Detection of Virulence Factors and Antibiotic Resistance among Klebsiella pneumoniae Isolates from Iran

The current study assessed the detection of virulence genes and drug resistance among Klebsiella pneumoniae isolates from Iran. During 2018 to 2020, 52 K. pneumoniae isolates were obtained from patients at Iran hospitals. By disk diffusion method, the antimicrobial susceptibility of K. pneumoniae isolates was assessed, and ESBL-producing K. pneumoniae isolates were detected by CDDT method. PCR analysis was done to detect virulence genes (iucB, iutA, iroN, kfu, allS, fimH, ybtS, mrkD, and entB); ESBL-encoding genes (blaTEM, blaPER, blaCTX-M, blaVEB, and blaSHV); and class D (blaOXA-48), class B (blaVIM, blaNDM, and blaIMP), and class A (blaKPC and blaGES) carbapenemase genes. Among all isolates, 84.6%, 13.5%, and 1.9% isolates were multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR), respectively. Also, 84.6% were ESBL-producing and 71.2%, 53.8%, 40.4%, and 9.6% of all isolates were carrying blaTEM, blaSHV, blaCTX-M, and blaOXA-48 genes, respectively. Six isolates (11.5%) were positive for blaNDM gene. In contrast, no isolates were positive for the presence of blaKPC, blaIMP, and blaVIM. Virulence factor genes including iucB, iutA, iroN, kfu, allS, fimH, ybtS, mrkD, and entB were carried by 24%, 46.2%, 25%, 11.5%, 17.3%, 86.5%, 75%, 88.5%, and 100% isolates, respectively. This study evaluated the distribution and prevalence of virulence factor genes among K. pneumoniae isolates. The treatment of these infections is challenging due to the existence of particular virulence factors and the rise of antibiotic resistance. Therefore, the current study accentuates the necessity of finding new and efficient solutions for stopping the increase of antibiotic resistance.


Introduction
K. pneumoniae is a facultative anaerobic bacillus which is Gram-negative, encapsulated, and ferment lactose [1,2], and as it may cause serious infections and life-threatening disorders, this opportunistic pathogen remains a serious concern among public health.
K. pneumoniae is a common pathogen among nosocomial infections, including bacteremia, urinary tract infections (UTIs), pneumonia, pyogenic liver abscesses, burn, and wound infections [3,4]. Additionally, the treatment of K. pneumoniae infections is demanding due to the emergence of particular virulence factors as well as the rise of antibiotic resistance [1].
K. pneumoniae's pathogenicity is related to the existence of virulence genes that encode specific virulence factors which let the bacterium target the immune system, resulting in a variety of disorders. Among K. pneumoniae isolates, quite a few virulence factors, such as iron acquisition systems and adhesions, which contribute to virulence and pathogenicity, have been introduced [5,6].
Bacteriocin biosynthesis (yersiniabactin (ybts) and enterobactin (entB)), type 3 and type 1 adhesins (fimH and mrkD), aerobactin synthase genes (iuc and iut), iron acquisition system-related genes (iron and kfu), and allantoin metabolism-associated gene (allS) are virulenceassociated genes which have a critical impact in invasion and adhesion of K. pneumoniae isolates to host tissues, playing an essential part in the pathogenicity of K. pneumoniae isolates collected from the infections which are acquired from the hospital [1,[5][6][7].
Above all, antibiotic resistance is growing among K. pneumoniae isolates. Gram-negative bacteria, like K. pneumoniae, have acquired a variety of antibiotic resistance mechanisms, including the gaining of antibiotic resistance genes, to combat routinely used antimicrobials. The rate of resistance to widely used antibiotic groups, including tetracyclines, aminoglycosides, penicillins, lincosamides, macrolides, folic acid inhibitors, phenicols, and fluoroquinolones, according to epidemiological studies, was high among K. pneumoniae strains which were isolated from the infections which are sorted as hospital-acquired [5,6,8,9].
Due to the major impact of virulence genes on the pathogenesis of K. pneumoniae isolates and concerning increasing rates of antibiotic resistance among them, this study was to detect the distribution of virulence genes and drug resistance among K. pneumoniae isolates from Iran.

Ethical Statement.
This report is ethically approved and verified with the code "IR.SBMU.MSP.REC.1398.755," by SBMU Ethics Committee.
The privacy of the patients was preserved by keeping participants confidential, and identifiable private data was neither acquired nor involved in the research.

Bacterial
Isolates. Totally, 52 K. pneumoniae isolates were obtained from Iran between October 2018 and September 2020. The identification of bacterial isolates was performed by standard microbiological and biochemical techniques, including the ornithine decarboxylase (OD) test, urease test, methyl red/Voges-Proskauer (MR/VP) test, triple sugar iron agar media (TSI), citrate utilization test, and reactions on SH2/motility/indole (SIM) [10]. Then, bacteria isolates were kept and stored in TSB (HiMedia, India) accompanied with 20% glycerol at -70°C until they were needed.

Antimicrobial Susceptibility
Testing. According to the Clinical and Laboratory Standards Institute's standards (CLSI 2020), antimicrobial susceptibility of K. pneumoniae isolates was examined and assessed using the disk diffusion method [11]. In this study, antimicrobial disks ( Since CLSI has not published any approved guideline for fosfomycin breakpoints in K. pneumoniae, FDA fosfomycin susceptibility breakpoints for Enterobacteriaceae were used as disk diffusion interpretation criteria.
The CLSI recommendations 2020 were used to evaluate the MICs of 7 antibiotics, including colistin, meropenem, imipenem, ciprofloxacin, cefepime, ceftazidime, and cefotaxime, using the broth microdilution technique [11]. The quality of the test was confirmed using Escherichia coli ATCC 25922 in the role of control.

Detection of Extended-Spectrum β-Lactamase (ESBL).
We used the method of the combination disk diffusion (CDDT) to investigate ESBL production. Antimicrobial disks containing ceftazidime (CAZ) and cefotaxime (CTX) alone and combined disks consisting of ceftazidime 30 μg and clavulanic acid 10 μg and cefotaxime 30 μg and clavulanic acid 10 μg were also used. This test was validated by K. pneumoniae ATCC700603 and E. coli ATCC 25922 in the role of positive and negative standard controls for the production of ESBL, respectively [12].

PCR Detection of the Genes Linked to Pathogenicity.
With the usage of the DNA extraction kit (Roche, Germany, Lot. No. 10362400), genomic DNA was extracted from single bacterial colonies, which were cultured on primary culture plates as stated in the manufacturer's instructions. With the use of specific primers used in prior researches, PCR analysis was done to detect virulence genes (iucB, iutA, iroN, kfu, allS, fimH, ybtS, mrkD, and entB) ( Table 1), and the results were authenticated by sequencing. A total volume of 25 μl reaction solution was used for PCR which contained 12.5 μl of 2× Master Mix (SinaClon, Iran, CAT. No., PR901638), 1 μl of 10 pmol of each primer, 2 μl (20 ng) of DNA template, and 8.5 μl of sterile distilled water. The amplification reactions were done by thermal cycler (Eppendorf, MasterCycler Gradient, Germany). In the first phase, denaturation at 94°C for 5 min was done. Then, 36 cycles of denaturation at 94°C for 45 s were performed, and in the next stage, annealing at 50-60°C for 45 s was done. Then, extension at 72°C for 45 s and a final extension at 72°C for 5 min were carried out [13].
The electrophorese of PCR products was done by 1-1.5% agarose gel; then, they were visualized by the use of DNA SafeStain (SinaClon, Tehran, Iran), and in the next stage, they were photographed under UV light. Positive control isolates were generously provided.

PCR Detection of Carbapenemases and Genes Related to
Extended-Spectrum β-Lactamases. PCR assay with specific primers (used in prior studies) was done to investigate genes linked to ESBL-encoding (bla SHV , bla TEM , bla PER , bla CTX-M , and bla VEB ), class D (bla OXA-48 ), class B (bla VIM , bla NDM , and bla IMP ), and class A (bla KPC and bla GES ) carbapenemase gene primers (Table 1). Subsequently, PCR results were validated by sequencing. The Medical Microbiology Department of SBMU generously donated positive control isolates. ) of isolates were positive for genes linked to bla TEM , bla SHV , and bla CTX-M . The bla OXA-48 gene was carried by 5 isolates (9.6%), although none of the isolates were positive for the existence of bla KPC , bla IMP , and bla VIM . Additionally, 6 isolates (11.5%) were positive bla NDM gene. Besides, the resistance to imipenem was observed among all of the NDM-1 producing K. pneumoniae (p ≤ 0:05).

Discussion
K. pneumoniae results in a broad spectrum of infections by colonizing and spreading in the human body with the utilization of quite a few virulence factors.
In this study, the patients' ages varied from one to eighty-six years of age. However, there was no noteworthy relationship among the age of the patients and getting infected with K. pneumoniae.
The scientific community is concerned about this bacteria's rising antibiotic resistance in recent years.
Above all, the rise in resistance to quite a few antibiotics among K. pneumoniae isolates is posing a huge concern in the world.
Heidary et al. (Iran) [14] in a meta-analysis and systematic review article reported that the isolates which were drugresistant K. pneumoniae are highly prevalent in Iran. Due to that report, resistance to ampicillin (82.2%) and aztreonam (55.4%) was indicated to be the most prevalent resistance among K. pneumoniae isolates [14]. On the contrary, in BioMed Research International the current study, 63.5% of isolates were resistant against aztreonam, and the greatest proportion of resistance among the K. pneumoniae isolates was found against piperacillin (96.2%), cefotaxime (92.3%), ceftazidime (90.4%), nalidixic acid (88.5%), and ciprofloxacin (82.7%).
In this study, antimicrobial susceptibility tests revealed that the resistance against imipenem was prevalent in 65.4% of isolates (Table 2) [17], was lower than those of our study. Therefore, the percentage of resistance against imipenem has fluctuated from 2018.
In this research, antimicrobial susceptibility tests revealed that 28.8% of isolates were resistant to colistin ( Table 2) which is lower rather than the result of a study performed by Rad et al. (31.7%) in 2020, Iran [18], and is more than the result of a research performed by Bir et al. (14.6%) in 2022, India [19], and a study done by Ballen et al. (2%) in 2021, Spain [20]. Thus, resistance to colistin has relatively decreased between 2018 and 2022.
Additionally, another global issue is the prevalence of ESBL-producing isolates of K. pneumoniae that its rate differs among different countries. Besides, the reports show that the prevalence of ESBL-producing K. pneumoniae is increasing over time in Iran [21]. In this research, 84.6% of isolates were ESBL-producing K. pneumoniae which was almost similar to results of a study performed by Mogha-  [15].
In the current study, among all K. pneumoniae isolates investigated (n = 52), 1 (1.9%), 7 (13.5%), and 44 (84.6%) isolates were PDR, XDR, and MDR, respectively. Generally, according to other studies, a considerable increase in the percentage of multidrug resistance can be seen from 2019, but the amount of extensive drug resistance and pandrug resistance has shown some variations which can be due to the differences in geographical locations, sanitation levels, and antibiotic prescribing trends in hospitals. By way of    [28]. In 2014, Azimi et al. published the initial report of OXA-48 generating K. pneumoniae isolates in Iran [29].
Moreover, a study was done by Sleiman et al. (Lebanon) [30], and they reported the highest number of AMR genes (47 genes) in a K. pneumoniae isolate which bring about resistance to all antimicrobial agents which are used widely; hence, this has caused a huge health concern.
Initially, NDM-1 was detected and found in New Delhi, India, by Yong et al. in 2009 [31], and then, numerous case reports were reported in Pakistan and the United Kingdom.
In the current study, 9.6% and 6% of isolates were positive for bla OXA-48 and bla NDM genes, respectively. In a research done by Hashemi et al. (Iran), the prevalence of bla OXA-48 gene among K. pneumoniae isolates was reported 2.4% while the prevalence of bla OXA-48 among K. pneumoniae isolates was reported 58% [9] in a study performed by Candan et al. in 2015, Turkey [32], and 20% in a study performed by Ballen et al. in 2021, Spain [20]. These differences are due to the high prevalence of bla OXA-48 in Turkey and European countries. Also, the prevalence of bla NDM among K. pneumoniae isolates reported 5% in a study performed by Ahmed et al. in 2020, Bangladesh [26]; 10% in a study performed by Moghadampour et al. in 2018, Iran;and 23.3% in a study performed by Rad et al. in 2020, Iran [18].
Prior studies demonstrate that entB gene which is responsible for enterobactin metabolism is highly prevalent in K. pneumonia isolates as it is one of the most essential virulence factors among Enterobacteriaceae. The prevalence of entB was above 85% in a research performed by Fatima [16]; and Ali and Al-kakei (85.7%, Iraq) [34]. In our research, all of the isolates were positive for entB gene.
Earlier reports demonstrate that YbtS is the second common siderophore in K. pneumoniae after entB [7]. In the present study, this gene was observed in 75% of isolates. In China and Algeria, the presence of YbtS gene was 83.7% and 46.3%, respectively [35]. Also, the prevalence of ybts gene was reported 82.85% by Ali and Al-kakei (Iraq) [34].

Conclusions
This study evaluated the detection of virulence factor genes among K. pneumoniae isolates. The treatment of these infections is challenging due to the presence of particular virulence factors and the rise of antibiotic resistance. Therefore, the current study accentuates the necessity of finding new and efficient solutions for stopping the increase of antibiotic resistance considering the high prevalence of antibiotic resistance, especially high prevalence of β-lactamase-encoding genes, among these isolates with high rate of virulence factors. Above all, new and dependable diagnostic methods for detecting XDR and PDR K. pneumoniae isolates should be established.

Data Availability
All the data generated or analyzed during this study were included in this article.

Conflicts of Interest
There are no conflicts of interest.