Investigation for some Aminoglycosides Modifying Enzymes- Encoding Genes and Co-Resistance to Fluoroquinolones among Klebsiella pneumoniae Isolates from Different Clinical Cases

In this study, we investigated the prevalence of aminoglycosides modifying enzymes (AMEs)-encoding genes, including aac(3′)-ΙΙ, ant(3′′)-Ι, aph(3′)-VΙ, and aac(6′)-Ιb-cr and their potential effect on the development of resistance to aminoglycosides and fluoroquinolones in clinical isolates of Klebsiella pneumoniae. According to the phenotypic and biochemical characteristics of 150 clinical samples, 50 (33%) isolates were identified as K. pneumoniae. These isolates were collected from different clinical sources, including urine (15, 30%), blood (12, 24%), sputum (9, 18%), wounds (9, 18%), and burns (5, 10%). The minimum inhibitory concentrations (MICs) assay revealed that the resistance values of isolates were 25 (50%) to gentamicin (≥16μg/ml), 21 (42%) to amikacin (≥64 μg/ml), 15 (30%) to ciprofloxacin (≥4 μg/ml), and 11 (22%) to levofloxacin (≥8 μg/ml). Genotypic detection revealed that aac(3′)-ΙΙ, aac(6′)-Ιb-cr, aph(3′)-VΙ, and ant(3′′)-Ι were found in 47 (94%), 38 (76%), 18 (36%), and 8 (16%) of K. pneumoniae isolates, respectively. The co-resistance pattern for both aminoglycosides and fluoroquinolones was detected in 14 (28%) isolates, of these 10 (71.4%) harbored aac(6′)-Ιb-cr. DNA sequencing for some isolates revealed the presence of point and frameshift mutations in the studied genes. Our study findings suggest that the presence of missense and frameshift mutations may contribute to the elevated resistance to amikacin and gentamicin. The increased prevalence of AMEs-encoding genes among K. pneumoniae isolates could contribute in reducing susceptibility to amikacin and gentamicin. The co-resistance pattern for aminoglycosides and fluoroquinolones was highly associated with the presence of the aac(6′)-Ιb-cr gene.


Introduction
Klebsiella pneumoniae is a Gram-negative bacillus which is classified as a member of the family Enterobacteriaceae, with cells that are usually surrounded by a polysaccharide capsule that interferes with the host defense by inhibiting phagocytosis [1]. Once humans acquire K. pneumoniae from environment or hospital, it can colonize the mucosal membranes of the nasopharynx and gastrointestinal tract [2]. This colonization can be progressed into serious infections, especially in hospitalized immune-compromised individuals, making them more susceptible to develope urinary tract infections (UTIs), bloodstream infections, pneumonia, surgical and burn infections, meningitis, cellulitis, and liver abscess [2].
Throughout the last decade, K. pneumoniae represented a major worldwide burden concerning antibiotics resistance. As a result of the selective pressure of the antibiotics, K. pneumoniae continuously undergoes mutations and acquires antibiotic resistance genes by horizontal gene transfer [3]. This enables K. pneumoniae to evolve multiple resistance mechanisms against most of the common antibiotics in clinical use. Such effect could lead to the emergence of the multidrug resistant (MDR) and extremely drug resistant (XDR) strains that pose a high risk to public health and are often associated with prolonged hospitalization along with high rates of morbidity and mortality [3].
Aminoglycosides and fluoroquinolones become among the last antimicrobial options used to treat infections with K. pneumoniae. Aminoglycosides act by binding to the highly conserved A site, a part of the 16S rRNA within the small subunit 30S of bacterial ribosome, leading to the blockage of protein synthesis [4]. On the other hand, fluoroquinolones act by preventing the synthesis and replication of the bacterial DNA by targeting topoisomerases (DNA gyrase and topoisomerase IV) [5].
The aminoglycosides modifying enzymes (AMEs) are the most common mechanism employed by K. pneumoniae to interfere with the action of aminoglycosides [6]. Three families of the AMEs were detected in K. pneumoniae, including aminoglycosides acetyltransferases (AACs), aminoglycosides phosphotransferases (APHs), and aminoglycosides nucleotidyltransferases (ANTs). These are known to inhibit the active role of aminoglycosides by N-acetylation, O-phosphorylation, and Onucleotidylation of the aforementioned drugs, respectively [4,6]. Interestingly, the frequency of mutations in the AMEs-encoding genes increases the diversity of these genes through the contribution in the emergence of new alleles. This results in reducing the active role of common used aminoglycosides which imposes a great health challenge for both patients and physicians that can be inevitably difficult to be confronted and controlled [4,6].
aac(6′)-Ιb-cr is an allele of the aac(6′)-Ιb gene that belongs to the AACs family and confers coresistance for both aminoglycosides and fluoroquinolones [7]. Initially, it was detected on the plasmid in K. pneumoniae as one of the plasmid mediated quinolone resistance (PMQR) mechanisms, but recently was found on the chromosome of this species [5]. aac(6′)-Ιb-cr deactivates fluoroquinolones that have amino nitrogen on the piperazinyl ring, the substrate of the encoded enzyme AAC(6′)-Ιb-cr. This enzyme is distinguished from the other AAC(6′)-Ιb enzymes by the presence of two unique amino acid substitutions, Trp102Arg and Asp179Try, that are required for acetylating the fluoroquinolones [5,7].

Materials and Methods Sampling
One hundred fifty clinical samples were recovered from patients at different hospitals in Baghdad during the period between July 2018 to October 2018. The samples were collected from different specimen types, including urine, blood, sputum, wounds, and burns. All urine and sputum samples were collected in sterile disposable containers (Himedia, India), while blood samples were collected in EDTA tubes (Apex Bio Medicals, India). Also, sterile transport swabs (Pal Surgical Works, India) were used for collecting the samples from wound and burn infections. The instructions of the Ethics Committee of the Iraqi Ministry of Health were followed to conduct this study according to the official ethical approval numbered 31864.

Laboratory Diagnosis
Initially, the clinical samples were cultured on Gram negative selective media, including MacConkey agar and Eosin Methylene Blue agar (Oxoid, UK), at incubation conditions of 37°C for 24 hrs [8]. Then, lactose fermenting colonies were identified by performing several conventional biochemical tests, including oxidase, catalase, urease, and IMVIC tests [8]. Ultimately, the Vitek 2 Compact System (BioMerieux, France) was used to confirm the identified bacteria [9].

DNA Extraction
DNA was extracted from bacterial isolates using the Wizard Genomic DNA Purification Kit (Promega, USA) according to the information of the manufacturing company. Briefly, 1400 µl of overnight K. pneumoniae broth culture was centrifuged (ThermoScientific, USA) for 2 min at 1300 rpm, then the cell pellet was collected in an Eppendorf tube (Promega, USA). The subsequent steps of DNA extraction included the addition of nuclei lysis solution, RNase solution, and protein precipitation solution. After that, DNA was precipitated by adding isopropanol and 70% ethanol. Finally, DNA pellet was rehydrated by adding rehydration solution and kept on ice at -20˚C. The concentrations and purity of the extracted DNA were measured by Nanodrop spectrophotometer (BioDrop, UK) [11].

Molecular Screening
The extracted DNA was used as a template to detect the presence of aac(3′)-ΙΙ, ant(3′′)-Ι, aph(3′)-VΙ, and aac(6′)-Ιb-cr, utilizing Polymerase Chain Reaction (PCR). The lyophilized primers (Alpha DNA, USA, reported in Table-1) were dissolved in sterilized deionized distilled water (ddDW) (Promega, USA) to obtain 100 pmol/µl and then diluted to 10 pmol/µl according to the instructions of the manufacturing company. The PCR mixture was prepared at a volume of 20 µl, which contained 10 µl Go Taq Green Master Mix (Promega, USA), 2 µl template DNA, 1 µl of both forward and reverse primers, and 6 µl ddDW [11]. The amplification was achieved using Thermal Cycler (BioRad, USA) and started by initial denaturation at 95°C for 15 min, followed by 30 cycles of denaturation at 95˚C for 1 min, annealing at 55˚C for 1 min, and elongation at 72˚C for 5 min, while it was finalized with a final extension at 72°C for 5 min [12]. All PCR products were loaded into wells of 1% agarose gel and stained with 0.5 µg/ml ethidium bromide in 1X TAE buffer (Promega, USA) at 100 V for 80 min. The DNA ladder (100-1500 bp) (Promega, USA) was used as a marker for the molecular weight of DNA bands. The DNA bands were photographed under 320 nm UV light using UV-Transilluminator (Major Science, Taiwan) [11].

DNA Sequencing
Two K. pneumoniae PCR products from each of aac(3′)-ΙΙ, ant(3′′)-Ι, and aph(3′)-VΙ were sent to Macrogen company in South Korea for sequencing the DNA fragments from both directions using the Genetic Analyzer (Macrogen Inc., South Korea). These isolates were selected for sequencing due to show resistance to the aminoglycosides used in this study. Consequently, the potential mutations in these isolates may be associated with the high amikacin and/or gentamicin resistance rates. Only clear chromatographs obtained from ABI sequence files were further analyzed, ensuring that the annotations and variations are not because of PCR or sequencing artifacts. By comparing the observed DNA sequences of local isolates with the most homologous reference DNA sequences of K. pneumoniae retrieved from the National Center for Biotechnology Information (NCBI), the virtual positions and other details of the studied PCR fragments were identified using the NCBI BLASTn program (https://www.ncbi.nlm.nih.gov) and BioEdit Sequence Alignment Editor Software Version 7.1 (DNASTAR, Madison, WI, USA) (http://bioedit.software.informer.com/7.1/), respectively. The observed variations in each sequenced sample were numbered in PCR amplicons as well as in its corresponding position within the referring genome.
Regarding protein translation, the amino acid sequences of the targeted proteins were also retrieved from NCBI. The observed variants in the coding portions were translated into a reading frame, that were 3ʹ-5ʹ frame3 for aac(3′)-ΙΙ and 5ʹ-3ʹ frame1 for ant(3′′)-Ι and aph(3′)-VΙ, corresponding to the referring amino acid residues, using the Expasy online program (http://web.expasy.org/translate/).
The results of MICs, listed in Table-2, exhibited that 25 (50%) of K. pneumoniae isolates were resistant to gentamicin, with MIC ≥16 µg/ml, while 21 (42%) isolates were resistant to amikacin, with MIC ≥64 µg/ml. The resistance rates reported by Zafer and colleagues [16] were nearly in line with those observed by this study for both antibacterial agents. In contrast, the investigation of Mokhtari et al. [17] reported lower resistance rates to gentamicin and amikacin. The ability of K. pneumoniae to produce a wide variety of AMEs confers high resistance against aminoglycosides, in addition to overexpression of efflux pumps and loss of porins from cell membrane [4,6]. The emergence of 16S rRNA methylases provides high resistance to all common aminoglycosides [4].  (22) In respect to fluoroquinolones, 15 (30%) of K. pneumoniae isolates were resistant to ciprofloxacin (MIC ≥4 µg/ml), while 11 (22%) isolates were resistant to levofloxacin (MIC ≥8 µg/ml). Higher ciprofloxacin and levofloxacin resistance values were demonstrated by previous findings [16,18]. It was found that the resistance for ciprofloxacin and/or levofloxacin among K. pneumoniae isolates was correlated with the high prevalence of aac(6′)-Ιb-cr in these isolates. Alongside, there are other mechanisms that contribute to fluoroquinolones resistance. These include the accumulation of chromosomal mutations in the topoisomerases encoding genes (gyrA and parC), acquisition of other PMQR (qnr and oqxAB) that become widely spread even in other genera of Gram negative bacteria, particularly these involved in severe nosocomial infections [19], and the alternations of cell permeability through the loss of porins and overexpression of the MDR efflux pumps [5,7].
aph(3′)-VΙ gene was observed in 18 (36%) of K. pneumoniae isolates with a PCR product of 780 bp ( Figure-3). This rate of prevalence was lower than that observed by previously published articles [20,22]. aph(3′)-VΙ encodes for aminoglycoside 3´-O-phosphotransferases type VI, which confers resistance to aminoglycosides by catalyzing the transfer of the γ-phosphoryl group from ATP to aminoglycoside molecules, causing a lack of their effectiveness against K. pneumoniae [4].
Furthermore, ant(3′′)-Ι was identified in 8 (16%) of K. pneumoniae isolates with a PCR product size of 787 bp ( Figure-4). However, earlier studies reported this gene at higher rates among their K. pneumoniae isolates [20,23]. ant(3′′)-Ι encodes for aminoglycoside 3′′-O-nucleotidyltransferases type I, which confers resistance to aminoglycosides by catalyzing the transfer of AMP from MgATP to aminoglycoside molecules, leading to the loss of their activity against K. pneumoniae [4].  The difference in the prevalence rates of AMEs-encoding genes among the present study and the earlier reports can be attributed to the number and source of the studied isolates, working conditions in the laboratory, silent colonization by K. pneumoniae carrying AMEs-encoding genes , long term health care, misuse of antibiotics, and contact with the possible reservoir animals [26]. More serious consequences can arise from the fact that all these factors may facilitate the horizontal gene transfer of AMEs-encoding genes between high risk resistant strains of K. pneumoniae and normal flora of these bacteria that colonize gastrointestinal tract of healthy humans [3] The results of the association between phenotype (MICs) and genotype (PCR) of aminoglycosides resistance, listed in Table-3, exhibited that 25 (50%) of K. pneumoniae isolates were resistant for amikacin and/or gentamicin, of these 23 (88%) isolates harbored one or more AMEs-encoding genes. Alongside, the phenotype (MICs)/ genotype (PCR) association value for fluoroquinolones resistance was 17 (34%) of K. pneumoniae isolates that showed resistance to ciprofloxacin and/or levofloxacin, among which 13 (76.4%) possessed aac(6′)-Ιb-cr genes. The co-resistance pattern for both aminoglycosides and fluoroquinolones was detected in 14 (28%) isolates, among which 10 (71.4%) harbored aac(6′)-Ιb-cr. As with other PMQR, aac(6′)-Ιb-cr confers low or moderate level resistance against fluoroquinolones, but it enhances the occurrence of chromosomal mutations in the topoisomerases encoding genes (gyrA and parC), resulting in high level fluoroquinolones resistance [5,7]. The co-resistance pattern plays an influential role in the continuous emergence of epidemic clones that employ various modes of resistance. Thus, pandemic clones can be evolved that constitute a substantial challenge for healthcare due to limited options of antimicrobial treatment [3].  The results of DNA sequencing in the present study exhibited the presence of point mutations in the selected isolates (Figures-5,6, and 7). Some point mutations were observed to be of transition type, in which substitution occurs to pyrimidine with another pyrimidine or purine with another purine, whereas others were of transversion type, with replacement of pyrimidine with purine and vice versa [27]. Furthermore, the occurrence of frameshift mutation was observed in the DNA sequence of ant(3′′)-I gene that led to the insertion of the triple CTT in the mutant sequence (Figure-7). The translation of protein revealed that some point mutations were silent, in which the amino acid sequences were not affected, but one point mutation was observed to be a missense mutation in which one amino acid is replaced with another, as illustrated in Table-4

Conclusions
Overall, the present study concluded that AACs were the most prevalent AMEs among the investigated K. pneumoniae isolates, followed by APHs and ANTs. The increasing spread of AMEsencoding genes reduced the susceptibility for amikacin and gentamicin, making the control on MDR K. pneumoniae inevitably very difficult. The co-resistance pattern for aminoglycosides and fluoroquinolones was found at low prevalence among K. pneumoniae isolates, but it was highly associated with the presence of the aac(6′)-Ιb-cr gene. Since K. pneumoniae serves as a worldwide source for the dissemination of AMEs-encoding genes and PMQR, even to other genera of non-Enterobacteriaceae, further surveys should be accomplished in this field to control the development of antibiotic resistance as much as possible, given the limited options of antimicrobial therapy.