Distribution of fluoroquinolone resistance determinants in Carbapenem-resistant Klebsiella pneumoniae clinical isolates associated with bloodstream infections in China

Background The rate of fluoroquinolone (FQ) resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) is high. The present study aimed to investigate the distribution of fluoroquinolone resistance determinants in clinical CRKP isolates associated with bloodstream infections (BSIs). Results A total of 149 BSI-associated clinical CRKP isolates collected from 11 Chinese teaching hospitals from 2015 to 2018 were investigated for the prevalence of fluoroquinolone resistance determinants, including plasmid-mediated quinolone resistance (PMQR) genes and spontaneous mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes. Among these 149 clinical CRKP isolates, 117 (78.5%) exhibited resistance to ciprofloxacin. The GyrA substitutions (Ser83 → IIe/Phe) and (Asp87 → Gly/Ala) were found among 112 (75.2%) of 149 isolates, while the substitution (Ser80 → IIe) of ParC was found in 111 (74.5%) of the 149 isolates. In total, 70.5% (105/149) of the CRKP isolates had at least two mutations within gyrA as well as a third mutation in parC. No mutations in the QRDRs were found in 31 ciprofloxacin susceptible CRKP isolates. Eighty-nine (56.9%) of 149 were found to carry PMQR genes including qnrS1 (43.0%), aac(6′)-Ib-cr (16.1%), qnrB4 (6.0%), qnrB2 (2.7%), and qnrB1 (1.3%). Nine isolates contained two or more PMQR genes, with one carrying four [aac(6′)-Ib-cr, qnr-S1, qnrB2, and qnrB4]. The co-existence rate of PMQR determinants and mutations in the QRDRs of gyrA and parC reached 68.5% (61/89). Seventy-four (83.1%, 74/89) PMQR-positive isolates harbored extended-spectrum beta-lactamase (ESBL)-encoding genes. Multilocus sequence typing (MLST) analysis demonstrated that the ST11 was the most prevalent STs in our study. Conclusions Mutations in the QRDRs of gyrA and parC were the key factors leading to the high prevalence of fluoroquinolone resistance among BSI-associated CRKP. The co-existence of PMQR genes and mutations in the QRDRs can increase the resistance level of CRKP to fluoroquinolones in clinical settings. ST11 CRKP isolates with identical QRDR substitution patterns were found throughout hospitals in China.


Introduction
Klebsiella pneumoniae is a commonly detected pathogen in hospital settings, causing nosocomial and communityacquired infections in the lung, urinary tract, surgical sites, soft tissue infections and the bloodstream [1]. CRKP has emerged as a worldwide problem, posing major challenges for its clinical management and public health, through its ability to cause severe and untreatable infections in otherwise healthy individuals [2,3]. In particular, BSIs caused by CRKP is associated with high mortality due to the ineffectiveness of antibacterials used to treat them [4]. CRKP usually shows high levels of resistance to many types of antibiotics [5]. The optimal treatment options for CRKP infections are not well defined. They currently include the use of older agents either as monotherapy or in combination with drugs such as fluoroquinolones (FQs) [6,7].
FQs are important synthetic antimicrobial agents extensively used in clinical and veterinary medicine. They exhibit broad-spectrum activity against a wide of important clinical pathogens and exhibit excellent tissue penetration [8][9][10]. To reduce the use of carbapenems, FQs have been proposed as first-choice alternatives in the treatment of FQ-susceptible, ESBL-producing enterobacterial organisms in pyelonephritis [11]. Carbapenems with FQ are also used to treat carbapenem nonsusceptible K. pneumoniae infections [12,13]. However, resistance to FQs has increased rapidly due to their overuse, thereby limiting the available treatment options or leading to treatment failure [14,15]. FQs target DNA gyrase A and topoisomerase IV, which are encoded by gyrA and parC, respectively. The biological mechanisms of resistance to FQs include impermeability, active efflux, target modification, and antibiotic neutralization. Two major mechanisms involved in the development of quinolone resistance are the acquisition of plasmidmediated quinolone resistance (PMQR) genes [such as aac(6′)-Ib-cr and qnr] and spontaneous mutations in quinolone resistance-determining regions (QRDRs) of gyrA and parC [16][17][18]. PMQR genes have recently been shown to confer low-level resistance to FQs and can be horizontally transferred [19]. The co-carriage of mutations in QRDRs and PMQR genes has been reported in clinical isolates of Enterobacteriaceae exhibiting highlevel quinolone resistance [18,20,21]. Alterations in both gyrA and parC often confer high-level resistance and are reported more frequently than those in gyrB or parE [16]. However, relatively few studies have assessed the prevalence of PMQR determinants and the diversity of DNA gyrase and topoisomerase IV mutations in clinical isolates of CRKP associated with BSIs in China [14,22]. Accordingly, the current study aimed to investigate the prevalence, molecular characteristics, and distribution of PMQR determinants and mutations in the QRDRs of gyrA and parC BSI-associated clinical CRKP isolates from 11 hospitals in China.

Materials and methods
Collection and identification of clinical K. pneumoniae isolates From April 2015 to November 2018, a total of 149 CRKP isolates were cultured from the blood of patients with BSIs in 11 hospitals in eight provinces of China, including Zhejiang (n = 22), Fujian (n = 9), Shandong (n = 26), Hubei (n = 10), Henan (n = 16), Shanghai (n = 18), Jiangxi (n = 40), and Hunan (n = 8). These K. pneumoniae isolates were identified by Gram-staining and a VITEK-2 automated platform (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions, as well as by additional biochemical testing. CRKP isolates were selected based on resistance to imipenem or meropenem according to Clinical and Laboratory Standards Institute (CLSI) guidelines [23]. Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as control isolates for the identification and antimicrobial susceptibility testing of bacterial clinical isolates.

PCR detection and DNA sequence analyses of QRDR and PMQR
Genomic DNA (gDNA) of the 149 CRKP isolates was extracted using the Ezup Column Bacteria Genomic DNA Purification Kits (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. The Qubit and Nanodrop were used to determine the concentrations and purity of the extracted gDNA. The nucleotide mutations in the QRDRs of gyrA and parC were further tested by PCR and nucleotide sequencing using previously primers described [24]. Nucleotide mutations were identified based on the available nucleotide sequences of gyrA and parC genes of K. pneumoniae ATCC 13833. Sequence alignment and analysis were performed online using the BLAST program (http:// www.ncbi.nlm.nih.gov). Plasmids were extracted from the 149 CRKP isolates using a Plasmid Midi Kit (Qiagen, Germany), and all the isolates were screened for the presence of the PMQR genes, including qnrA, qnrB, qnrS, qepA, and aac(6′)-Ib-cr, by PCR and DNA sequencing [25].

Statistical analysis
The data obtained for all CRKP isolates harboring different FQs resistance mechanisms were analyzed by SPSS software (version 20, IBM SPSS Statistics). The chisquare test was used for categorical variables. P-values < 0.05 were considered significant.
All the experiments were carried out following the relevant guidelines and regulations.
The prevalence of mutations in the QRDRs of gyrA and parC among clinical CRKP isolates Among the 149 CRKP isolates, the nucleotide mutations in QRDRs were detected in 112 (75.2%) isolates. Substitutions of QRDRs were observed at position 83 (102 isolates with Ser83 → IIe and 10 isolates with Ser83 → Phe) and position 87 (97 isolates with Asp87 → Gly and 10 isolates with Asp87 → Ala) of GyrA. The substitutions of Ser83 → Phe and Asp87 → Ala substitutions co-existed in 10 ciprofloxacin-resistant isolates. Mutations in parC were only found at position 80 (Ser80 → IIe) among 111 (74.5%) of the 149 isolates (Table 1). No gyrB or parE mutations were observed in any of the CRKP isolates.
Among the 149 CRKP isolates, PMQR-positive isolates were more sensitive to gentamicin and amikacin but had higher resistance rates to ceftazidime/avibactam,   tetracycline, minocycline, and sulfamethoxazole compared with PMQR-negative isolates(p < 0.01). PMQRnegative isolates were more resistant to ciprofloxacin due to mutations in QRDRs (p < 0.05).

Discussion
The emergence of CRKP over the past few decades has posed an increasing threat to public health worldwide [2]. FQs have broad-spectrum antimicrobial activities against both Gram-positive and Gram-negative bacteria and have been widely used since the 1980s [29]. In our study, we collected a total of 149 CRKP isolates from clinical patients with BSIs from 11 teaching hospitals across China, and 78.5% (117/149) of the isolates exhibited resistance to ciprofloxacin. Notably, most of the CRKP isolates tested showed high-level resistance to ciprofloxacin and were distributed in eight provinces surveyed in China. The most prevalent mechanism underlying FQ resistance in K. pneumoniae involves mutations in QRDRs. Resistance to FQs is associated with alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV [30]. Additionally, in 1996, Georgiou et al. reported that some key mutations identified in gyrA and parC were associated with high-level resistance to ciprofloxacin [31]. In our study, among the 117 ciprofloxacin-resistant isolates, 94.8% had mutations in QRDRs, and all the high levels of resistance (MICs of 16 and 32) were associated with QRDR mutations. Ser80 → IIe in ParC (111/149, 74.5%) was the most common substitution among the 149 CRKP isolates, while Ser 83 → Ile/Phe and Asp87 → Ala/Gly in GyrA were also frequently observed. These GyrA and ParC substitutions observed in this study have already been reported [16]. None of the CRKP isolates harboring mutations in QRDRs were sensitive to ciprofloxacin, suggesting that mutations in QRDRs are the primary cause of FQ resistance. Multiple amino acid substitutions in QRDRs are needed for the acquisition of high-level resistance to FQs [9,32]. In the present study, isolates possessing double or more amino acid substitutions in QRDRs were highly prevalent, with 105 FQ-resistant CRKP isolates exhibiting at least two mutations within gyrA as well as a third mutation in parC. The rate of FQs resistance in K. pneumoniae has become very high in some parts of Europe, CRKP usually belongs to several genetic lineages, such as the high prevalence of ST11 [33]. Also, ST11 CRKP has become the dominant clone in many China provinces [34,35]. Similarly, ST11 was the most frequently identified ST among the 117 ciprofloxacin-resistant isolates in our study. Almost all the ST11 isolates had identical mutation patterns in QRDRs and were detected in 8 provinces in China, indicative of wide distribution. This suggests that these isolates, harboring the same FQ resistance gene profiles, may have disseminated vertically by clonal and multiclonal expansion. Similar patterns of the mutation have been reported in clinical Enterobacteriaceae isolated in Warsaw, Poland, but not such large accumulations in clinical CRKP isolates [22]. PMQR determinants (qnr and aac(6′)-Ib-cr genes) have been found in plasmids and are generally thought to confer only low levels of FQ resistance [36]. A higher rate of PMQR (59.7% 89/149) was detected with CRKP in the current study, which may explain the predominance of PMQR genes among the K. pneumoniae isolates [22]. The most frequently detected PMQR gene among all the isolates was qnrS1, followed by aac(6′)-Ib-cr, qnrB4, qnrB2, and qnrB1, which was not consistent with the results of previous Muggeo et.al results that reported aac(6′)-Ib-cr dominance [13]. Nine isolates contained two or more PMQR genes, 1 of which carried four PMQR genes (aac(6′)-Ib-cr, qnr-S1, qnrB2, qnrB4). To the best of our knowledge, this is the first study to report the co-existence of four PMQR genes (aac(6′)-Ibcr, qnr-S1, qnrB2, qnrB4) in CRKP. Among the 31 fluoroquinolone-sensitive CRKP isolates in our study, those positive for PMQR had higher MIC values than their PMQR-negative counterparts, suggesting that PMQR can indeed mediate low levels of drug resistance or increase the MICs of fluoroquinolone-sensitive isolates. Notably, 4 ciprofloxacin-resistant isolates investigated in the present study had no amino acid substitutions in their QRDRs, but all of them had at least one PMQR gene, and all showed considerable resistance levels to FQs (ciprofloxacin MICs of 8 μg/L) by possessing wild-type gyrA and parC. This suggests that PMQR can also mediate drug resistance, although the MIC value was not as high as that seen with some of the CRKP isolates harboring mutations in QRDRs (30 with ciprofloxacin MICs of 16 μg/ml, 14 with ciprofloxacin MICs of 32 μg/ml). Remarkably, in the present study, 1 ciprofloxacin-resistant isolate had no PMQR genes or mutations in QRDRs. We speculate that, in addition to the PMQR genes, other undetected mechanisms may be involved in conferring increased resistance, such as altered permeability or the presence of efflux pump systems.
Nagasaka et al. reported that cephalosporin-resistant K. pneumoniae isolates, including those producing ESBL, tend to display resistance to FQs [22]. Additionally, mutations in double-serine residues have often been observed in isolates of the major international STs of ESBL-producing K. pneumonia (gyrA Ser83 → Phe/Ile; parC Ser80 → Ile) [9].
In conclusion, we characterized 149 BSI-associated clinical CRKP isolates from 11 hospitals located in 8 provinces of China and found that PMQR genes and mutations in QRDRs were highly prevalent. Mutations in the QRDRs of gyrA and parC were key factors underlying FQ resistance in CRKP, while PMQR genes could also increase the level of FQ resistance in the CRKP isolates. Furthermore, the co-existence of PMQR genes and mutations in QRDRs found to be common in this study, led to high levels of FQ resistance. ST11 was the most prevalent ST, while ST11 isolates with identical resistance mechanisms (mutations in QRDRs) were distributed across the eight provinces we investigated, highlighting the need to remain vigilant to prevent its further spread. Additionally, antibiotics, especially quinolones, should be used reasonably in the treatment of clinical CRKP infections.