Introduction

Urinary tract infection (UTI) is one of the frequently recognized bacterial infections, striking 150 million humans every year. It results in high morbidity and mortality and also costly health and medical issues [1]. Among the numerous bacterial agents causing UTI, Uropathogenic Escherichia coli (UPEC) is one of the etiologic agents of UTIs, accounting for 80–90%, and 50% of the community and hospital-acquired UTIs, respectively [1,2,3,4,5]. Among the pathogenesis characterization of UPEC isolates, biofilm formation is crucial as it facilitates their colonization in the urethra [6,7,8]. Moreover, recurrence of UTI by UPEC isolates might be due to the capability of virulent strains in biofilm production [9]. Biofilm formation can affect both the activity of antimicrobial agents and the host immune response, leading to the persistence of UPEC in the urinary tract and emergence of severe symptoms and antimicrobial resistance [7,8,9,10]. Several antibiotics are used for treatment of UTI, including Nitrofurantoin, Trimethoprim/Sulfamethoxazole, Oral β-lactams, fosfomycin and Fluoroquinolones [11]. Nitrofurantoin is the best recommended treatment for cystitis. This drug inhibits bacterial enzymes involved in the synthesis of DNA, RNA, cell wall protein synthesis, and other metabolic enzymes [12]. Trimethoprim/Sulfamethoxazole has been considered as the standard for therapy of acute and recurrent UTIs. These two agents act synergistically in inhibiting folic acid synthesis [11]. Fosfomycin inhibits an enzymatic-catalyzed reaction in the first step of the synthesis of bacterial cell wall [13]. Quinolones, as one of the frequently used antibiotics, are critical drugs for therapy of UTIs caused by Gram-negative bacteria in clinical settings [14, 15]. The extensive and inappropriate use of quinolones and other antimicrobial agents for the management of bacterial infections has led to a remarkable increase in resistant isolates which are recognized as a great public health concern [16, 17]. Four main groups of qnr determinants, namely qnrA, qnrB, qnrC, and qnrS, have been diagnosed. These plasmid-mediated quinolone resistance (PMQR) determinants act through protection of DNA gyrase and topoisomerase IV from antibiotic action [10, 18]. The increased antibiotic resistance could be attributed to horizontal transfer of genes for formation of multi-drug resistant (MDR) strains; therefore, biofilms are considered as an ideal niche for this transfer especially plasmid exchange [19, 20]. To study the population genetics of UPEC isolates, researchers have extensively used phylogenetic groups for their simplicity [21]. Both virulent and commensal isolates of E. coli have been categorized into four major phylogenetic sets consisting of A, B1, B2, and D, which are common in humans. Accordingly, human virulent extraintestinal pathogenic Escherichia coli (ExPEC) are commonly classified as phylogroup B2 or D, while A or B1 groups mostly belong to commensal strains and less pathogenic strains [22, 23]. Determination of resistance pattern and phylogenetic groups and their association is important in characterization of the isolates [21]. Due to lack of studies on quinolone resistance genes, phylogenetic groups and biofilm formation among UPEC isolates in our region, this study aimed to investigate these characteristics among UPEC isolates from inpatients in Shiraz, southern Iran.

Materials and methods

Bacterial isolates and study population

In this study, a total of 126 non-duplicate UPEC which were recovered from a previous study in 2016 were used [16]. The participants were hospitalized patients with symptomatic UTI, including cystitis, pyelonephritis and urosepsis. Exclusion criteria were patients with asymptomatic UTI, and not taking antibiotics during 1 month ago. The Ethics Committee of Shiraz University of Medical Sciences approved this study (Approval No. EC IR.SUMS.REC.1397.688).

Quinolone susceptibility testing

The antibiotic susceptibility pattern was performed by standard disk diffusion method on Mueller–Hinton agar (Himedia, India) plate according to the Clinical and Laboratory Standards Institute (CLSI) recommendation for nalidixic acid, ciprofloxacin, levofloxacin, norfloxacin and ofloxacin (Mast Co., UK) [24]. In our study E. coli ATCC 25922 was used as the control strain.

Phylogenetic grouping and the presence of quinolone resistance genes

After recovery of the isolates from freeze − 70 °C by plating onto blood agar, DNA extraction of the studied isolates was conducted using the boiling method as described previously [25]. PCR for detection of qnrA, qnrB, and qnrS genes [26] was carried out on a T100™ thermal cycler (Bio-Rad, Hercules, CA, USA) in a total volume of 25 µL containing 3 µL DNA template, 2.5 µL PCR buffer (1×), 1 µL deoxyribonucleotide triphosphates solution (dNTPs, 200 µM), 1.5 µL MgCl2 (1.5 mM), 0.25 µL Taq DNA polymerase (1 Unit), and 1 µL each specific primer (1 µM). Indeed, PCR was used for determining the phylogenetic groups (A, B1, B2 and D) through amplification of chuA and yjaA genes and the DNA fragment TspE4.C2 [21]. PCR amplification consisted of initial-denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, primer annealing at 54 °C and extension at 72 °C for 60 s, and a final extension at 72 °C for 7 min. The amplicons were analyzed on agarose gel 1.5%, stained with safe stain (CinnaGen Co. Iran) and finally visualized under ultraviolet light.

Biofilm formation assay

A microtiter plate method was performed to investigate the biofilm formation based on the protocol of O’Toole et al. [27]. Briefly, after adding 100 µL of the 1:100 dilutions of bacterial cultures into 96 well plates, they were incubated for 24 h at 37 °C. After washing of the wells with distilled water, the wells were stained with 0.1% solution of crystal violet (CV) and incubated for 15 min at room temperature. The microplates were rinsed and dried; then, 30% acetate was added as solvent of CV. After 15 of min incubation, the contents of wells were moved to a new microplate and their absorbance was read at 550 nm by ELISA reader. All tests were performed in triplicate.

DNA sequence analysis

To confirm the accuracy of amplified genes, the amplicons (four samples) were submitted for sequencing (Bioneer Co., Munpyeongseoro, Daedeok-gu, Daejeon, South Korea) and the sequences were compared using online BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/).

Statistical analysis

The analysis was performed using SPSS™ software, version 21.0 (IBM Corp., USA). The Chi-square test was used and differences were considered significant when the P value was ≤ 0.05.

Results

Study population

Our archive (126 UPEC) isolates were obtained from fifty females and seventy six males with an age range from 1 to 100 years old with a mean age of 48.9 ± 28.8 years. No significant association was shown in the age and gender of cases in relation to the three studied clinical groups (cystitis, pyelonephritis and urosepsis).

The wards of UPEC isolation were from Intensive Care Unit (ICU) with a frequency of 76 (60.4%), followed by Internal ward (36; 28.6%), Surgery ward (7; 5.6%), and Transplantation center (7; 5.6%). Furthermore, the frequency of the cases in different wards was as follows: cystitis (ICU = 23, Internal ward = 12, Surgery = 2, Transplantation = 5), pyelonephritis (ICU = 44, Internal ward = 22, Surgery = 5, Transplantation = 2) and urosepsis (ICU = 9, Internal ward = 2, Surgery = 0, Transplantation = 0).

Quinolone resistance among UPEC isolates

The results of antibiogram pattern revealed that the least susceptibility was against nalidixic acid (19%), followed by ofloxacin with 42.1% (Table 1).

Table 1 The antibiotic susceptibility testing results of 126 UPEC isolates
Full size table

Characterization of quinolone resistance genes

The analysis of PCR results showed that 22.2% (28/126) and 13.5% (17/126) of the isolates were positive for the qnrS and qnrB genes, respectively. Meanwhile, 2.4% of the isolates were positive for both qnrB and qnrS genes simultaneously, and qnrA gene was not detected in any of UPEC isolates. Descriptive results of the association between quinolone resistance and the presence of qnr genes showed that, among qnrS negative and positive isolates, the highest resistance rate was toward nalidixic acid, while the most effective antibiotic was ciprofloxacin. Statistical analysis indicated that there was no significant association between antibiotic resistance and the existence of qnr genes (Tables 2 and 3) (Fig. 1).

Fig. 1
figure 1

Gel electrophoresis of PCR products for qnrB, qnrS, chuA, TspE4C2 and yjaA genes in UPEC isolates. Lane 1 = chuA gene, Lane 2 = chuA Positive control, Lane 3 = TspE4C2 gene, Lane 4 = TspE4C2 Positive control, Lane 5 = yjaA gene, Lane 6 = yjaA Positive control, Lane M = DNA ladder (100 bp), Lane 7 = qnrS gene, Lane 8 = qnrS Positive control, Lane 9 = qnrB gene, Lane 10 = qnrB Positive control, Lane 11: Negative control

Full size image
Table 2 The antibiotic susceptibility pattern according to results of qnrS genes
Full size table
Table 3 The antibiotic susceptibility pattern according to results of qnrB genes
Full size table

Phylogenetic grouping

PCR method for the investigation of phylogroups showed that UPEC isolates were separated into four phylogenetic groups (A, B1, B2, and D) and seven subgroups. Out of 126 UPEC isolates, the commonest phylogroup was group B2 (106 isolates; 84.1%), followed by group D (13, 10.3%), group A (4, 3.2%) and group B1 (3, 2.4%) (Fig. 1). Furthermore, the main phylogenetic groups were divided into seven subgroups, namely B23 (81%), D2 (7.8%), B22 (3.2%), B1 (2.4%), D1 (2.4%), A1 (2.4), and A0 (0.8%). Distribution of phylogroups according to the ward’s, clinical disease, and biofilm formation capability is shown in Table 4.

Table 4 Distribution of phylogenetic group according to infections, wards and biofilm formation
Full size table

Biofilm production

Out of the 126 UPEC isolates, 80.2% (101/126) were positive to the ability of biofilm formation using the microtiter plate assay and were categorized into four groups based on their ability for biofilm production. According to the results, 42.1%, 16.7% and 21.4% of UPEC isolates were classified as weak, moderate and strong biofilm producers.

Discussion

Today, due to antibiotic resistance to first-line drugs, quinolones and fluoroquinolones are the preferential options for curing UTIs caused by UPEC isolates [28]. In this study, quinolone susceptibility pattern, biofilm formation and the distribution of quinolone resistance genes and phylogenetic groups among 126 UPEC isolated from a symptomatic population in southern Iran were assessed.

In the current survey, antibiotic susceptibility pattern showed that more than 50% and 81% of the isolates were resistant to fluoroquinolones and quinolones (nalidixic acid), respectively. In a recent survey from Shiraz, an increase in quinolone-resistant UPEC isolates (more than 40%) was reported; this in the same line with our study [29]. Shenagari and co-workers from north of Iran reported that 45.3% of UPEC isolates were resistant to norfloxacin, 48.9% to ofloxacin, 50.2% to ciprofloxacin, and 61.9% to nalidixic acid [30]. In the neighboring countries, reports indicate a high resistance to fluoroquinolone and quinolone. For instance, Muhammad et al. from Pakistan reported that the resistance rate to ciprofloxacin and nalidixic acid among UPEC isolates was 36.45% and 84.16%, respectively [31]. Because quinolones and fluoroquinolones are used for empirical therapy of complicated UTI, there is a major challenge for physicians to treat this infection [32].

Increased resistance to fluoroquinolones among Enterobacterales is associated with a high rate of PMQR genes which is related to diversity of PMQR genes or mutations in gyrA and parC genes, or both of them in PMQR-positive strains [33]. Accordingly, in the current study the frequency of qnrS and qnrB genes was found 22% and 13.5%, respectively, indicating no significant difference between the presence of fluoroquinolone genes and antibiotic resistance to them. In investigation of Malekzadegan et al. 33.1% and 12.4% of the isolates were qnrS and qnrB positive, respectively, and no significant difference was found between higher antibiotic resistance and qnr genes [29]. In contrast to our research, in studies conducted by Sedighi and colleagues [34] and Rezazadeh et al. [35]., low rates of qnrS and qnrB genes among quinolone-resistant UPEC isolates were identified, along with a significant association between qnr genes and quinolone resistance. In contrast, in a study from north of Iran, regarding to UPEC, a high prevalence of qnrB (71.3%) and qnrA (62.8%) genes together a considerable correlation with resistance to quinolones has been reported [36]. In the current work, no isolate harbored qnrA gene which is consistent with the result of the studies conducted by Sedighi and co-workers and Rezazadeh et al. [34, 35]. Although there is relative agreement on the high prevalence of qnrS and qnrB genes in UPEC isolates, the distribution of predominant genes varies in different areas.

According to Clermont classification, E. coli strains based on chuA, yjaA and TspE4.C2 genetic determinants have been divided into four phylogenetic groups, including A, B1, B2, and D [37].

In consistent with the literature, all of the major phylogroups were determined in the current work. In line with prior studies [8, 21], most of our isolates belonged to group B2, followed by D group. Conversely, in studies from Romania and India after B2 as the dominant group, B1 and A groups were the second phylogroup, respectively [38, 39]. Distribution of phylogenetic groups varies according to geographical regions, even within each country. In accordance with our results, Yazdanpour et al. and Mostafavi and colleagues revealed that among 248 their UPEC isolates, 67.3%, and 45.4% of the isolates were associated with the B2 phylogenetic group, followed by D (21.4%, 25.3%), A (6.5%, 10.5%) and B1 (4.8%, 16.8%) groups, respectively [40, 41]. Iranpour et al. and Derakhshan et al. also showed that the B2 group was the most prevalent group among UPEC isolates and phylogenetic group of D had a very low rate (2.9% and 3.3%) [42, 43]. Even though previous investigations demonstrate that commensal strains are associated with group A or group B1[37], Hashemizadeh and co-workers revealed that B2 and D were the frequent phylogroups in fecal isolates [44]. Therefore, distribution of various types of phylogroups among E. coli isolates may be caused by the heterogeneity of ExPEC isolates in various studies.

It has been suggested that B2 phylogenetic group isolates reveal higher quinolone-susceptibility than those of other groups [39]. However, in our survey the isolates belonging to B2 phylogroup demonstrated higher resistance against fluoroquinolones. Likewise, in a report from Iran, B2 and D phylogroups represented the most resistant UPEC isolates to antimicrobials [45].

In 80% of infections, biofilm formation in bacteria is documented to account for a serious problem in UTIs [46]. Biofilm production by UPEC strains in the urinary tract, as well as on the urinary catheters, is one of the major causes of the persistence of these isolates, so that in most cases it leads to recurrent infections [9]. Our results showed that 80.2% (101/126) of the isolates were positive for biofilm production, of which, 42.1%, 16.7% and 21.4% of UPEC isolates were classified as weak, moderate and strong positive in their ability to form biofilms, correspondingly. In the study of Tajbakhsh et al., out of 80 biofilm-producing E. coli strains, 15 (18.75%) were strong, 20 (25%) moderate, and 45 (56.25%) weak positive isolates [47]. Also, Ponnusami and colleagues observed that among 100 UPEC isolates, 17.23%, 26.3% and 50% were strong, intermediate and weak biofilm formers, respectively [48]. These findings are relatively similar to our results. Likewise, Poursina et al. studied the biofilm formation assay of 100 UPEC isolates, of which 80% had capability of this characteristic and among them 29% were strong producers [32]. Jomezadeh and co-workers recovered 98 UPEC isolates, 42.85% of which were phenotypically biofilm formers, among them the majority (38%) formed moderate biofilms [49]. Taken together, these results imply that due to the high rate of biofilm-producing UPEC isolates, it is necessary to pay attention to these isolates for fast treatment of UTIs. The current work had a limitation, so that we did not evaluate the correlation of UPEC virulence determinants within phylogenetic groups, because it was beyond the scope of this work.

Conclusion

In summary, UPECs isolated from symptomatic hospitalized patients were highly resistant to fluoroquinolone and quinolones and biofilm formation capability was considerable among them. Additionally, we determined a remarkable rate of the isolates belonging predominantly to B2 and D phylogenetic groups. These findings strengthen the importance of these types of investigations for epidemiological surveillance and the therapeutic or prophylactic purposes of nosocomial UTIs.