Molecular characterization of carbapenem and ceftazidime-avibactam-resistant Enterobacterales and horizontal spread of bla NDM-5 gene at a Lebanese medical center

Introduction In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. Methods This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). Results Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. Discussion The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

NDM is among the most common and clinically significant carbapenemase in Enterobacterales capable of efficiently hydrolyzing a wide range of b-lactams, including penicillins, carbapenems, and cephalosporins (Nordmann et al., 2011;Nordmann and Poirel, 2014).It has rapidly spread across the Middle East, the Indian subcontinent, and the Balkan region (Dortet et al., 2014;Wu et al., 2019).In the United Kingdom, in 2011, NDM-5 was first detected, and two amino acid substitutions at positions 88 and 154 lead to increased NDM enzymatic activity (Hornsey et al., 2011).
Furthermore, there has been a notable spread of genes encoding OXA-48-like carbapenemases.In 2001, a K. pneumoniae strain resistant to all available b-lactams was reported in Turkey.A subsequent study by Poirel et al. in 2004 identified this isolate as carrying a novel bla variant, bla OXA-48 (Poirel et al., 2004).Isolates harboring bla OXA-48-like genes have garnered attention due to their rapid horizontal transmission and increasing detection across the Middle East and North Africa showing resistance to b-lactamase inhibitors (Poirel et al., 2004;Nordmann and Poirel, 2014;Al-Zahrani and Alsiri, 2018;Mairi et al., 2018).Co-production of NDM-and OXA-48-like enzymes has been increasingly reported, particularly in patients with travel histories from the Middle East, Southeast Asia, Europe, China, and Africa (Doi et al., 2014;Avolio et al., 2017;van Duin and Doi, 2017;Otlu et al., 2018;Solgi et al., 2020;Chudejova et al., 2021;Lorenzin et al., 2022).
This study, thus, was warranted at expanding our understanding of the resistance and dissemination by performing a whole genome characterization of CR-and CZA-resistant E. coli and K. pneumoniae strains producing NDM and/or OXA-48-like carbapenemases, at a major Lebanese tertiary care medical center.

Bacterial isolates and determination of carbapenems minimal inhibitory concentrations
This study encompassed characterization a total of 60 Enterobacterales isolates (consisted of 34 isolates of K. pneumoniae and 26 isolates of E. coli) recovered at the Clinical Microbiology Laboratory of the Department of Pathology and Laboratory Medicine at the American University of Beirut Medical Center, between 2019 and 2021.These isolates as were identified by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the MALDI-biotyper software (Bruker Daltonics, Bremen, Germany).The MICs determination to three carbapenems and CZA was carried out as reported earlier (Sobh et al., 2021).
For further characterization, all isolates were sent to Charles University, Faculty of Medicine in Pilsen, Czech Republic.Carbapenemase production and activity were assessed using MALDI-TOF MS with the meropenem hydrolysis assay, as described by Rotova et al. (Rotova et al., 2017), and the double disk synergy test incorporating EDTA, phenylboronic acid, and temocillin, following previously described methodologies (Lee et al., 2003).In addition antimicrobial susceptibility testing was conducted using the broth microdilution method, adhering to the EUCAST guidelines, with results interpreted based on the EUCAST 2024 criteria (https://www.eucast.org/clinical_breakpoints)(Clinical breakpoints -breakpoints and guidance v13.0, 2023).

Short-reads sequencing and analysis
Based on MLST and MIC results, 17 K. pneumoniae and 10 E. coli isolates were selected as representatives for whole genome sequencing.Genomic DNA was extracted using NucleoSpin Microbial DNA kit (Machery-Nagel, Germany).A multiplexed library was prepared using Nextera XT library preparation kit, and sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States) using the MiSeq v3 reagent kit using 600 cycle cartridges.Initial paired-end reads were quality assessed using fastqc, then trimmed and filtered to high quality using fastp (Andrews, 2010;Chen et al., 2018).Subsequent reads were assembled by a de Bruijn graph-based de novo assembler SPAdes (Prjibelski et al., 2020).The assembled contigs were analyzed to detect sequence types (STs) using MLST software, virulence genes using VFanalyzer, resistance genes using ResFinder, and plasmid incompatibility (Inc) types using PlasmidFinder (Carattoli et al., 2014;Liu et al., 2019;Florensa et al., 2022).Additionally, these contigs were also uploaded to PHASTER to detect phage genomes, CRISPRfinder to detect CRISPR-cas systems, and ISfinder to detect insertion sequences (Siguier et al., 2006;Arndt et al., 2016).

Long-read sequencing and analysis
Based on the previous short-read results, seven and four representative isolates corresponding to K. pneumoniae and E. coli isolates, respectively, were selected for long-read sequencing using PacBio Sequel I (Pacific Biosciences, CA, United States).Briefly, the extracted DNA was subjected to shearing using the Megaruptor 2 (Diagenode, Liege, Belgium); this will generate sheared fragments of ~15 kb each.Library preparation was performed using the microbial multiplexing protocol according to the manufacturer's recommendation.During the library preparation, no size selection was performed.Using SMRT Link v10.1, the microbial assembly pipeline was used for the assembly and the circularizing of contigs using a minimum seed coverage of ×30.The assembled contigs were then analyzed using the same procedures as the short-read assembly.Finally, all genomes were annotated using the NCBI's Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016;Haft et al., 2018;Li et al., 2021).

Phylogeny and single nucleotide polymorphism
To study the genetic diversity and phylogenetic relationship of the studied isolates and global isolates, genomes of K. pneumoniae, specifically of the ST383 type, were retrieved from the NCBI assembly database, encompassing both complete and draft genomes, totaling 105 genomes.Using parsnp v1.7.4 (available in the harvest suit), a core genome phylogeny of the model Ge neral Time Reversible (GTR) based on SNP and recombination, an SNP-based phylogeny was constructed between the sequenced genomes in this study and the 105 genomes downloaded from the NCBI database using KP1674 as a reference (complete circular genome) and K. quasipneumoniae as an outgroup (Edgar, 2004;Bruen et al., 2006;Price et al., 2010;Treangen et al., 2014).Briefly, SNPs identified in local collinear blocks were subsequently used for reconstructing an approximate maximum-likelihood tree using FastTree, while including the general time reversible (GTR) model of nucleotide substitution.The Shimodaira-Hasegawa test implemented in FastTree2 was used to assess the support for significant clustering in the observed phylogeny.The interactive tree of life or iTOL (https:// itol.embl.de/)was used for the graphic illustration of the trees along with relative annotations.
Moreover, the SNPs between the ST383 genomes were detected to determine the extent of clonal dissemination in the hospital.SNPs were detected using snippy multicommand (snippy-base application v4.5.0) (Seeman, 2015), which generates a core genome multiple alignment against the common reference (KP1674).The pipeline detects the variants and generates a single file for each isolate listing the different variations.

Comparison of antimicrobial multidrugresistant region
The MDR sequence from KP1633 isolate (complete circular genome) was used as a reference to compare with the rest of the isolates.Easyfig v2.2.0 was used for generating the figure (Sullivan et al., 2011).It uses blastn from the package blast + by NCBI to generate a similarity report between the fragments (Camacho et al., 2009).An alignment between the short-read sequences and the MDR sequence characterized with Pacbio was used to generate a consensus and then annotated using Prokka to include in the generation of the figure (Seemann, 2014).

Phenotypic antimicrobial resistance profile of the isolates
All 34 K. pneumoniae isolates showed resistance against CZA, carbapenems, tazobactam, ciprofloxacin, and tetracycline.Moreover, all isolates showed resistance against amikacin and gentamicin (except one isolate) (Table 1).Similarly, all E. coli isolates (n=26) were also resistant against CZA, carbapenem, and   showed resistance to ciprofloxacin and tazobactam and tetracycline (except two isolates).However, all isolates were susceptible against amikacin apart from two resistant isolates (Table 2).

Molecular characteristics of the isolates
PCR screening showed that all the K. pneumoniae isolates were positive for bla NDM (n=34), while 27 of them were simultaneously positive for the presence of bla OXA-48 .On the other hand, the E. coli isolates carried bla NDM-5 gene.
Based on the short-read sequencing results, seven K. pneumoniae isolates (one isolate each from ST307, ST15, ST147, ST39, and ST101 and two isolates from ST383) and four E. coli (one isolate each from ST405, ST131, ST2450, and ST617) were selected for long-read sequencing to have complete circular chromosomes and plasmids for downstream analysis.
In K. pneumoniae, the long-read sequencing of the two ST383, KP1633, and KP1674 showed that the bla NDM-5 was harbored on a fusion plasmid (IncFIB and IncHI1B) that was 372,845 bp (pKP1633LB_IncFIB_IncHI1B_NDM5) and 372,828 bp (pKP1674LB_IncFIB_IncHI1B_NDM5) in size, respectively.Upon blasting, the plasmid against the NCBI database showed 100% query coverage and 99.9% sequence identity with pFQ61_ST383_NDM_5 that was 376,754 bp in size (accession number CP091814), a plasmid isolated from a clinical K. pneumoniae strain in Qatar in 2016.In silico plasmid analysis of the rest of the ST383 K. pneumoniae isolates that were sequenced using short reads showed that all ST383 isolates carried the same plasmid type.Furthermore, pKP1633LB_IncFIB_IncHI1B_NDM5 carried a 35-kb MDR sequence (from 286,445 bp to 323,218 bp).The MDR region was composed of a transposase from IS1, followed by two additional transposases and ends with a transposase.Furthermore, the same MDR region was also detected in isolate KP1674 on the plasmid pKP1674LB_IncFIB_IncHI1B_NDM5 from position 52,157-85,730.In the case of short-read sequenced isolates, the MDR presence was confirmed using blast, and later, a consensus was made for subsequent analysis.Data revealed that this MDR sequence was present in most ST383 isolates, with the exception of isolate KP1679 lacking four resistance genes [bla TEM- 1 , bla OXA-9 , ant(3′)-I, and aac(6′)-Ib].Similarly, isolate KP1655 noted the absence of bla TEM-1 .In addition to finding missing resistance genes across the isolates, some IS transposases were also found missing (IS1380-like only found in three isolates).Interestingly, the starting and ending IS transposases were found in all isolates (Figure 1).Furthermore, all ST383 K. pneumoniae strains also carried the IncL plasmid harboring bla OXA-48 , which had the same plasmid size (68,942 bp).This plasmid has been reported extensively, and high similarity scores were noticed upon blasting against the NCBI database (Poirel et al., 2012a;Poirel et al., 2012b;Alousi et al., 2018).
Other STs such as ST111 KP1859 carried the bla NDM-5 on an IncX3 plasmid, while ST307 KP1608 carried the bla NDM-5 on an IncFII plasmid.On the other hand, the bla NDM-1 was carried on different Inc plasmids; IncM2 plasmid (pKP1880LB_IncM2_NDM1 87,450 bp) was detected in KP1880 ST 101.This plasmid exhibited 100% query coverage and 100% sequence identity with AR_0127 plasmid (87,450 bp) identified in a Salmonella enterica clinical strain in USA in 2018.Moreover, ST39 KP1851 and ST15 KP1917 both carried the bla NDM -1 on the same IncC plasmid (pKP1851LB_IncC_NDM1 137,593 bp and pKP1917LB_IncC_NDM1 140,300 bp, respectively) (99% query coverage and 99.9% sequence identity).This plasmid exhibited 100% sequence identity and 98% query coverage with pNDM-US (accession number CP006661.1),which was isolated from a clinical strain in 2016 in the USA.Finally, ST147 KP1734 carried the bla NDM- 1 on an IncFIB-IncHI1B plasmid (269,122 bp), while bla OXA-232 was carried on a ColKP3 plasmid (6,141 bp).The IncFIB-IncHI1B plasmid had similar sequence to those identified in ST383 isolates, KP1633 and KP1674, and the ColKP3 showed the same nucleotide sequence as that of pC06114_4 (accession number CP016038.1)(100% identity and 100% query coverage), a plasmid isolated in 2016 in Germany.
All the characterized E. coli isolates carried the bla NDM-5 gene.Long-read sequencing revealed that ST131 EC1918 and ST2450 EC1811 isolates carried the bla NDM-5 gene on the same IncX3 plasmid (79,672 bp).Moreover, short-read sequencing showed that ST1284 EC1733 carried an IncX3 plasmid, identical to pKP1859LB_IncX3_NDM5.Moreover, the bla NDM-5 gene was carried on IncFIA-IncFII (pEC1609LB_IncFIA_IncFII_NDM5, 118,953 bp) in ST405 EC1609 and on IncFII-IncFIA-IncFIB (pEC1856LB_IncFII_IncFIA_IncFIB_NDM5, 118 140,386 bp) in ST617 EC1856.Short-read sequences scaffolding showed that IncF plasmids (with different structures and sequences) carrying bla NDM- 5 gene was found in the rest of E. coli despite STs.ST383 K. pneumoniae virulence factor analysis revealed the presence of virulence genes responsible for acriflavine resistance, type VI secretion system, synthases, transporters, esterase, adherence, regulation, immune modulation, fimbriae, and delivery.Some genes were found to be missing, for example, the regulatory gene for the mucoid phenotype rmpA2 (KP1710, KP1679, KP1663, KP1657, KP1658, KP1655, KP1645, KP1639, and KP1601).In E. coli, the presence of the virulence genes was associated with acriflavine resistance, invasion, secretion, adherence, fimbriae, enterobactin, and yersiniabactin.Notably in ST648 EC1930 in comparison with ST648 EC1726, some virulence factors were found missing: fim genes for fimbriae, gsp genes for secretion, irp genes for capsule and invasion characteristics, and ybt genes for yersiniabactin.In addition, in comparison between the ST405 EC735 and ST405 EC1609, genes of esp variations responsible for secretion and two genes for adherence (papE and papF) were found to be missing (Supplementary Tables S1, S2).

Phylogeny and clonality
To evaluate the relatedness of the K. pneumoniae ST383 isolates in the study, global SNPs phylogeny was performed on all K. pneumoniae ST383 isolates in the NCBI database.The phylogenetic tree showed that our ST383 isolates clustered together along with previously detected NDM-5 producing ST383 isolates from Lebanon (Figure 2).Furthermore, SNP analysis was performed on all isolates belonging to this clade and showed high similarity with SNPs ranging from 11 to 73 (read, Table 5 and Supplementary Table S3).Plasmid analysis of the Lebanese clade showed that all the clustered isolates carried the bla NDM-5 gene on the same Inc plasmid (IncFIB-IncHI1B) previously detected.Remarkably, these clustered isolates were all NDM-5/OXA-48 co-producers, and their bla OXA-48 gene was similarly carried by an IncL plasmid (Figure 2).
Moreover, the clonality of the K. pneumoniae ST383 strains was assessed through analyzing the CRISPR array sequences.A population of any isolates that went through the same environmental conditions (subjected to phages or plasmids) will have a similar CRISPR array, since these sequences (correlated with foreign sequences) are added in chronological order (Gagaletsios et al., 2022).CRISPRCasFinder showed that all genomes have the CRISPR/Cas I-E type.The sequences were then aligned, and a SNPsbased phylogenetic tree was executed (Figure 3).The tree showed that the isolates were divided between two distinct clades (A and B) and

Discussion
Lebanon confronts significant antibiotic resistance, notably with the recent emergence of resistance to ceftazidime-avibactam in CRE treatments (Araj et al., 2018;Iskandar et al., 2021;Rima et al., 2022;Zakhour et al., 2024),.This study investigated CR K. pneumoniae and E. coli strains exhibiting resistance to last line antibiotic choices, including carbapenems and CZA, as reported by (Sobh et al., 2021).It intended to explore mechanisms involved in their resistance and dissemination especially through characterizing the genetic features of bla OXA-48-like and/or bla NDM -positive K. pneumoniae and E. coli isolates recovered at a major Lebanese medical center.Sequence data showed the presence of bla NDM on plasmids of different Inc groups, in K. pneumoniae belonging to different STs (Table 3).Most of the K. pneumoniae isolates belonged to ST383 and carried a multireplicon plasmid (IncFIB-IncHI1B) with bla NDM-5 .This sequence type has been reported in different parts of the world, including countries around the Mediterranean basin, where it contributes to the burden of CRE (Turton et al., 2018;Edward et al., 2022;Tsui et al., 2023).The detection of bla NDM-5 positive isolates raises significant concerns due to its potent ability to break down a broad spectrum of b-lactam antibiotics, including carbapenems.Recognized globally for its contribution to antibiotic resistance (Daaboul et al., 2023;Linkevicius et al., 2023;Simner et al., 2023;Tao et al., 2023), the prevalence of bla NDM-5 underscores the urgent challenge it poses to treating bacterial infections.The concurrent presence of bla NDM-5 and bla OXA-48 in our strains severely limits treatment options with last-resort antibiotics like ceftazidime-avibactam.This synergy of resistance genes, documented across the MENA region (Dandachi et al., 2016;Aqel et al., 2017;Rojas et al., 2017;Solgi et al., 2020;Ahmed El-Domany et al., 2021) and mirrored in findings from Italy (Lorenzin et al., 2022), emphasizes the global nature of this threat and underscores the need for international efforts in surveillance and containment.This finding highlights the important role of horizontal transfer in the worldwide spread of bla NDM resistance determinants (Paskova et al., 2018).Additionally, the close clonal phylogenetic relationship of ST383 isolates was confirmed by SNPs phylogeny.ST383 is a high-risk clone associated with the spread of diverse resistance mechanisms (Papagiannitsis et al., 2010) like bla KPC and bla VIM .Nevertheless, CRISPR-Cas array analysis revealed genetic diversity within ST383 K. pneumoniae isolates, identifying distinct clades and indicating diverse ancestral origins, suggesting varied exposure to environmental pressures and selective forces.
On the other hand, bla NDM-5 was also found in E. coli isolates.E. coli isolates, which belonged to diverse STs, harbored a variety of bla NDM-5 -carrying plasmids.The variation in plasmids involved in the spread of bla NDM-5 underlines the important role of mobile elements, like insertion sequences and transposons, in the horizontal spread of resistance mechanisms.However, short-read sequencing data suggested that, in most of the E. coli, which belonged to diverse STs, the bla NDM-5 gene was localized on IncF-type plasmids.Additionally, Prophage harboring bla NDM-5 region comparison, found in ST383 K. pneumonia plasmids and in E. coli EC1609, with the prophage sequence detected in the long-read sequenced KP1633 isolate.Red represents the resistance genes, green the insertion sequences, transposases in blue DUF domains; and in gray, other genes.
WGS data confirmed the presence of similar bla NDM-5 -carrying plasmids, like pKP1674LB_IncFIB_IncHI1B_NDM5 (IncFIB and IncHI1B) and pEC1609LB_IncFIA_IncFII_NDM5 (IncF-type), into different STs or species.These findings highlight the important role of plasmids in the horizontal gene transfer (HGT) of resistance genes in the institution and community where such strains exist.

Conclusion
In conclusion, the current study confirmed the fact that production of NDM carbapenemases is one of the most clinically significant resistance mechanisms among CRE isolates in Lebanon, including ceftazidime-avibactam.This finding is in line with previous studies highlighting the importance of bla NDM spread, canceling last-line therapeutic options worldwide (Chudejova et al., 2021).Additionally, our study showed the contribution of divers STs and mobile genetic elements to the success of NDM resistance mechanism.Furthermore, the high-risk clone ST383 and the broad host range plasmids IncH and IncF has contributed to the successful prevalence and dissemination of NDM resistance.Thus, such findings emphasize the need of scrutinizing the implementation of infection control aspects to curb/control the spread of these "superbugs".

FIGURE 2 SNP
FIGURE 2 SNP-based phylogeny for global K. pneumoniae ST383 with 105 isolates downloaded from NCBI database.Red nodes highlight the isolates used in this study.

TABLE 1
MIC and disk diffusion test results for the K. pneumoniae isolates.

TABLE 2
MIC and disk diffusion test results for the E. coli isolates.

TABLE 3
WGS data representing resistance gene content and plasmid replicons of the K. pneumoniae isolates.

TABLE 4
WGS data representing resistance gene content and plasmid replicons of the E. coli isolates.