Genetic Plurality of OXA/NDM-Encoding Features Characterized From Enterobacterales Recovered From Czech Hospitals

The aim of this study was to characterize four Enterobacterales co-producing NDM- and OXA-48-like carbapenemases from Czech patients with travel history or/and previous hospitalization abroad. Klebsiella pneumoniae isolates belonged to “high risk” clones ST147, ST11, and ST15, while the Escherichia coli isolate was assigned to ST167. All isolates expressed resistance against most β-lactams, including carbapenems, while retaining susceptibility to colistin. Furthermore, analysis of WGS data showed that all four isolates co-produced OXA-48- and NDM-type carbapenemases, in different combinations (Kpn47733: blaNDM–5 + blaOXA–181; Kpn50595: blaNDM–1 + blaOXA–181; Kpn51015: blaNDM–1 + blaOXA–244; Eco52418: blaNDM–5 + blaOXA–244). In Kpn51015, the blaOXA–244 was found on plasmid p51015_OXA-244, while the respective gene was localized in the chromosomal contig of E. coli Eco52418. On the other hand, blaOXA–181 was identified on a ColKP3 plasmid in isolate Kpn47733, while a blaOXA–181-carrying plasmid being an IncX3-ColKP3 fusion was identified in Kpn50595. The blaNDM–1 gene was found on two different plasmids, p51015_NDM-1 belonging to a novel IncH plasmid group and p51015_NDM-1 being an IncFK1-FIB replicon. Furthermore, the blaNDM–5 was found in two IncFII plasmids exhibiting limited nucleotide similarity to each other. In both plasmids, the genetic environment of blaNDM–5 was identical. Finally, in all four carbapenemase-producing isolates, a diverse number of additional replicons, some of these associated with important resistance determinants, like blaCTX–M–15, arr-2 and ermB, were identified. In conclusion, this study reports the first description of OXA-244-producing Enterobacterales isolated from Czech hospitals. Additionally, our findings indicated the genetic plurality involved in the acquisition and dissemination of determinants encoding OXA/NDM carbapenemases.


INTRODUCTION
The increased incidence of multidrug-resistant (MDR) Gramnegative bacteria worldwide over the last decade has been worrisome (Bassetti et al., 2019). Carbapenems are considered the drug of choice in treating such infections. However, the increased usage of these antibiotics has led to the emergence of carbapenem-resistant strains (Roberts et al., 2020). Center for Disease Control (CDC) considers carbapenem-resistant Enterobacterales (CRE) as a serious global threat to patient health that limits treatment options, especially in chronically ill patients in intensive care units (ICU) and long-term care facilities (McConville et al., 2017;Gupta et al., 2019). Resistance to carbapenems is caused by various mechanisms, such as porin loss, increased efflux pump activity and most importantly the production of carbapenemases (Ye et al., 2018).
Also, OXA-48-producing Enterobacterales pose an important public threat, mainly due to their challenging detection and the rapid horizontal transfer of pOXA-48-like plasmids . OXA-48-like enzymes hydrolyze penicillins at a high level and carbapenems at a low level, sparing broad-spectrum cephalosporins, and are not susceptible to β-lactamase inhibitors (Poirel et al., 2004). Since its first report in Turkey in 2004, 11 variants with few amino acid substitutions or deletions emerged globally (Bakthavatchalam et al., 2016;Mairi et al., 2018). In 2006-2007, OXA-181 was reported for the first time in India and since then it is considered one of the most disseminated OXA-48like enzymes worldwide especially in patients with travel history to the Indian continent (Castanheira et al., 2011;Rojas et al., 2017). In 2011, OXA-244 was reported for the first time in Spain, since then there has been only limited number of reports, indicating limited dissemination (Oteo et al., 2013;Fursova et al., 2015;Potron et al., 2016;van Hattem et al., 2016). Recently, coproduction of NDM-and OXA-48-like carbapenemase has been increasingly described, especially in patients with travel history to Italy (Marchetti et al., 2019), South Korea (Baek et al., 2019), Turkey (Otlu et al., 2018), Singapore (Balm et al., 2013) and the United States (Doi et al., 2014;Contreras et al., 2020).
Thus, the aim of this study was to genomically characterize four isolates (three Klebsiella pneumoniae and one E. coli) co-producing NDM-and OXA-48-like carbapenemases from Czech patients with travel history or/and previous hospitalization abroad.

Case Presentations
The first case was reported, in December 2018, from a Czech patient admitted to the Neuro Intensive Unit Care (ICU) of the Military University Hospital in Prague for head injury and concussion. The patient had traveled shortly before admission to India, where he/she was hospitalized due to a motorcycle accident, and then transferred back to Prague. A rectal swab was collected, highlighting a K. pneumoniae isolate (Kpn47733) co-producing NDM-and OXA-48-like carbapenemases.
The second case referred to an inpatient of the Rehabilitation Unit of the Malvazinky Clinic in Prague, who underwent an orthopedic surgery for hip replacement in May 2019. During rehabilitation, the patient developed a urinary tract infection. Urine culture confirmed the presence of a K. pneumoniae isolate (Kpn50595), coproducing NDM-and OXA-48-like carbapenemases. The patient had a travel history 2 weeks before admission (April 2019) to Mauritius, but didn't have any history of hospitalization there.
The third case was a patient admitted to Hepatogastroenterology Unit of the Institute of Clinical and Experimental Medicine in Prague for bile duct obstruction, in June 2019. As a part of the screening process, a rectal swab was performed, and culture highlighted the presence of a K. pneumoniae isolate (Kpn51015), co-producing NDM-and OXA-48-like enzymes. The patient had a travel history to Egypt in December 2018, without hospitalizations.
The fourth case was reported in August 2019, when a female patient was admitted to the Nephrology Ambulatory of the University Hospital of Ostrava, showing urinary tract infection symptoms. Urine sample culture identified the presence of an E. coli isolate (Eco52148), co-producing NDM-and OXA-48-like enzymes. The patient's hospitalization history showed that she had kidney transplantation shortly before being admitted. Additionally, the patient was recently repatriated from northern part of Africa.

Carbapenem Production and Susceptibility Testing
These four isolates, mentioned above, were selected to be further characterized, since they were the only Enterobacterales, coproducing NDM-and OXA-48-like enzymes, referred from local microbiological laboratories to our lab, during 2018-2019. Species identification of the four strains was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) through MALDI Biotyper software (Bruker Daltonics, Bremen, Germany). MALDI-TOF MS meropenem hydrolysis assay was used to confirm carbapenem production . Production of carbapenemases (metallo-β-lactamase, OXA-48 and KPC) was assessed using the double-disc synergy test with EDTA, temocillin disc test and phenylboronic acid test (Lee et al., 2003;Doi et al., 2008;Glupczynski et al., 2012). The isolates were screened by PCR for the presence of bla NDM -like bla VIM -like, bla IMP -like, bla KPC -like and bla OXA−48 -like genes (Papagiannitsis et al., 2015). Antimicrobial susceptibility was performed using broth microdilution according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Susceptibility to fosfomycin was performed using agar dilution based on EUCAST guidelines. Susceptibility data were interpreted according to the criteria (version 10.0) of the EUCAST 1 .

Transfer of Carbapenemase-Encoding Genes
The conjugal transfer of carbapenemase-encoding genes was tested in liquid medium using the E. coli A15 strain (Azd R ) as recipient. Transconjugants were selected on MacConkey agar (Scharlab, SL, Barcelona, Spain) plates containing sodium azide (100 mg/L) (Sigma-Aldrich, St. Louis, MO, United States) and ampicillin (100 mg/L) (Sigma-Aldrich). The presence of bla NDMlike and bla OXA−48 -like was confirmed by PCR.

Whole-Genome Sequencing and Analysis
Genomic DNA was extracted from the four clinical isolates using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 15kb long inserts. No size selection was performed during the library preparation. Microbial Assembly pipeline offered by the SMRT Link v8.0 software was used to perform the assembly and circularization with minimum seed coverage of 30×. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Antibiotic resistant genes, plasmid replicons, mobile elements and multilocus sequence types (MLST) were determined through uploading the assembled sequences to ResFinder 4.1 and CARD (Zankari et al., 2012;Alcock et al., 2020), PlasmidFinder (Carattoli et al., 2014), ISfinder (Siguier et al., 2006), and MLST 2.0 (Larsen et al., 2012), respectively. Comparative genome alignment was done using Mauve v.2.3.1. 2 and BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011). Diagrams and gene organization were sketched using Inkscape 0.92.4 3 .
WGS performed on the Sequel I platform and assembly performed on Microbial Assembly pipeline resulted in complete, closed chromosomes and plasmids shown in Table 2. WGS revealed that K. pneumoniae isolates Kn47733, Kpn50595, and Kpn51015 belonged to sequence types (STs) 147, 11 and 15, respectively. All these three STs have been considered as "high risk" clones (Woodford et al., 2011). The E. coli isolate Eco52418 was assigned to ST167. Several studies have reported the association of ST167 E. coli with the dissemination of resistance genes, especially of the carbapenemase-encoding gene bla NDM−5 (Mani et al., 2017;Sánchez-Benito et al., 2017;Sun et al., 2018;Xu et al., 2019).
The carbapenem resistance phenotypes of all clinical strains were transferred to azide-resistant E. coli A15 by conjugation (Supplementary Table 1). For isolates Kpn47733, Kpn50595 and Kpn51015, all transconjugants carried both carbapenemaseencoding genes, while only the bla NDM−5 gene was identified in the transconjugants of the E. coli isolate Eco52148.
Analysis of contigs carrying carbapenemase-encoding genes showed that, in isolate Kpn51015, the bla OXA−244 gene was found on a plasmid (p51015_OXA-244) of 71402 bp in size, while the respective gene was localized in the chromosomal contig of E. coli isolate Eco52418. In both isolates, the bla OXA−244 gene was bounded by two copies of IS1 insertion sequence in parallel orientation (Supplementary Figure 1), forming a composite transposon, named Tn51098. In the E. coli isolate Eco52418, the  in GenBank accession no. CP050382), as described previously (Potron et al., 2016;Hoyos-Mallecot et al., 2017). Direct repeats of 9 bp (TGAATTGCT) were found at the boundaries of the bla OXA−244 -carrying composite transposon, suggesting its transposition into the E. coli chromosome. However, unlike the isolate Eco52418, an ORF encoding a LysR transcriptional regulator wasn't found between bla OXA−244 (downstream) and IS1, in plasmid p51015_OXA-244. Plasmid p51015_OXA-244, which belonged to the incompatibility group FII (IncFII), exhibited extensive similarity with IncFII plasmids from E. coli strains D181 and F5176C6 (GenBank accession nos. CP024250 and CP024669, respectively) (Supplementary Figure 2A). Unlike p51015_OXA-244, those plasmids were negative for the presence of bla OXA−244 gene. Among p51015_OXA-244, no resistance genes other than bla OXA−244 were identified. On the other hand, the bla OXA−181 carbapenemase-encoding gene was identified on a ColKP3 plasmid (p47733_OXA-181) of 6812 bp in size, in isolate Kpn47733, while a bla OXA−181 -carrying plasmid (p50595_OXA-181; 51140 bp), being an IncX3-ColKP3 fusion, was identified in isolate Kpn50595. In both plasmids, the bla OXA−181 genes were surrounded by identical sequences (Supplementary Figure 3). In comparison with the archetypal ColE2-type plasmid pKP3-A carrying bla OXA−181 (Potron et al., 2011), p47733_OXA-181 was composed only of repA and bla OXA−181 genes (Supplementary Figure 2A). The mob genes, encoding proteins that form a plasmid mobilization system, were not found in plasmid p47733_OXA-181. One additional difference between the two plasmids was the presence of Tn5403 transposon in p47733_OXA-181. The Tn5403 transposon has been previously found in bla NDM−1 -positive IncN2 plasmids, like plasmid pJN24NDM1 characterized from a ST405 E. coli from China (Hao et al., 2019). On the other hand, plasmid p50595_OXA-181 was almost identical to plasmids p1-Ec-BERN-042 (100% coverage, 99.99% identity; GenBank accession no. CP042935) and pOXA181_29144 (100% coverage, 99.99% identity) (Supplementary Figure 2A). Plasmid pOXA181_29144 was previously characterized from a ST18 K. pneumoniae strain (Kpn-29144) isolated, in 2015, in the Czech Republic . Similar to pOXA181_29144, which was transferable by conjugation , a complete tra locus was found in the sequence of p50595_OXA-181. Also, the qnrS1 gene, conferring low-level resistance to fluoroquinolones, was identified in the sequence of p50595_OXA-181.
Finally, in all four carbapenemase-producing isolates, a diverse number of additional replicons were identified ( Table 2). Some of these replicons were associated with important resistance determinants, like bla CTX−M−15 , arr-2 and ermB.

DISCUSSION
Previous studies have reported the spread of bla OXA−48like and bla NDM -like genes in Enterobacterales recovered from Czech hospitals Paskova et al., 2018). However, based on our knowledge, this study reports the first description of OXA-244-producing Enterobacterales isolated from Czech hospitals. Both OXA-244-producing isolates, also expressed NDM-1 or NDM-5 MβLs. Additionally, two K. pneumoniae isolates, co-producing OXA-181 and NDMtype carbapenemases, were identified. Overall, horizontal gene transfer is the main mechanism involved in the dissemination of bla NDM -like and bla OXA−48 -like genes, but certain clones have been associated with the spread of these resistance genes (Pitout et al., 2019). Carbapenemase-producing K. pneumoniae and E. coli isolates, characterized during this study, belonged to clones (STs 11, 15, and 147 in K. pneumoniae, and ST167 in E. coli; Table 2), which have been previously characterized as "high risk clones, " and have been reported in association with these carbapenem-resistance mechanisms (Woodford et al., 2011;Pitout et al., 2019). In all cases, carbapenemase-producers were recovered from patients with travel history or previous hospitalization abroad. The endemicity of OXA-181 and NDM-5 carbapenemases among Enterobacterales isolated in the Indian subcontinent has been reported in several studies (Lascols et al., 2013;Krishnaraju et al., 2015;Ahmad et al., 2019; FIGURE 2 | Linear maps of the multidrug resistance regions (MDRs), carrying bla NDM−5 genes. Arrows show the direction of transcription of open reading frames (ORFs), while truncated ORFs appear as rectangles (arrows within rectangles indicate the direction of transcription). Resistance genes are shown in red. IS elements and transposases are shown in yellow and green, respectively. intI1 genes are shaded purple. The remaining genes are shown in white. Homologous segments (representing ≥99% sequence identity) are indicated by light blue shading, while pink shading shows inverted homologous segments. Pitout et al., 2019). Interestingly, a recent report from the United States described the characterization of a ST147 K. pneumoniae harboring bla NDM−5 and bla OXA−181 from a patient, who was previously hospitalized in India (Rojas et al., 2017). Also, North African countries, and especially Egypt, represent a geographical region, where bla OXA−48like and/or bla NDM -like genes are highly disseminated among Enterobacterales (Tafoukt et al., 2017;Soliman et al., 2020a,b). Studies from Italy have described the import of NDM-1producing K. pneumoniae isolates from Egypt (Principe et al., 2016;Nucleo et al., 2020). Additionally, the import of OXA-244-producing E. coli isolates from countries in Northern Africa was observed in a surveillance from Denmark (Hammerum et al., 2020). Finally, two studies have documented the spread of NDM-1-producing K. pneumoniae isolates in Mauritius Holman et al., 2017), speculating a link with India, due to the geographical and cultural links between the two countries. In 2018, another study described the characterization of a K. pneumoniae isolate co-producing NDM-1 and OXA-181 carbapenemases, recovered from a patient, who had previously went to Mauritius (Allyn et al., 2018). These findings highly underline that import of carbapenemase-producing isolates via travel or/and hospitalization abroad could represent a risk for a further dissemination of these isolates in Czech hospitals. However, epidemiological data don't confirm the scenario regarding the spread of Enterobacterales co-producing NDMand OXA-48-like carbapenemases in Czech hospitals (Hrabak, unpublished results).
Although the main limitation of this study was the small number of Enterobacterales isolates co-producing OXA-and NDM-type carbapenemases, which were collected in clinical microbiology laboratories, analysis of WGS data revealed that all four isolates harbored a huge variety of genes conferring resistance to several categories of antibiotics. Additionally, inspection of contigs carrying carbapenemase-encoding genes showed that different genetic structures and replicon types were involved in the dissemination of these resistance determinants. These contigs also included a huge variety of insertion sequences that might be involved in the organization of MDR regions, conferring resistance to several antibiotic categories thus, limiting therapeutic options. Thus, in addition to different combinations of carbapenemase-encoding genes, the variability of replicon types, genetic structures, resistance genes and mobile elements observed among the studied isolates indicated the genetic plurality involved in the acquisition and dissemination of determinants encoding OXA/NDM carbapenemases.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/genbank/, CP050360-CP050384.

AUTHOR CONTRIBUTIONS
CP and IB played an important role in interpreting the results and in writing the manuscript. KC, LK, and JH helped to acquire data. KC, LK, VM, and IB carried out experimental work. CP supervised the experiments and revised the final manuscript, which was approved by all authors.