Prevalence of qnrA, qnrB, and qnrS Genes in Klebsiella pneumoniae Isolates From Patients in Hospitals in Sabzevar, Iran: A Cross-sectional From February 2022 to October 2023

1.Introduction
Klebsiella pneumoniae is commonly found in the human body as a commensal organism, particularly in the gastrointestinal tract and oropharynx. Despite its presence in these areas, it can become pathogenic and cause serious infections, especially in critically ill and immunocompromised patients [1]. It is the second most common cause of bloodstream infections (BSIs) attributed to gram-negative bacteria, after Escherichia coli [2]. While BSI can occur as a primary infection with no clear source, it is more often the result of the bacterium spreading into the bloodstream from a known site of infection [1, 3]. In addition, K. pneumoniae is an important pathogen associated with a wide variety of other infections, the most common of which are pneumonia, meningitis, pyogenic liver abscesses, and urinary tract and wound infections [4]. K. pneumoniae has become a significant clinical concern over the past 20 years due to its increasing antibiotic resistance and ability to cause serious infections, particularly in critically ill and immunocompromised patients [5]. The emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains is a major challenge, as these strains are resistant to all current antimicrobial therapies, largely due to the accumulation of antibiotic resistance genes [6]. Quinolones, especially fluoroquinolones, are commonly used to treat gram-negative bacterial infections. However, resistance to these antibiotics has been increasing, particularly in Enterobacteriaceae. The main mechanism of resistance is mutations in genes encoding key enzymes such as topoisomerase IV and DNA gyrase, as well as alterations in efflux pumps and porins [7]. In addition, plasmid-mediated quinolone resistance (PMQR) contributes to the problem, involving several genes, including qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, aac(6′)-Ib-cr, qepAB, and oqxA. The qnr genes produce proteins that interact with DNA gyrase and topoisomerase IV, thereby inhibiting quinolone activity [8]. The second mechanism of resistance is the acetylation of fluoroquinolones by a variant of the gene associated with aminoglycoside acetyltransferase, AAC(6′)-Ib [9]. The third PMQR mechanism involves quinolone efflux pumps (QepAB and OqxAB), which confer resistance to hydrophilic quinolones, particularly ciprofloxacin, enrofloxacin and norfloxacin [10]. The link between quinolone resistance and resistance to other antimicrobials, particularly aminoglycosides and beta-lactams, poses a significant challenge in the management of these infections [10]. In vitro studies indicate that isolates with PMQR genes can develop high levels of resistance after antibiotic exposure. Therefore, monitoring for PMQR genes in Enterobacteriaceae is essential. Data on the prevalence of qnrA, qnrB, and qnrS PMQR genes among clinical isolates of K. pneumoniae from human samples in Sabzevar, Iran are limited. Therefore, the aim of this study was to investigate the prevalence of these genes in K. pneumoniae isolates from patients in a hospital in Sabzevar, Iran.

2. Materials and Methods
2.1. Bacterial isolation from clinical samples
The present study was conducted from February 2022 to October 2023. In this cross-sectional study, a total of 100 specimens of urine, respiratory secretions, blood, tracheal lavage, pleural fluid and trachea were collected from patients referred to Emdad Hospital (n=1), Zargarian Laboratory (n=7), Rooyesh Laboratory (n=7), Heshmatiyeh Hospital (n=6), Mobini Hospital (n=8) and Wasei Hospital (n=71) in Sabzevar, northeastern Iran. Identification of bacteria was performed by gram stain, culture characteristics and biochemical methods. Positive control strain was K. pneumoniae ATCC700603. Trypticase soy broth containing 20% glycerol was used for preservation of bacteria at -70 °C.

2.2. Antimicrobial susceptibility testing
Bacterial susceptibility to quinolone antibiotics (nalidixic acid (30 μg), ciprofloxacin (5 μg) and ofloxacin (5 μg)) and other antibiotics (trimethoprim-sulfamethoxazole (1.25/23.75 μg), ceftriaxone (30 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), imipenem (10 μg), cefazolin (30 μg), ampicillin (10 μg), nitrofurantoin, cefotaxime (30 μg), ceftizoxime (30 μg), cephalothin (30 μg), ceftazidime (30 μg), and cefoxitin) using the Kirby-Bauer disk diffusion method according to Clinical Laboratory Standards Institute (CLSI) guidelines [11]. The discs were ordered from MAST co. England. E. coli ATCC25922 and K. pneumoniae ATCC700603 were used for antibiotic susceptibility quality control. 

2.3. DNA extraction and PCR assay
Total DNA extraction was performed by the boiling method: in brief, colonies suspected of being Acinetobacter baumanii were suspended in 500 μL TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0), boiled at 95 °C for 10 min and centrifuged at 14,000×g for 5 min. The supernatants were collected as DNA templates and stored at -20 °C for the PCR. The extracted DNA was subjected to polymerase chain reaction (PCR) assay targeting three genes of qnrA, qnrB and qnrS using specific primers (Table 1).

Reaction mixtures were prepared in a total volume of 20 μL containing 10 μL PCR master mix plus, 1 μL template DNA and 5 ng genomic DNA, 0.5 μL of each primer (0.5 μM) and 8 μL water. PCR amplification was performed in a thermocycler as follows: 95 °C for 5 minutes and 30 cycles of 50 seconds at 94 °C, 45 seconds at a specific annealing temperature for each primer, and 40 seconds at 72 °C. A final extension step of 5 minutes at 72 °C was performed. The analysis of the PCR products was checked on 1.5% gel electrophoresis with safe DNA stain and the results were visualized under the gel document system.

2.4. Statistical analysis
The data were analyzed using the chi-squared test and the Fisher’s exact test with the IBM SPSS Statistics version 26 software. P<0.05 were also considered statistically significant.

3. Result
In this cross-sectional study, 100 non-duplicate K. pneumoniae isolates were collected from various clinical specimens, including urine (n=62; 62%), respiratory tract secretions (n=6), trachea (n=18; 18%), blood (n=10; 10%), tracheal lavage (n=3; 3%), and pleural fluid (n=1; 1%) (Table 2).

The mean age of the patients was 62.2±3.25 years. In addition, 25 isolates were from outpatients and 75 isolates were from inpatients. According to antibiotic susceptibility testing, the highest resistance rate of the isolates was to ampicillin (92%), followed by cephalothin (n=68; 68%), cefazolin (n=64; 64%), ceftriaxone, meropenem, nalidixic acid and trimethoprim-sulfamethoxazole (n=62; 62%), cefoxitin and ofloxacin (n=57; 57%), ceftizoxime (n=55; 55%), ciprofloxacin (n=60; 60%), cefotaxime (n=52; 52%), gentamicin (n=43; 43%), ceftazidime (n=42; 42%), amikacin (n=39; 39%), imipenem (n=38; 38%) and nitrofurantoin (n=22; 22%). There was no statistically significant association of resistance to different antibiotics with sex and age (P>0.739). Table 2 described the characterizations of K. pneumoniae isolates from clinical samples. The qnrA, qnrB and qnrS genes were present in 61% (n=61), 56% (n=56) and 47% (n=47) of the isolates, respectively. The qnrA, qnrB and qnrS genes were present simultaneously in 16(16%) isolates, while 9(9%) isolates had none of the genes. Also, 21(21%) isolates had both qnrA and qnrB genes while 11(11%) isolates had both qnrA and qnrS genes. Table 3 indicated the relationship between qnr(s) genes and fluoroquinolone resistance.

There is a statistically significant association between the presence of qnr genes and resistance to fluoroquinolones (P<0.002). In addition, of 59 norfloxacin-resistant isolates, 56(56%) isolates had at least one of the qnr genes. Also, out of 57 ofloxacin resistant isolates, 53(53%) isolates had at least one of the qnr genes. Of 60 nalidixic acid-resistant isolates, 60(60%) isolates had at least one of the qnr genes. In addition, of 60 ciprofloxacin-resistant isolates, 57(57%) isolates had at least one of the qnr genes.

4. Discussion
K. pneumoniae is a gram-negative, non-motile, facultative anaerobic bacterium. K. pneumoniae frequently colonizes various mucosal surfaces, including the upper respiratory tract and the intestines, where colonization rates vary greatly among individuals based on habitat and exposure to disease. This bacterium is one of the most important causes of nosocomial infections globally [10]. Klebsiella species cause infections in several sites, including the lungs, urinary tract, bloodstream, wound or surgical sites, and brain. These infections are more likely to occur in people with pre-existing conditions. The discrimination between colonization and infection has always been obscure for clinicians and researchers, making subsequent intervention strategies a stubborn problem. Nonetheless, several factors can be considered to discriminate between colonization and infection [4]. In this study, urine had the highest concentration of K. pneumoniae by 62%. Similarly, in the study by Beyene et al., 50% of K. pneumoniae cases were isolated from the urine of patients [12]. In the study by Kot et al., 87.1% of K. pneumoniae were isolated from urine [5]. Jalal et al. isolated the most K. pneumoniae from blood, followed by sputum [13]. Selden et al. found evidence from sources of gastrointestinal infection that K. pneumoniae infections often have the same serotypes as the bacteria inhabiting the intestinal tract [14]. More recent studies have confirmed the association between K. pneumoniae colonization and strains obtained from infection sites [15]. Chang et al. suggest that because of the widespread presence of K. pneumoniae in humans, gastrointestinal colonization serves as a major reservoir for transmission and infection to other sites [4]. In this study, 25 isolates were obtained from outpatients and 75 from inpatients. The ICU ward showed the highest rate of isolates with 21 cases. And subgroup analysis of the antibiotic resistance patterns in ICU ward compared to non-ICU inpatients showed that strains isolated in the ICU ward were more resistant than those isolated in other wards. Similar to our study, the prevalence of Klebsiella in inpatients was higher in the study by Jalal et al. [13]. In the study by Hafiz et al., the ICU ward had the highest prevalence of K. pneumoniae among inpatients. Hafiz et al. stated that ICUs are considered as a setting where antibiotic resistance is developed, strengthened, and spread [1]. Jalal et al. also mentioned the weakening of the immune system due to aging and prolonged hospitalization, especially in the ICU, as a potential risk factor for K. pneumoniae infection [13]. Thus, it can be concluded that patients admitted to the ICU are mainly more susceptible to infection due to a attenuated immune system [16]. Pathogens use several mechanisms to develop antibiotic resistance, including beta-lactamase production, biofilm formation, creation of genetic and phenotypic diversity, and loss of sensitive outer membrane proteins, efflux pumps, and integrons. Exposure to antibiotics at sublethal concentrations may lead to the development of resistance among pathogens. In addition to the inappropriate clinical use of antimicrobial agents, human populations are often exposed to a wide range of non-iatrogenic antibacterial drugs in everyday life, including exposure to livestock antibiotics in the meat processing industry, leading to increased drug resistance in pathogens. It appears that the use of quinolones at a higher level in animal and fish farm treatment have played a significant role in the development of this type of resistance [17]. The increased use of antibiotics in both clinical and nonclinical settings is associated with an increased number of drug-resistant strains [4]. K. pneumoniae infection is often treated with beta-lactam antibiotics which are one of the most widely-used resistant antibiotics, thence causing a major crisis in medical clinics in the last two decades [18]. Based on the results of this study, the highest and lowest resistance rates of K. pneumoniae isolates were against ampicillin (92%) and nitrofurantoin (22%), respectively. In the study by Beyene et al., the highest resistance was against ampicillin and amoxicillin, and the lowest resistance was against nitrofurantoin [4]. Jalal et al. reported ampicillin (97.6%) as the most-resisted antibiotic in their study [15]. Moreover, in a study by Amraie et al. conducted on 195 Klebsiella isolates, the highest and lowest resistance rates were against amoxicillin (79.19%) and ciprofloxacin (15.6%), respectively [18]. Considering these results, in most studies, isolates were resistant against amoxicillin and ampicillin, implying that these antibiotics cannot be used as empirical treatment. On the other hand, very low levels of resistance to antibiotics such as nitrofurantoin have been reported in previous studies [14]. It can also be said that the regional variations of resistance to antibiotics may be partially explained by different local antibiotic practices. Further studies in different geographical areas are required to investigate the susceptibility of organisms to antibiotics due to genetic differences, hospital practices, and environmental factors [18]. The impact of excessive or inappropriate use of antibiotics has been shown to lead to the emergence of antibiotic-resistant strains, especially broad-spectrum agents that are administered practically. Reduced frequency of prescriptions for certain antibiotics can lead to reduced resistance. The transmission of resistant isolates between people or the consumption of foods derived from animals that have received antibiotics has also contributed to the spread of antibiotic resistance [18]. In this study, the prevalence rate of three genes qnrA, qnrB, and qnrS was investigated. qnrA was the most frequent gene isolated from isolates with 61%. Besides, 16% of isolates entailed all three genes and 9% of isolates contained none of the genes. In the study by Nourozi et al., the qnrB gene was the most frequent gene isolated from the isolates with 43%, followed by the qnrS and qnrA genes with 34% and 23%, respectively [19]. Further, in the study by Moghadasi et al., the qnrB gene showed the highest prevalence with 46.66% [17]. In the study by Dehghan Banadkouki et al., the qnrB gene was isolated in 21 isolates (45.7%), the qnrS gene in 7 isolates (15.2%), and the qnrA gene was not detected in any of the isolates [16]. In this study, a statistically significant association was observed between the presence of qnr genes and resistance to fluoroquinolones (P<0.05). Resistance to nalidixic acid was higher in isolates containing the qnrA gene compared to other antibiotics (41%). Besides, in the study by Eftekhar et al., isolates containing both qnrB and aac(6’)-Ib-cr genes were significantly more resistant to quinolones (P<0.05) compared to isolates containing either gene alone. Eftekhar et al. suggested that additional resistance mechanisms, such as mutations in the chromosomal genes gyrA and gyrC or the presence of efflux pumps such as QepA, are responsible for the high levels of resistance [20]. These findings confirm that although qnr genes are not solely involved in quinolone resistance, they do reduce susceptibility to nalidixic acid and fluoroquinolones [20]. The qnr genes protect quinolone targets in bacteria, and the coding genes are widely distributed in the Enterobacteriaceae family. The qnr genes are assumed to promote low to moderate quinolone resistance, while strains with mutations in the gyrA and parC genes or the plasmid-mediated aac(6’)-Ib-cr and qnr genes exhibit high levels of quinolone resistance. Nourozi et al. reported that the purified qnrB gene, like the qnrA gene, protects DNA gyrase from quinolone action. Resistance to quinolones is also associated with loss of purine, specific efflux pump, or DNA gyrase alterations and a four- to eight-fold increase in expression [19]. Amraie et al. further suggested that some genes are mobile among isolates and spread in the environment. A different mechanism of gene transfer, such as horizontal gene transfer between serotypes, could possibly cause the spread of resistance genes [18]. 

5. Conclusion
In conclusion detection of virulence factors in K. pneumoniae isolates, especially combined with antibiotic resistance, is very important as it allows for assessing the possible course and site of infection in the human body and is essential for development of treatment strategy. Hence, today, much attention has been paid to molecular studies of resistance genes in K. pneumoniae. Although molecular methods exhibit very high sensitivity and specificity, their application is limited because these methods are not available in every laboratory and require specific skills and standards. Based on these results, qnrA-producing K. pneumoniae strains were isolated from patients. Thus, prescribing appropriate antibiotics and detection of qnr genes is required and it can be useful in tracking, treating and knowledge of K. pneumoniae infection prevalence rate.

Acknowledgements
The authors would like to express their gratitude to the Sabzevar University of Medical Sciences, Sabzevar, Iran for their invaluable support in conducting this study.

Compliance with ethical guidelines
The study was approved by the Research Ethics Committee of the Sabzevar University of Medical Sciences, Sabzevar, Iran (Code: IR.MEDSAB.REC.1400.133).

Data availability
The data that support the findings of this study are available on request from the corresponding author.

Funding
This research was supported by the research project Funded by the Sabzevar University of Medical Sciences, Sabzevar, Iran (No.: 400139).

Authors' contributions
Conceptualization: Faezeh Kheirabadi; Methodology: Faezeh Kheirabadi and Ehsan Javaheri; Data collection: Maryam Rostazadeh, Mohsen Heidary, and Reza Faraji; Data analysis: Ehsan Javaheri and Saeed Khoshnood; nvestigation: Maryam Rostazadeh; Writing the original draft: Tahereh Navidifar and Reza Faraji; Review and editing: Mohammad Keyvanloo Shahrestanaki and Mahta Mojahedifar; Final approval: All authors.

Conflict of interest
The authors declared no conflict of interest.

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