Molecular Epidemiology of OXA-48 and NDM-1 Producing Enterobacterales Species at a University Hospital in Tehran, Iran, Between 2015 and 2016

Carbapenem-resistant Enterobacterales (CRE) is an increasing problem worldwide. Here, we examined the clonal relatedness of 71 non-repetitive CRE isolates collected in a university hospital in Tehran, Iran, between February 2015 and March 2016. Pulsed-field gel electrophoresis (PFGE) and MLST were used for epidemiological analysis. Screening for antibiotic resistance genes, PCR-based replicon typing, conjugation experiments, and optical DNA mapping were also performed. Among all 71 isolates, 47 isolates of Klebsiella pneumoniae (66.2%), eight Escherichia coli (11.2%), five Serratia marcescens (7%), and two Enterobacter cloacae (2.8%) harbored blaNDM–1 and blaOXA–48 genes together or alone. PFGE analysis revealed that most of the OXA-48- and NDM-1-producing K. pneumoniae and all of OXA-48-producing S. marcescens were clonally related, while all eight E. coli and two E. cloacae isolates were clonally unrelated. The predominant clones of carbapenemase-producing K. pneumoniae associated with outbreaks within the hospital were ST147 (n = 13) and ST893 (n = 10). Plasmids carrying blaNDM–1 and blaOXA–48 were successfully transferred to an E. coli K12-recipient strain. The blaOXA–48 gene was located on an IncL/M conjugative plasmid, while the blaNDM–1 gene was located on both IncFII ∼86-kb to ∼140-kb and IncA/C conjugative plasmids. Our findings provide novel epidemiologic data on carbapenemase-producing Enterobacterales (CPE) in Iran and highlight the importance of horizontal gene transfer in the dissemination of blaNDM–1 and blaOXA–48 genes. The occurrence and transmission of distinct K. pneumoniae clones call for improved infection control to prevent further spread of these pathogens in Iran.


INTRODUCTION
Carbapenems are broad-spectrum beta-lactam agents that are frequently used as a last resort to treat serious infections caused by multidrug-resistant Enterobacterales. Resistance to carbapenems mainly depends on the production of carbapenemase enzymes. Carbapenemase-producing Enterobacterales (CPE) are increasingly reported and represent a major public health threat (Tängdén and Giske, 2015). The most clinically significant carbapenemases in Enterobacterales include the class A (KPC type), class B (metallo-β-lactamases [MBLs] [i.e., VIM, IMP, and NDM types]), and class D carbapenemhydrolyzing β-lactamases (OXA-48-like enzymes) Tängdén and Giske, 2015). NDM-1 and OXA-48 β-lactamases were initially identified in India and Turkey, respectively, and then spread to various countries worldwide including India, the Middle East, and Mediterranean countries (Yong et al., 2009;Johnson and Woodford, 2013;Sartor et al., 2014;Jamal et al., 2016;Solgi et al., 2017b). There is a lot of pilgrimage tourism and business travel between Iran and neighboring countries such as Iraq, Afghanistan, Pakistan, Turkey, and the Persian Gulf, so travelers with CPE colonization may be the vectors for spread of resistant strains. In the scope of outbreaks in Iran, diverse sequence types (STs) of dominant OXA-48-and NDM-producing Klebsiella pneumoniae have been identified in outbreaks or solitary case reports (STs 11,893,147,and 915) (Solgi et al., 2017a(Solgi et al., , 2018. VIM-2-producing K. pneumoniae ST23 has been reported in Iran more recently (Mohammad Ali Tabrizi et al., 2018).
The dissemination of OXA-48 and NDM-1 among Enterobacterales is mediated by the rapid spread of broad host-range conjugative plasmids. The bla NDM−1 gene has been detected on plasmids of various incompatibility groups: IncF, IncA/C, IncL/M, IncH, IncN, and IncX3 or untypeable (Voulgari et al., 2014). The bla OXA−48 gene has also been carried by various plasmids types including IncL/M, IncN, and IncA/C (Guo et al., 2016). Up until today, only one study has reported the finding of the prevalence and distribution of carbapenem resistance among Enterobacterales isolates in Iran (Shahcheraghi et al., 2017). However, limited data about the sequence type of CRE isolates that has spread in Iran were available.
Here, we investigated the prevalence of ESBL and carbapenemase genes, to explore the distribution of plasmid replicons, and molecular epidemiology of CPE isolated in an Iranian hospital.

Bacterial Strains
In this cross-sectional study, a total of 71 non-repetitive carbapenem-resistant Enterobacterales (CRE) clinical isolates resistant to at least one of the carbapenems (imipenem, meropenem, or ertapenem) were collected at the Loghman Hakim Educational Hospital, a 496-bed university hospital in Tehran (Iran) between February 2015 and March 2016. All isolates were identified by standard biochemical tests and API 20E (bioMérieux, Marcy-l'Etoile, France).

Molecular Typing
The genetic relatedness of CPE isolates was investigated by pulsed-field gel electrophoresis (PFGE). The genomic DNA of the CPE isolates and reference marker Salmonella serotype Braenderup strain H9812 were digested by XbaI endonuclease, which was performed with a CHEF-DRIII system (Bio-Rad Laboratories) as previously described (Tenover et al., 1995). A similarity coefficient was obtained using Dice coefficients. Cluster analysis was done with the unweighted pair group method with arithmetic averages (UPGMA). Isolates that exhibited similarity cut-off ≥80% of their banding patterns were considered to belong to the same clonal lineage (pulsotypes). Multilocus sequence typing (MLST) was performed according to the protocol described on the Pasteur Institute MLST website 1 for K. pneumoniae, MLST website for E. coli 2 , and MLST website for Enterobacter cloacae 3 .

Plasmid Extraction
Plasmid DNA was prepared from an overnight culture with NucleoBond R Xtra Midi kit for isolates according to the manufacturer's description for high-copy plasmid purification (Müller et al., 2016a). Eluted plasmid DNA is then precipitated with isopropanol and washed with 70%; the dried pellet was reconstituted TE buffer, pH 8.0. The DNA concentration and purity were determined using the Qubit 3.0 Fluorometer.

Optical DNA Mapping in Nanochannels for Plasmid Analysis
The presence of the bla NDM−1 gene on plasmids from isolates LO94, LO204, LO271, LO247, LO64, LO63, LO89, and LO149 was investigated using optical DNA mapping (Müller and Westerlund, 2017). For this, Cas9 enzyme (PNA Bio Inc., Newbury Park, CA, United States) was used to make a sitespecific cut at the bla NDM−1 gene (target gene sequence was 5 -CGGTATGGACGCGCTGCATG-3 , RNA was synthesized by Dharmacon Inc., Lafayette, CO, United States) on the plasmids (Müller et al., 2016b). Cas9 will cut all the bla NDM−1 genecarrying plasmids in each isolate at the same location which would show as a consensus cut site in the ODM data. For the plasmids not carrying the bla NDM−1 gene, we expected randomly distributed cuts.
After the Cas9 reaction, the plasmids were stained using YOYO-1 and Netropsin which created an emission intensity pattern along the DNA, with dark AT-rich regions and bright GC-rich regions (Nyberg et al., 2012;Nilsson et al., 2014). Netropsin prevents the binding of the fluorescent YOYO-1 to AT-rich regions which results in the formation of a variation in intensity, a DNA barcode. Plasmids were stretched to their full contour lengths by confining them in 100 × 150-nm 2 nanofluidic channels and imaged using an EMCCD camera. For each of the eight isolates, hundreds of plasmids were imaged and analyzed. The barcodes were aligned, clustered based on similarity, and compared among the isolates using custom-built MATLAB routines (Müller et al., 2016a). Lambda phage DNA was used as an internal control to correlate the length in pixels with the length in base pairs, and this correlation factor was then used to estimate plasmid sizes.

Antimicrobial Susceptibility
Susceptibility profiles against ten antimicrobials agents are listed in Table 1. As expected, the majority of the CRE isolates exhibited resistance to most β-lactams. Most of the isolates were also resistant to ciprofloxacin (70/71 98.6%) and gentamicin (42/71 59.1%). On the other hand, most of them were susceptible to amikacin (46/71 64.8%), and all isolates were susceptible to colistin, with MICs ≤ 1 mg/L. Based on phenotypic detection, 40 out of the 62 isolates (64.5%) were positive for MHT.

Plasmid Replicon Typing and Conjugation Assay
The bla NDM−1 gene was identified on an IncFII-type plasmid for twenty-six K. pneumoniae and two E. cloacae isolates and on an IncA/C-type plasmid for a single E. coli isolate, while the bla OXA−48 gene was identified on an IncL/M-type plasmid for nineteen K. pneumoniae, seven E. coli, and five S. marcescens isolates. In the six K. pneumoniae isolates, we could not identify the incompatibility group.
Conjugation experiments revealed that all of the NDM-1 and OXA-48 plasmids were successfully transferred to E. coli K12, conferring resistance to carbapenems and cephalosporins in transconjugants. In addition, co-transfer of bla NDM−1 , bla OXA−48 , and other resistance determinants (bla CTX−M , bla TEM , and bla SHV ) was observed in several isolates ( Table 2). Plasmid gel extraction followed by PCR amplification of the transconjugants revealed that the bla OXA−48 gene was harbored on transferable plasmids belonging to the IncL/M incompatibility group, while the bla NDM−1 gene was located on conjugative plasmids. Transconjugant Tc-Lo204 had two different plasmids, and the size of one plasmid was ∼140 kb with bla NDM−1 and the other one was ∼135 kb with bla OXA−48 . Notably, all bla OXA−48positive conjugative plasmids co-harbored beta-lactamase gene bla CTX−M−15 .

Optical DNA Mapping
The presence of the bla NDM−1 gene on plasmids of isolates LO94, LO204, LO271, LO247, LO64, LO63, LO89, and LO149 was characterized using optical DNA mapping (ODM). Table 3 presents a summary of the ODM data for the bla NDM−1 -carrying plasmids in these eight K. pneumoniae strains. DNA barcodes for each isolate were clustered based on similarity, and clusters with consensus cut sites (with at least nine barcodes) were used to infer the Cas9 cutting, suggesting the presence of the bla NDM−1 gene on the plasmids (Müller and Westerlund, 2017). For isolate LO271, two plasmids (∼86 kb and ∼107 kb) carrying the bla NDM−1 gene were identified. For isolate LO204, two plasmids of length ∼140 kb and ∼135 kb were found; however, only the ∼140-kb plasmid carried the bla NDM−1 gene. The remaining six isolates carried only one plasmid in the size range ∼110 kb to ∼130 kb carrying the bla NDM−1 gene.
After plasmid size estimation and bla NDM−1 gene detection, we compared the consensus barcodes among the eight isolates (Figure 4). The ODM assay showed that identical plasmids with the same size (∼125 kb) and the same location of the bla NDM−1 were found in LO63 and LO64 (Figure 4A). These isolates belong to sequence types ST147 and ST893, respectively (Figure 1), suggesting a possible transmission of plasmid from one strain to the other. Similarly structured plasmids were found in LO89 and LO271 (∼107 kb) ( Figure 4A); they both belong to the same sequence type, ST147. By visual inspection, it appears that large regions of the plasmids of isolates LO63, LO64, LO89, and LO271 ( Figure 4A) are similar, further accentuated by the fact that the bla NDM−1 gene is located at the same position. There are however, other regions that are not the same, and the size differs (plasmids from LO63, LO64, and LO89 were ∼125 kb while the plasmid from LO271 was ∼107 kb). The plasmids from the other isolates do not match among each other ( Figure 4B) or with the plasmids in Figure 4A. In total, we therefore found seven different plasmids carrying the bla NDM−1 gene.

DISCUSSION
Herein, we found 71 CRE in a period of 1 year with a lot of CPE species from patients in the same hospital in Tehran, Iran, and major dissemination of the bla NDM−1 and bla OXA−48 genes, which might be considered endemic in the geographical area, through the spread of conjugative plasmids.
The co-occurrence of NDM-1-and OXA-48-producing Enterobacterales species is also considerable since the identification of NDM-1 and OXA-48 producers in Iran (Solgi et al., 2017b), Lebanon (Dandachi et al., 2016), and Kuwait (Jamal et al., 2015) shows that these carbapenemases, known to be widespread in the Indian subcontinent, may also   be widespread in the Middle East. In our study, the majority of the NDM-1-and OXA-48-producing Enterobacterales isolates co-harbored at least one ESBL gene which is concordant with previous reports (Torres-González et al., 2015;Solgi et al., 2017a). In this study, nine carbapenem-resistant K. pneumoniae were identified; this may be due to other resistance mechanisms (e.g., more rare carbapenemases, porin loss, AmpC enzymes) that were not investigated in detail in this study. The plasmid incompatibility types IncFII and IncA/C were identified among the NDM-1-producing isolates, while only IncL/M was detected among OXA-48 producers ( Table 1). These replicon types have been reported in Enterobacterales species in many regions of the world (Brañas et al., 2015;Guo et al., 2016;Kieffer et al., 2016;Solgi et al., 2017b). Also, Weber et al. (2019) demonstrated that the potential transmission of mobilized Tn125-like transposons with bla NDM−1 into different plasmids among Enterobacterales species (Weber et al., 2019). Conjugation assays were successful for all CPE isolates and allowed the identification of bla OXA−48 -carrying plasmids belonging to the IncL/M incompatibility group in all transconjugants, with the exception of Tc-LO-149 (Table 2). Also, analysis of transconjugants showed that the bla NDM−1 carried on transferable plasmids belonging to the IncFII and IncA/C incompatibility group, respectively.
The identification of conjugative plasmids harboring bla NDM−1 and bla OXA−48 genes in CRE isolates shows that these plasmids contribute to the dissemination of carbapenemase genes among Enterobacterales species. Therefore, resistance to carbapenems in CRE isolates is likely to be associated with the spread of these genes in this hospital, which is consistent with previous studies (Jamal et al., 2016;Kieffer et al., 2016;Solgi et al., 2017a).
Pulsed-field gel electrophoresis revealed that different clones of carbapenemase-producing K. pneumoniae (CPKP) were present, and there were two predominated clones that were identified as ST147 and ST893, comprising 13 and 10 isolates, respectively. ST147 and ST893 have been circulating in this hospital setting during the period of investigation, indicating two separate outbreaks, with the ICU poisoning acting as the epicenter. Indeed, hospital outbreaks of ST147 NDM-1-producing K. pneumoniae are common in Europe (Bogaerts et al., 2011;Giske et al., 2012), whereas the outbreak of OXA-48-producing ST893 K. pneumoniae was only reported from Isfahan, Iran (Solgi et al., 2018).
The four NDM-1-and one OXA-48-producing K. pneumoniae in our study belonged to ST16 and were positive for bla CTX−M−15 and bla SHV−199 genes. It is noteworthy that OXA-48-producing ST16 have also been described in K. pneumoniae that caused outbreaks in two hospitals in different regions of Spain (Oteo et al., 2013). Furthermore, two OXA-48-and one NDM-1producing K. pneumoniae were isolated from three patients in two different ward. They belonged to ST377, which has previously not been reported as a carbapenemase producer. Finally, one OXA-48-producing K. pneumoniae isolate that cocarried bla CTX−M−15 , bla TEM−1 , and bla SHV−182 was identified as ST11. The bla OXA−48 -harboring IncL/M plasmids have been mainly described in K. pneumoniae ST11 in different countries including, Spain (Brañas et al., 2015), Taiwan (Ma et al., 2015), and Greece (Voulgari et al., 2013).
Considering this study and our previous study in Isfahan province (Solgi et al., 2018), the main K. pneumoniae STs that were identified in Iran were ST893, ST11, and ST147. This scenario suggests that these STs have likely been circulating in Iran in recent years. Our results show that, in general, the population structure of CP E. coli is more diverse than that of CPKP, which is essentially similar to the findings of other studies (Sartor et al., 2014;Kieffer et al., 2016;Solgi et al., 2017b). We detected E. coli ST410, ST1431, ST3134, and ST5114 which have been reported as harboring bla OXA−48 and ESBL genes. Moreover, we identified only one ST131 of E. coli which harbored bla NDM−1 , bla CTX−M−15 , and bla TEM−1 genes. The association of NDM-1 and ESBL genes with the pandemic clone ST131 has been previously reported from several countries (Peirano et al., 2011(Peirano et al., , 2014. The two NDM-1-positive E. cloacae isolates were genetically not related and belonged to two STs, ST78 and ST175, both also carried bla CTX−M−15 and bla TEM−1 genes, while the five S. marcescens isolates were considered identical (>99% similarity). Interestingly, looking at the hospitalization ward from which the patients originated, several infections were detected at the ICU poisoning, with a total of five patients harboring this OXA-48producing S. marcescens strain which co-carried further betalactamase genes (bla CTX−M−15 , bla SHV−12 , and bla TEM−1 ). Our results showed that this OXA-48-producing S. marcescens strain was isolated among inpatients who shared a room. Therefore, it is possible that the spread of this strain from patient to patient occurred. To the best of our knowledge, this is the first report of an outbreak of OXA-48-producing S. marcescens that coharbored ESBL genes in Iran. A small hospital outbreak linked to OXA-48-producing S. marcescens has been previously reported in Lebanon (Hammoudi et al., 2014). The exact mechanism of CPE spread in Iran is not well understood. Our previous study in July to November 2015 in two university hospitals in Iran showed that the rate of fecal carriage of CRE among inpatients is high (37.9%) and predominant species were K. pneumoniae, E. coli, E. cloacae, and Proteus mirabilis, which harbored the bla NDM−1 and bla OXA−48 genes (Solgi et al., 2017a). The circulation of bla NDM−1 and bla OXA−48 carbapenemase genes in the general population may result in a further spread by traveling and continuous introduction into the hospitals.
In conclusion, findings of extensive analysis of plasmids in the present study showed the enormous potential of spread of carbapenemase genes by horizontal gene transfer via plasmids and we identified the conjugative plasmids carrying the bla NDM−1 and bla OXA−48 genes in different Enterobacterales species that co-produce ESBLs. Here, in one Tehran hospital, we report two separate outbreaks of NDM-1-producing ST147 and OXA-48producing ST893 K. pneumoniae STs. Furthermore, an outbreak with OXA-48-producing S. marcescens was observed. It is necessary to continue epidemiological and active surveillance to improve the control and prevention of infections associated with CPE isolates in healthcare facilities.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The study was approved by the research and the Ethics Committee of the Pasteur Institute of Iran (No. 1395.51). No ethical approval was obtained for using the clinical isolates since they were collected during the routine diagnostic laboratory at our hospital.