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Revista da Sociedade Brasileira de Medicina Tropical

versão impressa ISSN 0037-8682versão On-line ISSN 1678-9849

Rev. Soc. Bras. Med. Trop. vol.52  Uberaba  2019  Epub 27-Jun-2019

http://dx.doi.org/10.1590/0037-8682-0499-2018 

Major Article

Determination of antibiotic resistance genes and virulence factors in Escherichia coli isolated from Turkish patients with urinary tract infection

Azer Özad Düzgün1 
http://orcid.org/0000-0002-6301-611X

Funda Okumuş2 

Ayşegül Saral3 

Ayşegül Çopur Çiçek4 

Sedanur Cinemre2 

1Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Gumushane University, Gümüşhane, Turkey.

2Department of Biotechnology, Institute of Natural Sciences, Gumushane University, Gümüşhane, Turkey.

3Department of Nutrition and Dietetics, Faculty of Health Sciences, Artvin Coruh University, Artvin, Turkey.

4Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdoğan University, Rize, Turkey.


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 issue1-4. Many virulence factors are responsible for the pathogenicity of E. coli strains4-5. 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 cell6.

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 factors7. S fimbrial adhesion factors, encoded by sfa genes, represent another type of virulence factor8. A fimbrial adhesion factors in E. coli are encoded by afa genes9. In addition, the main fimbrial subunit of type 1 fimbriae is encoded by fimA in E. coli10. 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, CNF15, and aer genes, respectively11,12.

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 worldwide13 and have been mainly identified in Enterobacteriaceae of the IMP and VIM types14-17.

Quinolones are widely used to treat UTIs caused by E. coli. This extensive use of quinolones has led to increased resistance in E. coli18. 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 qnrdeterminants19.

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 integrons20-21.

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 assays22.

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.

TABLE 1: Prevalence of virulence factors and antibiotic resistance genes among strains. 

Antibiotic resistance genes and integrons Virulence factor genes
afa hly aer cnf fim
class 1 integron 9 3 25 11 48
bla CTXM-1 7 - 11 7 42
bla CTXM-2 7 - 16 7 44
qnrS - - - - 2
qnrA 4 1 12 3 23
qnrB 1 1 1 1 4

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).

TABLE 2: Prevalence of virulence patterns among 90 E. coli isolates. 

Pattern codes Virulence Patterns Number of Isolates
E1 afa-aer-fım 6 (6.7%)
E2 fım 35 (38.9%)
E3 aer-fım 12 (13.3%)
E4 afa-fım 6 (6.7%)
E5 hly-fım 1 (1.1%)
E6 afa-aer-cnf-fım 1 (1.1%)
E7 hly-aer-cnf-fım 2 (2.2%)
E8 aer-cnf-fım 10 (11.1%)
E9 aer-cnf 1 (1.1%)
E10 afa-aer 1 (1.1%)
E11 afa-cnf-fım 1(1.1%)
No virulence factor 14 (15.6%)

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 factors4.23.

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 strains24-27. 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 results28. 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 isolates29. In our study, we did not find the pap gene in any E. coli isolates. In another study30, 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 study24. 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 gene31. 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 tract32. In addition, these results showed that the geographical region can affect the prevalence of these genes12. 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 options19. 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 qnrS18. 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 found33.

Plasmid-mediated quinolone resistance (PMQR) genes are usually found in association with the ESBL genes18. CTX-M enzymes have been identified in both hospital and community settings and belong to one group of ESBLs34. Co-expression of bla CTXM and PMQR genes has been reported in E. coli isolated from UTIs18,35. 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 genes36.

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 UTIs19.

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Recebido: 04 de Dezembro de 2018; Aceito: 24 de Abril de 2019

Corresponding author: Azer Özad Düzgün. e-mail:azerozad@windowslive.com

Conflict of interest: The authors declare no conflicts of interest

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