Molecular Characterization of Efflux Pump and Porin Related Genes in Multidrug Resistance Klebsiella pneumoniae Isolates Recovered from Erbil Hospitals

Background: Multidrug-resistant efflux pumps are one of the most significant methods by which bacteria can evade the effects of numerous antimicrobials. The aim of this study was to determine the prevalence of multidrug-resistant MDR, extremely drug-resistant XDR, and pan-drug-resistant PDR K. pneumoniae phenotypes in clinical isolates, as well as to examine the detection and prevalence of efflux pump genes ompK35


INTRODUCTION
Klebsiella pneumoniae (K. pneumoniae) is a rod-shaped, Gram-negative, nonmotile, encapsulated, facultatively anaerobic, bacterium [1 and 2]. It ferments lactose and exhibits a mucoid phenotype on culture media [3]. It is associated with extraintestinal infections and life-threatening illnesses, including septicemia and endocarditis [4]. Continual and unrestricted use of antimicrobial medications for treatment of bacterial infections, in addition to horizontal gene transfer among K. pneumoniae correlates with the spread of MDR bacteria in clinical settings [5 and 6]. The efflux pump system is one of the main reasons of the appearance of MDR bacteria in clinical isolates [7].
MDR is described as resistance to at least one agent across three or more antimicrobial classes. Whereas XDR is described as resistance to at least one antimicrobial agent in all antimicrobial categories other than two or hardly any antimicrobial classes [8]. Efflux pumps are transport proteins responsible for innate or induced resistance to various antibiotics, based on the chromosomal or plasmid origins of efflux genes, accordingly. Such proteins are responsible for the expulsion of hazardous substrates, including antibiotics, from cells into the surrounding environment [9]. Consequently, reducing antibiotic concentrations within the cell [10]. Gram-negative bacteria have acquired multiple methods of tolerance to currently used antimicrobials [11].
Genes encoding for the MDR efflux pump system AcrAB-TolC and MdtK, as well as major outer membrane porin (OmpK35 and OmpK36), have significant clinical aspects on the incidence of bacterial drug resistance in K. pneumoniae isolates [12]. The AcrAB and mdtK systems belong to the K. pneumoniae resistant nodule (RND) and multi-antimicrobial extrusion (MATE) efflux pump families [13]. AcrAB-TolC pumps is accountable for tetracycline, quinolone, and chloramphenicol resistance in several Strains isolated [14]. Some of these antimicrobials are transported by MATE pumps, namely the mdtK system [15]. Membrane proteins like OmpK35 and OmpK36 are essential for antibiotic perforation inside cells and sensitivity to carbapenems and cephalosporins [11]. Data on the prevalence of efflux pump-related genes of K. pneumoniae strains are limited in the Kurdistan region. Accordingly, the present study has been conducted to find out the multi-drug resistance profiles, and frequency of MDR, XDR, and PDR phenotypes as well as to investigate the existence and prevalence rate of efflux pump-related genes in K. pneumoniae strains recovered from different types of nosocomial infections.

Specimen's collection and sources
Between July 2021 and February 2022, 60 unique isolates of K. pneumoniae were isolated from different hospitals in Erbil city, Kurdistan, Iraq. The purified colonies were recognized as K. pneumoniae by different biochemical and standard diagnostic tests such as urease and catalase test, as previously mentioned [16]. To affirm the identification of all isolates, Vitek II automated system (bioMérieux Marcy l''Etoile, France) (Vitek Systems Version: 06.01) was applied. The purified isolates were stored in brain heart infusion broth (BHI) (Oxoid) supplemented with 15% glycerol at − 20 °C for further study. This research project was allowed in all likelihood by the Scientific and Research Ethics Committee at the College of Health Sciences, Hawler Medical University/ Iraq, ethic approval number 69 which was accepted in 26/11/2022. All persons had given their acceptance to share in the study.

Genomic DNA extraction
Using the Beta Byern Biotech DNA isolation kit (Beta Byern, Germany) in agreement with the manufacturer's protocol, whole genomic DNA was extracted from the LBgrown colonies of every isolate. To evaluate the extracted DNA's purity, the absorbance ratio A260/A280 was measured with a Nanodrop spectrophotometer. Until further usage, the crude DNA extracts were refrigerated at -20°C.

Molecular methods
Primer sequences for K. pneumoniae efflux pumps (acrAB and tolC) and porin genes (ompK35 and ompK36) were used as depicted previously in (Table 1). Each PCR tube consisted of a total volume of 20 µL of a reaction mixture as mentioned previously  [19]. Conventional PCR for amplification of targeted genes (acrAB and tolC) were performed under the conditions prescribed in [14], regarding ompK35 and ompK36 genes the condition mentioned in [19] were followed using the BioRad thermal cycler (MJ Mini, BioRad, USA). The amplified products were separated on a 2.0% agarose gel and electrophoresed at 80 V, 30 mA for 1 hour. DNA fragments were visualized under an ultraviolet (UV) transilluminator. A PCR DNA ladders with 100 bp augmentation were used as a DNA standards.

Results and Discussion
A total of sixty (40%) non-repetitive K. pneumoniae strains were isolated from 150 different clinical specimens, isolates were identified based on bacterial colony morphology on agar media, isolates which showed positive catalase and urease test were confirmed by VITEK 2 Compact. Urine samples (25,41.6%) were the most common source of K. pneumoniae, the second largest source was sputum samples (22,36.6%), followed by blood samples (11,18.3%), then wound swabs (2, 3.3%) as shown in (Figure 1). [12] observed that the total incidence of K. pneumoniae in healthcare illnesses in Iran was (44%). They determined that the frequency of K. pneumoniae in urine, blood, sputum, wound and rectal swab samples was (64 %), (38%), (35% ), and  [20] observed that 20% of healthcare infections in Iran health facilities were caused by K. pneumoniae strains. They identified urinary tract infections (UTIs) among the most predominant infections caused by K. pneumoniae isolates (63%). Previously, comparable outcomes discovered in Pakistan and India respectively [21 and 22]. In addition, a greater proportion of K. pneumoniae variants in UTIs have been documented in the United States [22]. The antimicrobial sensitivity profile of all K. pneumoniae isolates upon 13 antimicrobial drugs from 8 antimicrobial classes is shown in (Table 2). According to the results of the present study, all isolates were resistant with varied rates to tested antimicrobials. It has been observed that K. pneumoniae strains possess the highest frequency of resistance against ampicillin (95%), piperacillin (83.3%), ceftazidime (81.7%), piperacillin-tazobactam (75%), amoxicillin-clavulanic acid (73.3%), ceftriaxone (68.3%), ciprofloxacin (66.7%), amikacin (63.3%), gentamicin (61.7%) and trimethoprim-sulphamethoxazole (61.7%). However, K. pneumoniae isolates possess the lowest frequency of resistance against imipenem (41.7%), meropenem (38.3%), and ertapenem (36.7%). All Klebsiella pneumoniae isolates have undergone antibiotic susceptibility testing to multiple classes of antibiotics, including antipseudomonas, folate pathway inhibitors, penicillins, beta-lactamase inhibitors, aminoglycosides, carbapenems, fluoroquinolones, and cephalosporins extended spectrum. As determined, it was resistant to at least three classes of antibiotics.
The current study found that (38%) of the isolates were multidrug resistant (MDR) K. pneumoniae. It is characterized by resistance to at least one agent of three or  [8]. The high rate of antimicrobial resistance found in our study may be attributed to the lack of stringent policies governing antimicrobial use in the city of Erbil. Our study demonstrates that most isolates were resistant to Ampicillin (95%). Similar investigations have discovered a high ampicillin resistance in China [23]. Significantly higher rates of antimicrobial resistance have been identified in strains recovered from urine (26.7%) and sputum (26.6%) samples. Furthermore, the results of this study demonstrated grouping of the Klebsiella pneumoniae phenotype according to the antimicrobial resistance profile, as shown in (Figure 2). The study found that 15 (25%) isolates were resistant to each of the eight antimicrobial categories identified as PDR K. pneumoniae. Twenty-two (37.0%) isolates identified as XDR K. pneumoniae were unsusceptible to any agent except for two or fewer antimicrobial categories. In addition, resistance to one or more of this agents of three or more antimicrobial categories was observed in 23 (38%) isolates classified as MDR K. pneumoniae.  PCR assay using specific primers for efflux pump and porin genes demonstrated that the most prevalent observed efflux pump genes of the K. pneumoniae were acrAB (95%) followed by tolC (93%). Nevertheless, the frequency of porin-coding genes was less abundant regarding ompK36 (82%) and ompK36 (80%) as shown in (Figure 3) and (Table 3). Furthermore, It has been observed that acrAB gene coding for the efflux pump system was determined in (100%) of urine samples. Besides tolC gene was found in all samples recovered from sputum while it is not detected in any isolates recovered from the wound. All isolates obtained from wound samples had the ompK35 and ompK36 porin-coding genes.
PCR assays demonstrated that both ompk 35 and ompK36 porin-encoding genes were discovered in all K. pneumoniae isolates obtained from wounds. However, its frequency varied in sputum, blood, and urine samples; this was consistent with a study conducted in Egypt by [14].   K. pneumoniae contributes significantly to the global incidence of antibiotic resistance [4]. Antibiotic efflux pumps are among the most important antibiotic resistance mechanisms utilized by clinical strains of K. pneumoniae [24]. The higher efflux of the antimicrobial drug reduces its intracellular levels, hence promoting microbial viability [25]. Both ompK35 and ompK36 contribute to the virulence and infection of K. pneumoniae. ompK36 or ompK35/ompK36 deletions can reduce the pathogenicity of hypervirulent bacteria and make them more vulnerable to neutrophil clearance [26].
In the current study, the prevalence of the AcrAB efflux pump system was 95%. The acrAB gene, encoding the efflux pump system, was found in 100% of urine samples. This was consistent with an Egyptian study [14]. Another study was then conducted in Saudi Arabia [27]. Several genes important for intracellular invasion and maintenance have been found to be deregulated in mutant strains lacking the AcrAB-tolC efflux pump [28]. Additionally, the tolC gene was identified in all sputum samples. This was the same as a study reported in Saudi Arabia [27].
The antibiotic efflux pump is one of the most important mechanisms of antibiotic resistance postulated by clinical isolates of K. pneumoniae. Accessory genomes are primarily responsible for carbapenem resistance and can also be associated with alterations in coding regions. Carbapenem resistance in K. pneumonia can be affected  [29], mutations of outer membrane proteins in the nuclear genome [30], and overproduction of ESBL enzymes or AmpC-β-lactamase in accessory genomes [31].
It has been concluded from the present study that K. pneumoniae isolates express a high prevalence of resistance against ampicillin, piperacillin, ceftazidime, piperacillin-tazobactam, and amoxicillin-clavulanic acid antibiotics. Furthermore, observation of a high prevalence of MDR, XDR, and PDR K. pneumoniae strains in Erbil city is worrisome in hospital wards.
AcrAB efflux system is one of the primary antibiotic resistance pathways in multidrug-resistant K. pneumoniae strains. We detected high distribution of acrAB, tolC, ompk35, and ompK36 genes in our isolates, which increase the survival of these pathogens when they are exposed to different antimicrobial agents.