Introduction

Fluoroquinolones (FQs) are potent and broad-spectrum agents extensively used to treat a wide range of Gram-positive/negative bacterial infections by inhibiting the activity of both DNA gyrases (GyrA and GyrB) and the topoisomerase IV enzymes (ParC and ParE)1. Unfortunately, despite prescribing guidelines that now recommend reserving FQ use, over the last decade, worldwide spread of FQ-resistant organisms has reduced their therapeutic effectiveness and emerged as an important threat to global health2.

Organisms resistant to FQs can occur via several mechanisms, including intrinsic mutations under selection pressure or harboring transferable plasmid-mediated quinolone resistance (PMQR) determinants2. The most common mechanism of high-level FQ resistance is due to mutation in one or more of the genes that encode the targets of FQs. Kishii et al. showed the mutations that alter the expression and function of outer membrane protein, OmpF, can also lead to FQ resistance in Escherichia coli3. In addition, resistance can be conferred by upregulation of chromosomal multidrug efflux pumps (for example, AcrAB-TolC) (by mutations in regulatory proteins), increasing the capability of actively removing FQs and other drugs from the bacterial cell4.

Although FQ resistance can arise by a range of mechanisms, the greatest concern is placed on these bacteria harboring transferable PMQR genes; for example qnr alleles, oqxAB, qepA and aac(6′)Ib-cr5,6,7,8. The binding of the Qnr protein to the topoisomerase physically prevents the intercalation of the FQs with the target enzyme and thus causes drug resistance5. A variant of an aminoglycoside acetyl transferase, aac(6′)-lb-cr, is able to confer decreased susceptibility to FQs by acetylating the amino nitrogen on the piperazinyl substituent present in these antimicrobial agents6. Moreover, two plasmid borne efflux systems, oqxAB and qepA, which encode transporters that can export FQs and other drugs, have become increasingly prevalent among Enterobacteriaceae over the past decade7,8.

Although most E. coli are harmless, some pathogenic E. coli isolates can cause diverse gastrointestinal or urinary tract diseases and even bacteremia and thus cause millions of death every year. The characterization of FQ-resistant E. coli was reported worldwide; however, isolates in most studies were enrolled over a relatively short duration. As a result, the longitudinal evolution and epidemiologic trends FQ-resistant E. coli isolates are possibly hidden. The aim of this study was to investigate the molecular epidemiology of FQ-resistant E. coli isolated from patients with bloodstream infections in Taiwan, 2001–2015.

Results

Long-term surveillance and antimicrobial susceptibility of FQ-resistant E. coli

During the study period, 2001–2015, we randomly selected 1,171 E. coli isolates from patients with bacteremia, of which 248 (21.2%) were identified as levofloxacin-resistant by using the disk diffusion method (Table 1). The trend in the prevalence of FQ-resistant invasive isolates remained stable during the 15-year surveillance (19.2–24.3%) (Table 1). The phylogenetic analysis revealed five groups (A, B1, B2, D and F) in 248 FQ-resistant isolates. Ninty-six (38.7%) of the FQ-resistant isolates belonged to phylogenetic group B2. Phylogenetic group B1 was the second most common, representing in 23.4% of the isolates, followed by group A (22.6%), group D (14.9%) and group F (0.4%) (Table 1). The dramatically increasing ratio of phylogenetic group B2 among FQ-resistant isolates was revealed during the study period (Table 1).

Table 1 Distribution of phylogenetic group, PMQR genes and β-lactamase genes in 248 FQ-resistant bacteremia E. coli isolates.
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The susceptibilities of the 248 FQ-resistant isolates to 15 antimicrobial agents are shown in Table 2. All isolates were resistant to levofloxacin and ciprofloxacin, as determined by the agar dilution method. However, the entire collection was highly susceptible to cefepime (91.5%), imipenem (96.0%), meropenem (98.8%), amikacin (98.0%) and fosfomycin (99.6%) (Table 2). One isolate showed resistance to tigecycline and all isolates were susceptible to colistin. Moreover, a total of 89 (35.9%) and 223 (89.9%) isolates were defined to be ESBL-producers and multidrug resistant (MDR) strains, respectively. The trends of resistance of FQ-resistant invasive isolates to 11 selected antimicrobial agents were generally stable during this 15-year surveillance (Fig. 1). The prevalence of antimicrobial resistance to tetracycline decreased from 86.7% to 55.6% during this period (Fig. 1).

Table 2 In vitro activity of 15 antimicrobial agents against 248 FQ-resistant bacteremia E. coli isolates.
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Figure 1
figure 1

Trends of antimicrobial resistance among 248 FQ-resistant E. coli, 2001–2015.

AMP, ampicillin; AN, amikacin; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; FOS, fosfomycin; FOX, cefoxitin; GM, gentamicin; KN, kanamycin; SAM, ampicillin-sulbactam; TET, tetracycline.

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Characterization of antimicrobial resistance genes

The numbers of β-lactamase- and PMQR-producers among the 248 FQ-resistant isolates are shown in Table 1. The results showed that the dominant β-lactamase was blaTEM (66.5%), followed by blaCMY (19.0%), blaCTX-M (4.8%), blaDHA (1.6%) and blaSHV (1.6%) in FQ-resistant E. coli isolates (Table 1). Sequence analysis revealed that 6 blaCTX-M-14, 3 blaCTX-M-174, 2 blaCTX-M-15, 1 blaCTX-M-13 and 1 blaCTX-M-55 genes were identified among 12 isolates producing blaCTX-M type extended spectrum β-lactamases (ESBLs) (isolate 1902 harbored blaCTX-M-14 and blaCTX-M-15). Only the blaSHV-12 ESBL was found in 4 blaSHV-porducers. In addition, all blaCMY and blaDHA genes were identified as blaCMY-2 and blaDHA-1, respectively.

The prevalence of PMQR genes, including qnr alleles, aac(6′)-Ib-cr, qepA and oqxAB were determined by PCR and direct sequencing and the results showed that 37 FQ-resistant isolates (14.9%) harbored at least one PMQR gene (Table 1). qnrB2, qnrB4, qnrS1 and the coexistence of qnrB4 and qnrS1 were found in 1, 4, 4 and 1 isolates, respectively (Table 1). oqxAB and aac(6′)-Ib-cr genes were identified in 15 (isolate 1315 harbored only oqxA but not oqxB) and 14 isolates (2 isolates also harbored qnrB4), respectively (Table 1). qnr alleles, including qnrA, qnrC, qnrD, qnrVC and qepA, were not found in any of the detected isolates. This survey also showed a trend of increase in the prevalence of aac(6′)-Ib-cr and oqxAB among FQ-resistant isolates between 2004–2006 and 2010–2012 (Table 1). Among 10 qnr-producers, blaSHV-12, blaDHA-1 blaCMY-2 were found in 3, 4 and 2 isolates, respectively. However, no blaSHV-12 or blaDHA-1 were detected in oqxAB- or aac(6′)-Ib-cr-producers. In contrast, blaCMY-2 was found in 7 oqxAB-producers (7/15, 46.7%) and 6 aac(6′)-Ib-cr-producers (6/14, 42.9%), respectively.

Characterization of QRDR mutations in PMQR-harboring isolates

Thirty-seven PMQR-harboring E. coli isolates were distributed into each of the four main phylogroups: A, 12 isolates (32.4%); B1, 15 isolates (40.5%); B2, 6 isolates (16.2%); and D, 4 isolates (10.9%) (Table 3). Chromosomal QRDR mutations were determined by PCR and direct sequencing and the results showed that only 1 (isolate 1019) and 2 (isolate 1706 and 1763) isolates contained wild-type GyrA and ParC, respectively (Table 3). The most common point mutations in PMQR-harboring isolates were GyrA S83L/D87N (31 isolates, 83.8%) and S83L (4 isolates, 10.8%) and those in ParC were S80I (23 isolates, 62.2%) and S80I/E84V (6 isolates, 16.2%) (Table 3).

Table 3 Phylogenic group, MICs, PMQR genes and QRDR mutations of 37 E. coli isolates harboring PMQR genes.
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PMQR gene transfer and plasmid analysis

E. coli isolates harboring PMQR genes were further analyzed by conjugation tests to determine whether there was horizontal plasmid spread in Taiwan. Transfer of PMQR genes by conjugation to recipient cells of E. coli C600 was successful for 11 (29.7%) of the 37 selected isolates (2, 4, 1, 2 and 2 parental isolates harbored qnrB, qnrS, qnrB/qnrS, oqxAB and aac(6′)-Ib-cr/qnrB, respectively) (Table 4). Plasmid numbers and sizes present in parental isolates and transconjugants were verified according to the method of Kado and Liu9 and the results showed that 14 transconjugants (except 1962-3) contained only a single plasmid with a size over 50 kb (Table 4). The antimicrobial resistance genes in transconjugants were further verified by PCR and the results showed that the aac(6′)-Ib-cr and qnrB4 genes were located on the same plasmid in 1377-3. Two and three transconjugants harboring different plasmid profiles were selected from parental isolates 1426 and 1962, respectively (Table 4). No co-transference of qnrB4 and qnrS1 were found in 30 randomly selected transconjugants from isolate 1426. In contrast, transference of aac(6′)-Ib-cr, qnrB and aac(6′)-Ib-cr/qnrB from isolate 1962 was found in 6 (20%), 4 (13.3%) and 20 (66.7%) of transconjugants.

Table 4 MICs, antimicrobial resistance genes and plasmid profiles of E. coli isolates used in conjugation experiments.
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Co-transference of blaDHA-1 and qnrB to recipient cells was found in 3 of 5 qnrB-producers (isolates 1377, 1426 and 1962) (Table 4). No blaCMY-2 was detected in oqxAB- or aac(6′)-Ib-cr-harboring transconjugants. Transconjugant 1649-2 showed resistance to ampicillin and cefoxitin with an un-identified β-lactamase gene. In addition, transconjugants 1377-3, 1706-2 and 1962-2 showed increased MICs to tetracycline. The results indicated the co-transference of the tetracycline resistance gene with PMQR determinants. Moreover, 6 of 11 transconjugants showed high resistance to trimethroprim (MIC > 256 μg/mL) (Table 4). PCR-based replicon typing results revealed that IncN, IncFII and IncHII were identified in 4, 4 and 2 PMQR-plasmids of transconjugants harboring only a single plasmid. However, 3 plasmids (613-3, 1377-3 and 1426-4) were nontypable by PCR-based replicon typing (Table 4).

Discussion

In this study, we present the characteristics of 248 FQ-resistant bacteremia isolates of E. coli from Taiwan, 2001–2015. Among them, 37 isolates harbored at least one PMQR gene. oqxAB and aac(6′)-Ib-cr genes were most prevalent among PMQR-producers. In addition, horizontal transmission of PMQR genes is often accompanied by transmission of genes conferring resistance to other antimicrobial agents.

Antimicrobial resistance in Gram-negative bacteria is on the rise worldwide, particularly in E. coli, which constitutes a majority of invasive Gram-negative isolates. Wong et al. showed that ciprofloxacin resistance in E. coli isolated from bacteremia in Canada peaked in 2006 at 40% and subsequently stabilized at 29% in 2011, corresponding to decreasing ciprofloxacin usage after 200710. In this study, we showed the prevalence of FQ-resistant invasive E. coli isolates is lower compared with Canada (Table 1). In addition, the prevalence of FQ resistance in bacteremia-causing E. coli was lower than urinary-tract-related E. coli in Taiwan (21.2% vs. 32%)11. Moreover, the entire collection was highly susceptible to cefepime, imipenem, meropenem, amikacin and fosfomycin (Table 2). Fosfomycin is found active against Enterobacteriaceae, particularly E. coli, regardless of source (urinary tract infections or bacteremia), ciprofloxacin resistance and ESBL production12,13,14. In addition, fosfomycin is recommended as one of the first-line agents for treatment of urinary tract infections (UTIs) in the latest guidelines endorsed by the Infectious Diseases Society of America and the European Society for Clinical Microbiology and Infectious Diseases15. As a result, the clinical usefulness of fosfomycin, as a first-line treatment agents of bacteremia E. coli infections, should be evaluated further, especially in regions where ciprofloxacin resistance rates are high.

The phylogenetic group B2 was the most common pathogenic E. coli in many countries and group A and group B1 were usually isolated as commensals16,17. Massot et al. showed a parallel and linked increase in the frequency of the B2 group strains (from 9.4% in 1980 to 22.7% in 2000 and 34.0% in 2010) and of virulence factors18. Here, we showed 38.7% of the FQ-resistant bacteremia E. coli isolates belonged to phylogenetic group B2, followed by group B1 (23.4%), group A (22.6%), group D (14.9%) and group F (0.4%) (Table 1). Moreover, based on the 15-year epidemiologic analysis, we further showed that the increasing trend of group B2 among bacteremia E. coli isolates (Table 1). Phylogenetic group B2 dominates the bacteremia E. coli isolates during the period 2007–2015, but group B1 was most prevalent among bacteremia E. coli isolates during the period 2001–2006 (Table 1). As a result, the longitudinal collection of clinical isolates provides the opportunity to characterize the dynamics of the epidemiologic trend and evolution in infectious pathogens over long periods.

Zhao et al. showed that qnr, aac(6′)-Ib-cr, qepA and oqxAB were found in 2.7%, 24.5%, 11.9% and 6.3% of ciprofloxacin-resistant E. coli isolates in China, respectively19. Yang et al. showed that PMQR genes were detected in 59 of 80 (73.8%) ciprofloxacin-nonsusceptible bacteremia E coli isolates from Korea20. In this study, we revealed the prevalence of PMQR genes among FQ-resistant E. coli in Taiwan (14.9%) was relatively lower than in China (37.3%)19 or in Korea (73.8%)20. In addition, the dominant PMQR genes among FQ-resistant E. coli in Taiwan is oqxAB (40.5%), followed by aac(6′)-Ib-cr (37.8%) and qnr alleles (27.0%). No qepA-producer was found in this study. Although PMQR genes provide a low level of FQ resistance, they have been reported to favor the selection of additional chromosome-encoded resistance mechanisms21. Moreover, all of the PMQR-positive isolates had QRDR mutations (Table 3). These results suggest that along with high-level resistance mediated by QRDR mutations, selection pressure from FQs was absent and in this case PMQR genes may be lost21. It is possible that evolution by natural selection may explain the higher level of FQ resistance and the relatively lower prevalence of PMQR genes in FQ-resistant invasive E. coli from Taiwan. As a result, continual epidemiologic surveillance of PMQR genes is necessary to evaluate whether there are specific plasmids disseminated in Taiwan.

Previous studies showed the most common point mutations in ciprofloxacin-resistant E. coli isolates from China were GyrA S83L/D87N (263 isolates, 87.1%) and S83L (21 isolates, 7.0%) and those in ParC were S80I (233 isolates, 77.2%) and S80I-E84V (35 isolates, 11.6%)19. Our results regarding the distribution of QRDR mutations among FQ-resistant isolates were consisted with previous studies (Table 3). Isolate 1019 showed low-level FQ resistance presented S129A/S134G/A141V/L151M substitutions in ParC in the absence of GyrA substitutions raised the possibility that these mutations were not associated with FQ resistance. However, the direct evidence to demonstrate the association of specific QRDR mutations with FQs susceptibility is still limited and thus worth investigating.

A striking association between blaDHA-1 and qnrB4 was reported in Korea and Taiwan22,23 and this tight association was also observed in our study (Table 4). The co-transference of the blaDHA-1 and qnrB4 genes was identified by conjugation assay (3/4, 75%) (Table 4). In contrast, although 7 oqxAB- (7/15) and 6 aac(6′)-Ib-cr-producers (6/14) also carried blaCMY-2, the results of the conjugation assay showed that no blaCMY-2 was located on oqxAB- or aac(6′)-Ib-cr-containing plasmids (Table 4). To our knowledge, this is the first description of the high co-occurrence of blaCMY-2 in oqxAB or aac(6′)-Ib-cr-producing E. coli.

Highly transferable PMQR genes were observed in this study (11/37, 29.7%) (Table 4). Additional phenotypically expressed resistances were co-transferred with PMQR genes by 12 plasmids (92.3%, except 613-3), resulting in diverse resistance patterns (Table 4). Overall, the most frequently co-transferred resistances were to ampicillin (69.2%), trimethoprim (42.6%), ceftazidime (38.5%), cefotaxime (30.8%), cefoxitin (30.8%), kanamycin (30.8%) and tetracycline (23.1%) (MICs > 4-fold change) (Table 4). These results indicated the high co-existence of antimicrobial resistance genes on the PMQR-plasmids.

In summary, plasmid profiling of E. coli isolates exhibiting the co-existence of both PMQR genes and other antimicrobial resistance genes on a single plasmid shows how they contribute to the rapid spread and increase in bacterial resistance, which is important to public health. The plasmid backgrounds of the PMQR genes were variable, ruling out the hypothesis for the spread of specific plasmids in Taiwan, however, continual epidemiologic surveillance and monitoring antimicrobial prescriptions and consumption would decrease the prevalence of FQ-resistant organisms and PMQR spread.

Methods

Sampling and isolation of E. coli

Bacteremia E. coli isolates were recovered in National Cheng Kung University hospital, 2001 to 2015. The Ethics Committee approved that no formal ethical approval was needed to use these clinically obtained materials, because the isolates were remnants from patient samples and the data were analyzed anonymously. A total of 1,171 non-duplicate clinical isolates were randomly selected and stored at −80 °C in Luria-Bertani (LB) broth containing 20% glycerol (v/v) until used. E. coli was identified in the clinical laboratory by colony morphology, Gram stain, biochemical tests and the Vitek system (bioMérieux, Marcy l′Etoile, France) according to the manufacturer’s recommendations. Susceptibility to levofloxacin for E. coli isolates was determined by the disk diffusion method (5 μg/disc, BD BBL™ Sensi-Disc™, Sparks, MD, USA) on Mueller-Hinton (MH) agar (Bio-Rad, Marne la Coquette, France) based on the CLSI guidelines24. A total of 248 levofloxacin-nonsusceptible bacteremia E. coli isolates were identified for further analysis.

Antimicrobial susceptibility testing

Antimicrobial susceptibilities to ampicillin, ampicillin-sulbactam, gentamicin, colistin and tigecycline (BD BBL™ Sensi-Disc™) were determined by the disk diffusion method on Mueller-Hinton agar24. MICs of selected antimicrobial agents (from Sigma-Aldrich: amikacin, cefepime, cefotaxime, ceftazidime, ciprofloxacin, fosfomycin, kanamycin, levofloxacin; from USP Standards: cefoxitin, imipenem, meropenem) were determined by the agar dilution method in accordance with CLSI guidelines24. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. The interpretation of resistance to these antimicrobial agents was determined according to the recommendations of the CLSI25. Tigecycline and colistin susceptibilities were interpretated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)26 and previous study27, respectively. MDR E. coli was defined as isolates that were resistant to at least 3 classes of the tested antimicrobial agents28.

Characterization of antimicrobial resistance genes

All 248 FQ-resistant E. coli isolates were further screened for selected β-lactamases (blaTEM, blaSHV, blaCTX-M, blaDHA and blaCMY) and PMQR genes (qnr alleles, oxqAB, qepA and aac(6′)Ib-cr) by PCR amplification with specific primers (Supplmentary Table S1). DNA sequencing was further carried out on β-lactamases (except blaTEM) and PMQR genes and the DNA sequences and deduced amino acid sequences were compared with genes in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) to confirm the subtypes of antimicrobial resistance genes.

Screening for mutations in quinolone resistance-determining regions

GyrA and ParC QRDRs of 37 isolates harboring PMQR genes were examined by amplifying and sequencing gyrA (490 bp) and parC (470 bp) genes using primers (Supplmentary Table S1) described by Zhao et al.19. Amplimers were sequenced and amino acid mutations were determined using the control strain E. coli K-12 (NZ_AKBV01000001.1) as a reference.

Determination of the phylogenetic origin of E. coli isolates

Phylogenetic grouping of E. coli isolates was performed using a previously published method29. Primers used are described in Supplmentary Table S1. The PCR-amplified products were separated by electrophoresis on 1.8% agarose gels, stained with ethidium bromide and assigned to one of the seven phylogenetic groups A, B1, B2, C, D, E and F.

Conjugation experiments and plasmid analysis

The liquid mating-out assay was carried out to transfer PMQR genes from 37 FQ-resistant E. coli isolates to rifampicin-resistant E. coli C600 as described previously30. Transconjugants were selected on LB plates containing 256 μg/mL rifampicin (Sigma) and 0.06 μg/mL ciprofloxacin. The plasmids were extracted as described previously9, followed by electrophoresis in a 0.6% agarose gel at 50 V for 3 h and compared by co-electrophoresis with plasmids of known sizes from Salmonella OU7526 and a GeneRularTM DNA ladder (Fermentas, Burlington, ON, Canada) to predict the plasmid sizes30. Plasmids were typed by PCR-based replicon typing according to the previous study31.

Additional Information

How to cite this article: Kao, C.-Y. 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. 6, 32281; doi: 10.1038/srep32281 (2016).