Co-transfer of plasmid-mediated blaAmpC and fluoroquinolone resistance genes in Klebsiella pneumoniae isolates causing nosocomial urinary tract infection

Abstract

Background: Klebsiella pneumoniae is a pathogen that frequently causes nosocomial urinary tract infection (UTI), and the prevalence of plasmid-mediated resistance determinants among clinical isolates of K. pneumoniae leads to the appearance of resistance to antibiotics. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in acquired AmpC (ac-AmpC) β‑lactamase‑producing K. pneumoniae isolates from patients with nosocomial UTI and to characterize the transmissibility of plasmids co-harbouring bla_AmpC and PMQR genes.

Methods: From January 2017 to June 2018, we collected 46 AmpC-producing K. pneumoniae isolates causing nosocomial UTI from a tertiary care hospital in China. β-lactamase, PMQR and virulence genes were detected by PCR and sequencing. Clonal relatedness was assessed using ERIC-PCR and multilocus sequence typing (MLST). Plasmids carrying multiple bla_AmpC and PMQR genes were characterized by PCR-based replicon typing (PBRT) and S1-PFGE. Conjugation and electroporation experiments were carried out to assess resistance transfer mediated by plasmids. Overlapping PCR was used to map the genetic context of the bla_AmpC genes. 

Results: In the studied isolates, non-susceptibility of third-generation cephalosporin and fluoroquinolone was very high (>80%). bla_CMY-2, bla_DHA-1, and quinolone resistance gene (qnr) were detected in 11, 41 and 33 isolates, respectively. Among the isolates, 6 strains co-harboured multiple AmpC and qnrB genes. The bla_AmpC and qnrB genes from these six isolates were co-transferrable to recipients via conjugation or electroporation, with IncFIA, IncFIB and IncA/C being the dominant replicons (sizes from ~78 to 217 kb). Forty-six isolates were categorized into 25 ERIC types, and the 6 isolates harbouring multiple bla_AmpC and qnrB genes belonged to ST1/STnew1. The conserved genetic structures in bla_CMY-2 and bla_DHA-1 were identical to those described in the pNF4656 and pSAL-1 plasmids, respectively.

Conclusion: This work reports that qnrB is highly prevalent in AmpC-producing K. pneumoniae isolates and illustrates the emergence of plasmids co-harbouring multiple acquired bla_AmpC and qnrB genes in K. pneumoniae causing UTI in China. We determined that the IncFIA, IncFIB and IncA/C plasmids carrying bla_AmpC with qnrB resistance genes and several mobile genetic elements mediate the local prevalence in K. pneumoniae UTI. The genetic context of bla_AmpC was highly conserved.

Background

Urinary tract infections (UTIs) are one of the most frequent infections among hospitalized patients and in critical care units [1]. Gram-negative Klebsiella pneumoniae in an opportunistic bacteria responsible for different nosocomial infections, such as pneumonia, UTI, liver abscesses and bloodstream infections [2]. β-lactam and fluoroquinolone antimicrobials continue to play important roles in the treatment of serious infections caused by gram-negative pathogens. However, the spread of plasmid-mediated drug resistance factors among gram-negative bacteria leads to the appearance of strains resistant to antibiotics. These strains cause the development of UTIs resistant to these antimicrobial therapies [1, 3].

The most common cause of bacterial resistance to broad-spectrum β-lactam antibiotics is the production of extended-spectrum broad-spectrum β-lactamases (ESBLs) and AmpC β-lactamase [4]. Recently, the prevalence of AmpC enzymes has been increasingly reported worldwide [5–7]. AmpC-type cephalosporinases are Ambler class C β-lactamases that are poorly inhibited by clavulanic acid and confer resistance to a variety of β-lactams but not fourth-generation cephalosporins or carbapenems [8]. Although most AmpC enzymes are intrinsic and chromosomally encoded, they can also be acquired through mobile genetic elements, such as plasmids. These plasmids encode acquired AmpC (ac-AmpC) β-lactamases and have been mainly detected in Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis [5, 9–11].

Based on sequence similarities with species-specific AmpC enzymes, ac-AmpC variants are typically derivatives of the DHA, ACC, FOX, EBC, and CIT (CMY-2) families and have broadly spread geographically, particularly in Europe, Canada, and Tunisia as well as in Korea and China [5, 12–15]. Several genetic elements surrounding ampC genes are involved in the mobilization of ac-AmpC β-lactamase genes, e.g., the insertion sequences IS26 (CMY-13), ISEcp1 (CMY-2-type, ACC-1-type), and ISCR1, associated with complex integrons (DHA-1 type) [8, 16].

Moreover, ac-AmpC β-lactamases are highly expressed and can be transferred to other bacteria through conjugation and transformation to frequently cause resistance to non-β-lactam antibiotics such as aminoglycosides, fluoroquinolones, sulphonamides, trimethoprim, and chloramphenicol, which makes anti-infective therapy more challenging [17]. Because these bla_AmpC genes can be located on large plasmids (> 30 kb in size) that, as principal vehicles, contain additional antimicrobial resistance genes, such as the plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6’)-Ib-cr, qepA and oqxAB, they facilitate the classic mechanisms of fluoroquinolone resistance [18, 19].

The occurrence of both ac-AmpC β-lactamases and PMQR genes on transmissible elements has also been observed in companion and food-producing animals and culinary herbs, but the co-existence of plasmid-borne AmpC β-lactamases and PMQR genes in bacterial isolates collected from patients with nosocomial infections is uncommon [18, 20]. The present study was conducted to investigate the prevalence of PMQR genes in ac-AmpC β‑lactamase‑producing K. pneumoniae isolated from patients with nosocomial UTI. The transmissibility of plasmids carrying multiple ac-AmpC and PMQR genes was a focus, the characterization of plasmids was performed, and the genetic context of AmpC genes was analysed. This study also highlights the possibility of co-transmission of multiple resistance genes in large individual plasmids.

Materials And Methods

Antimicrobial Susceptibility Testing

Susceptibility studies were carried out using a MicroScan (Siemens AG, Munich, Germany) and the standard disc diffusion method according to the CLSI guidelines for the following antimicrobials (Oxoid, Hampshire, England): ampicillin (AMP, 10 µg), cefazolin (KZ, 30 µg), cefuroxime (CXM, 30 µg), cefotaxime (CTX, 30 µg), ceftazidime (CAZ, 30 µg), ceftriaxone (CRO, 30 µg), cefepime (FEP, 30 µg), cefoxitin (FOX, 30 µg), aztreonam (ATM, 30 µg), amoxicillin/clavulanic acid (AMC, 20 µg/10 µg), imipenem (IPM, 10 µg), levofloxacin (LEV, 5 µg), ciprofloxacin (CIP, 5 µg) and amikacin (AMK, 30 µg). Quality control strains were included to monitor the performance of each test.

Detection of β-lactamase and PMQR genes

Total DNA preparations were obtained by thermolysis of isolates as described previously [21]. Multiplex PCR assays were performed on all isolates for the detection of ampC family (bla_MOX, bla_CMY, bla_FOX, bla_DHA, bla_ACC, bla_ACT, and bla_MIR) and PMQR genes (qnrA, qnrB, qnrS, aac(6’)-Ib and qepA). The primers used for multiplex PCR amplification are listed in Table 1. aac(6’)-Ib-cr was differentiated from its wild-type allele by digestion with the enzyme BtsCI (New England Biolabs, Massachusetts, USA). Other β-lactamase genes (bla_SHV, bla_TEM bla_CTX−M and bla_OXA) were analysed by PCR using specific primers and conditions we described previously [22].

Molecular Characterization Of Plasmids Carrying Multiple Ampc Genes

S1-nuclease (Takara, Dalian, China) digestion as well as pulsed-field gel electrophoresis (S1-PFGE) analysis was performed for donor strains and the corresponding transconjugants or transformants. For plasmid size estimation, comparison with the molecular weight marker Salmonella braenderup H9812 was performed. Plasmid replicons were determined using the PCR-based replicon typing (PBRT) scheme with 18 pairs in PCR for detecting F, FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y and FII replicons, as described by Carattoli et al. [24].

Virulence factors of multiple AmpC-producing K. pneumoniae isolates

K. pneumoniae isolates harbouring multiple ac-AmpC β-lactamases were screened for the presence of seven genes encoding putative virulence factors and six capsular serotypes by PCR as we previously described [25]. These virulence-associated genes included genes encoding regulators of exopolysaccharide synthesis (rmpA), fimbrial adhesins (mrkD), the ferric iron uptake system (entB, ybtS, iutA and kfu), allantoin metabolism (allS) and the virulent capsular serotype (K1, K2, K5, K20, K54 and K57).

Genetic context analysis of bla_ac−AmpC genes in multiple AmpC-producing K. pneumoniae isolates

The flanking regions of ac-AmpC genes were identified by overlapping PCR as previously described [26]. For bla_CMY−2, the presence of a composite genetic structure (bla_CMY−2-blc-sugE) originating from the Citrobacter freundii chromosome and ISEcp1 gene (responsible for the transfer of the bla_{CMY−2−like}-blc-sugE region) was explored. For the genetic organization of bla_DHA−1, the searched genes were ISCR1, IS26, orf2 (conserved region of unknown function in Morganella species), ampR, qacEΔ1 and sul1. PCR products were sequenced and compared with those available in GenBank (www.ncbi.nih.gov/BLAST).

Multilocus Sequence Typing (mlst)

MLST analysis of multiple AmpC-producing K. pneumoniae isolates was conducted by sequencing fragments of seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB), and sequence types (STs) were assigned using the K. pneumoniae MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae).

Results

Prevalence Of Various β-lactamase And Pmqr Genes

Multiplex PCR revealed two ac-AmpC-encoding genes, bla_CMY−2 (n = 11) and bla_DHA−1 (n = 41), in the 46 isolates studied. Single ampC genes were detected in 40 isolates, i.e., bla_CMY−2 (n = 5) or bla_DHA−1 (n = 35). Among these 40 isolates, 33 K. pneumoniae isolates also produced PMQR genes. In the 6 remaining strains (Kp1, Kp2, Kp7, Kp11, Kp13 and Kp20), two ampC genes (bla_CMY−2 and bla_DHA−1) were detected, and PMQR (qnrB) genes were co-harboured. The prevalence of qnrB and fluoroquinolone non-susceptibility was higher in the multiplex AmpC-producing isolates than in the simplex AmpC-producing isolates; however, the differences were not significant (100.00% and 82.50%, respectively, P = 0.570; 100.00% and 87.50%, respectively, P = 1.000; Table 3).

Among all isolates, the other β-lactamase genes bla_SHV, bla_CTX−M, bla_TEM and bla_OXA were detected in 39, 37, 8 and 26 isolates, respectively. DNA sequence analysis revealed five bla_SHV subtypes: bla_SHV−12 (n = 23), bla_SHV−11 (n = 9), bla_SHV−33 (n = 4), bla_SHV−28 (n = 2) and bla_SHV−27 (n = 1). Among the bla_CTX−M gene family, four variants were identified: bla_CTX−M−3 (n = 24), bla_CTX−M−15 (n = 6), bla_CTX−M−14 (n = 6) and bla_CTX−M−22 (n = 1). Of the 26 OXA-positive isolates, all harboured bla_OXA−10, while the 8 TEM-positive isolates harboured bla_TEM−1 (Table 2).

Resistance Transfer And Molecular Characterization Of Plasmids

Plasmids carrying ac-AmpC and qnrB genes were successfully transferred via conjugation (n = 2) and/or electroporation (n = 4) from the 6 multiple AmpC-producing K. pneumoniae isolates. The resistance profiles of the transconjugants/transformants were similar to those of the K. pneumoniae donor strains, demonstrating the transfer of antimicrobial resistance, including resistance to β-lactam-based antimicrobial compounds and fluoroquinolones.

S1-PFGE plasmid profiles and PCR replicon typing identified 3 replicons, IncFIA, IncFIB and IncA/C (ranging from ~ 78 to 217 kb), which were found in both donors and transconjugants/transformants and were associated with the transfer of bla_DHA−1, bla_CMY−2, qnrB and several other β-lactamase genes (Table 4 and Fig. 1).

Virulence Factors

Six K. pneumonia strains carrying multiple ac-AmpC and qnrB genes showed important virulence profiles, including the entB (n = 4), mrkD (n = 6) and ybtS (n = 6) genes, and had the capsular serotype K5 (n = 4). Two-three virulence factors were found per isolate and the results are presented in Table 5.

Genetic Context Of Ac-ampc-encoding Genes

Analysis of the genetic context of the 6 strains that harboured multiple ac-AmpC genes revealed that the bla_CMY−2 gene was associated with ISEcp1, which is responsible for the transfer of the bla_{CMY−2−like}-blc-sugE region from the chromosome of Citrobacter freundii to plasmids. In our study, six strains contained ISEcp1, the outer membrane lipoprotein gene blc and the drug efflux channel sugE upstream and downstream of the bla_CMY−2 gene. However, truncation at the 5' end of ISEcp1 was observed in the isolate Kp7 (Fig. 2a). 

We found a structure composed of the bla_DHA−1, ampR, qacEΔ1 and sul1 genes in 6 bla_DHA−1-carrying isolates. All isolates had the ISCR1 element upstream of bla_DHA−1, but no IS26 element was found upstream or downstream (Fig. 2b).

Clonal Relationships

The clonal relatedness of all isolates was determined by ERIC-PCR. By analysing the ERIC-PCR profiles (Fig. 3), the 46 isolates were categorized into two main phylogenetic groups including 25 ERIC types. In the major phylogenetic subgroup, 95.65% (44/46) of the isolates were clustered. The 25 distinct types were then labelled A01 to A25. We found that 6 isolates harboured multiple ac-AmpC β-lactamase and qnrB genes belonging to ST1 or STnew1 (Table 5).

Discussion

The spread of antimicrobial resistance is primarily caused by the dissemination of large plasmids carrying multiple antibiotic resistance genes [27]. In the last decade, the prevalence of plasmid-mediated AmpC-producing K. pneumoniae isolates has increased in nosocomial acquired and healthcare-associated infections [28, 29]. Plasmids carrying ampC genes are often co-harboured with different antibiotic resistance markers, such as ESBL and PMQR genes [30]. Co-transfer of these resistance genes may contribute to the emergence of multidrug-resistant strains, increasing the risk for antibiotic treatment failure. Our study focused on the dissemination of K. pneumoniae strains carrying multiple AmpC and PMQR genes in patients with nosocomial UTI. To the best of our knowledge, this is the first report on the co-occurrence of various plasmid-borne bla_AmpC and PMQR genes in Enterobacteriaceae causing nosocomial infections.

In the studied isolates, non-susceptibility of both third-generation cephalosporin and fluoroquinolone was very high (CTX, 100%; CAZ, 89.13%; CRO, 97.83%; LEV, 82.61%; and CIP, 89.13%), as non-susceptibility to at least one third-generation cephalosporin was one of the major criteria adopted in this study. However, with the increasing use of fluoroquinolone-class drugs in community- and hospital-acquired- UTI patients, the rates of resistance to fluoroquinolones have increased [31]. In this study, the resistance rates of two fluoroquinolones (LEV and CIP) in K. pneumoniae isolates were prominently higher than those reported in a prior Asia-Pacific survey (51–55%) of pathogens causing UTI [31]. And recent report mentioned above suggested that fluoroquinolones are not recommended as a UTI therapy empirically unless in vitro data show susceptible results [31].

The most commonly occurring gene encoding an ac-AmpC enzyme in this study was DHA-1, which was found in 89.13% of the K. pneumoniae isolates and is the most prevalent plasmid-mediated AmpC in China and other areas of Asia; this study also found a low prevalence of bla_CMY−2 (23.91%), which has been mostly reported in Europe. Notably, the simultaneous production of multiple AmpC β-lactamases in a single isolate (as observed in 6 of the 46 isolates), complicates the detection of both enzymes and treatment of infections caused by these isolates. The multiplex AmpC-producing isolates exhibited a higher percentage (100%) of PMQR prevalence than the simplex AmpC-producing isolates (82.5%), but the difference was not statistically significant. Multiplex PCR also revealed that qnrB had the highest prevalence (84.78%), followed by qnrA (4.35%) and qnrS (4.35%). Earlier studies also indicate that qnrB is the most prevalent PMQR gene in China and that its distribution is closely related to the presence of acquired AmpC β-lactamases [32, 33].

Co-transfer of PMQR genes along with bla_AmpC in large individual plasmids co-harbouring many other resistance genes has been shown in other studies [18]. The successful transfer of bla_AmpC along with qnrB genes was studied in the 6 isolates carrying multiple ac-AmpC β-lactamase genes. However, this study showed that not all multiple AmpC genes were co-transferred with the qnrB gene, which indicates that these genes are likely located on different plasmids. Previous studies on plasmid replicon types show that the IncI1, IncA/C, IncFII and IncX1 plasmids carry bla_AmpC [34, 35]. PMQR genes are associated with the IncN, IncL/M, IncFII, IncHI1, IncI1, IncR, and colE types [36]. In the present study, we found that in K. pneumoniae, plasmids carrying both bla_AmpC and PMQR genes were of the replicon type IncA/C, followed by IncFIB and IncFIA. IncF-group plasmids are highly conjugative and are widely distributed in Enterobacteriaceae, and the presence of any gene in this group of plasmids will escalate the spread of the gene to other organisms [36]. Furthermore, we found that bla_AmpC genes were generally located on plasmids of various sizes ranging from ~ 78 to 217 kb. Our results are in agreement with reports of ac-AmpC genes found to be encoded by plasmids of sizes varying from 7 to 180 kb [16].

Subsequently, we analysed the regions surrounding the ac-ampC genes. The genetic organization of bla_CMY−2 was highly conserved. All the isolates carried the mobile element ISEcp1 (ISEcp1-bla_CMY−2-blc-sugE), as documented in previous reports [26, 37]. A well-conserved structure was also found in all isolates co-harbouring the bla_DHA−1 and bla_CMY−2 genes. However, although IS26 or ISCR1 elements are commonly related to the transmission of DHA-1 enzymes [38], none of our isolates harboured the IS26 insertion sequence.

Based on the distinct patterns observed with ERIC-PCR typing, the dissemination of AmpC-producing K. pneumoniae strains is multifactorial and cannot be fully attributed to a predominant clone, as was reported by Thouraya et al [37]. In addition, we found that the six isolates co-harbouring multiple acquired bla_AmpC genes belonged to the ST1/STnew1 clone. This ST has been described recently in a clinical K. pneumoniae isolate of swine origin [39]. Moreover, we found that clonal K. pneumoniae strains had the capsular serotype K5 and showed important virulence profiles, including the three genes entB, mrkD and ybtS. mrkD and ybtS are widely distributed in K. pneumoniae and encode type 3 fimbriae and phenolate-type siderophores.

Conclusions

The present study indicates that third-generation cephalosporin and fluoroquinolone resistance is high in clinical isolates causing nosocomial UTI. PMQR genes were highly prevalent in AmpC-producing K. pneumoniae isolates, and qnrB was predominant. AmpC genes were co-transferred with qnrB and several other β-lactamase genes, conferring resistance to most antibiotics tested. All transconjugants/transformants were associated with IncFIA, IncFIB and IncA/C plasmids and a conservative genetic environment. This study has several limitations. First, this study was a retrospective analysis, and only limited UTI patient information was available. Another limitation of the study was the small number of multiple AmpC-producing K. pneumoniae isolates obtained from a single medical centre. However, the study focused on the transfer of plasmid-mediated resistance genes among clinical AmpC-producing K. pneumoniae strains to better guide the prevention and clinical treatment of nosocomial infections. Further studies are needed to address these limitations.

Abbreviations

UTI, urinary tract infection; PMQR, Plasmid-mediated quinolone resistance; ac-AmpC, acquired AmpC; ERIC-PCR, enterobacterial repetitive intergenic consensus-PCR; PBRT, PCR-based replicon typing; PFGE, Pulsed-field gel electrophoresis; ST, sequence type; AMP, ampicillin; KZ, cefazolin; CXM, cefuroxime; CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; FOX, cefoxitin; ATM, aztreonam; AMC, amoxicillin/clavulanic acid; IPM, imipenem; LEV, levofloxacin; CIP, ciprofloxacin; AMK, amikacin.

Declarations

Ethics approval and consent to participate

The study protocol was carefully reviewed and approved by the institutional Ethics Committee of the Dalian Medical University.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interest.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 81401697), the Natural Science Foundation of Liaoning Province (grant number 2019-MS-092) and the Program for High-Level Entrepreneurial and Innovative Talents of Dalian, China (2017RQ120). This work was also supported by the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences.

Authors’ contributions

YC conceived and designed the experiments and contributed to the writing of the manuscript. YLX, CZ, YQH and SYJ performed the experiments. ZZN and JW analysed the data. WTG and YM contributed reagents and materials. QQZ coordinated collection of specimens. All authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Hui Xu for providing PCR controls.

References

1. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. 2015;13(5):269–84.
2. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998;11(4):589–603.
3. 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.
4. Livermore DM. beta-Lactamases- the Threat Renews. Curr Protein Pept Sci. 2009;10(5):397–400.
5. Ribeiro TG, Novais A, Rodrigues C, Nascimento R, Freitas F, Machado E, et al. Dynamics of clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings over time. Int J Antimicrob Agents. 2019;53(5):650–6.
6. Maeyama Y, Taniguchi Y, Hayashi W, Ohsaki Y, Osaka S, Koide S, et al. Prevalence of ESBL/AmpC genes and specific clones among the third-generation cephalosporin-resistant Enterobacteriaceae from canine and feline clinical specimens in Japan. Vet Microbiol. 2018;216:183–9.
7. Dziri O, Dziri R, Maraoub A, Chouchani C. First Report of SHV-148-Type ESBL and CMY-42-Type AmpC beta-Lactamase in Klebsiella pneumoniae Clinical Isolates in Tunisia. Microb Drug Resist 2018.
8. Jacoby GA. AmpC beta-lactamases. Clin Microbiol Rev. 2009;22(1):161–82. Table of Contents.
9. Alonso N, Miro E, Pascual V, Rivera A, Simo M, Garcia MC, et al. Molecular characterisation of acquired and overproduced chromosomal blaAmpC in Escherichia coli clinical isolates. Int J Antimicrob Agents. 2016;47(1):62–8.
10. Doublet B, Praud K, Nguyen-Ho-Bao T, Argudin MA, Bertrand S, Butaye P, et al. Extended-spectrum beta-lactamase- and AmpC beta-lactamase-producing D-tartrate-positive Salmonella enterica serovar Paratyphi B from broilers and human patients in Belgium, 2008-10. J Antimicrob Chemother. 2014;69(5):1257–64.
11. Girlich D, Bonnin RA, Dortet L, Naas T. Genetics of Acquired Antibiotic Resistance Genes in Proteus spp. Front Microbiol. 2020;11:256.
12. Denisuik AJ, Lagace-Wiens PR, Pitout JD, Mulvey MR, Simner PJ, Tailor F, et al. Molecular epidemiology of extended-spectrum beta-lactamase-, AmpC beta-lactamase- and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolated from Canadian hospitals over a 5 year period: CANWARD 2007-11. J Antimicrob Chemother. 2013;68(Suppl 1):i57–65.
13. Luk S, Wong WK, Ho AY, Yu KC, To WK, Ng TK. Clinical features and molecular epidemiology of plasmid-mediated DHA-type AmpC beta-lactamase-producing Klebsiella pneumoniae blood culture isolates, Hong Kong. J Glob Antimicrob Resist. 2016;7:37–42.
14. Cheong HS, Chung DR, Lee C, Kim SH, Kang CI, Peck KR, et al. Emergence of serotype K1 Klebsiella pneumoniae ST23 strains co-producing the plasmid-mediated AmpC beta-lactamase DHA-1 and an extended-spectrum beta-lactamase in Korea. Antimicrob Resist Infect Control. 2016;5:50.
15. Ben Said L, Jouini A, Alonso CA, Klibi N, Dziri R, Boudabous A, et al. Characteristics of extended-spectrum beta-lactamase (ESBL)- and pAmpC beta-lactamase-producing Enterobacteriaceae of water samples in Tunisia. Sci Total Environ. 2016;550:1103–9.
16. Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother. 2002;46(1):1–11.
17. Meini S, Tascini C, Cei M, Sozio E, Rossolini GM. AmpC beta-lactamase-producing Enterobacterales: what a clinician should know. Infection. 2019;47(3):363–75.
18. Ma J, Zeng Z, Chen Z, Xu X, Wang X, Deng Y, et al. High prevalence of plasmid-mediated quinolone resistance determinants qnr, aac(6')-Ib-cr, and qepA among ceftiofur-resistant Enterobacteriaceae isolates from companion and food-producing animals. Antimicrob Agents Chemother. 2009;53(2):519–24.
19. Rodriguez-Martinez JM, Machuca J, Cano ME, Calvo J, Martinez-Martinez L, Pascual A. Plasmid-mediated quinolone resistance: Two decades on. Drug Resist Updat. 2016;29:13–29.
20. Veldman K, Kant A, Dierikx C, van Essen-Zandbergen A, Wit B, Mevius D. Enterobacteriaceae resistant to third-generation cephalosporins and quinolones in fresh culinary herbs imported from Southeast Asia. Int J Food Microbiol. 2014;177:72–7.
21. Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 2002;40(6):2153–62.
22. Xu H, Huo C, Sun Y, Zhou Y, Xiong Y, Zhao Z, et al. Emergence and molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates harboring bla CTX-M-15 extended-spectrum beta-lactamases causing ventilator-associated pneumonia in China. Infect Drug Resist. 2019;12:33–43.
23. El-Badawy MF, Alrobaian MM, Shohayeb MM, Abdelwahab SF. Investigation of six plasmid-mediated quinolone resistance genes among clinical isolates of pseudomonas: a genotypic study in Saudi Arabia. Infect Drug Resist. 2019;12:915–23.
24. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63(3):219–28.
25. Turton JF, Perry C, Elgohari S, Hampton CV. PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. J Med Microbiol. 2010;59(Pt 5):541–7.
26. Mata C, Miro E, Alvarado A, Garcillan-Barcia MP, Toleman M, Walsh TR, et al. Plasmid typing and genetic context of AmpC beta-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: findings from a Spanish hospital 1999–2007. J Antimicrob Chemother. 2012;67(1):115–22.
27. Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303(6–7):298–304.
28. Marques C, Menezes J, Belas A, Aboim C, Cavaco-Silva P, Trigueiro G, et al. Klebsiella pneumoniae causing urinary tract infections in companion animals and humans: population structure, antimicrobial resistance and virulence genes. J Antimicrob Chemother. 2019;74(3):594–602.
29. Caneiras C, Lito L, Melo-Cristino J, Duarte A. Community- and Hospital-Acquired Klebsiella pneumoniae Urinary Tract Infections in Portugal: Virulence and Antibiotic Resistance. Microorganisms 2019; 7(5).
30. Donati V, Feltrin F, Hendriksen RS, Svendsen CA, Cordaro G, Garcia-Fernandez A, et al. Extended-spectrum-beta-lactamases, AmpC beta-lactamases and plasmid mediated quinolone resistance in klebsiella spp. from companion animals in Italy. PLoS One. 2014;9(3):e90564.
31. Jean SS, Coombs G, Ling T, Balaji V, Rodrigues C, Mikamo H, et al. Epidemiology and antimicrobial susceptibility profiles of pathogens causing urinary tract infections in the Asia-Pacific region: Results from the Study for Monitoring Antimicrobial Resistance Trends (SMART), 2010–2013. Int J Antimicrob Agents. 2016;47(4):328–34.
32. Li P, Liu D, Zhang X, Tuo H, Lei C, Xie X, et al. Characterization of Plasmid-Mediated Quinolone Resistance in Gram-Negative Bacterial Strains from Animals and Humans in China. Microb Drug Resist. 2019;25(7):1050–6.
33. Pai H, Seo MR, Choi TY. Association of QnrB determinants and production of extended-spectrum beta-lactamases or plasmid-mediated AmpC beta-lactamases in clinical isolates of Klebsiella pneumoniae. Antimicrob Agents Chemother. 2007;51(1):366–8.
34. Naseer U, Haldorsen B, Simonsen GS, Sundsfjord A. Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway. Clin Microbiol Infect. 2010;16(2):171–8.
35. Wiesner M, Fernandez-Mora M, Cevallos MA, Zavala-Alvarado C, Zaidi MB, Calva E, et al. Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid. BMC Microbiol. 2013;13:264.
36. Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother. 2009;53(6):2227–38.
37. Cherif T, Saidani M, Decre D, Boutiba-Ben Boubaker I, Arlet G. Cooccurrence of Multiple AmpC beta-Lactamases in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in Tunisia. Antimicrob Agents Chemother. 2016;60(1):44–51.
38. Verdet C, Benzerara Y, Gautier V, Adam O, Ould-Hocine Z, Arlet G. Emergence of DHA-1-producing Klebsiella spp. in the Parisian region: genetic organization of the ampC and ampR genes originating from Morganella morganii. Antimicrob Agents Chemother. 2006;50(2):607–17.
39. Ma K, Feng Y, Liu L, Yao Z, Zong Z. A Cluster of Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Carrying blaNDM-1 and mcr-8.2. J Infect Dis. 2020;221(Supplement_2):237–42.
40. Kim HB, Park CH, Kim CJ, Kim EC, Jacoby GA, Hooper DC. Prevalence of plasmid-mediated quinolone resistance determinants over a 9-year period. Antimicrob Agents Chemother. 2009;53(2):639–45.