingwe A, Sordillo EM, et al. Community-associated extended-spectrum β-lactamase-producing Escherichia coli infection in the United States. Clin Infect Dis 2013;56:641-8.
• 32. Arana DM, Rubio M, Alós JI. Evolution of antibiotic multiresistance in Escherichia coli and Klebsiella pneumoniae isolates from urinary tract infections:A 12-year analysis (2003-2014). Enferm Infecc Microbiol Clin 2017;35:293‐8.
• 33. Aykan SB, Ciftci IH. Türkiye'de idrar kültürlerinden izole edilen Escherichia coli suşlarının antibiyotiklere direnç durumu:Bir meta-analiz. Mikrobiyol Bul 2013;47:603‐18.
• 34. Çelikbilek N, Gözalan A, Özdem B, Kırca F, Açıkgöz ZC. Ayaktan başvuran hastaların idrar kültürlerinde üretilen Enterobacteriaceae izolatlarında genişlemiş spektrumlu beta-laktamaz üretimi:Yedi yıllık izlem sonuçları. Mikrobiyol Bul 2015;49:259-65.
• 35. Temoçin F, Köse H. Poliklinik hastalarının idrar kültürlerinden izole edilen Escherichia coli ve Klebsiella pneumoniae suşlarının genişlemiş spektrumlu beta-laktamaz üretim oranları ve antibiyotik duyarlılıklarının değerlendirilmesi. ANKEM Derg 2018;32:79-86.
• 36. Sid Ahmed MA, Bansal D, Acharya A, Elmi AA, Hamid JM, Sid Ahmed AM, et al. Antimicrobial susceptibility and molecular epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae from intensive care units at Hamad Medical Corporation, Qatar. Antimicrob Resist Infect Control 2016;5:2-6.
• 37. Elhassan MM, Ozbazk HA, Hemeg HA, Ahmed AA. Dissemination of CTX-M extended-spectrum β-lactamases (ESBLs) among Escherichia coli and Klebsiella pneumoniae in Al-Madenah Al-Monawwarah region, Saudi Arabia. Int J Clin Exp Med 2016;9:11051-7.
• 38. Tutun H, Karagöz A, Altıntaş L, Koçak N. Determination of antibiotic susceptibility, ESBL genes and pulsed-field gel electrophoresis profiles of extended-spectrum β-lactamase- containing Escherichia coli isolates. Ankara Univ Vet Fak Derg 2019;66:407-16.
• 39. Peterson DL, Bonomo RA. Extended spectrum beta-lactamases:A clinical update. Clin Microbiol Rev 2005;18:657-86.
• 40. Calbo E, Romaní V, Xercavins M, Gomez L, Vidal CG, Quintana S, et al. Risk factors for community-onset urinary tract infections due to Escherichia coli harbouring extended-spectrum beta-lactamases. J Antimicrob Chemother 2006;57:780‐3.
• 41. Ortega M, Marco F, Soriano A, Almela M, Martinez JA, Munoz A, et al. Analysis of 4758 Escherichia coli bacteraemia episodes:Predictive factors for isolation of an antibiotic-resistant strain and their impact on the outcome. J Antimicrob Chemother 2009;63:568‐74.
• 42. Karamanlıoğlu D, Aysert-Yıldız P, Kaya M, Sarı N. Extended-spectrum β-lactamase production rates and antibiotic susceptibilities among Enterobacteriaceae isolated from urine. Klimik Derg 2019;32:233-9.
• 43. Mahesh E, Ramesh D, Indumathi VA, Punith K, Raj K, Anupama HA. Complicated urinary tract infection in a tertiary care center in South India. Al Ameen J Med Sci 2010;3:120-7.
• 44. Shah AA, Hasan F, Ahmed S, Hameed A. Extended-spectrum beta-lactamases in Enterobacteriaceae:Related to age and gender. New Microbiol 2002;25:363‐6.
• 45. Al-Garni SM, Ghonaim MM, Ahmed MMM, Al-Ghamdi AS, Ganai FA. Risk factors and molecular features of extended-spectrum beta-lactamase producing bacteria at southwest of Saudi Arabia. Saudi Med J 2018;39:1186-94.
• 46. Marković T, Jeinić L, Smitran A, Petkovic M. Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase producing urinary isolates of Escherichia coli in outpatients. Srp Arh Celok Lek 2013;141:775-9.