Molecular characterization of plasmid-mediated quinolone resistance (PMQR) and ESBLs-producing Klebsiella pneumonia isolated from an Iranian teaching hospital

Background The spread of plasmid-mediated multidrug resistance in Klebsiella pneumonia is a serious threat to the public health. We investigated the clinical characteristics and molecular epidemiology of K. pneumoniae isolated at a teaching hospital in Iran. Methods A total of 50 third-generation cephalosporins resistant K. pneumoniae strains were collected from patients’ clinical cultures. Antibiotic susceptibility testing and determination of MIC values for ceftazidime, cefotaxime and ciprooxacin were performed. PCR and DNA sequencing were used to assess the presence of ESBL genes (bla CTX−M , bla TEM , bla SHV ) and PMQR genes (qnrA, qnrB, qnrS, qepA, oqxA, oqxB and aac(6)-Ib-cr). Multilocus sequence typing (MLST) was performed on the strains to assess homology. of all of is high. All K. pneumoniae strains harboured at this study highlighted the crucial need for implementing strict control measures to prevent cross transmission of these endemic clones.


Introduction
Klebsiella pneumoniae is a common pathogen causing nosocomial and community-acquired infections that is responsible for various infections such as pneumonia, septicemia, liver abscess, meningitis, urinary tract infections, and bacteremia, whose mortality rates are high [1]. Most of such infections are caused by multidrug-resistant (MDR) strains that interrupt the treatment processes. MDR K. pneumoniae acquires various resistance mechanisms that confer antibiotic resistance to commonly used antibiotics [2]. One of the important resistance mechanisms of Enterobacterales, including K. pneumoniae, is the production of extended-spectrum beta-lactamases (ESBLs), and plasmid-mediated quinolone resistance (PMQR) genes that have been detected in clinical isolates. ESBLs are plasmid-mediated enzymes that hydrolyze oxyimino-β lactam agents such penicillins, cephalosporins, and monobactams [3,4]. ESBL genes are classi ed in several types including bla CTX−M , bla SHV , and bla TEM . CTX-M-15type ESBL-producing strains have especially increased in recent years [5][6][7]. Presence of PMQR determinants including qnr genes, aac (6')-Ib-cr, and e ux pumps genes qepA, qepA2, and oqxAB confer reduced susceptibility to uoroquinolones and facilitate selection of uoroquinolone resistance in K. pneumoniae [4,8].
In the last years, studies show that an increase in the prevalence of hospital-acquired ESBL-K. pneumoniae infections in our area [7,9,10]. Most of these clinical isolates harbored bla CTX M−15 gene. Previous reports have identi ed an association between ESBL-encoding genes and genes encoding PMQR, as they can sometimes be found on the same plasmids or mobile genetic elements [3,11]. The aim of this study was to investigate the presence of PMQR in ESBL-producing K. pneumoniae isolated from an Iranian teaching hospital. The secondary aim was to determine the genetic relatedness between these isolates by molecular typing.

Materials And Methods
Identi cation of strains The 50 independently isolates of third-generation cephalosporins resistant K. pneumoniae were collected from a local general hospital. This hospital has more than 220 beds and includes a medical education center in Isfahan,  [12]. The reference strain E. coli ATCC 25922 was used as a control.

Multilocus Sequence Typing (MLST)
Genetic relatedness of the isolates was investigated by multilocus sequence typing. MLST was conducted according to previously published methods using primers of seven housekeeping genes listed in the PubMLST website (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Alleles and sequence types were assigned by using the MLST database.

Antimicrobial susceptibility testing
All experimental strains were classified as MDR (Tables 1 and 2). All strains were resistant to cefotaxim, ceftazidim and ceftriaxone. Resistance was 98% for cefepim, 88% for gentamicin and 50% for amikacin. The resistance rates to uoroquinolones were 88% and 78% for cipro oxacin and levo oxacin, respectively. The range of MIC for cipro oxacin was 0.064 mg/L to > 32 mg/L. Whereas, MIC against ceftazidime and cefotaxime in all isolates was > 32 mg/L.

Discussion
Our study demonstrated that quinolone agents were not effective against ESBL-producing K. pneumoniae isolated inpatients in Iran. ESBL-producing K. pneumoniae showed high resistant rates not only to cephalosporins but also cipro oxacin (88%) and levo oxacin (78%). Amikacin was indicated to be effective for about 50% of the strains in our study. In Indonesia and China, ESBL-positive K. pneumoniae strains were > 90% and > 70% susceptible to amikacin, respectively [16,17]. We also found that, among the resistant strains bla CTX−M−15 were the predominant ESBL gene. Therefore, we demonstrated that bla CTX−M−15 , which is a widespread public health problem around the world, was the most common ESBL gene in ESBL-producing K. pneumoniae in Iran. The nding is in agreement with recent studies in Iran and other parts of the world [11,17,18]. In accordance to other studies [7,11], our results also showed that the prevalence rates of bla TEM and bla SHV genes were high.
In the present study, a signi cant number of isolates (96%) carried at least one of the PMQR genes. The aac(6′)-Ib-cr gene was the most prevalent PMQR gene, in agreement with other studies [9,19,20]. It is suggested that aac(6′)-Ib-cr has epidemiologically strong associations with CTX-M-15 [21]. Among the qnr genes, qnrS 20 (40%) was the most predominant, followed by qnrB 14 (28%), the occurrence of qnr alleles with aac(6′)-Ib-cr gene was in accordance with previous studies [9,20]. Analysis of the data also revealed that the prevalence of oqxA was highest (72%) followed by oqxB (68%). Frequencies of oqxA and B genes found in this study are higher than those reported by Azargun in Tabriz, Iran in 2018 which were 33.7 for oqxA and 20.6% for oqxB [9]. Previous studies have found that the prevalence of PMQR genes is more common in ESBL-producing K. pneumoniae [4,9,20]. We found that PMQR genes (aac(6′)-Ib-cr, qnrS, qnrB) were also be detected in strains containing ESBL determinants. Among the isolates containing ESBL genes, 96% were producers of PMQR. It is to be noted that ESBLs are highly prevalent in the study isolates and could have contributed to the spread of PMQRs.
In the present study MLST was used for homology analysis. ST11 international high-risk clone, the most prevalent sequence type in this collection, has been described in outbreaks of ESBL-producing K. pneumoniae in some countries such as Iran, China, Sweden and detected in a OXA-48-producing isolates from Iran and Spain [2,11,22,23]. In our study, ST11 co-carried multiple resistance determinants such as bla CTX−M−15, bla TEM , bla SHV and PMQR genes including qnrB, qnrS, oqxA, oqxB and aac(6)-Ib-cr, that correlates well with earlier reports as the dominant global ESBLs are the CTX-M type beta-lactamases in K. pneumoniae [2,22]. The presence of quinolone resistance genes aac(6′)Ib-cr and qnrB was recently reported in ST11 K. pneumoniae strains from Colombia [24]. The second most common endemic sequence type K. pneumoniae in our study was ST893 which co-harbored both ESBLs and PMQR genes. In recent years, the emergence of ST893 have been reported from several Iranian hospitals [11,25] which are strongly associated with the carriage of bla NDM , bla OXA−48 and ESBLs genes. Our results suggest that, ST893 is most likely endemic in Iran. ST11 and ST893 were mainly concentrated in the ICU ward, which suggests these strains may have originated in this ward and then spread to other wards in our hospital. These results suggest more attention is required in the ICU ward to avoid dissemination outbreaks of infection.
The two K. pneumoniae isolates in our study belonged to ST16 which is one of the two isolates were positive for bla CTX−M−15 , bla SHV , bla TEM , aac(6′)Ib-cr and qnrS genes, whereas other isolates carried bla CTX−M−15 , bla SHV and bla TEM . ST16 has been reported worldwide, showing multiple resistance determinant pro les. ST16 has been identi ed as a carbapenemase producer in many parts of the world and reported as an ESBL producer in Iran, Denmark and Sweden [6,11].
One isolate belonging to ST13 co-carried multiple antibiotic resistance genes such as bla CTX−M−15 , bla SHV , bla TEM and aac(6′)Ib-cr. ST13 is a SLV of ST327, that has been reported to harbour bla NDM−1 , bla CTX−M−15 and bla SHV in Iran [28].
The ST392, identi ed in this study in one patient, ST392 was sporadically observed in different countries related to NDM-1, OXA-48 and ESBLs in Iran [10] and to aac(6′)Ib-cr, oqxAB, bl SHV , bla CTX−M−15 , bla TEM−1 in Tunisia [29]. Finally, one SHV-producing K. pneumoniae in our study belonged to ST377 and were positive for qnrS, aac(6′)Ib-cr, oqxA and qepA genes. Previously, K. pneumoniae ST377 strain carrying bla OXA−48 and ESBLs was described in Russia and Iran [11,30]. The complexity and diversity of ESBLs and PMQR combinations detected among K. pneumoniae isolates especially successful international clones ST11 and endemic clone ST893 in this study and their potential for spread poses a real threat to the management of infections by this species in Iran.