Impact of co-existence of PMQR genes and QRDR mutations on fluoroquinolones resistance in Enterobacteriaceae strains isolated from community and hospital acquired UTIs

Urinary tract infections (UTIs) are common infectious diseases in both hospital- acquired UTI (HAUTI) and community-acquired UTI (CA-UTI) [1, 2]. UTI can be diagnosed by at least one of the following clinical symptoms or signs; temperature ≥ 38 °C, suprapubic pain, costovertebral angle pain, urinary urgency, dysuria or frequency. A quantitative urine culture with bacterial counts ≥10⁵ colony forming units per mL (CFU/mL) is essential for the diagnosis of UTI [3]. UTIs may be caused by gram-negative, gram-positive bacteria or by fungi. E. coli is the most common cause followed by Klebsiella and other Enterobacteriaceae in both CA-UTI [4] and hospital- acquired UTI (HAUTI) [5]. Fluoroquinolones (FQs) have been considered a highly effective treatment of UTIs. However, the development of resistance to these agents especially by gram-negative microorganisms complicates the treatment of infections caused by these organisms [6]. FQs resistance is mainly caused by spontaneous mutations in the quinolone resistance determining regions (QRDR) of gyr A and par C genes, either gyr A or par C, or both genes [7]. However, in the past few decades, plasmid-mediated quinolone resistance (PMQR) has been increasingly reported in Enterobacteriaceae species all over the world [8]. The co-existence of mutations in QRDR and PMQR genes carriage can occur together in Enterobacteriaceae species. Moreover, the presence of PMQR determinants may promote QRDR mutations increasing the FQs resistance rates [9]. Some studies in Egypt have previously investigated the FQs resistance [10, 11], but the study of contribution of various mechanisms of resistance in different Enterobacteriaceae species in immunocompetent patients was not addressed. Accordingly, the aim of the current study was to investigate antimicrobial resistance of different Enterobacteriaceae species isolated from CA-UTI and HAUTI against FQs and correlate its levels with the existing genetic mechanisms of quinolones (Qs) resistance.

UTIs are common bacterial infections in hospital settings and community [21]. In the current study, 440 Enterobacteriaceae isolates were isolated from UTIs with a percentage of (440/705, 62.4%). The isolation rates from inpatients and outpatients were (73.1%) and (55.1%) respectively. A higher frequency of isolation (86%) was recorded previously in Asia-Pacific region [22]. The frequency of isolation from outpatients agrees with a previous report in Ethiopia (57.75%) [23], and disagrees with another report in Korea (89%) [4]. E. coli was the most frequent followed by K. pneumoniae, Citrobacter spp., Proteus spp. and lastly Enterobacter spp. These findings agree with several previous studies [24, 25]. In spite of similarity with these reports, Citrobacter spp. isolation rate in the current study remains the highest. The highest antimicrobial resistance rates were recorded in the present study against SXT with a percentage of (57.5%) followed by CRO (49.3%), however, none of the isolates were resistant to imipenem. These findings agree with several previous studies [4, 5]. High resistance to CRO may be caused by extensive use in the locality. NA has been used for treatment of UTIs for more than five decades [26] so the resistance to NA is expected to be higher than to FQs. In the current study, NA has a resistance rate of (143/440, 32.5%), however higher rates were reported previously in several studies [23, 25, 27]. The fact that NA is not an empiric treatment of UTI in Egypt may be the cause of this difference. FQs have an overall resistance rate of (90/440, 20.5%). Near results were reported in Korea (24.8%) [4], while a higher resistance rate (54.9%) was recorded in Asian countries [28]. With the analysis of PMQR genes (qnr genes and qepA gene), the most frequent gene was qnrB, which was detected in (62.9%) of Q and FQs resistant isolates, and in (100%) of FQs resistant isolates while qnrS gene was detected in (46.9%) of Q and FQs resistant isolates and in (74.4%) of FQs resistant isolates. In other studies on Egyptian population, qnrB was the most prevalent qnr gene among K. pneumoniae isolates as denoted by El-Badawy et al., 2017 which agree with our results [29], while others reported that qnrS was the most prevalent gene among gram negative bacilli isolated from different clinical settings [10, 11]. QnrB and qnrS genes detection rate in our study was higher than that reported in several previous studies [27, 30]. Neither qnrA nor qnrC were detected at all, but qepA gene was detected in (10%) of FQs resistant isolates. In the same context with us, several studies also could not detect qnrA gene among Enterobacteriaceae isolates [27, 30, 31]. However, Szabó et al., 2018 could detect qnrA in their isolates but could not detect neither qnrC nor qepA genes [32]. The difference between our results and others may be caused by geographical distribution of qnr genes, type of clinical isolates or the used methods of detection. With considering the analysis of the QRDRs of gyrA and parC genes, this study reported that the mutation at position 83 of gyrA was the most frequent in Q and FQs resistant strains. Three types of amino acid changes resulted from this mutation. Change from serine to leucine in E. coli strains which was also reported by many reports [33, 34] and change from serine to tyrosine which was detected in K. pneumoniae and E. coli and was also reported previously [35]. The third change (from serine to isoleucine) was detected in Citrobacter strains and was reported in several studies [36, 37]. A double concomitant mutation in gyrA A (Lys154Arg and Ser171Ala) was observed in high FQs resistant Citrobacter spp. isolates with MIC of CIP ≥ 256 μg/mL and E. coli isolates with MIC of CIP ≥128 μg/mL. These mutations were reported previously in few studies [35, 38]. Asp-87 Asn mutation of gyrA gene was the least frequent mutation, where it was detected only in high resistant E. coli isolates with MIC ≥128 μg/mL. Therefor the mutation at position 87 of gyrA seemed to contribute to high resistance to FQs, while the mutation at position 83 could contribute to both Q and FQs resistance. This suggestion agrees with other results [34, 39]. Ser80Ile mutation of parC gene was frequent in both Q and FQs resistant strains that agrees with other studies [40, 41]. Single mutation in either gene occurred only in isolates with MIC of CIP ≤ 2 μg/mL, so the presence of double mutation in gyrA and parC genes seemed to be associated with high levels of resistance to FQs. These findings agree with other reports suggested that high levels of FQs resistance appeared to happen as a result of gradual accumulation of QRDR mutations [38, 42]. All isolates with high and intermediate resistance phenotypes harbored one or more PMQR gene, interestingly, isolates with FQs susceptible phenotype (resistant to NA only) harbored none of the tested PMQR genes. This agrees with Rodrı’guez-Martı’nez et al., 2016, who stated that resistance to NA only is not enough to suggest presence of PMQR genes [43], while Szabó and his colleagues could found PMQR genes among susceptible or low-level resistance to ciprofloxacin with (MIC = 0.06–1 mg/L) isolates [32]. Our study also agrees with Piekarska et al., 2015, who stated that combination of both PMQR genes and mutations in QRDRs of gyrA and parC contributes to high resistance to FQs [9].

In the current study Enterobactericeae remain the most common cause of UTIs. The resistance rate of Q is (32.5%), while the resistance to Q and FQs is (20.5%) among Enterobactericeae isolates. At least one of PMQR gene was detected in FQs resistant isolates. The most frequent gene was qnrB, which was detected in (62.9%) of Q resistant isolates followed by qnrS gene which was detected with a percentage of (46.9%). The co-existence of 2 PMQR genes in the same isolate was observed in (46.9%) of resistant isolates, while co-existence of 3 PMQR genes was reported in (6.3%). The presence of at least two PMQR genes together with simultaneous QRDR mutations in each of gyrA and parC genes can describe the mechanism of resistance in high resistance phenotype (highly resistant to all tested Q and FQs), while presence of at least one PMQR gene together with one QRDR mutation at either genes could be the cause of resistance in isolates with intermediate resistance phenotype (intermediate resistance to all tested Q and FQs). Presence of mutation in only one QRDR regions of gyrA or parC genes could be the cause of resistance to NA only. To our knowledge, the current study is the largest study that reported molecular epidemiology of quinolones resistance in different Enterobacteriaceae species in the study area and could suggest a phenotypic algorithm to describe genetic mechanisms of quinolone resistance.