Characterization of Transferrable Mechanisms of Quinolone Resistance (TMQR) among Quinolone-resistant Escherichia coli and Klebsiella pneumoniae causing Urinary Tract Infection in Nepalese Children
BMC Pediatrics volume 23, Article number: 458 (2023)
Transferrable mechanisms of quinolone resistance (TMQR) can lead to fluoroquinolone non-susceptibility in addition to chromosomal mechanisms. Some evidence suggests that fluoroquinolone resistance is increasing among the pediatric population. We sought to determine the occurrence of TMQR genes among quinolone-resistant E. coli and K. pneumoniae causing urinary tract infections among Nepalese outpatient children (< 18 years) and identify molecular characteristics of TMQR-harboring isolates.
We performed antimicrobial susceptibility testing, phenotypic extended-spectrum β-lactamase (ESBL) and modified carbapenem inactivation method tests, and investigated the presence of six TMQR genes (qnrA, qnrB, qnrS, aac(6’)-Ib-cr, oqxAB, qepA), three ESBL genes (blaCTX−M, blaTEM, blaSHV), and five carbapenemase genes (blaNDM, blaOXA−48, blaKPC, blaIMP, blaVIM). The quinolone resistance-determining region (QRDR) of gyrA and parC were sequenced for 35 TMQR-positive isolates.
A total of 74/147 (50.3%) isolates were TMQR positive by multiplex PCR [aac(6’)-Ib-cr in 48 (32.7%), qnrB in 23 (15.7%), qnrS in 18 (12.3%), qnrA in 1 (0.7%), and oqxAB in 1 (0.7%) isolate]. The median ciprofloxacin minimum inhibitory concentration of TMQR-positive isolates (64 µg/mL) was two-fold higher than those without TMQR (32 µg/mL) (p = 0.004). Ser-83→Leu and Asp-87→Asn in GyrA and Ser-80→Ile in ParC were the most common QRDR mutations (23 of 35). In addition, there was a statistically significant association between TMQR and two β-lactamase genes; blaCTX−M (p = 0.037) and blaTEM (p = 0.000).
This study suggests a high prevalence of TMQR among the quinolone-resistant E. coli and K. pneumoniae isolates causing urinary tract infection in children in this area of Nepal and an association with the carriage of ESBL gene. This is a challenge for the management of urinary infections in children. Comprehensive prospective surveillance of antimicrobial resistance in these common pathogens will be necessary to devise strategies to mitigate the emergence of further resistance.
Fluoroquinolone (FQ) antimicrobials are important in the treatment of a range of infections, including urinary tract infections (UTIs). They have a broad spectrum of activity, high bioavailability, convenient dosing regimens, and high potency [1, 2]. FQs are listed as an essential medicine by World Health Organization (WHO) . Their extensive use has led to a marked increase in FQ resistance globally [4,5,6,7].
Quinolone/fluoroquinolone (Q/FQ) resistance in Enterobacterales is commonly attributed to chromosomal mutations in the quinolone resistance-determining region (QRDR) of the genes encoding subunits of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) . The reduction of Q/FQ concentration in the cytoplasm by chromosomal efflux pumps or permeability alterations also contributes to resistance . Transferrable mechanisms of quinolone resistance (TMQR) can additionally confer low-level Q/FQ resistance and promote the development of full resistance . TMQR determinants include seven Qnr proteins, AAC(6’)-Ib-cr (an aminoglycoside acetyltransferase), and two efflux pumps, QepA and OqxAB. Qnr proteins (QnrA, QnrB, QnrC, QnrD, QnrE, QnrS, and QnrVC) are dimeric proteins belonging to the pentapeptide repeat protein (PRP) family and protect DNA gyrase and topoisomerase IV from the action of quinolones . The AAC(6’)-Ib-cr is a bifunctional variant of AAC(6’)-Ib that imparts resistance to aminoglycosides and fluoroquinolones having a piperazinyl substituent, such as ciprofloxacin and norfloxacin, via acetylation of amino nitrogen in the piperazinyl ring . These diverse mechanisms can act in concert to confer non-susceptibility to Q/FQ.
As the use of FQ is restricted in children , the increase in FQ resistance among the pediatric population is important [13, 14]. In a recent study from our institution, 66 (41.8%) of 158 E. coli and 7 (23.3%) of 30 K. pneumoniae isolates causing UTI among children were resistant to ofloxacin . Studies from tertiary care centers of Nepal focusing on pediatric UTI have reported ciprofloxacin resistance in 576 (78%) of 739 and 44 (63%) of 69 isolates, and ofloxacin resistance in 104 (62%) of 168 E. coli isolates [16,17,18]. We have investigated the occurrence of TMQR among quinolone-resistant E. coli and K. pneumoniae isolates causing UTI among children at our institution and sought to identify molecular characteristics of TMQR-harboring isolates.
Materials and methods
Study design and setting
This is a retrospective study conducted at Siddhi Memorial Hospital (SMH), Bhaktapur, Nepal. SMH is a 50-bedded secondary care maternal and pediatric hospital with 10 pediatric ICU beds, serving about 16,000 pediatric OPD visits annually. E. coli and K. pneumoniae isolates obtained from UTI patients less than 18 years old attending the outpatient department (OPD) of the hospital were included in the study. An anonymized dataset, with personal identifiers removed, containing the patient’s age, sex, name of the pathogen, and the susceptibility result to nalidixic acid from June 2018 to February 2021 was retrieved from the microbiology laboratory.
Microbiological methods at the time of isolation of the isolates
Clean catch mid-stream urines were collected from children suspected of UTI as per the pediatrician’s discretion as a part of routine patient diagnosis. Urine cultures were performed by semi-quantitative method on a cysteine-lactose-electrolyte deficient agar (CLED) plates which were then incubated at 37 oC for 18–24 h aerobically. Urine cultures with a growth of ≥ 105 CFU/mL were considered for further processing.
The presumptive identification of the pathogens was performed by Gram stain, colony morphology, and a panel of in-house biochemical tests. Susceptibility to nalidixic acid (NA) was performed by the Kirby Bauer disk diffusion method . Significant isolates were stored at -40 oC at the time of isolation.
E. coli and K. pneumoniae isolate resistant to NA were sub-cultured from the frozen stocks on a MacConkey agar and sheep blood agar till uniform well-isolated colonies were obtained. The investigations carried out in this study include antimicrobial susceptibility testing and molecular investigations for the detection of β-lactamases, TMQR genes, and mutations in gyrA and parC.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing (AST) of E. coli and K. pneumoniae isolates was performed by the Kirby Bauer disk diffusion method according to the CLSI guideline . The antimicrobial disks used for testing were ampicillin (10 µg) [tested only for E. coli], amoxicillin-clavulanic acid (20/10 µg), piperacillin-tazobactam (100/10 µg), cefazolin (30 µg), cefuroxime (30 µg). cefixime (5 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), imipenem (10 µg), ciprofloxacin (5 µg), trimethoprim sulphamethoxazole (1.25/23.75 µg), nitrofurantoin (300 µg), and amikacin (30 µg). Amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftazidime, ciprofloxacin, and imipenem disks were purchased from the manufacturer Mast (Mast group Ltd, Liverpool, UK) with the remainder from HiMedia (HiMedia, India). The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by E-test (0.002-32 µg/mL) (HiMedia, India), and those isolates with MIC ≥ 32 µg/mL were further tested by agar dilution method following the procedures described by CLSI . An isolate was defined to display multidrug resistance (MDR) if non-susceptible to ≥ 1 agent in ≥ 3 antimicrobial categories . Escherichia coli ATCC 25922 was used for quality control.
Nucleic acid extraction
The genomic DNA was extracted using Qiagen DNA mini kit (Qiagen, Hilden, Germany) following the procedures described by the manufacturer with the only exception that the final elution was made with 150 µl of nuclease-free water (NFW). The DNA extracts were quantified using Qubit 4 Fluorometer (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s recommendations.
Characterization of β-lactamases
The phenotypic determination of extended-spectrum β-lactamase (ESBL) production was first performed by a combination disc diffusion method with cefotaxime, cefotaxime-clavulanic acid, ceftazidime, and ceftazidime-clavulanic acid (D62C and D64C, Mast group Ltd, Liverpool, UK). The results were interpreted as described in the CLSI guideline . DNA samples of the ESBL-positive isolates were analyzed by PCR to detect blaCTX−M , blaSHV (for E. coli only), and blaTEM by the assays described elsewhere .
The modified carbapenem inactivation method (mCIM) was used to confirm carbapenemase production among imipenem non-susceptible isolates as described in the CLSI guideline . DNA samples of these isolates were analyzed by PCR to detect blaNDM, blaOXA−48, blaKPC, blaIMP, and blaVIM using the primers published previously .
Escherichia coli ATCC 25922 and clinical strains confirmed to harbor blaCTX−M, blaTEM, and blaSHV β-lactamase genes were used for quality control for ESBL phenotyping and genotyping. Previously characterized strains confirmed to harbor blaNDM and blaOXA−48 were used as positive controls for mCIM. DNA extracted from the control strains was used as a positive control, and Escherichia coli ATCC 25922 DNA was used as a negative control in PCR assays.
Detection of TMQR genes
Previously validated multiplex PCR assay for the detection of TMQR genes was used for the detection of qnrA, qnrB, qnrS, oqxAB (reported only for E. coli), qepA, and aac(6’)-Ib-cr . Briefly, the PCR reaction mixture of 50 µl was prepared with 25 µl of multiplex PCR master mix (2X) (Qiagen, Hilden, Germany), 5 µl of the pool of primers containing 2 µM of each primer, 5 µl of template of concentration of 20 ng/µl, and 15 µl of NFW (Ambion™ Nuclease-Free water, Invitrogen, Thermo Fisher Scientific). The PCR amplification was carried out in Veriti 96 Well Thermal Cycler (appliedbiosystems, Thermo Fisher Scientific) with 15 min of initial denaturation at 95 oC followed by 30 cycles of denaturation at 94 oC for 30 s, annealing at 63 oC for 90 s, and extension for 10 min at 72 oC. The amplification products were first resolved by gel electrophoresis (1.5%, w/v) at 100 V for 40 min and visualized in a gel documentation system (Major Science, California, USA). All PCR amplicons of qnr genes were sequenced and confirmed by BLAST (Basic Local Alignment Search Tool).
Detection of mutations in gyrA and parC
A convenience sample of thirty-five TMQR-positive isolates with representative ciprofloxacin’s interpretive categories (susceptible, intermediate, and resistant) for E. coli and K. pneumoniae were selected for the amplification of the gene fragment covering the QRDR of the gyrA  and parC . None of the K. pneumoniae isolates with a TMQR gene were susceptible to ciprofloxacin. The gyrA and parC amplicons were purified and subjected to bi-directional DNA sequencing by capillary electrophoresis (Macrogen, South Korea).
The chromatograms were visualized and processed in BioEdit software, and the sequences were then imported into MEGA11 software. In MEGA11 alignment explorer, the sequences were aligned by the ClustalW algorithm followed by codon-based nucleotide alignment. The substitutions in the QRDR of GyrA and ParC were determined by comparing the amino acid sequences of the isolates to the amino acid sequences of E. coli ATCC 25922 (GenBank Accession number NZ_CP032085 for gyrA, NZ_CP009072 for parC) and Klebsiella pneumoniae ATCC 13883 (GenBank Accession number DQ673325 for gyrA, KFJ75438 for parC).
The data were collected in a Microsoft Excel spreadsheet and imported to IBM SPSS Statistics for Windows v.20 (IBM Corp, Armonk, NY). A chi-squared test of independence or Fisher exact test was performed to determine whether there was a significant relationship between TMQR and other categorical variables. The difference in ciprofloxacin MIC among TMQR positive and negative isolates was investigated by the Mann-Whitney U test. A cutoff value of ≤ 0.05 for the P-value was considered for statistical significance.
There were 522 unique uropathogens isolated from children with a UTI within the study period. Of the 522 isolates, there were 362 E. coli isolates and 74 K. pneumoniae isolates. Nalidixic acid resistance was present in 130/362 (35.9%) of E. coli and 24/74 (32.4%) of K. pneumoniae. Five E. coli and two K. pneumoniae isolates were not recovered in the sub-culture. The final sample size of this study was 147 isolates. The isolates included in this study were obtained from 100 female (68%) and 47 (32%) male children. The median (inter-quartile range (IQR)) age of the children was 6 (2–9) years.
The susceptibility results are in the supplementary material (Additional file 1: Fig. 1). For E. coli, 116/125 (92.8%) isolates were ciprofloxacin-resistant, 8/125 (6.4%) were intermediate, and only 1/125 (0.8%) isolate was susceptible. The median (IQR) ciprofloxacin MIC was 32 (16–128) µg/mL with values ranging from 0.25 to 512 µg/mL. The proportion of ciprofloxacin resistance, intermediate phenotype, and susceptibility among K. pneumoniae was 16/22 (72.7%), 5/22 (22.7%), and 1/22 (4.6%), respectively. The median (IQR) ciprofloxacin MIC for K. pneumoniae was 64 (0.75–128) µg/mL.
The proportion of susceptible E. coli was highest for nitrofurantoin (120/125, 96.0%) followed by imipenem (111/125, 88.8%) and piperacillin-tazobactam (96/125, 76.8%). K. pneumoniae displayed the highest susceptibility towards nitrofurantoin (n = 15/22, 68.2%), imipenem (13/22, 59.1%), piperacillin-tazobactam and amikacin (9/22, 40.9%) (Additional file 1: Fig. 1). Overall, 96/147 (65.3%) of the isolates were ESBL positive and 137/147 (93.2%) were MDR.
Distribution of TMQR genes
Of 147 isolates, 74 were found to harbor TMQR genes (50.3%) (Tables 1 and 2). The prevalence of TMQR was slightly higher in males than females, 55.3% (26/47) vs. 48% (48/100), respectively. There were only 5 neonatal UTI cases caused by nalidixic acid-resistant organisms. Among the rest of the age groups, TMQR positivity was highest for adolescents (14/23, 60.9%) and lowest for children aged 6–12 years (24/58, 41.4%). The proportion of presence of TMQR was about 10% higher in K. pneumoniae (13/22, 59.1%) than in E. coli (61/125, 48.8%).
The different TMQR gene combinations detected are in Table 2. A gel electrophoresis picture of the PCR amplification products of the representative TMQR positive isolates is shown in the supplementary material section (Additional file 2 Fig. 2) The most frequently detected TMQR gene was aac(6’)-Ib-cr present in 48 (32.7%) isolates. The qnrB, qnrS, qnrA, and oqxAB genes were detected in 23 (15.7%), 18 (12.3%), 1 (0.7%), and 1 (0.7%) isolate, respectively. Among 23 qnrB genes, 4 were qnrB1 and the rest were qnrB4. All 18 qnrS and one qnrA gene were qnrS1 and qnrA1, respectively. Of the 74 TMQR-positive isolates, 13 had two, and 2 had three TMQR genes. In E. coli, aac(6’)-Ib-cr (n = 39), qnrB (n = 16), and qnrS (n = 16) were among the most frequent, and similarly, for K. pneumoniae it was aac(6’)-Ib-cr (n = 9) and qnrB (n = 7).
GyrA and ParC substitutions
The amino acid substitution profiles observed in the QRDR of GyrA and ParC of E. coli and K. pneumoniae along with ciprofloxacin MIC values and the presence of TMQR genes are presented in Table 3. Twenty of 30 E. coli and three of five K. pneumoniae isolates had double residue substitutions (Ser-83→Leu and Asp-87→Asn) in GyrA and single substitution in ParC (Ser-80→Ile). Three E. coli isolates had double mutations in ParC (Ser-80→Ile and Glu-84→Val) in addition to GyrA double mutations (Ser-83→Leu and Asp-87→Asn). One E. coli isolate also had double-double mutations, but the alteration at the 84th position in ParC was from glutamic acid to glycine (Glu-84→Gly). One K. pneumoniae isolate had an alteration from aspartic acid to glycine at the 87th position in addition to Ser-83→Tyr in GyrA and Ser-80→Ile in ParC.
Co-existence of TMQR with β-lactamase genes
The combinations of TMQR genes with β-lactamases are presented in Table 2. Among 74 TMQR-positive isolates, blaCTX−M, blaTEM, and blaSHV were found in 51 (68.9%), 35 (47.3%), and 1 (1.4%) isolates, respectively. Four K. pneumoniae and twenty-five E. coli that harbored TMQR had both blaCTX−M and blaTEM. A statistically significant association of TMQR positivity was observed with ESBL phenotype (p = 0.005) and the presence of blaCTX−M (p = 0.037) and blaTEM (p = 0.000) (Additional file 3: Table 1).
Of 23 imipenem non-susceptible isolates, carbapenemase production was observed in 21 isolates by mCIM. The proportion of blaOXA−48 and blaNDM among TMQR positive and negative isolates were 12.2% (9 of 74) vs. 6.8% (5 of 73) and 9.5% (7 of 74) vs. 5.5% (4 of 73), respectively. This association was not statistically significant (Additional file 3: Table 1). No isolate was found to be positive for blaKPC, blaIMP, and blaVIM.
This study demonstrates alarmingly high levels of FQ resistance among E. coli and K. pneumoniae isolates causing UTI in children attending the outpatient department of Siddhi Memorial Hospital, Bhaktapur, Nepal. Half of the isolates were TMQR positive which suggests that TMQR genes may have an important role in the emergence of quinolone resistance in E. coli and K. pneumoniae isolates within our study population. TMQR genes were found to have a statistically significant association with two β-lactamases, blaCTX−M and blaTEM.
We found a high prevalence of TMQR in diverse gene combinations among study isolates. Similar high proportions of the TMQR genes have been reported in previous studies, while few studies have comparatively lower proportions. The proportion of TMQR positivity among FQ-resistant isolates we report, 68/132 (51.5%) of ciprofloxacin-resistant isolates, is similar to a study from the Netherlands (29/ 56, 51.8%) , higher than in Korea (13/122, 10.7%) , Taiwan (37/248, 14.9%) , and China (137/302, 45.4%) , and lower than in Iran (54/60, 90%) , South Africa (47/48, 98%) , and Egypt (90/90, 100%) . Studies from China, Korea, and Taiwan investigated solely the E. coli isolates and the Iran study included E. coli and K. pneumoniae. The rest of the three studies had various Enterobacterales isolates. The proportion and distribution of TMQR genes vary among different studies possibly due to the heterogeneity in the isolate selection criteria, the specific TMQR genes investigated, and the study population. Also, the actual proportion of TMQR could be slightly higher than reported in this study among uropathogens at our institution because they can be present even among nalidixic acid-susceptible Enterobacterales . Since we only included nalidixic-resistant isolates, we might have missed isolates with such phenotype.
The distribution of the TMQR genes observed in this study is consistent with the general distribution reported by previous studies. We found the highest prevalence for three TMQR genes; aac(6’)-Ib-cr (n = 48, 32.7%), qnrB (n = 23, 15.7%), and qnrS (n = 18, 12.3%) (Table 2). In line with this study, the aac(6’)-Ib-cr gene was the most common TMQR gene among FQ-resistant E. coli in the investigation in South Korea (11/122, 9%) , China (74/302, 24.5%) , and Netherlands (23/56, 41.1%) . Studies from Iran, South Africa, China, Taiwan, and Egypt found qnrB and qnrS as the most common compared to other qnr genes investigated among FQ-resistant clinical isolates, in agreement with our findings [30,31,32,33,34]. On the other hand, the prevalence of oqxAB or oqxA/B in Iran [oqxA: 22/60 (36.7%), oqxB: 31/60 (51.7%)], South Africa [oqxA: 20/48 (41.7%), oqxB: 43/48 (89.6%)], China [oqxAB: 19/302 (6.3%)], and Taiwan [oqxAB: 15/248 (6.1%)] is contrary to our findings; we only found one E. coli isolate with oqxAB gene among 125 isolates investigated [30,31,32,33]. No isolate was found to harbor qepA gene similar to the study in the Netherlands and Taiwan [28, 30], but 3/60 (5.0%), 9/90 (10.0%), and 36/302 (11.9%) of FQ-resistant isolates were found to possess qepA in Iran, Egypt, and China, respectively [31, 32, 34]. Overall, our data in conjunction with the previous findings suggest that aac(6’)-Ib-cr and the two qnr genes, qnrB and qnrS, are the most prevalent TMQR genes among Q/FQ-resistant Enterobacterales in general. A recent study demonstrated that possession of aac(6’)-Ib-cr gives a selective advantage to E. coli ST131 in the presence of ciprofloxacin . In addition, alone or in combination with chromosomal mutations, QnrS1 has been shown to increase bacterial fitness while QnrA1 and QepA2 decrease fitness . These observations could explain the predominance of aac(6’)-Ib-cr and qnrS, and the low prevalence of qnrA and qepA.
Our data show that TMQR-positive isolates have higher FQ MIC than those that lack them similar to studies from Iran and Korea [36, 37]. The ciprofloxacin MIC values were significantly higher in TMQR positive isolates (Median = 64 µg/mL, n = 74) compared to TMQR negative isolates (Median = 32 µg/mL, n = 73) (Mann-Whitney U = 1969, Z=-2.871, p = 0.004, but with a small effect size of r = 0.24). Results from the analysis of 35 representative isolates suggest that the concomitant presence of GyrA and ParC substitutions accompanies TMQR genes to result in high levels of FQ resistance (Table 3). Similar findings of multiple substitutions in GyrA and ParC along with TMQR genes leading to high FQ resistance have been shown in other studies [34, 38].
The statistically significant association of TMQR with ESBL observed in this study mirrors previous findings from several other studies [36, 37, 39, 40]. The β-lactamase genotypes, blaTEM and blaCTX−M, showed an independent association with TMQR, but the difference in the proportion of blaTEM was remarkably high between TMQR positive and TMQR negative group (47.3% vs. 1.4%) (Additional file 3: Table 1). Notably, of 57 ESBL-producing TMQR positive isolates, 29 (50.9%) co-harbored both blaTEM and blaCTX−M while no TMQR negative isolate had more than one β-lactamase gene (Table 2). These observations suggest that the association of β-lactamase and TMQR is driven by the co-existence of multiple β-lactamase genes rather than a single genotype in the study population. In a study from Iran investigating UTI caused by Enterobacterales, 72 (43.6%) isolates had the co-existence of blaCTX−M and blaTEM among 165 ESBL-producing TMQR positive E. coli and K. pneumoniae isolates . In contrast, among 155 ESBL-positive TMQR harboring K. pneumoniae (originating from various clinical specimens) in a study in Algeria, all 155 isolates had blaCTX−M only . Geographic, demographic, and differences in clinical specimens could account for this disparity. The predominance of blaCTX−M as the most common ESBL gene associated with TMQR is in an agreement with both Iranian and Algerian studies. We also demonstrate carbapenemase genes among TMQR-positive isolates, in contrast to previous findings; most studies either had no or negligible TMQR-positive isolate resistant to carbapenem [31, 34, 39, 42]. Two-thirds (14/21, 66.7%) of the carbapenemase-producing isolates were TMQR-positive. Although, this was not a statistically significant association (Additional file 3: Table 1).
WHO’s GLASS report 2022 showed that more than 90% of antimicrobial use in Nepal in 2018 was attributed to oral administration reflecting their use in the community setting . Ciprofloxacin and cefixime were the second and third most consumed oral antimicrobials, respectively. Pathogens harboring resistance mechanisms for either or both of these two antimicrobials most likely thrived under such high selective pressure in the community. A recent study from a tertiary care center in Nepal with a large sample size (n = 2153) showed a high prevalence of isolates with overlapping resistance to extended-spectrum cephalosporin and fluoroquinolone in both inpatient and outpatient settings that is consistent with the hypothesis that these two groups of genes are co-spreading in Nepal . TMQR and ESBL genes can be located in the same conjugative plasmid with other antimicrobial-resistant determinants, and this facilitates their simultaneous spread and contributes to the emergence of MDR . Such co-localization implies that the use of either quinolones or β-lactams could also promote the selection of these strains as suggested in a study from Vietnam . Considering that fluoroquinolone is typically avoided in children, high levels of fluoroquinolone resistance may be explained by the high prevalence of such strains, promoted by selective pressure in the community, and by the spread of strains with co-localization of TMQR and β-lactamases within the same conjugative plasmids.
TMQR genes seem to have community origin [42, 46], and several studies have shown that commensal gut microbiota frequently harbors these genes [46, 47], as do isolates from other body surfaces . With the growing appreciation of the involvement of the gut microbiome  and urinary microbiome in causing UTI , the detection and characterization of TMQR genes from the microbiome of these niches could be a future investigation at our institution. Characterization of the genetic background of TMQR (such as TMQR copy number, and expression level), conjugation experiments, phylogrouping, and MLST could be another aspect of focus for further research. The lack of data on whether the patients had consumed antimicrobials prior to hospital visits is a limitation of this study. In addition, we have not characterized other mechanisms of quinolone resistance, such as chromosomal efflux pumps, permeability alterations, and the role of biofilms.
Our study demonstrates alarmingly high levels of FQ resistance among E. coli and K. pneumoniae causing UTI in Nepalese children indicating the presence of high selective pressure in the community to promote their resistance. High prevalence and diversity in combinations of TMQR genes among quinolone-resistant E. coli and K. pneumoniae suggest an important role of these genes in the emergence of Q/FQ resistance. Also, the findings of this study highlight the dissemination of TMQR along with β-lactamases among the pediatric population in Nepal, amplifying multidrug resistance. The increase in FQ resistance is a challenge for the management of UTIs. Comprehensive prospective surveillance of antimicrobial resistance in these common pathogens will be necessary to understand the origin and spread of TMQR genes and to devise strategies to mitigate the emergence of further resistance.
The datasets generated and/or analysed during the current study are available in the GenBank repository (https://www.ncbi.nlm.nih.gov/genbank/), accession numbers OR271074 to OR271143.
Urinary tract infection
Quinolone resistance-determining region
Intensive care unit
Colony forming unit
Transferrable mechanisms of quinolone resistance
White blood cells
Clinical Laboratory Standards Institute
Hooper DC, Jacoby GA. Topoisomerase inhibitors: Fluoroquinolone Mechanisms of Action and Resistance. Cold Spring Harb Perspect Med. 2016;6(9):a025320. https://doi.org/10.1101/cshperspect.a025320.
Kim ES, Hooper DC. Clinical importance and epidemiology of Quinolone Resistance. Infect Chemother. 2014;46(4):226–38. https://doi.org/10.3947/ic.2014.46.4.226.
World Health Organization Model List of Essential Medicines – 22nd List., 2021. Geneva: World Health Organization; 2021 (WHO/MHP/HPS/EML/2021.02). Licence: CC BY-NC-SA 3.0 IGO. https://www.who.int/publications/i/item/WHO-MHP-HPS-EML-2021.02.
Fasugba O, Gardner A, Mitchell BG, Mnatzaganian G. Ciprofloxacin resistance in community- and hospital-acquired Escherichia coli urinary tract infections: a systematic review and meta-analysis of observational studies. BMC Infect Dis. 2015;15:545. https://doi.org/10.1186/s12879-015-1282-4.
Ong A, Mahobia N, Browning D, Schembri M, Somani BK. Trends in antibiotic resistance for over 700,000 Escherichia coli positive urinary tract infections over six years (2014–2019) from a university teaching hospital. Cent Eur J Urol. 2021;74(2):249–54. https://doi.org/10.5173/ceju.2021.0053.
Durkin MJ, Jafarzadeh SR, Hsueh K, Sallah YH, Munshi KD, Henderson RR, et al. Outpatient antibiotic prescription Trends in the United States: A National Cohort Study. Infect Control Hosp Epidemiol. 2018;39(5):584–9. https://doi.org/10.1017/ice.2018.26.
Williamson DA, Roos R, Verrall A, Smith A, Thomas MG. Trends, demographics and disparities in outpatient antibiotic consumption in New Zealand: a national study. J Antimicrob Chemother. 2016;71(12):3593–8. https://doi.org/10.1093/jac/dkw345.
Drlica K, Hiasa H, Kerns R, Malik M, Mustaev A, Zhao X. Quinolones: action and resistance updated. Curr Top Med Chem. 2009;9(11):981–98. https://doi.org/10.2174/156802609789630947.
Ruiz J. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J Antimicrob Chemother. 2003;51(5):1109–17. https://doi.org/10.1093/jac/dkg222.
Ruiz J. Transferable mechanisms of Quinolone Resistance from 1998 onward. Clin Microbiol Rev. 2019;32(4):e00007–19. https://doi.org/10.1128/CMR.00007-19.
Rodríguez-Martínez JM, Machuca J, Cano ME, Calvo J, Martínez-Martínez L, Pascual A. Plasmid-mediated quinolone resistance: two decades on. Drug Resist Updat. 2016;29:13–29. https://doi.org/10.1016/j.drup.2016.09.001.
Grady RW. Systemic quinolone antibiotics in children: a review of the use and safety. Expert Opin Drug Saf. 2005;4(4):623–30. https://doi.org/10.1517/147403220.127.116.113.
Bryce A, Hay AD, Lane IF, Thornton HV, Wootton M, Costelloe C. Global prevalence of antibiotic resistance in paediatric urinary tract infections caused by Escherichia coli and association with routine use of antibiotics in primary care: systematic review and meta-analysis. BMJ. 2016;352:i939. https://doi.org/10.1136/bmj.i939.
Nateghian AR, Karaji S, Zamani K. A decade of trends in the distribution and antimicrobial susceptibility of prevalent uropathogens among pediatric patients from Tehran, Iran during 2005–2016. Asian J Urol. 2021;8(3):253–9. https://doi.org/10.1016/j.ajur.2020.05.008.
Raya GB, Dhoubhadel BG, Shrestha D, Raya S, Laghu U, Shah A, et al. Multidrug-resistant and extended-spectrum beta-lactamase-producing uropathogens in children in Bhaktapur, Nepal. Trop Med Health. 2020;48:65. https://doi.org/10.1186/s41182-020-00251-6.
Parajuli NP, Maharjan P, Parajuli H, Joshi G, Paudel D, Sayami S, et al. High rates of multidrug resistance among uropathogenic Escherichia coli in children and analyses of ESBL producers from Nepal. Antimicrob Resist Infect Control. 2017;6:9. https://doi.org/10.1186/s13756-016-0168-6.
Shrestha LB, Baral R, Poudel P, Khanal B. Clinical, etiological and antimicrobial susceptibility profile of pediatric urinary tract infections in a tertiary care hospital of Nepal. BMC Pediatr. 2019;19(1):36. https://doi.org/10.1186/s12887-019-1410-1.
Thapaliya J, Khadka P, Thapa S, Gongal C. Enhanced quantitative urine culture technique, a slight modification, in detecting under-diagnosed pediatric urinary tract infection. BMC Res Notes. 2020;13(1):5. https://doi.org/10.1186/s13104-019-4875-y.
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, Thirty first Informational Supplement in M100-S30, Wayne. PA., USA; 2020.
Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That Grow Aerobically Document in M07-A9, 10th Edn. Wayne, PA., USA; 2015.
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–81. https://doi.org/10.1111/j.1469-0691.2011.03570.x.
Lewis JS, Herrera M, Wickes B, Patterson JE, Jorgensen JH. First Report of the emergence of CTX-M-Type extended-spectrum β-Lactamases (ESBLs) as the predominant ESBL isolated in a U.S. Health Care System. Antimicrob Agents Chemother. 2007;51(11):4015–21. https://doi.org/10.1128/AAC.00576-07.
Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65(3):490–5. https://doi.org/10.1093/jac/dkp498.
Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2011;70(1):119–. https://doi.org/10.1016/j.diagmicrobio.2010.12.002. https://doi.org/https://doi.org/. 23.
Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham DW. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J Med Microbiol. 2013;62(Pt 12):1823–7. https://doi.org/10.1099/jmm.0.064428-0.
Weigel LM, Steward CD, Tenover FC. gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrob Agents Chemother. 1998;42(10):2661–7. https://doi.org/10.1128/AAC.42.10.2661.
Vila J, Ruiz J, Goñi P, De Anta MT. Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob Agents Chemother. 1996;40(2):491–3. https://doi.org/10.1128/AAC.40.2.491.
Paltansing S, Kraakman MEM, Ras JMC, Wessels E, Bernards AT. Characterization of fluoroquinolone and cephalosporin resistance mechanisms in Enterobacteriaceae isolated in a dutch teaching hospital reveals the presence of an Escherichia coli ST131 clone with a specific mutation in parE. J Antimicrob Chemother. 2012;68(1):40–5. https://doi.org/10.1093/jac/dks365.
Kim B, Seo M-R, Kim J, Kim Y, Wie S-H, Ki M, et al. Molecular epidemiology of ciprofloxacin-resistant Escherichia coli isolated from community-acquired urinary tract infections in Korea. Infect Chemother. 2020;52(2):194–203. https://doi.org/10.3947/ic.2020.52.2.194.
Kao C-Y, Wu H-M, Lin W-H, Tseng C-C, Yan J-J, Wang M-C, et al. Plasmid-mediated quinolone resistance determinants in quinolone-resistant Escherichia coli isolated from patients with bacteremia in a university hospital in Taiwan, 2001–2015. Sci Rep. 2016;6:32281. https://doi.org/10.1038/srep32281.
Zhao L, Zhang J, Zheng B, Wei Z, Shen P, Li S, et al. Molecular epidemiology and genetic diversity of fluoroquinolone-resistant Escherichia coli isolates from patients with community-onset infections in 30 Chinese County Hospitals. J Clin Microbiol. 2015;53(3):766–70. https://doi.org/10.1128/JCM.02594-14.
Azargun R, Soroush Barhaghi MH, Samadi Kafil H, Ahangar Oskouee M, Sadeghi V, Memar MY, et al. Frequency of DNA gyrase and topoisomerase IV mutations and plasmid-mediated quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolated from urinary tract infections in Azerbaijan, Iran. J Glob Antimicrob Resist. 2019;17:39–43. https://doi.org/10.1016/j.jgar.2018.11.003.
Osei Sekyere J, Amoako DG. Genomic and phenotypic characterisation of fluoroquinolone resistance mechanisms in Enterobacteriaceae in Durban, South Africa. PLoS ONE. 2017;12(6):e0178888. https://doi.org/10.1371/journal.pone.0178888.
Kotb DN, Mahdy WK, Mahmoud MS, Khairy RMM. Impact of co-existence of PMQR genes and QRDR mutations on fluoroquinolones resistance in Enterobacteriaceae strains isolated from community and hospital acquired UTIs. BMC Infect Dis. 2019;19(1):979. https://doi.org/10.1186/s12879-019-4606-y.
Phan M-D, Peters KM, Fraga LA, Wallis SC, Hancock SJ, Nhu NTK, et al. Plasmid-mediated Ciprofloxacin Resistance imparts a selective advantage on Escherichia coli ST131. Antimicrob Agents Chemother. 2022;66(1):e02146–21. https://doi.org/10.1128/AAC.02146-21.
Azargun R, Sadeghi MR, Soroush Barhaghi MH, Samadi Kafil H, Yeganeh F, Ahangar Oskouee M, et al. The prevalence of plasmid-mediated quinolone resistance and ESBL-production in Enterobacteriaceae isolated from urinary tract infections. Infect Drug Resist. 2018;11:1007–14. https://doi.org/10.2147/idr.S160720.
Kim NH, Choi EH, Sung JY, Oh CE, Kim HB, Kim E-C, et al. Prevalence of plasmid-mediated Quinolone Resistance genes and Ciprofloxacin Resistance in Pediatric Bloodstream isolates of Enterobacteriaceae over a 9-Year period. Jpn J Infect Dis. 2013;66(2):151–4. https://doi.org/10.7883/yoken.66.151.
Araújo BF, Campos PAd, Royer S, Ferreira ML, Gonçalves IR, Batistão DWdF, et al. High frequency of the combined presence of QRDR mutations and PMQR determinants in multidrug-resistant Klebsiella pneumoniae and Escherichia coli isolates from nosocomial and community-acquired infections. J Med Microbiol. 2017;66(8):1144–50. https://doi.org/10.1099/jmm.0.000551.
Xue G, Li J, Feng Y, Xu W, Li S, Yan C, et al. High prevalence of plasmid-mediated Quinolone Resistance Determinants in Escherichia coli and Klebsiella pneumoniae isolates from Pediatric Patients in China. Microb Drug Resist. 2016;23(1):107–14. https://doi.org/10.1089/mdr.2016.0004.
Akgoz M, Akman I, Ates AB, Celik C, Keskin B, Ozmen Capin BB, et al. Plasmidic Fluoroquinolone Resistance genes in fluoroquinolone-resistant and/or extended Spectrum Beta-Lactamase-Producing Escherichia coli strains isolated from Pediatric and adult patients diagnosed with urinary tract infection. Microb Drug Resist. 2020;26(11):1334–41. https://doi.org/10.1089/mdr.2020.0007.
Zemmour A, Dali-Yahia R, Maatallah M, Saidi-Ouahrani N, Rahmani B, Benhamouche N, et al. High-risk clones of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital establishment of Oran, Algeria (2011–2012). PLoS ONE. 2021;16(7):e0254805. https://doi.org/10.1371/journal.pone.0254805.
Logan LK, Medernach RL, Rispens JR, Marshall SH, Hujer AM, Domitrovic TN, et al. Community Origins and Regional differences highlight risk of plasmid-mediated Fluoroquinolone resistant Enterobacteriaceae infections in children. Pediatr Infect Dis J. 2019;38(6):595–9. https://doi.org/10.1097/INF.0000000000002205.
Global antimicrobial resistance and use surveillance system (GLASS) report. 2022. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO. https://www.who.int/publications/i/item/9789240062702.
Manandhar S, Zellweger RM, Maharjan N, Dongol S, Prajapati KG, Thwaites G, et al. A high prevalence of multi-drug resistant gram-negative bacilli in a Nepali tertiary care hospital and associated widespread distribution of extended-spectrum beta-lactamase (ESBL) and carbapenemase-encoding genes. Ann Clin Microbiol Antimicrob. 2020;19(1):48. https://doi.org/10.1186/s12941-020-00390-y.
Vien LTM, Minh NNQ, Thuong TC, Khuong HD, Nga TVT, Thompson C, et al. The Co-Selection of Fluoroquinolone Resistance genes in the Gut Flora of Vietnamese Children. PLoS ONE. 2012;7(8):e42919. https://doi.org/10.1371/journal.pone.0042919.
Saksena R, Gaind R, Sinha A, Kothari C, Chellani H, Deb M. High prevalence of fluoroquinolone resistance amongst commensal flora of antibiotic naïve neonates: a study from India. J Med Microbiol. 2018;67(4):481–8. https://doi.org/10.1099/jmm.0.000686.
Li B, Chen Y, Wu Z, Zhao Z, Wu J, Cao Y. Prevalence of plasmid-mediated Quinolone Resistance genes among Escherichia coli in the gut of healthy people in Fuzhou, China. Ann Lab Med. 2018;38(4):384–6. https://doi.org/10.3343/alm.2018.38.4.384.
Minh Vien LT, Baker S, Phuong Thao LT, Phuong Tu LT, Thu Thuy C, Thu Nga TT, et al. High prevalence of plasmid-mediated quinolone resistance determinants in commensal members of the Enterobacteriaceae in Ho Chi Minh City, Vietnam. J Med Microbiol. 2009;58(Pt 12):1585–92. https://doi.org/10.1099/jmm.0.010033-0.
Magruder M, Sholi AN, Gong C, Zhang L, Edusei E, Huang J, et al. Gut uropathogen abundance is a risk factor for development of bacteriuria and urinary tract infection. Nat Commun. 2019;10(1):5521. https://doi.org/10.1038/s41467-019-13467-w.
Perez-Carrasco V, Soriano-Lerma A, Soriano M, Gutiérrez-Fernández J, Garcia-Salcedo JA. Urinary microbiome: Yin and Yang of the urinary tract. Front Cell Infect Microbiol. 2021;11:617002. https://doi.org/10.3389/fcimb.2021.617002.
We would like to thank Mr. Anil Rajbhandari for supporting this project and the laboratory staff of Siddhi Memorial Hospital for providing the study isolates. We would also like to extend our special thanks to Associate Professor Chanwit Tribuddharat, Faculty of Medicine Siriraj Hospital, Mahidol University, for kindly sending us the positive controls for qnrA.
No external funding was received for this project.
The authors declare no competing interests.
Ethics approval and consent to participate
The study protocol was approved by the ethical review committee of the Nepal Health Research Council (NHRC) (Reg. no. 100/2020). The ethics committee of NHRC waived the need for informed consent due to the retrospective nature of this study. The bacterial isolates were obtained as a part of the hospital’s routine patient diagnosis, and the current work was conducted as a part of the hospital’s routine surveillance. Data used in this retrospective study were accessed from microbiology laboratory records which were devoid of any personal identifiers. Thus, individual written informed consent was not applicable and was waived by the ethical review committee of NHRC. All methods were carried out in compliance with National Ethical Guidelines for Health Research in Nepal 2022.
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Shrestha, R.K., Thapa, A., Shrestha, D. et al. Characterization of Transferrable Mechanisms of Quinolone Resistance (TMQR) among Quinolone-resistant Escherichia coli and Klebsiella pneumoniae causing Urinary Tract Infection in Nepalese Children. BMC Pediatr 23, 458 (2023). https://doi.org/10.1186/s12887-023-04279-5
- Transferrable mechanisms of quinolone resistance (TMQR)
- Quinolone resistance
- Urinary tract infection