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 sfagenes, represent another type of virulence factor8. A fimbrial adhesion factors in E. coli are encoded by afagenes9. 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. coliisolates 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. coliisolates 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 (blaIMP, 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. coliisolates 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.
