Abstract

INTRODUCTION

: Escherichia coli ranks among the most common sources of urinary tract infections (UTI).

METHODS:

Between November 2015 and August 2016, 90 isolates of E. coli were isolated from patients at Rize Education and Research Hospital in Turkey. Antibiotic susceptibility was determined for all isolates using the Kirby-Bauer disk diffusion method. These E. coli isolates were also screened for virulence genes, β-lactamase coding genes, quinolone resistance genes, and class 1 integrons by PCR.

RESULTS:

With respect to the antibiotic resistance profile, imipenem and meropenem were effective against 98% and 90% of isolates, respectively. A high percentage of the isolates showed resistance against β lactam/β lactamase inhibitor combinations, quinolones, and cephalosporins. PCR results revealed that 63% (57/90) of the strains carried class 1 integrons. In addition, a high predominance of extended-spectrum β-lactamases (ESBLs) was observed. The qnrA, qnrB, and qnrS genes were found in 24 (26.6%), 6 (6.6%), and 3 (3.3%), isolates, respectively. The most common virulence gene was fim (82.2%).The afa, hly, and cnf1 genes were detected in 16.6%, 16.6%, and 3.3% of isolates, respectively. Moreover, we observed eleven different virulence patterns in the 90 E. coli isolates. The most prevalent pattern was fım, while hly-fım, afa-aer-cnf-fım, aer-cnf, afa-aer, and afa-cnf-fım patterns were less common.

CONCLUSIONS:

Most of the E. coli virulence genes investigated in this study were observed in E. coli isolates from UTI patients. Virulence genes are very important for the establishment and maintenance of infection.

Keywords:
Quinolones; Virulence genes; UTI

INTRODUCTION

Escherichia coli are among the most common etiological agents that cause urinary tract infections (UTI); this makes E. coli infection an important public health issue¹1. Farell DJ, Morrissay I, De Rubeids D, Robbins M, Felmingham D. A UK multicentre study and the antimicrobial susceptibility of bacterial pathogens causing urinary tract infection. J Infect. 2003;46(2):94-100.

2. Matute AJ, Hake E, Schurink C. Resistance of uropathogens in symptomatic urinary tract infection in Leon, Nicaragua. Int J Antimicrob Agents. 2004;23(5):506-09.

3. Zhanel G, Hisanaga T, Laing N. Antibiotic resistance in Escherichia coli outpatient urinary isolates: Final results from the North American Urinary Tract Infection Collaborative Alliance (NAUTICA). Int J Antimicrob Agents . 2006;27(6):468-75.^-44. Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J. Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. Int J Infect Dis. 2013;17(6):e450-53.. Many virulence factors are responsible for the pathogenicity of E. coli strains⁴4. Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J. Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. Int J Infect Dis. 2013;17(6):e450-53.^-55. Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev. 1991;4(1):80-128.. There are two main types of E. coli virulence factors; these include (i) virulence factors that are produced within the cell and released at the site of action, and (ii) virulence factors that are displayed on the surface of the cell⁶6. Emody L, Kerényi M, Nagy G. Virulence factors of uropathogenic Escherichia coli. Int J Antimicrob Agents . 2003;22 (Suppl 2):29-33..

The most important E. coli virulence factors are the surface virulence factors (adhesins). P fimbriae are encoded by pap genes and are the main adherence factors⁷7. Jadhav S, Hussain A, Devi S, Kumar A, Parveen S, Gandham N, et al. Virulence characteristics and genetic affinities of multiple drug resistant uropathogenic Escherichia coli from a semi urban locality in India. PLoS One 2011;6(3):e18063.. S fimbrial adhesion factors, encoded by sfa genes, represent another type of virulence factor⁸8. Pobiega M, Wojkowska-Mach J, Chmielarczyk A, Romaniszyn D, Adamski P, Heczko PB, et al. Molecular characterization and drug resistance of Escherichia coli strains isolated from urine from long-term care facility residents in Cracow, Poland. Med Sci Monit. 2013;19:317-26.. A fimbrial adhesion factors in E. coli are encoded by afa genes⁹9. Servin AL. Pathogenesis of Afa/Dr diffusely adhering Escherichia coli. Clin Microbiol Rev . 2005;18(2):264-92.. In addition, the main fimbrial subunit of type 1 fimbriae is encoded by fimA in E. coli¹⁰10. Gally DL, Leathart J, Blomfield IC. Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12. Mol Microbiol. 1996;21(4):725-38.. Toxins are another important type of virulence factor in E. coli. The a-hemolysin (HlyA) virulence factor, cytotoxic necrotizing factor, and aerobactin are encoded by the hly, CNF1⁵5. Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev. 1991;4(1):80-128., and aer genes, respectively¹¹11. Slavchev G, Pisareva E, Markova N. Virulence of uropathogenic Escherichia coli. J Cult Collect. 2009;6(1):3-9.^,1212. Firoozeh F, Saffari M, Neamati F, Zibaei M. Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis. Int J Infect Dis . 2014;9:219-22..

The β-lactamases (enzymes that hydrolyze β-lactam antibiotics) are classified into four groups depending on their amino acid sequences: class A (e.g., KPC, CTX-M, and GES), class B (e.g., IMP, VIM, SPM, GIM, NDM, and SIM), class C (e.g., AmpC), and class D (e.g., OXA-type β-lactamase). All four classes of β-lactamase have been identified in E. coli. Metallo-β-lactamases (MBLs) are disseminated worldwide¹³13. Cornaglia G, Giamarellou H, Rossolini GM. Metallo-β-lactamases: A last frontier for β-lactams? Lancet Infect Dis. 2011;11(5):381-93. and have been mainly identified in Enterobacteriaceae of the IMP and VIM types¹⁴14. Siarkou V, Vitti D, Protonotariou E, Ikonomidis A, Sofianou D. Molecular epidemiology of outbreak-related Pseudomonas aeruginosa strains carrying the novel variant blaVIM-17 metallo-beta-lactamase gene. Antimicrob Agents Chemother. 2009;53(4):1325-30.

15. Bebrone C. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol. 2007;74(12):1686-701.

16. Sacha P, Wıeczorek P, Hauschıld T, Zorawskı M, Olszanska D, Trynıszewska E. Metallo-β- lactamases of Pseudomonas aeruginosa - a novel mechanism resistance to β-lactam antibiotics. Folia Histochem Cyto. 2008;46(2):137-42.^-1717. Igbinosa IH, Igbinosa EO, Okoh AI. Molecular detection of metallo-β-lactamase and putative virulence genes in environmental isolates of Pseudomonas species. Pol J Environ Stud. 2014;23(6):2327-31..

Quinolones are widely used to treat UTIs caused by E. coli. This extensive use of quinolones has led to increased resistance in E. coli¹⁸18. Yanat B, Vinuesa T, Viñas M, Touati A. Determinants of quinolone resistance inEscherichia colicausing community-acquired urinary tract infection in Bejaia, Algeria. Asian Pac J Trop Med. 2014;7(6):462-7.. Target modification, and changes in membrane permeability can confer resistance to quinolones. Moreover, plasmid-mediated qnr (quinolone-resistance) genes can facilitate quinolone resistance, with the qnrA, qnrB, andqnrS groups comprising the major qnrdeterminants¹⁹19. Rezazadeh M, Baghchesaraei H, Peymani A. Plasmid-mediated quinolone-resistance (qnr) genes in clinical isolates of Escherichia coli collected from several hospitals of Qazvin and Zanjan Provinces, Iran. Osong Public Health Res Perspect. 2016;7(5):307-12..

Integrons are mobile genetic elements that contribute to the spread of antibiotic resistance. Many gene cassettes emerged when class 1 integrons were first discovered in clinical strains. The role of integrons in promoting bacterial multidrug resistance is significant. A number of studies investigating the prevalence of integrons in E. coli isolates from UTI patients have reported a significant link between antimicrobial resistance and integrons²⁰20. Khoramrooz SS, Sharifi A, Yazdanpanah M, Asghar SA, Hosseini M, Emaneini M, et al. High frequency of class 1 integrons in Escherichia coli isolated from patients with urinary tract infections in Yasuj, Iran. Iran Red Crescent Med J. 2016;18(1):e26399.^-2121. Nilsson AI, Berg OG, Aspevall O, Kahlmeter G, Andersson DI. Biological costs and mechanisms of fosfomycin resistance in Escherichia coli. Antimicrob Agents Chemother . 2003;47(9):2850-8..

The purpose of this study was to investigate the presence of virulence genes, β-lactamase coding genes, quinolone resistance genes, fosfomycin resistance genes, and class 1 integron gene cassettes in E. coli isolates from patients with UTI.

METHODS

A total of 90 E. coli isolates were investigated in this study. All strains were isolated at the Rize Education and Research Hospital in Turkey between November 2015 and August 2016. Urine samples were cultured on blood agar and Eosin Methylene Blue (EMB) agar, then incubated at 37ºC for 18-24 h. Bacteria were identified using colony morphology and biochemical tests in urine cultures with high levels of viable bacteria (≥10⁵ CFU/mL). Antibiotic susceptibility of each isolate was determined by Kirby-Bauer disk diffusion and was based on the criteria recommended by the Clinical Laboratory Standards Institute (CLSI, 2014).

Genomic DNA was obtained from bacterial suspensions grown overnight in Luria Broth (LB) at 37°C. Bacterial suspensions were centrifuged. Pellets were resuspended in 500 µL of distilled water, then boiled in a water bath for 10 min. Boiled suspensions were centrifuged at 11,357 g for 5 min. Five hundred microlitres of each supernatant were used as a template for PCR assays²²22. Copur Cicek A, Saral A, Ozad Duzgun A, Yasar E, Cizmeci Z, Ozlem Balci P, et al. Nationwide study of Escherichia coli producing extended-spectrum β-lactamases TEM, SHV and CTX-M in Turkey. J Antibiot (Tokyo). 2013;66(11):647-50..

All strains were isolated from adult patients with uncomplicated community-acquired UTIs. Ninety E. coli isolates were screened for genes encoding β-lactamases, quinolone resistance factors, fosfomycin resistance factors, and virulence factors via polymerase chain reaction (PCR). Primers for β-lactamase-encoding genes (bla _IMP, bla _VIM, bla _NDM, bla _CTXM-1, bla _CTXM-2, bla _GES, bla _SIM, bla _AmpC, and bla _SPM), quinolone resistance genes (qnrA, qnrB, and qnrS), fosfomycin resistance genes (fosA, fosC2, and fosA3), and virulence genes (pap, sfa, afa, hly, aer, cnf, and fim) were used in these experiments. All PCR results were analyzed by electrophoresis in 1% agarose containing 0.5 μg/mL ethidium bromide, followed by examination under UV light.

PCR was performed on all isolates to detect class 1 integron gene cassettes using the primers 5′-GGCATCCAAGCAGCAAG-3′ (5′CS) and 5′-AAGCAGACTTACCTGA-3′ (3′CS). The PCR conditions were 3 min at 94°C for initial denaturation, followed by 34 cycles of 45 s at 94°C , 1 min at 55°C, and 3 min at 72°C, with a final extension at 72°C for 5 min.

RESULTS

Ninety E. coli isolates were investigated in this study. Of the 90 patients diagnosed with community-acquired UTIs, 62 (68.9%) were women and 28 (31.1%) were men. The extended-spectrum β-lactamase (ESBL) positivity rate was 18.9%. All 90 strains were isolated from urine samples. Results of antibiotic susceptibility test revealed that these isolates had low resistance rates for fosfomycin (2.7%), imipenem (3.2%), and meropenem (3.2%). However, resistance rates for ciprofloxacin (62.2%), trimethoprim sulfamethoxazole (75.6%), and ampicillin (61.1%) were high. Rates of resistance against amikacin, nitrofurantoin, ceftriaxone, ceftazidime, gentamycin, amoxicillin with clavulanic acid, aztreonam, cefazolin, and cefepimine were found to be 9.9%, 8.9%, 22.2%, 21.1%, 27.8%, 27.8%, 18.9%, 18.9%, and 20%, respectively.

More specifically, we found that fim was the most common virulence gene and was found in 74 isolates (82.2%). The afa and cnf1 genes were detected in 16.6% of the isolates, and hly was found in only three (3.3%) of the 90 isolates. The sfa and pap genes were not detected. In addition, the aer gene was found in 33 (36.6%) of the isolates. PCR results revealed that 63% (57/90) of the strains carried class 1 integron gene cassettes. We also observed a high prevalence of ESBLs, with 52 strains (57%) carrying a CTX-M-2, and 52 isolates (57%) carrying a CTX-M-1 group β-lactamase. No other β-lactamase-encoding genes (bla _IMP, bla _VIM, bla _NDM, bla _GES, bla _SIM, bla _AmpC, or bla _SPM) were identified. We also demonstrated that the qnrA, qnrB, and qnrS quinolone resistance genes-present on the plasmid-were present in 26.6% (24/90), 6.7% (6/90) and 3.3% (3/90) of the isolates, respectively. No fosfomycin resistance genes (fosA, fosC2, or fosA3) were found.

The prevalence of virulence factors differed among isolates that produced a class 1 integron, bla _{CTXM-1 ,} bla _{CTXM-2 ,} qnrS, qnrA, and qnrB (Table 1). Class 1 integron and CTX-M harboring isolates were more commonly positive forfim than for other virulence factors.

Eleven different virulence factors were observed among the 90 E. coli isolates. The most common virulence factor was fım (n = 35 isolates; 8.9%); hly-fım, afa-aer-cnf-fım, aer-cnf, afa-aer, and afa-cnf-fım were less commonly observed. No virulence factor was detected in fourteen of the isolates (Table 2).

DISCUSSION

Urinary tract infections (UTIs) are a major public health problem worldwide. E. coli is the most prevalent etiologic agent of UTIs. The virulence of UTI inducing E. coli strains is due to their expression of virulence factors⁴4. Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J. Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. Int J Infect Dis. 2013;17(6):e450-53.^.2323. Djuikoue IC, Woerther PL, Toukam M, Burdet C, Ruppé E, Gonsu KH, et al. Intestinal carriage of extended spectrum beta-lactamase producing E. coli in women with urinary tract infections, Cameroon. J Infect Dev Ctries. 2016;10(10):1135-9..

P fimbriae (pap), a fimbrial adhesin I (afaI), hemolysin (hly), cytotoxic necrotizing factor 1 (cnf1), aerobactin (aer), S fimbriae (sfa) and type 1 fimbriae (fimH) are the most important virulence factor genes found in these E.coli strains²⁴24. Momtaz H, Karimian A, Madani M, Safarpoor Dehkordi F, Ranjbar R, Sarshar M, et al. Uropathogenic Escherichia coli in Iran: Serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob. 2013;12:8.doi: 10.1186/1476-0711-12-8.
https://doi.org/10.1186/1476-0711-12-8....

25. Soto SM, Guiral E, Bosch J, Vila J. Prevalence of the set-1B and astA genes encoding enterotoxins in uropathogenic Escherichia coli clinical isolates. Microb Pathog. 2009;47(6): 305-7.

26. Bauer RJ, Zhang L, Foxman B, Siitonen A, Jantunen ME, Saxen H, Marrs CF. Molecular epidemiology of 3 putative virulence genes for Escherichia coli urinary tract infection-usp, iha, and iroN(E. coli). J Infect Dis. 2002;185(10):1521-4.^-2727. Johnson JR, Russo TA, Tarr PI, Carlino U, Bilge SS, Vary JC Jr, Stell AL. Molecular epidemiological and phylogenetic associations of two novel putative virulence genes, iha and iroN (E. coli), among Escherichia coli isolates from patients with urosepsis. Infect Immun. 2000;68(5):3040-7.. The bacterial adhesin fimH (which plays an integral role in the pathogenesis of E. coli) is a virulence factor that is located on the type 1 pili of E. coli. Of the seven virulence genes examined in this study, the fim gene was detected most frequently (82.2%). Kot et al. (2016) reported similar results²⁸28. Kot B, Wicha J, Grużewska A, Piechota M, Wolska K, Obrębska M. Virulence factors, biofilm-forming ability, and antimicrobial resistance of urinary Escherichia coli strains isolated from hospitalized patients. Turk J Med Sci. 2016;46(6):1908-14.. Moreover, the fimH adhesion gene was the most common virulence gene in both UTIs and asymptomatic bacteriuria (ABU) isolates studied by Yun et al. (2014). Their results showed that the pap gene family was also prevalent in UTI and ABU isolates²⁹29. Yun KW, Kim HY, Park HK, Kim W, Lim IS. Virulence factors of uropathogenic Escherichia coli of urinary tract infections and asymptomatic bacteriuria in children. J Microbiol Immunol Infect. 2014;47(6):455-61.. In our study, we did not find the pap gene in any E. coli isolates. In another study³⁰30. Arabi S, Tohidi F, Naderi S, Nazemi A, Jafarpour M, Naghshbandi R. The common fimbarie genotyping in uropathogenic Escherichia coli. Ann Biol Res. 2012;3(10):4951-4., sfa was the most common virulence gene; by contrast, we found no sfa in our isolates. The presence of afaI, hly, and cnf1 virulence factor genes was estimated to be 8.13%, 50.4%, and 50.4%, respectively, by another study²⁴24. Momtaz H, Karimian A, Madani M, Safarpoor Dehkordi F, Ranjbar R, Sarshar M, et al. Uropathogenic Escherichia coli in Iran: Serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob. 2013;12:8.doi: 10.1186/1476-0711-12-8.
https://doi.org/10.1186/1476-0711-12-8.... . In our study, we determined the presence of the afa, hly, and cnf1 virulence genes to be 16.6%, 3.3%, and 16.6%, respectively. Among the seven virulence genes that we studied, aer (36.6%) was the second most common virulence factor. Similar to our results, another study reported aer as the second most frequently detected virulence factor coding gene after the highly prevalent fimH gene³¹31. Usein CR, Damian M, Tatu-Chitoiu D, Capusa C, Fagaras R, Tudorache D, et al. Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases. J Cell Mol Med. 2001;5(3):303-10.. The elevated levels of type 1 fimbriae may be correlated with the pathogenicity of the isolated strains, as type 1 fimbriae (fimH) play a crucial role in the colonization of the urinary tract³²32. López-Banda DA, Carrillo-Casas EM, Leyva-Leyva M, Orozco-Hoyuela G, Manjarrez-Hernández ÁH, Arroyo-Escalante S, et al. Identification of virulence factors genes in Escherichia coli isolates from women with urinary tract infection in Mexico. Biomed Res. 2014; Int. :959206. doi: 10.1155/2014/959206.
https://doi.org/10.1155/2014/959206... . In addition, these results showed that the geographical region can affect the prevalence of these genes¹²12. Firoozeh F, Saffari M, Neamati F, Zibaei M. Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis. Int J Infect Dis . 2014;9:219-22.. The studied strains exhibited 11 virulence gene patterns. The E2 was characterized by the presence of only the fim gene and was the most commonly seen pattern, found in 35 isolates. A small number of isolates with four virulence factors were detected, and fim was the most common virulence factor. Most of the isolates contained different combinations of resistance determinants.

Inappropriate and unnecessary application of quinolones has led to the emergence of resistant E. coli isolates that limit treatment options¹⁹19. Rezazadeh M, Baghchesaraei H, Peymani A. Plasmid-mediated quinolone-resistance (qnr) genes in clinical isolates of Escherichia coli collected from several hospitals of Qazvin and Zanjan Provinces, Iran. Osong Public Health Res Perspect. 2016;7(5):307-12.. Our PCR results showed that qnrA, qnrB, and qnrS genes were present in 26.6% (24/90), 6.6% (6/90), and 3.3% (3/90) of studied isolates, respectively. In contrast to our results, one study reported that the most prevalent qnr determinant was qnrB, followed by qnrS¹⁸18. Yanat B, Vinuesa T, Viñas M, Touati A. Determinants of quinolone resistance inEscherichia colicausing community-acquired urinary tract infection in Bejaia, Algeria. Asian Pac J Trop Med. 2014;7(6):462-7.. In another study, 120 isolates of E. colifrom UTIs were investigated for the presence of qnrA, B, and S, and qnrB(2.18%) andqnrS (1.12%) genes were detected, butqnrA was not found³³33. Sedighi I, Arabestani MR, Rahimbakhsh A, Karimitabar Z, Alikhani MY. Dissemination of extended-spectrum β-lactamases and quinolone resistance genes among clinical isolates of uropathogenic Escherichia coli in children. Jundishapur J Microbiol. 2015;8(7):e19184..

Plasmid-mediated quinolone resistance (PMQR) genes are usually found in association with the ESBL genes¹⁸18. Yanat B, Vinuesa T, Viñas M, Touati A. Determinants of quinolone resistance inEscherichia colicausing community-acquired urinary tract infection in Bejaia, Algeria. Asian Pac J Trop Med. 2014;7(6):462-7.. CTX-M enzymes have been identified in both hospital and community settings and belong to one group of ESBLs³⁴34. Pitout JD, Laupland KB, Church DL, Menard ML, Johnson JR. Virulence factors ofEscherichia coli isolates that produce CTX-M-type extended-spectrum β-lactamases. Antimicrob Agents Chemother. 2005;49(11):4667-70.. Co-expression of bla _CTXM and PMQR genes has been reported in E. coli isolated from UTIs¹⁸18. Yanat B, Vinuesa T, Viñas M, Touati A. Determinants of quinolone resistance inEscherichia colicausing community-acquired urinary tract infection in Bejaia, Algeria. Asian Pac J Trop Med. 2014;7(6):462-7.^,3535. Nazik H, Bektöre B, Öngen B, İlktaç M, Özyurt M, Kuvat N, et al. Plasmid-mediated quinolone resistance genes in Escherichia coli urinary isolates from two teaching hospitals in Turkey: Coexistence of TEM, SHV, CTX-M and VEB-1 type β-lactamases. Trop J Pharm Res. 2011;10(3):325-33.. Multi drug resistance (MDR) rates were significantly higher in PMQR-positiveK. pneumoniaeandE. cloacaeisolates (17-28 times) than in PMQR-negative isolates. This finding, which has been observed by other researchers, may indicate a link betweenqnrBand other antibiotic resistance genes. In this study, however, this association was not found in E. coliisolates retaining PMQR genes³⁶36. Kim HB, Park CH, Kim CJ, Kim EC, Jacoby GA, Hooper DC. Prevalence of plasmid-mediated quinolone resistance determinants over a 9-year period. Antimicrob Agents Chemother . 2009;53(2):639-45..

The pattern of virulence factors and antibiotic resistance genes is constantly changing in organisms isolated from UTIs, so this and similar studies are necessary to stay abreast of local and national antimicrobial resistance trends for the empirical treatment of UTIs¹⁹19. Rezazadeh M, Baghchesaraei H, Peymani A. Plasmid-mediated quinolone-resistance (qnr) genes in clinical isolates of Escherichia coli collected from several hospitals of Qazvin and Zanjan Provinces, Iran. Osong Public Health Res Perspect. 2016;7(5):307-12..

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