Identification of Virulence Factors Genes in Escherichia coli Isolates from Women with Urinary Tract Infection in Mexico

E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed.


Introduction
Urinary tract infections (UTI) are one of the most common infections worldwide. Uropathogenic Escherichia coli (UPEC) is the primary pathogen causing UTIs; it colonizes the human intestine a few hours after birth and is considered part of the normal microbiota. However, it can cause various diseases such as diarrhea, UTI, and meningitis [1]. It is classified into three groups: (i) commensal, (ii) intestinal pathogenic, and (iii) extraintestinal pathogenic [2], and phylogenetically it has been classified into four classic groups (A, B1, B2, and D) [3]. Uropathogenic E. coli is located within the extraintestinal pathogenic E. coli (ExPEC), classified primarily into the phylogenetic group B2 and to a lesser extent to group D, whereas commensal strains are within the phylogenetic groups A and B1 [4][5][6][7][8].
The ability of E. coli to colonize different anatomical sites is due in part to genome plasticity and remodeling by acquisition or loss of genetic material from which it acquired resistance or virulence factors. Therefore, horizontal transfer is an important factor in the evolution and adaptation of E. coli to different niches [9,10]. The interaction between bacteria and epithelial cells is a multifactorial and complex phenomenon which involves several adhesins produced according to the stage of infection, while adherence to epithelial cells is essential for successful colonization and establishment; the expression of other genes encoding toxins, siderophores, lipopolysaccharide (LPS), capsule, and invasins determines the disease severity and the strain's virulence [8]. UPEC strains can cause acute infections and recurrent infections that do not respond to common antimicrobial treatments. UTI treatment generally includes -lactam antibiotics, fluoroquinolones, or trimethoprim/sulfamethoxazole [11][12][13] but may vary according to patient age, sex, Pathogen involved, course of disease, and the urinary tract anatomic area involved [5]. The increased resistance may be related to changes in the bacterial genome by mutation or acquisition by horizontal transfer of an extrachromosomal or chromosomal material [14][15][16].
Urinary tract successful invasion depends on the bacteria virulence, inoculums size, and the host's defense mechanisms [18]. However, women have higher UTI's prevalence and incidence mainly due to their anatomical characteristics such as the proximity between the anus and the urethral opening, hormone effects, and changes in the genital microbiota [14,19]. Clinically a UTI is defined by a bacteriuria with a count in midstream urine culture ≥10 5 CFU/mL and pyuria or the presence of white blood cells in the urine, more than five leukocytes per field [19].
Globally it is estimated that about 150 million UTIs occur annually [20]. In the United States and Spain the current situation and treatment of urinary tract infections had been thoroughly described [6][7][8]18]; it is estimated that 11% of women experience at least one diagnosis of urinary tract infection (UTI) per year, and 60% of women will have or have had an UTI or more during their lifetime [4]. In Mexico UTI's status has not been described. However, to our knowledge it is E. coli one of the pathogenic agents of UTI and it is more frequent in women, with high incidence and prevalence, representing a costly problem for the health sector [4,21]. In 2008, 3,244,994 cases were reported, which represents an incidence of 3,041.7/100,000 inhabitants, from which 75.6% (2,453,608/100,000 inhabitants) were women, representing an incidence of 4,508.6/100,000.
The present study aimed to describe the profile of E. coli from Mexican women with urinary tract infection by the identification of virulence genes (fimH, papC, sfa/focDE, sfaS, focG, ecpA, ecpR-B, hlyA, cnf-1, cdt-B, cvaC, iutA, ibeA, rfc, tratT, and fyuA), phylogenetic group, and their resistance to antibiotics to guide better diagnosis and treatment of UTI.

Material and Method
2.1. Bacteria and Culture. Bacterial isolates (108) were obtained from urine samples from women diagnosed with acute urinary tract infection and confirmed by the clinical laboratory of the General Hospital "Dr. Manuel Gea Gonzalez" during 2008 and until 2010. All samples with counts over 100,000 UFC/mL were included. Patients were within an age range between 12 and 58 years and mean age  [17].

Statistical Analysis.
To establish the results significance, the Fisher exact test was used. The level of significance was set at a P value of ≤0.05.

Results
The overall results of the isolates regarding the virulence genes, the phylogenetic group, and resistance profile are shown in Table 3. Regarding the phylogenetic group, most of the isolates were (60) grouped into the B2 group (55.6%), 33 isolates were classified as part of the A group (30.6%), 10 isolates (9.3%) to group B1, and 5 isolates (4.6%) to group D.  Table 5. Isolates which displayed resistance to more than ≥3 chemotherapeutic groups were considered multiresistant isolates, which represents 58%. However no statistical relation was observed among multiresistance and phylogenetic group (Table 6).

Discussion
In this work 108 E. coli isolates were screened from female patients with an average age of 39 years; women were regarded as a productive population, for which urinary tract infections are considered a major cause of morbidity in our country and represent a huge economic impact [23,24].
BioMed Research International Phylogenetic group (PG).  The predominant phylogenetic group was B2 (55.6%), widely associated with pathogenic strains. In Spain and the United States similar results had been reported and also a lower percentage related to the phylogenetic group D [6-8, 18, 25]. The phylogenetic group A, associated with commensal strains, represents a 30.6%, higher than in other studies, suggesting that the gastrointestinal tract is the main reservoir of strains that may be able to colonize the urinary tract in accordance to previous observations [6,7,18,25]. The B1 group (9.3%) as a cause of urinary tract infections points out the high plasticity of the E coli genome which allowed the presence of the fimH, papC, and ecp (A and RB) in percentages of 70%, 60%, and 100%, respectively. These genes are related to the ability to colonize the urinary tract epithelium [18,22,[26][27][28].
Adhesins genes were present in high percentages: fimH (86.1%), ecp (A y R-B) (98.1%), and papC (62%), this result could be related to the pathogenicity of the isolated strains as adherence is the most important pathogenicity determinant [4]. The fimH geneonce again was highly conserved in UTI isolates which confirms its crucial role during colonization of the urinary tract [4,[29][30][31][32].  The ecp (A and RB) gene is associated with commensals and enteropathogenic strains; it was present in 98.1% of this study isolates and according to a similar observation in Portugal it was found in 100% of their isolates; it may be associated with UPEC [17,22,24,28]. The papC gene encodes an outer membrane protein essential for the fimbriae P biogenesis regulation. pap genes presence had been associated with pyelonephritis; therefore, higher percentages (over 50%) suggest that the strains isolated from the Mexican population have greater capabilities to colonize kidneys and generate pyelonephritis [32,33].
The hlyA and cnf-1 genes showed a positive relationship with the B2 group; also they are associated with pathogenicity island PAI II J96 , and the iutA gene is associated with pathogenicity island PAI I CFT073 as well as with hlyA and pap operon [8,19,[34][35][36].
The sfaS gene was found exclusively in hlyA and cnf-1 positive isolates which could be linked to cystitits cases. This observation is in accordance with the previous report by Lloyd et al. [19].
The cvaC gene was present in only three isolates traT positive. These genes are both located at the colV plasmid. traT is related to the phylogenetic group B2, and presumes an animal source. The iutA and fyuA genesalso showed a relation with the phylogenetic group B2 [37][38][39]. The ibeA gene, related to the B2 group, was found in an isolate identified as B1, a result that may start to change the previous assumption [40]. The rfc gene was identified in just one isolate which indicates that the serogroup O4 was not the predominant serogroup in the population studied and that this result may need further serological confirmation [5,41].
The treatment of choice for ITU is in order of importance: fluoroquinolones (ciprofloxacin), the trimethoprim/ sulfamethoxazole, cephalosporins, and penicillins (ampicillin) to which an increasingly developed resistance has been reported due mainly to the indiscriminated antibiotic use [13,14]. In this work it is confirmed the resistance previously reported values for trimethoprim/sulfamethoxazole (56.1%) [14,42]; for ciprofloxacin (62.3%), gatifloxacin, levofloxacin, and moxifloxacin, resistance was always above 50%. Positive isolates to hlyA, cnf-1, and/or papC genes were susceptible to fluoroquinolones, results similar to those of Piatti et al. [43]. Besides in Mexico, the previously reported E. coli resistance profile included ampicillin, piperacillin, fluoroquinolones, and trimethoprim/sulfamethoxazole which are considered to be first-line choices [13,44,45]. Additionally, serotype 025b-ST131 has been reported to be within the Mexican population which has been associated with plasmid mediated quinolone resistance [46]. We had identified multidrug resistance of the E. coli strains causing UTI in 58% of the isolates which belonged mainly to group B2, result which kept our attention.

Conclusion
This work confirms that most of the isolates associated with urinary tract infections belong to the phylogenetic group B2 and in a lesser extent to group D. Also they displayed a great number of virulence genes. However, commensal strains may also be the cause of UTI. According to our results most parts of the isolates have the ability to colonize the kidneys as they have a high incidence of the papC gene. The hlyA and cnf-1 genes encoding toxins and fyuA iutA and siderophores encoding genes are tightly associated with the phylogenetic group B2.
E. coli has successfully adapted to host's conditions and to the general medical practices as we may observe the high resistance to trimethoprim/sulfamethoxazole and fluoroquinolones, especially on the most frequently isolated phylogenetic groups.
Finally, these results reinforce international knowledge on antimicrobial resistance and the high rate of multidrug resistance found invites us to encourage population awareness of the proper use of antimicrobials.