The Abundance of Plasmid-Mediated Quinolone Resistance Genes in Enterobacter cloacae Strains Isolated from Clinical Specimens in Kermanshah, Iran

Background Enterobacter cloacae (E. cloacae) is one of the most common Enterobacteriaceae causing nosocomial infections. Plasmid-mediated quinolone resistance (PMQR) determinants have been considered recently. This study evaluated the abundance of PMQR genes in strains of E. cloacae obtained from clinical samples in Kermanshah, Iran. Methods In this descriptive cross-sectional study, after collecting 113 isolates of E. cloacae, their identity was confirmed using specific biochemical tests. After determining their drug resistance patterns using disc diffusion, the phenotypic frequency of extended-spectrum beta-lactamase (ESBL)-producing isolates was measured by the double-disk synergy test (DDST) method. The isolates were examined for the presence of qnrA, qnrB, qnrS, and aac(6′)-Ib-cr genes by the polymerase chain reaction (PCR) assay. Results The antibiotic resistance rate of E. cloacae isolates varied from 9.7% to 60.2%; among them, 78% were multidrug-resistant (MDR). The highest quinolone resistance was observed in ESBL-producing strains of E. cloacae. The frequency of positive isolates for PMQR and ESBL was 79.6% and 57.5%, respectively. The genes aac(6′)-ib-cr (70.8%) and qnrB (38.1%) had the highest frequency among other genes. The number of isolates simultaneously carrying 2 and 3 genes was 64 and 5 isolates, respectively. Conclusion The obtained results indicate a high degree of quinolone resistance among ESBL-producing E. cloacae strains. Nevertheless, there was a significant relationship between the PMQR gene and ESBL-positive isolates. Therefore, special attention should be paid to molecular epidemiological studies on antibiotic resistance to quinolones and beta-lactamases in these strains.


Introduction
Enterobacter is one of the causes of nosocomial infections and belongs to the family Enterobacteriaceae.It exists as saprophytes everywhere and is part of the natural fora in the human intestine [1].E. cloacae and Enterobacter aerogenes have been identifed as major nosocomial pathogens among these bacterial species.E. cloacae is responsible for over 70% of these bacterial infections [2].Due to various virulence factors, such as bioflm-forming ability, toxins, cytotoxicity, and hemolysin release, this bacterium can lead to several nosocomial infections, including pneumonia, urinary tract infections, surgical wounds, skin and soft tissue infections, and bacteremia [3,4].Inappropriate antibiotic prescribing, overuse, and the underdevelopment of new antibiotics have rapidly caused antibiotic-resistant bacteria to emerge, causing nearly 700,000 deaths annually [5].Many broad-spectrum antibiotics are used to treat bacterial infections, and their improper use has led to widespread resistance, reducing the efectiveness of these antibiotics.Recent studies have reported cases of increased drug resistance in Enterobacter strains and the emergence of multidrug-resistant (MDR) strains of this bacterium [6,7].Enterobacter strains are often associated with the MDR phenotype due to their ability to acquire mobile genetic factors containing resistance genes and adaptability to the hospital environment.Tese bacteria are intrinsically resistant to ampicillin, amoxicillin, frst-generation cephalosporins, and cefoxitin due to the expression of AmpC beta-lactamase.Additionally, extended-spectrum betalactamase (ESBL) production in these bacteria makes them challenging to treat [8].Quinolones and fuoroquinolones are highly applied for the treatment of various bacterial infections.Resistance to quinolones can be caused by chromosomal mutations in bacterial genes encoding quinolone target proteins, mutations that cause decreased drug accumulation due to decreased uptake or increased efux, or plasmid-localized genes associated with quinolone resistance [9,10].Tree classes of plasmidmediated quinolone resistance (PMQR) genes have been identifed based on their mode of action, including qnr proteins, the aac(6′)-Ib-cr genes, and efux pump genes oqxA, oqxB, and qepA [11].Te qnr genes are one of the major PMQR factors, increasing drug resistance in bacteria due to their location on genetic factors [12].In addition to inducing quinolone resistance, PMQR might play an important role in resistance to other antibiotics, particularly aminoglycosides and beta-lactamases [13].Currently, seven groups of qnr genes have been identifed, including qnrA, qnrB, qnrS, qnrC, qnrD, and qnrVC [14].Te qnr gene induces quinolone resistance by blocking deoxyribonucleic acid (DNA) gyrase and topoisomerase IV.Quinolone resistance is also induced by an aminoglycoside acetyltransferase called aac(6)-ib-cr, which reduces sensitivity to ciprofoxacin.Additionally, the aac(6)-ib-cr gene, besides causing aminoglycoside resistance, confers resistance to fuoroquinolones [15].Quinolones are the most important antibiotics used in the treatment of various bacterial infections.Unfortunately, PMQR-mediated resistance has been widely reported in Enterobacteriaceae worldwide in recent decades.On the other hand, the presence of PMQR determinants can increase QRDR mutations, which increases fuoroquinolone resistance [16].As a result, determining the frequency of PMQR genes in diferent species of Enterobacteriaceae can provide important information to determine the epidemiology and understand the abundance and distribution of PMQRs to prevent the irrational use of these antibiotics and the spread of drug resistance.Considering that there is no study on the frequency of PMQR genes in Enterobacter strains isolated in this province, the present study evaluated the frequency of PMQR genes in E. cloacae strains collected from patient samples in Kermanshah City, Iran.

Extended-Spectrum Beta-Lactamases Detection.
Te isolates characterized by minimum inhibition zone diameters of 22, 25, and 27 mm for ceftazidime, ceftriaxone, and cefotaxime, respectively, were evaluated for ESBL genes.To confrm ESBL production, the combined disk (CD) approach was used, employing 30 μg ceftazidime and cefotaxime disks impregnated with 10 μg clavulanic acid (MAST, UK) on Mueller-Hinton agar (HiMedia Co., India), following the disk difusion method.After 24 hours of incubation at 37 °C, strains with a minimum inhibition zone diameter of ≥5 mm compared to a single disc of the same antibiotic were considered ESBL producers.

Detection of PMQR Genes.
Te isolates' genomes were extracted through boiling, and the frequency of the target genes (Supplementary fle (available here)) was determined using their specifc primers (Tekapo Biot Company, Iran) via polymerase chain reaction (PCR) [12,17].Te level of extracted DNA specimens was measured at 260 nm using a Nanodrop Synergy HTX (BioTek Instrument, Inc. USA), resulting in a concentration of 34 pmol/uL.Te purity of the DNA extracted at 280/260 nm wavelength was 1.82.Te PCR reaction, with a volume of 25 μL, included Master Mix (12.5 μL) (Sinocloon Company, Iran), 1 μL of primer, bacterial DNA (2 μL), and sterilized distilled water up to 25 μL.Te PCR procedure comprised initial denaturation (94 °C/ 5 min), 35 basic cycles, and extension (10 min/72 °C).In this study, in addition to using E. coli J53 strains containing pMG252, pMG298, and pMG306 as positive controls for qnrA, qnrB, and qnrS genes, respectively, we also used isolates carrying quinolone resistance genes from a previous study.Te fnal products were detected through electrophoresis on 1% agarose gel (70 V, 1 h) with ethidium bromide (0.5 μg/mL) in the Tris-EDTA bufer.An ultraviolet illuminator (ProteinSimple, USA) was employed to observe the gel, with distilled water serving as a negative control for each PCR.

Statistical Analysis.
Te data were analyzed using SPSS software (version 20) with the chi-square and Fisher's exact tests.A P value less than 0.05 was considered signifcant.

Results
A total of 113 clinical strains of E. cloacae were collected from 113 patients, including 47 (41.6%) male and 66 (58.4%) female subjects, with a mean age of 36.42 ± 11.59 years, ranging from 16 to 72 years.Tese strains were obtained from patients at Imam Reza Hospital in Kermanshah, Iran.Te highest and lowest frequencies of E. cloacae isolates were found in urine samples (43 : 38.1%) and BAL samples (5 : 4.4%), respectively.Furthermore, most isolates were obtained from the urology, intensive care unit (ICU), and outpatient wards (Table 1).Te results of the antibiotic resistance pattern of E. cloacae indicated that the highest antibiotic resistance was observed for nalidixic acid (68 : 60.2%) and ciprofoxacin (66 : 58.4%); however, the highest sensitivity was found for colistin (11 : 9.7%).Te frequency of MDR strains was 78% (89 isolates).Among the 113 isolates, the frequency of quinolone-resistant isolates was determined to be 53.7%, with the highest and lowest levels of resistance to nalidixic acid (60.2%) and gatifoxacin (33.6%), respectively.Notably, quinolone-resistant isolates exhibited signifcantly higher drug resistance than quinolone-sensitive isolates, particularly against cephalosporins and aminoglycosides (Table 2).Based on the results of the double-disk synergy test (DDST) method, the frequencies of positive and negative ESBL isolates were determined to be 65 (57.5%) and 48 (42.5%), respectively.A high degree of resistance to all tested quinolones was observed in ESBL-producing isolates compared to non-ESBL-producing isolates.Statistically, there was a signifcant relationship between the drug resistance properties of ESBL-positive and ESBL-negative strains (Table 3).Te frequency of PMQR in the 113 E. cloacae isolates was 77% (87 cases), and among these strains positive for ESBL, it was 93.8% (61/65 cases).Te highest frequency of quinolone resistance genes was identifed for the aac(6′)-Ib-cr gene (80 : 70.8%).Te frequencies of the remaining genes were 20 (17.7%) and 43 (38.1%) for qnrS and qnrB genes, respectively.None of the strains possessed the qnrA gene.Te frequency of the qnrB gene was the highest in the age group of 16-30 years (14/19 cases: 73.7%), which was statistically signifcant.However, in other cases, there was no signifcant relationship between the PMQR gene frequency and patient gender or diferent age groups (P > 0.05).Te frequencies of isolates with two concurrent genes included 35.4%, 4.4%, and 13.8% for qnrB + aac(6′)-Ib-cr, qnrB + qnrS, and qnrS + aac(6′)-Ib-cr, respectively.In addition, 5 isolates simultaneously possessed 3 genes: qnrB + qnrS + aac(6′)-Ib-cr.Te highest frequency of ESBL-and PMQR-positive strains was related to E. cloacae isolates obtained from clinical urine and blood samples in the urology and hospitalized wards (Table 2).From a statistical point of view, there was a signifcant relationship between the presence of the studied resistance genes and the drug resistance patterns (P < 0.05) (Supplementary fle (available here)).Te majority of the genes studied were detected in the ESBL strains, and in some cases, this relationship was statistically signifcant (Table 4).Figure 1 shows the PCR results for the qnrB, qnrS, and aac(6′)-Ib-cr genes.

Discussion
In recent years, there has been a global increase in infections caused by MDR E. cloacae strains [18].Te highest frequency of identifed strains was isolated from urine and wound samples.Previous studies have also reported isolating many strains of these bacteria from urine samples [2,5,19].However, Liu et al. reported the highest frequency of E. cloacae isolation from sputum samples [20].Among the studied strains, the highest resistance was observed against nalidixic acid (60.2%), ciprofoxacin, and tobramycin (58.4%).Te overall resistance rates to quinolones, cephalosporins, and aminoglycosides were determined to be 53.7%,50.4%, and 49.2%, respectively.Of 113 E. cloacae isolates, 89 (78%) exhibited multidrug resistance.Te frequency of MDR E. cloacae isolates has been reported to range from 69.9% to 75% in other studies [3,7,21].In Uzunović et al.'s study, over 66% and 28% of E. cloacae isolates were resistant to cephalosporins and aminoglycosides, respectively [22].In 2022, the resistance rates to cephalosporins, gentamicin, ciprofoxacin, and imipenem in these isolates were 100%, 82.3%, 60.8%, and 7.5%, respectively [23].Colistin (9.7%) and imipenem (14.2%) exhibited the lowest drug resistance rates among the isolates in this study.Other studies have reported lower antibiotic resistance rates in E. cloacae isolates against imipenem and colistin [20][21][22][23].However, in Ebomah and Okoh survey, more than 70% of these bacterial strains were resistant to carbapenems [24].Diferences in resistance outcomes across studies might be attributed to variations in bacterial populations, their prevalence in hospital settings, the distribution of resistance genes, variations in antibiotic use patterns, and patient management practices.Te frequency of ESBL production among E. cloacae strains in this study was determined to be 57.5%.Te ESBL-positive isolates of this bacterium have been reported at rates ranging from 59.1% to 100% in other studies [6,7,25].In ESBLproducing isolates, higher drug resistance was observed for all tested quinolones (P < 0.05).Azargun et al. reported a signifcant relationship between ESBL activity and fuoroquinolone resistance [26].Discrepancies in these fndings might be due to prolonged hospital stays and inappropriate and increased antibiotic use.
Te frequency of PMQR in E. cloacae strains and ESBLpositive strains in this study was 77% and 93.8%, respectively.Te high prevalence of PMQR and ESBL genes in this study can be indicative of the indiscriminate use of various antibiotics, including quinolones and betalactamases, followed by the spread of antibiotic resistance among bacterial isolates, which was consistent with the results of the drug resistance patterns.Te highest frequency of the qnr gene was found in E. cloacae strains related to the aac(6′)-Ib-cr gene (70.8%).Given the location of the qnr gene in plasmids containing many resistant genes, including beta-lactamases, the higher frequency of quinolone-resistant isolates in ESBL-producing E. cloacae strains in the present study might be reasonable.Markovska et al. reported a PMQR gene frequency of 59% in Enterobacter strains [27].In Azargon et al.'s study, similar to the results of the present study, the highest PMQR gene frequencies were found in ESBL-producing isolates.Tey also observed a signifcant 4 Canadian Journal of Infectious Diseases and Medical Microbiology correlation between the PMQR gene frequency and ESBLpositive isolates, which is consistent with the results of the present study [26].In another study performed in 2020, the highest PMQR gene frequency among ESBL-producing Klebsiella pneumoniae strains was linked to aac(6′)-Ib-cr (55.6%) and qnrB (34.9%) [12].Te aforementioned results suggest a high frequency of quinolone-resistant genes, the presence of resistant bacterial strains, and the widespread prevalence of resistant genes among them.In Huang et al.'s study, 68.8% of E. cloacae isolates had the PMQR gene.Tey reported that the highest frequency of PMQR-positive and aac(6′)-Ib-cr-positive isolates was among the ESBLproducing isolates of this bacterium, which is consistent with the results of the current study [25].Previous studies have shown that the presence of the PMQR gene is signifcantly associated with other antibiotic-resistant genes,   including the ESBL gene, in bacterial isolates [28].[30].In another study, the frequency of E. cloacae isolates carrying the qnr gene was reported at 60.3%, with qnrB1 being the most common (38.8%), followed by qnrS1 (24.1%).None of the isolates of the present study contained qnrA, which is consistent with studies conducted in Iran [10,19].However, in a study conducted in China, all 4 strains of E. cloacae had this gene [25].In the current study, only 4.4% of isolates simultaneously possessed both qnrB and qnrS genes.In Peymani et al.'s study, there was a higher frequency (8.6%) of these isolates containing these genes together [19].Tis is the frst study on the prevalence of quinolone resistance mediated by the plasmid aac(60)-Ib-cr in E. cloacae in Kermanshah City.According to the results of a previous study and the fndings of this study, it was shown that the frequency of the aac(6′)-Ib-cr gene was higher than that of the qnr gene in isolated Enterobacteriaceae strains.Te main limitations of this study were the lack of research on other quinolone resistance mechanisms and the lack of plasmid DNA analysis.

Conclusions
In the present study, a high level of antibiotic resistance was observed in E. cloacae strains capable of producing PMQR and ESBL.Tis resistance can be attributed to the presence of plasmids in ESBL-producing isolates, which often carry various resistance genes.Considering the signifcant prevalence of the PMQR gene in ESBL-producing isolates among the samples in the current study, it is essential to emphasize the rational and appropriate use of various fuoroquinolones in the treatment of bacterial infections.Te dissemination of PMQR and ESBL gene-containing plasmids is a cause for concern, as it can facilitate the selection and proliferation of MDR strains within the community.Terefore, conducting regular and comprehensive studies to gather more information about isolated E. cloacae strains in this context is imperative.

Table 1 :
Distribution of PMQR gene and ESBL-positive E. cloacae strains by sex, age group, specimens, and wards.

Table 2 :
Antibiotic resistance rates of quinolone-resistant and quinolone-susceptible in E. cloacae isolates.

Table 3 :
Association between antibiotic resistance in ESBL-positive and ESBL-negative E. cloacae isolates.

Table 4 :
Te frequency of PMQR genes in positive and negative ESBL-producing E. cloacae isolates.