Molecular Epidemiology of Ciprofloxacin-Resistant Extended-Spectrum β-Lactamase–Producing Klebsiella pneumoniae in Taiwan

Abstract

Fluoroquinolone resistance in extended-spectrum β-lactamases (ESBL)–producing isolates results in very few antimicrobial treatment options. In Taiwan's Surveillance of Antimicrobial Resistance (TSAR) III program, 124 (52.8%) cases of ESBL-producing Klebsiella pneumoniae (ESBL-KP) were resistant to ciprofloxacin. The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and chromosomal quinolone resistance-determining regions (QRDR) of gyrA and parC genes among ESBL-KP isolates was assessed via PCR sequencing. Chromosomal QRDR mutations were present in most of the 123 (96.8%) cases of ciprofloxacin-resistant ESBL-KP isolates. Sixty-six (53.2%) isolates had at least one PMQR gene. qnrB2, qnrB4, and qnrS1 were detected in 26, 19, and 13 isolates, respectively, whereas qnrA, qnrC, and qnrD were not detected. ESBL genes were transferable via conjugation with either aac(6′)Ib-cr or qnrB in 63.6% of the isolates carrying PMQR genes. QnrB was associated with either CTX-M-15 or SHV-12, and aac(6′)Ib-cr was linked to CTX-M-3 or CTX-M-14 in plasmids. qnrS did not co-transfer with ESBL genes. Clonal spread of PMQR genes harboring ESBL-KP isolates was observed in three hospitals. QnrA, which is common in Asia, was unexpectedly absent in ESBL-KP in Taiwan. Aside from transmission via clonal spread for ciprofloxacin-resistant ESBL-KP, concomitant transference of PMQR genes with either bla_CTX-M or bla_SHV via plasmid was common.

Introduction

The prevalence of extended-spectrum β-lactamase–producing Klebsiella pneumoniae (ESBL-KP) has been increasing worldwide.⁶ β-Lactams and fluoroquinolones are commonly prescribed antimicrobial agents, which are usually prescribed prior to the identification of the pathogen.²² Consequently, fluoroquinolone resistance in ESBL-producing isolates results in very few antimicrobial treatment options.^12,23

The mechanisms of quinolone resistance could either be chromosome-mediated or plasmid-mediated. Aside from mutations within the quinolone resistance-determined region (QRDR) in gyrA and parC chromosome genes,^2,12 plasmid-mediated quinolone resistance (PMQR) genes have been reported increasingly.^25,26,34 The emergence of PMQR has been reported since 1998.^19,21,25 Three known mechanisms for quinolone resistance include Qnr proteins, aminoglycoside acetyltransferase aac(6′)Ib-cr, and efflux pump QepA.^29,34 The Qnr proteins protect DNA gyrase and type IV topoisomerase from inhibition by quinolone. Five types of Qnr proteins have been reported: QnrA (six variants), QnrB (19 variants), QnrC (one variant), QnrD (one variant), and QnrS (three variants). QnrC and QnrD were recently identified in China in Proteus mirabilis and Salmonella isolates, respectively.^3,31 The aac(6′)Ib-cr, which contains two amino acid substitutions of aac(6′)Ib, acetylates norfloxacin and ciprofloxacin, but has no effects on enrofloxacin and pefloxacin.²⁶

In Taiwan, the prevalence rate of ESBL among cases of K. pneumoniae has increased from 8% to 18.3% between 2002 and 2009 (data from various surveillance projects).^8,9 In Taiwan's Surveillance of Antimicrobial Resistance (TSAR) III program, a high rate (i.e., 52.8%) of ESBL-KP cases were resistant to ciprofloxacin.¹⁸ We postulate that both PMQR determinants and ESBL genes may be on the same plasmid, and thereby result in co-resistance to both important classes of antimicrobials. However, the prevalence of PMQR genes associating with ESBL genes at the plasmid level in Taiwan is unknown. In the present study, we explored the molecular epidemiology and mechanisms of fluoroquinolone resistance in ESBL-KP isolates collected from a Taiwanese nationwide surveillance program. We also determined the extent of clonal spread of ciprofloxacin resistant ESBL-KP in Taiwan.

Materials and Methods

Bacterial isolates

In 2002, a total of 26 hospitals participated in the TSAR III project, a Taiwanese nationwide study on antimicrobial resistance. Nonduplicate isolates were collected, including 50 isolates from the outpatient department, 20 isolates from pediatric patients, 30 isolates from the adult intensive care unit (ICU), and 100 isolates from the adult non-ICU. A total of 235 ESBL-KP isolates were collected from this surveillance program. ESBL production was also screened in participating hospitals. Confirmation of ESBL production was performed at the National Health Research Institutes, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.⁵

Susceptibility testing and confirmation of ESBL production

Antimicrobial susceptibility was determined by the broth microdilution method, as per CLSI guidelines.^4,5 The following antimicrobial agents were used: Ampicillin, cefazolin, amoxicillin/clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, amikacin, gentamicin, ciprofloxacin, and trimethoprim/sulfamethoxazole. All drugs were incorporated into a Mueller–Hinton broth (Trek Diagnostic System Ltd., West Sussex, United Kindom) in serial two-fold concentrations of 0.025–64 mg/L. Two control strains, Escherichia coli ATCC 35218 and ATCC 25922, were included in each test run. The minimum inhibitory concentration (MIC) of each antimicrobial agent was defined as the lowest concentration that inhibited visible growth of the organism.

ESBL producers were confirmed using a disc diffusion method. The pairs of discs tested, included discs containing cefotaxime–clavulanic acid (30/10 μg), cefotaxime (30 μg) and ceftazidime (30 μg) (Becton Dikinson). A ≦ 5-mm increase in the zone diameter of the clavulanic acid–supplemented discs compared with the plain discs was considered indicative of ESBL production.

PCR amplification and sequencing

Detection of β-lactam and quinolone resistance genes and the ISEcp1 element was performed via PCR amplification. The designed primers for bla_SHV, were as follows: SHV-F, GGG TTA TTC TTA TTT GTC GC, and SHV-R, TTA GCG TTG CCA GTG CTC (AF124984). The published primers for bla_CTX-M,¹⁸ gyrA,² parC,² aac(6′)Ib,¹⁴qnrA,¹⁴ qnrB,¹⁴ qnrS,¹⁴ qnrC,³¹ qnrD,³ and ISEcp1²⁸ were also used. Bacterial DNA was prepared by suspending fresh colonies on a loop in 500 μl of sterile distilled water and heating at 95°C for 10 min. The reaction was carried out in a total volume of 50 μl. Amplification conditions were, as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min. After 35 cycles of amplification, samples were exposed to 72°C for 10 min to terminate primer extension.

Amplicons were sequenced with an automated sequencer (ABI Prism 377 sequencer; PerkinElmer). For further confirmation of aac(6′)Ib-cr, the amplicons of the aac(6′)Ib gene were digested with NdeI and FokI restriction enzymes; aac(6′)Ib is only digested by FokI, whereas the cr-variant is only digested by NdeI.¹⁶

Conjugation experiments

A rifampicin-resistant strain of E. coli, JP-995 or an azide-resistant strain E. coli, J53 were used as the recipients. The MIC of ciprofloxacin in the two recipients was 0.03 mg/L. All donor ESBL-KP isolates were not able to grow in MacConkey agar with either rifampicin (100 mg/L) or azide (100 mg/L). Recipients and donors were inoculated separately into brain heart infusion broth (Oxoid, Basingstoke, Hampshire, England) and incubated at 37°C for 4 hr. They were then mixed together at a volume ratio of 1:10 and incubated overnight at 37°C. A 0.1-ml volume of the overnight broth mixture was then spread onto a MacConkey agar plate containing rifampicin (100 mg/L) or azide (100 mg/L), as appropriate, and either cefotaxime (5 mg/L) or ceftazidime (5 mg/L). Then, lactose-fermenting transconjugants were selected from the agar plate.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was performed using the Bio-Rad CHEF MAPPER apparatus (Bio-Rad Laboratories, Richmond, CA). The restriction enzyme XbaI (New England Biolabs, Beverly, MA) was used. The resulting genomic DNA profiles were interpreted according to previously established guidelines.³⁰

Statistical analysis

Statistical differences between categorical variables were analyzed by the chi-squared test. All tests were two-tailed, and a p value of <0.05 was considered to be significant.

Results

Antimicrobial susceptibility

The antimicrobial susceptibilities of the 235 ESBL-KP isolates collected from a Taiwanese nationwide surveillance program are presented in Table 1. All isolates were resistant to ampicillin and cefazolin. Most of the isolates were intermediate or resistant to trimethoprim/sulfamethoxazole (91.5%), followed by gentamicin (91.1%), ciprofloxacin (59.1%), cefoxitin (56.6%), and amikacin (54.9%). Five (2.1%) isolates were found to be nonsusceptible to imipenem. Sixty-two (50%) ciprofloxacin-resistant ESBL-KP isolates were resistant to cefoxitin, whereas 32 (28.8%) isolates, which in addition to possesing ciprofloxacin susceptibility or intermediate susceptibility, were resistant to cefoxitin (p=0.01). Among the 124 ciprofloxacin-resistant isolates, one isolate could not be recovered from storage. Thus, a total of 123 isolates were included for further analysis with respect to their resistance mechanisms.

Table 1.

Antimicrobial Susceptibility to Various Antibiotics of Ciprofloxacin-Resistant and Ciprofloxacin–Susceptible/Intermediate Extended-Spectrum β-Lactamase–producing Klebsiella pneumoniae Isolates

	Ciprofloxacin-resistant (n=124)				Ciprofloxacin-susceptible/intermediate (n=111)
Antibiotics	MIC₅₀	MIC₉₀	MIC range	Resistance rate	MIC₅₀	MIC₉₀	MIC range	Resistance rate
Cefazolin	≥32	≥32	≥32	100 % (124/124)	≥32	≥32	≥32	100 % (111/11)
Cefoxitin	≥32	≥32	≤2 to ≥32	50 % (62/124)	8	≥32	≤2 to ≥32	28.8 % (32/111)
Imipenem	0.5	2	≤0.25 to 2	0.8 % (1/124)	≤0.25	1	≤0.25 to ≥16	0.9 % (1/111)
Ciprofloxacin	≥4	≥4	≥4	100 % (124/124)	0.25	2	≤0.03 to 2	0 % (0/111)
Gentamicin	≥16	≥16	≤0.5 to ≥16	87.9 % (109)	≥16	≥16	≤0.5 to ≥16	85.6 % (95/111)
Amikacin	32	≥64	≤2 to ≥64	43.5 % (54/124)	32	≥64	≤2 to ≥64	43.2 % (48/111)
SXT	≥4	≥4	≤0.5 to ≥4	93.5 % (116/124)	≥4	≥4	≤0.5 to ≥4	88.3 % (98/111)

SXT, trimethoprim/sulfamethoxazole; MIC, minimum inhibitory concentration (mg/L).

GyrA, parC, qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)Ib-cr, ISEcp1, and β-lactamases

With the exception of four isolates that had no mutations in QRDR of both the gyrA and parC genes, 119 (96.7%) isolates had a mutation in gyrA, and 104 isolates had a mutation in parC. The mutation profiles of the QRDR are listed in Table 2. Aside from the detected non-ESBL β-lactamases (i.e., SHV-1, SHV-11, LEN-1, TEM-1, and TEM-31), at least one of the following ESBL-encoding genes, including SHV-5, SHV-12, CTX-M-3, CTX-M-15, and CTX-M-14, were detected in all 123 ciprofloxacin-resistant (MIC ≥ 4 mg/L) K. pneumoniae isolates. All isolates had an ISEcp1 gene that can serve as a promoter for the bla_CTX-M gene. Among these ciprofloxacin-resistant isolates, 66 (53.7%) also had PMQR genes. The qnrB2, qnrB4, and qnrS1 genes were detected in 26, 19, and 13 isolates, respectively. No qnrA, qnrC, and qnrD genes were detected. Five isolates carried only the aac(6′)Ib cr-variant, and 15 isolates carried both the aac(6′)Ib and cr-variants.

Table 2.

Mutations of gyrA and parC in 124 Ciprofloxacin-Resistant Extended-Spectrum β-lactamase–Producing Klebsiella pneumoniae Isolates

	gyrA (n)
parC (n)	Ser 83	Asp87
None (19)	None (4)	None (4)
	S83F (7)	None (7)
	None (1)	D87G (1)
	S83Y (7)	None (4), D87E (3)
S80R (3)	S83F (3)	None (3)
S80I (101)	S83F (11)	D87A (10), D87H (1)
	S83I (82)	None (73), D87G (5), D87N (1), D87Y (3)
	S83L (1)	D87N (1)
	S83Y (7)	D87A (7)
N.A.^a	(1)

Not available for further molecular studies because the isolate was not recovered from storage.

The distribution of PMQR and ESBL genes is summarized in Table 3. Isolates with PMQR genes all co-existed with either a CTX-M-1 (CTX-M-3 or CTX-M-15) or a CTX-M-9 (CTX-M-14) group. Among the 45 qnrB-positive isolates, 24, 6, and 15 isolates harbored CTX-M-3, CTX-M-14, and CTX-M-15, respectively. In the 13 qnrS positive isolates, 8 isolates harbored CTX-M-3, and 5 had CTX-M-14. In the 20 aac(6′)Ib-cr harboring isolates, 13 and 7 isolates expressed CTX-M-3 and CTX-M-14, respectively.

Table 3.

Plasmid-Mediated Quinolone-Resistant Genes (i.e., qnrB, qnrS, and/or aac(6′)Ib-cr) in 66 Ciprofloxacin-Resistant Extended-Spectrum β-Lactamase–Producing Klebsiella pneumoniae Isolates

	Co-existence of ESBL-genes
Genes (n)	bla_CTX-M(n)	bla_SHV(n)
qnrB4 (13)	CTX-M-3 (6)	SHV-5 (2), SHV-12 (3)
	CTX-M-14 (3)	—
	CTX-M-15 (4)	SHV-5 (1), SHV-12 (1)
qnrB2 (24)	CTX-M-3 (12)	SHV-12 (11)
	CTX-M-14 (1)	SHV-12 (1)
	CTX-M-15 (11)	SHV-5 (5), SHV-12 (6)
qnrS1 (6)	CTX-M-3 (3)	SHV-12 (2)
	CTX-M-14 (3)	SHV-12 (1)
qnrB2+qnrS1 (1)	CTX-M-3 (1)	SHV-12 (1)
qnrB4+qnrS1 (2)	CTX-M-3 (1)	SHV-12 (1)
	CTX-M-14 (1)	SHV-12 (1)
qnrB4+qnrS1+aac(6′)Ib-cr (1)	CTX-M-3 (1)	ESHV-5 (1)
aac(6′)Ib-cr (12)	CTX-M-3 (7)	SHV-5 (1), SHV-12 (3)
	CTX-M-14 (5)	SHV-12 (1)
aac(′)Ib-cr+qnrB2 (1)	CTX-M-3 (1)	SHV-5 (1)
aac(6′)Ib-cr+qnrB4 (3)	CTX-M-3 (2)	—
	CTX-M-14 (1)	—
aac(6′)Ib-cr+ qnrS1 (3)	CTX-M-3 (2)	—
	CTX-M-14 (1)	—

ESBL, Extended-spectrum β-lactamase.

Furthermore, regional differences in Taiwan are present with respect to the distribution of the PMQR harboring isolates. The qnrB2 gene was predominant in the southern and eastern regions (p<0.01), whereas the qnrB4 gene was only detected in the northern and southern regions (p=0.01). There was a higher prevalence of the qnrS gene detected in the northern region than any other region (p=0.014). Ten isolates had two co-existing PMQR genes, and 1 isolate harbored three PMQR genes. The 11 isolates with at least two PMQR genes were from five hospitals, including two from the northern region, two in the western region, and one in southern region of Taiwan. No co-existence of two PMQR genes was detected in any eastern region hospitals of Taiwan.

PFGE analysis

PFGE was performed to characterize the clonality of 66 PMQR harboring isolates. Fifty-nine isolates were typeable by PFGE, and PFGE analysis revealed that eight clusters were related clonally (Fig. 1). Twenty-seven (45.8%) of the 59 isolates were spread clonally. Clusters A and H were found in a hospital from the northern region of Taiwan (N-4), clusters D and E were found in a hospital from the eastern region (E-1), and cluster G was found in a hospital from the southern region (S-2). These clusters comprised 22.7% (5 of 22), 87.5% (7 of 8), and 52.6% (10 of 19) of the PMQR carrying isolates from N-4, E-1, and S-2, respectively. These findings indicate that a high percentage of the PMQR gene carrying ciprofloxacin-resistant ESBL-KP isolates was spread by clonal dissemination in certain hospitals.

FIG. 1.

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA for 59 plasmid-mediated quinolone-resistant (PMQR) genes carrying extended-spectrum β-lactamase (ESBL)–producing Klebsiella pneumoniae isolates. Seven hospitals in the northern part of Taiwan were designated as N1 through N7; six in the south were designated as S1 through S6; five in the west were designated as W1 through W6; and two in the east were designated as E1 and E2.

Transfer of ESBL resistance and antimicrobial resistance in transconjugants

Conjugation experiments were carried out on 66 isolates carrying PMQR genes, of which, only 9 (13.6%) strains were not transferable. All 57 transconjugants either had a CTX-M or SHV gene, and, of these, 48 (84.2%) harbored either the aac(6′)Ib or aac(6′)Ib-cr gene. The qnrB gene was detected in 28 transconjugants, whereas the qnrS gene was not detectable in any transconjugant. ESBL genes were transferable by conjugation with either the aac(6′)Ib-cr or qnrB gene in 63.6% (42/66) isolates carrying PMQR genes (i.e., 28 from a qnrB and 14 from a aac(6′)Ib-cr gene-carrying host). The MIC of ciprofloxacin was less than 0.015 mg/L against 26 of the transconjugants, of which, 9 had the aac(6′)Ib-cr gene and 2 had the qnrB gene. The MIC of ciprofloxacin against the other 31 transconjugants ranged from 0.03 to 0.5 mg/L. Of these, 26 harbored qnrB, 4 carried aac(6′)Ib-cr, and 1 had none of the tested PMQR genes. In the 31 transconjugants with decreased ciprofloxacin susceptibility (MIC, 0.03–0.5 mg/L), CTX-M-3, CTX-M-14, and CTX-M-15 were detected. Furthermore, SHV-12 and SHV-5 were detected in 20 and 6 of these transconjugants, respectively.

Discussion

Most (96.7%) of our isolates had a gyrA and/or parC chromosomal QRDR mutation. Because chromosomal QRDR mutations may result in higher MICs to quinolones, having both QRDR mutations and PMQR genes would not significantly influence the level of fluoroquinolone resistance. Although PMQR contributed to low-level fluoroquinolone resistance, it may provide an optimal environment for certain isolates to develop or acquire resistant determinants to fluoroquinolone, and consequently facilitate the selection of higher-level quinolone resistance.¹⁹ Given that a high proportion (53.2%) of our isolates had both PMQR genes and chromosomal QRDR mutations, then PMQR genes may be associated with the development of a higher level of quinolone resistance. Another explanation for the high proportion of isolates containing both PMQR genes and QRDR mutations may be that these PMQR genes were acquired via ESBL gene-containing plasmids, which carried these PMQR genes to an isolate that already had QRDR mutations.

Only a few previous studies have found bacteria harboring more than one qnr gene. In the present study, we found an isolate harboring both qnrB2 and qnrS1 and 2 isolates harboring qnrB4 and qnrS1. In comparison to the isolates harboring two qnr genes, the co-existence of a qnr gene with a aac(6′)Ib-cr gene was more common. That is, 8 isolates (12.1%) were found to carry aac(6′)Ib-cr with either a qnrB or qnrS gene in the present study. Whether multiple PMQR determinants have an additive effect on the MIC of fluoroquinolones is unknown and warrants further investigation.

Isolates containing qnr genes are emerging worldwide; however, the predominant qnr type depends on the location. In North America, qnrA, qnrB, and qnrS are more prevalent, whereas qnrA and qnrS are predominantly found in Europe. In Asia, qnrA was once thought to be predominant.²⁵ However, recent reports demonstrate that both qnrB and qnrS are now more common than qnrA in Asia.³³ Contrary to most of the reports from Asian countries, qnrA was not detectable in K. pneumoniae isolates in the present study. Interestingly, qnrA is also not detectable in Enterobacter cloacae in Taiwan.³² The prevalence of qnrB and qnrS was 45 (36.6%) and 13 (10.6%) for ciprofloxacin-resistant ESBL-KP, respectively. Recent studies revealed that plasmids carrying the qnr gene may have ESBL genes and aac(6′)Ib-cr.¹⁴ Furthermore, qnrB1, bla_CTX-M-28, bla_TEM-1, and aac (6′)Ib-cr can be co-transferred via plasmids with high molecular weights.²⁰ Jacoby et al. reported that both ESBL and qnrB genes can be co-tranferred.¹³

In the present study, qnrB and aac(6′)Ib genes were co-transferred with ESBL genes. However, qnrS was not detectable in any of the transconjugants. In 63.6% of the isolates carrying PMQR genes, ESBL genes were transferable by transconjugation with either a aac(6′)Ib-cr or a qnrB gene in the same plasmid. This proportion (63.6%) was higher than the proportion (25%) of ESBL-KP carrying both qnrA and bla_SHV-12 in the United Kingdom between 2003 and 2005.⁷ All qnrB and qnrS in the present study co-existed with bla_CTX-M in plasmids, and some plasmids also contained bla_SHV. These observations corroborate recent reports that qnrB co-exists with bla_SHV-12 or bla_CTX-M-15.^21,25 Additionally, qnrS is usually found in nonconjugative plasmids.²⁹ The co-existence of qnrB and qnrS was only reported in one clinical strain found in China, and these two genes were found in different plasmids.¹¹ We observed a similar phenomenon, where only qnrB transferred with ESBL, whereas qnrB and qnrS co-existed in clinical isolates. Moreover, Strahilevitz et al. reported that the insertion sequence of ISCR1 was in the upstream of qnrA and qnrB, but not of qnrS.²⁹ These findings demonstrate the unique genetic characteristics of qnrS. The higher prevalence rate of qnrB than that of qnrS may be related to the co-transference ability of qnrB with ESBL genes.

Recent reports indicated that aac(6′)Ib-cr is associated with bla_CTX-M-1-, bla_CTX-M-14-, and bla_CTX-M-15-harboring isolates.^24,27 In the present study, aac(6′)Ib-cr was linked with either bla_CTX-M-3 or bla_CTX-M-14. Co-mobilization of PMQR genes with the bla_CTX-M gene may be efficient for the spread of multiple drug-resistant pathogens, as the selection pressure may result from either fluoroquinolones or β-lactams. Given that ESBL genes were transferable via conjugation with either the aac(6′)Ib-cr or qnrB gene in 63.6% of the isolates carrying PMQR genes, there could be an increasing trend of co-resistance to fluoroquinolones and ESBLs, and, thus, infection control measures are warranted to prevent the expanding scale of resistance to more antimicrobial agents.

Furthermore, it was found that the predominant PMQR gene was different among the four regions of Taiwan. Although there is no nationwide epidemic of specific PMQR dissemination, PFGE analysis revealed that there are eight clusters of isolates, and some of these clusters are related to intrahospital transmission. Approximately 45.8% PMQR-positive isolates were related clonally, indicating that clonal spread may play a role in PMQR gene dissemination. Our results suggest that infection control measures are necessary to avoid person-to-person transmission and could be effective in preventing the dissemination of PMQR genes, despite the dissemination of PMQR determinants via plasmid transmission.

The rate (52.8%) of ciprofloxacin resistance among Taiwanese ESBL-KP in the 2002 TSAR study was higher than that found between 1998 and 2000 (18.5%).³⁵ This rate is also higher than the ciprofloxacin resistance rate (16.6%) among ESBL-KP in Japan between 2000 and 2003, as well as the rate (18%) of bacteremia isolates found by Paterson et al. in 2000.²³ However, this was not the highest rate, because the ciprofloxacin-resistant rate among ESBL-producing isolates in Slovenia was 83.7%.¹ Studies revealed that prior exposure to quinolones is the most common risk factor for the emergence of quinolone resistance.^15,17,23 Given that the annual amount of fluoroquinolone used in Taiwanese hospitals between 1997 and 2001 has increased, this may contribute to the high fluoroquinolone resistance rate found in the present study, in addition to the co-transference of ESBL and PMQR genes, and clonal spread.¹⁰

In conclusion, there was a high prevalence rate of PMQR genes among ciprofloxacin-resistant ESBL-KP. Furthermore, qnrB can co-transfer with either bla_CTX-M-15 or bla_SHV-12, whereas qnrS is a nontransferable plasmid. Additionally, aac(6′)Ib-cr can co-transfer with either bla_CTX-M-3 or bla_CTX-M-4. Although PMQR genes are located in plasmids, they can be disseminated via clonal spread. Thus, the high prevalence of ciprofloxacin resistance in ESBL-KP, and the various mechanisms and transmission modes, should be taken into account in developing treatment strategies with empirical antibiotics, as well as infection control measures for ESBL-producing pathogens.

Footnotes

Acknowledgment

This work was supported by the National Health Research Institutes, Taiwan.

Disclosure Statement

No competing financial interests exist.

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