Prevalence of Quinolones Resistance Proteins Encoding Genes (qnr genes) and Co-Resistance with β-lactams among Klebsiella pneumoniae Isolates from Iraqi Patients

This study investigated the prevalence of quinolones resistance proteins encoding genes (qnr genes) and co-resistance for fluoroquinolones and β-lactams among clinical isolates of Klebsiella pneumoniae. Out of 150 clinical samples, 50 isolates of K. pneumoniae were identified according to morphological and biochemical properties. These isolates were collected from different clinical samples, including 15 (30%) urine, 12 (24%) blood, 9 (18%) sputum, 9 (18%) wound, and 5 (10%) burn. The minimum inhibitory concentrations (MICs) assay revealed that 15 (30%) of isolates were resistant to ciprofloxacin (≥4μg/ml), 11 (22%) of isolates were resistant to levofloxacin (≥8 μg/ml), 21 (42%) of isolates were resistant to ertapenem (≥8 μg/ml), 18 (36%) of isolates were resistant to imipenem (4≥16μg/ml), 43 (86%) of isolates were resistant to ceftriaxone (4≥64 μg/ml), 42 (84%) of isolates were resistant to ceftazidime (16-64 μg/ml), and 40 (80%) of isolates were resistant to cefepime (4≥16μg/ml). The results revealed that all fluoroquinolone resistant K. pneumoniae isolates were resistant for β-lactams that used in this study. Genotypic detection of qnr genes revealed that qnrS and qnrB were found in 38 (76%) and 18 (36%) of K. pneumoniae isolates, respectively. On the other hand, qnrA, qnrC, and qnrD were not found among K. pneumoniae isolates. DNA sequencing of qnrB gene revealed that the presence of silent and missense mutations that may have led to increase the resistance values of MICs for ciprofloxacin and levofloxacin. These variants were registered in NCBI at the accession numbers LC373260 and LC381730. The phylogenetic tree of qnrB variants showed a significant deviation of these variants from K. pneumoniae species. The spread of qnr genes among clinical isolates of K. pneumoniae and high association observed between resistance to fluoroquinolones and βlactams have led to a major threat to public health through development of MDR K. pneumoniae.


Introduction:
Klebsiella pneumoniae is a gram negative, rod shaped, encapsulated, and non-motile bacterium, which belongs to the family Enterobacteriaceae (1). It is an opportunistic pathogen that can colonize mucosal surfaces of human and causes serious nosocomial and community acquired infections include urinary tract infections (UTIs), pneumonia, bacteremia, liver abscess, cystitis, and wound and burn infections (2). The development of resistance mechanisms of K. pneumoniae against most common usage antibiotics has led to emergence of multidrug resistant (MDR) K. pneumoniae that associated with high rates of morbidity and mortality due to the limited options of clinical treatment (3).
Department of Biology, College of Education for Pure Science/Ibn Al-Haitham, University of Baghdad, Baghdad, Iraq. * Corresponding author: mustavasuhel@gmail.com * ORCID ID: https://orcid.org/0000-0002-  Fluoroquinolones are one of the last resorts of antimicrobial options used to treat infections of K. pneumoniae. These agents act by preventing the synthesis and replication of the bacterial DNA by targeting topoisomerases (DNA gyrase and topoisomerase IV) (4). The resistance of K. pneumoniae for fluoroquinolones is partially attributed to harbor qnr genes, which encode quinolones resistance proteins that prevent fluoroquinolones to bind with their topoisomerases target (5). qnrA was first determined in a multiresistant K. pneumoniae strain in USA in 1994 and then became widely spread throughout world. The detection of qnrA was then followed by determination of qnrS, qnrB, qnrC, and qnrD, respectively (5). Although qnr genes confer low resistance against fluoroquinolones, they enhance selection of chromosomal mutations in the topoisomerases encoding genes gyrA and parC, leading to increase resistance of K. pneumoniae (6).
qnr genes are usually carried on plasmids that can transfer horizontally, ensuring the wide dissemination of resistance to fluoroquinolones, so that K. pneumoniae can survive in the face of these agents (4,5). Furthermore, qnr genes can be found on the chromosome (5).
The presence of qnr genes together with βlactamases encode genes on the same plasmid provides cross-resistance for both agents, leading to emergence of extremely drug resistant (XDR) K.pneumoniae (3).
This study aims to investigate the prevalence of quinolones resistance proteins encoding genes (qnrA, qnrB, qnrC, qnrD, and qnrS) among clinical isolates of K.pneumoniae and association between qnr genes with resistance to fluoroquinolones and β-lactams.

Collection of Samples:
A total of 150 clinical samples (urine, blood, sputum, wounds, and burns) were collected from patients who suffered from different cases, including UTIs, bacteremia, pneumonia, burns, and wounds infections from different hospitals in Baghdad. The collection of samples was done according to instructions of the ethics committee at the Ministry of Health in Baghdad according to the official approval that numbered 31864.

Bacterial Isolation and Identification:
The clinical samples were cultured on MacConkey agar, Blood agar, Eosin Methylene Blue agar (Oxoid, UK), and CHROMagar Orientation (Pioneer, France). All culture media were incubated at 37°C for 24 hrs (7). Then, the pure colonies were selected and identified by performing biochemical assays, including oxidase, catalase, and IMVIC tests (7). The Vitek 2 Compact System (BioMerieux, France) was used for confirmation of identified bacteria (8).

DNA Extraction:
The Wizard Genomic DNA Purification Kit (Promega, USA) was used to extract DNA from bacterial isolates according to information of manufacturing company. The concentrations and purity of DNA were measured using Nanodrop (Biogroup, UK) (10).

Gel Electrophoresis:
The PCR products were run on 1% agarose gel stained with 0.5 µg/ml ethidium bromide in 1X TAE buffer (Promega, USA) using DNA ladder (100-1500 bp) supplied by Promega (USA), as a marker of DNA size. The electrophoresis was performed at 100 V for 80 min. The UV-Transilluminater (Major Science, Taiwan) was used for observation of PCR products under 320 nm UV light (15).

DNA Sequencing:
The sequencing of two PCR products that referred to the qnrB was done by Genetic Analyzer  (17).

Phylogenetic Tree Construction:
The observed PCR amplicons variants of qnrB genetic loci were compared with the neighbor

Results and Discussion:
Fifty isolates were identified as K. pneumoniae ( Table 7). The bacteria were found at high prevalence in urine 15 (30%) followed by blood 12 (24%), whereas sputum and wounds were obtained 9 (18%) isolates, and finally burns were obtained 5 (10%) isolates. Several studies revealed that the most common site of K. pneumoniae infections is UTIs followed by bloodstream infections, pneumonia, and burns and wounds infections (2,18,19). Other studies collected K. pneumoniae from other cases, including pus, stool, cerebrospinal fluid, and catheters (18,19,20,21,22). The results of MICs listed in Table 8 exhibited that K. pneumoniae isolates showed high resistance to third and fourth generation cephalosporins. It was found that 43 (86%) of K. pneumoniae isolates were resistant to ceftriaxone with MIC 4-≥64 µg/ml, 42 (84%) of isolates were resistant to ceftazidime with MIC 16-64 µg/ml, and 40 (80%) of isolates were resistant to cefepime with MIC 4-≥16 µg/ml. On the other hand, K. pneumoniae isolates exhibited low resistance for carbapenems, in which 21 (42%) of K. pneumoniae isolates were resistant to ertapenem with MIC ≥8 µg/ml and 18 (36%) of isolates were resistant to imipenem with MIC 4-≥16 µg/ml. The production of extended spectrum beta-lactamases (ESBLs) and AmpC-β-lactamases, in addition to carbapenemases is responsible for evolving high resistance to cephalosporins and carbapenems (23). The emergence of carbapenemases represents a great problem in modern medicine due to confer resistance not only for carbapenems, but also for non β-lactams, particularly aminoglycosides and fluoroquinolones (23,24). K. pneumoniae isolates exhibited lower resistance against fluoroquinolones than β-lactams (cephalosporins and carbapenems). Consequently, fluoroquinolones were more effective against K. pneumoniae isolates. The results revealed that 15 (30%) of K. pneumoniae isolates were resistant to ciprofloxacin with MIC ≥4 µg/ml, while 11 (22%) of isolates were resistant to levofloxacin with MIC ≥8 µg/ml. The acquisition of plasmid mediated quinolone resistance (PMQR) enables K. pneumoniae to exhibit low resistance against fluoroquinolones through three mechanisms include protection of topoisomerases by the action of Qnr proteins, acetylation of fluoroquinolones by expression of aac(6′)-Ιb-cr, and extruding of fluoroquinolones through the action of efflux pumps OqxAB and QepA (3,5,6).
Association between resistance to fluoroquinolones and β-lactams revealed that all fluoroquinolones resistant K. pneumoniae isolates were resistant for β-lactams. This may indicate the co-existence of qnr genes and β-lactams resistance encoding genes on the same plasmid, leading to confer co-resistance for both agents (3,5). This contributes to emergence of XDR K. pneumoniae that represents a great challenge in medicine with the limited options of clinical treatment (3).
Screening of qnr genes revealed that qnrS was most prevalent. It was found in 38 (76%) of K. pneumoniae isolates with 428 bp (Fig.1). Moreover, qnrB was detected in 18 (36%) of K. pneumoniae isolates with 469 bp (Fig.2). On the other hand, qnrA, qnrC, and qnrD were not found among K. pneumoniae isolates. Although these genes have low prevalence, they can easily spread among clinical isolates of K. pneumoniae and even to other species by horizontal gene transfer because these genes are mainly carried on plasmids. (3,5,6).  Correlation between phenotype (MIC) and genotype (PCR) of fluoroquinolones resistance revealed that 17 (34%) of K. pneumoniae isolates were resistant for ciprofloxacin and/or levofloxacin, of these 15 (88.23%) isolates possessed qnrS and/or qnrB. On the other hand, 33 (66%) isolates were susceptible for ciprofloxacin and levofloxacin, in spite of most of these harbored qnrS and/or qnrB. This can be explained due to qnr genes confer low resistance in which increased MIC values did not exceed the breakpoint of susceptibility that mentioned in the CLSI (5,6). DNA Sequencing of qnrB exhibited that the presence of point mutations in both selected isolates (qnrB-A and qnrB-B) (Fig.3). Some point mutations were observed to be transition, which led to substitution of pyrimidine with another pyrimidine or purine with another purine and others were transversion, which led to replacement of pyrimidine with purine and vice versa (25,26). The translation of protein revealed that some point mutations didn't affect on the amino acid sequences (silent mutations), but others led to replacement of one amino acid with another (missense mutations), as illustrated in Table (    412 Phylogenetic tree (Fig.4) indicated the presence of at least twenty three species that harbored qnrB variants sequence. The observed high number of point mutations that noticed in both qnrB variants had led to a significant deviation from K. pneumoniae species. However, both qnrB variants were located near K. pneumoniae portion, in which the majority of K. pneumoniae species were localized. Nevertheless, such mutations were still confined within Citrobacter freundii and K. pneumoniae without deviated toward other distant organisms. This tree had given nearly high biodiversity of variable organisms. This may indicate a potential evidence about the restricted power of qnrB based tree in yielding a final determination of the current local K. pneumoniae isolates.
In conclusion, although qnr genes provide low resistance against fluoroquinolones, they enhance chromosomal mutations in gyrA and parC, leading to evolve high resistance for these agents. The high association observed between resistance to β-lactams and fluoroquinolones has led to a major threat to public health through development of MDR K. pneumoniae.

Acknowledgment:
Special Thanks go to the laboratory staff of Hospitals of Medical City in Baghdad for their assistance during the collection of samples.