The emergence of carbapenemase-producing Enterobacterales in hospitals: a major challenge for a debilitated healthcare system in Lebanon

Background Carbapenem- and extended-spectrum cephalosporin-resistant Enterobacterales (CR-E and ESCR-E, respectively) are increasingly isolated worldwide. Information about these bacteria is sporadic in Lebanon and generally relies on conventional diagnostic methods, which is detrimental for a country that is struggling with an unprecedented economic crisis and a collapsing public health system. Here, CR-E isolates from different Lebanese hospitals were characterized. Materials and methods Non-duplicate clinical ESCR-E or CR-E isolates (N = 188) were collected from three hospitals from June 2019 to December 2020. Isolates were identified by MALDI-TOF, and their antibiotic susceptibility by Kirby-Bauer disk diffusion assay. CR-E isolates (n = 33/188) were further analyzed using Illumina-based WGS to identify resistome, MLST, and plasmid types. Additionally, the genetic relatedness of the CR-E isolates was evaluated using an Infrared Biotyper system and compared to WGS. Results Using the Kirby-Bauer disk diffusion assay, only 90 isolates out of the 188 isolates that were collected based on their initial routine susceptibility profile by the three participating hospitals could be confirmed as ESCR-E or CR-E isolates and were included in this study. This collection comprised E. coli (n = 70; 77.8%), K. pneumoniae (n = 13; 14.4%), Enterobacter spp. (n = 6; 6.7%), and Proteus mirabilis (n = 1; 1.1%). While 57 were only ESBL producers the remaining 33 isolates (i.e., 26 E. coli, five K. pneumoniae, one E. cloacae, and one Enterobacter hormaechei) were resistant to at least one carbapenem, of which 20 were also ESBL-producers. Among the 33 CR-E, five different carbapenemase determinants were identified: blaNDM-5 (14/33), blaOXA-244 (10/33), blaOXA-48 (5/33), blaNDM-1 (3/33), and blaOXA-181 (1/33) genes. Notably, 20 CR-E isolates were also ESBL-producers. The analysis of the genetic relatedness revealed a substantial genetic diversity among CR-E isolates, suggesting evolution and transmission from various sources. Conclusion This study highlighted the emergence and broad dissemination of blaNDM-5 and blaOXA-244 genes in Lebanese clinical settings. The weak AMR awareness in the Lebanese community and the ongoing economic and healthcare challenges have spurred self-medication practices. Our findings highlight an urgent need for transformative approaches to combat antimicrobial resistance in both community and hospital settings.


Introduction
The order Enterobacterales includes the most common human bacterial pathogens responsible for community-and healthcareassociated infections.These species have the ability to rapidly evolve through horizontal gene transfer (e.g., mobile genetic elements) (1).This includes the ability to develop resistance to multiple antibiotics, which complicates the treatment of infections and increases, potentially mortality and morbidity in patients.Of particular concern is the emergence of carbapenem-resistant Enterobacterales (CR-E), which poses a major concern in clinical as well as community settings all across the globe (2).Carbapenems referred to as last-resort antibiotics, possess a broad spectrum of activity against most clinically-relevant Gram-negative bacteria (3,4).Consequently, it is crucial to continuously monitor and assess the spread of carbapenem resistance, especially in low-and middle-income countries (LMICs) that face established challenges in antimicrobial stewardship and public health systems.
Available observations suggest that antimicrobial resistance (AMR) has been precipitously increasing in Lebanon, a country with a plethora of issues resulting from an unprecedented economic collapse (5)(6)(7).The latter has amplified critical issues such as access to medical care, sanitation, and nutritious and safe food, as well as promoted lax medical practices, including self-medication and the reliance on widely and easily available antibiotics as cheaper alternatives across the country.This is important because excessive and inappropriate use of antimicrobials in human and veterinary medicine and agriculture has been well-documented in Lebanon, even before the economic collapse (8)(9)(10).Taken together, these challenging conditions have been predicted to enhance the emergence of AMR and the cycle of complicated infections, especially in the most vulnerable populations in Lebanon (5-7, 10, 11).Nevertheless, studies on AMR in Lebanon are generally scant and, when available, can be limited, in scope or (e.g., low sample number) and/or descriptive (e.g., phenotypic AMR evaluation and absence of in-depth genomic analysis).Despite this, available studies have reported rates of extended-spectrum cephalosporin-resistant Enterobacterales (ESCR-E; ~50% of tested isolates) and CR-E (~3%) among clinical isolates in Lebanon between 2015 and 2019 (12,13).The enzymatic nature of the carbapenem-resistance has also been evidenced, by the detection of several carbapenemase genes, including bla OXA-48 , bla NDM-5 , and bla NDM-19 , in Escherichia coli, bla OXA-48 , bla OXA-181 , and bla NDM-5 in Klebsiella pneumoniae, bla OXA-48 , bla VIM-1 , bla VIM-4 , and bla NDM-1 in Enterobacter cloacae, and bla OXA-48 in Citrobacter freundii isolated in Lebanese hospitals (12).In addition, high rates of multidrug-resistant (MDR) Enterobacterales (60.7%) and the dissemination of extendedspectrum ß-lactamase (ESBL) producing-, carbapenemase-producing (CP)-, and colistin-resistant E. coli isolates among healthy people in the Lebanese community (14), animals (15), and the environment (16) has been documented.Hence, in-depth studies to evaluate the emergence and spread of AMR in Lebanon, focusing on the determinants that contribute to the dissemination of ESBL-E and/or CP-E is now mandatory.Specifically, details of the sequence types of these bacteria, their resistome, and the plasmids carrying these genes may be crucial to identify the transmission routes and propose intervention strategies to limit their spread.To fill these gaps, we evaluated the occurrence of ESBL-and/or CP-E in three different hospitals in Lebanon and determined the underlying mechanisms of resistance, the population structure of the isolates, and the associated plasmid types using whole genome sequencing analysis (WGS), a powerful technique for investigating AMR, but still not commonly available in LMICs, such as Lebanon, due to its relative expensiveness and requirement of specialized equipment and skills (17).
This study focused only on hospital isolates, as (1) anecdotal evidence suggests that hospitals play an important role in the transmission of ESBL-E-and CP-E, and (2) hospitalized patients are more susceptible to infections/colonization with these species.

Ethical approval
This study was approved by the Azm Center/Lebanese University ethical committee and the Lebanese Ministry of Public Health (CE-EDST-1-2020).All the specimens were analyzed anonymously, without any patient identifiers, and the patients were not physically involved in this study.

Isolation and identification of bacteria from clinical samples
A total of 188 clinical Enterobacterales isolates being either resistant to Expanded Spectrum Cephalosporin (ESCR) or Carbapenem Resistant were collected between 2019 and 2020 by the bacteriology laboratories of three hospitals, including El Youssef Hospital Center (50 isolates), the Nini Hospital (137 isolates), and the Tripoli Governmental Hospital (1 isolate), which are located in the Akkar and North governorates of Lebanon, respectively.These isolates were collected based on their susceptibility profile established in the hospitals as part of routine clinical testing.These clinical isolates were recovered from different sample types, including urine, pus, wound, rectal, axillary, pleural fluid, gastric fluid, and bronchial fluid.They were identified at the hospitals using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) with VITEK MS protocol (bioMérieux, Version 3.0, Marcy L'Etoile, France).The isolates were subsequently stored at the Lebanese University bacterial bank (CMUL).

Evaluation of the ESCR-and CR-E isolates using enzymatic assays
The β LACTA™ test (Bio-Rad, Marne-la-Coquette, France) was used to further evaluate the ESCR-E isolates.Briefly, this test is based on the hydrolysis of a chromogenic cephalosporin that turns red upon hydrolysis.Notably, the chromogenic cephalosporin is not hydrolyzed by acquired penicillinases (e.g., SHV-1, TEM-1) but by ESBL, carbapenemase, and acquired AmpC (19).Furthermore, the NG-Test ® CTX-M MULTI (NG Biotech, Guipry, France) immunochromatographic assay (ICA) was performed on the β LACTA™ positive isolates to infer the presence of CTX-M-type ESBLs (20).The CR-E isolates were also evaluated using the Carba NP hydrolysis test, which detects carbapenemase activity based on in vitro hydrolysis of imipenem (21).The NG-Test ® CARBA-5 ICA (NG Biotech) was used to detect members of the five main families of carbapenemases (i.e., KPC-, NDM-, VIM-, IMP-, and OXA-48-like enzymes) produced by the CR-E isolates as described in the manufacturers' instructions (22).

Molecular characterization of the ESCR-and CR-E isolates
The ESCR-E isolates (positive using the β-LACTA™ and NG-Test ® CTX-M MULTI tests) were screened by PCR and subsequent Sanger sequencing to identify the bla CTX-M allele as described previously (23,24).For the ESCR-E isolates (positive using the β-LACTA™ but negative with the NG-Test ® CTX-M MULTI) and the CR-E isolates, the total DNA was extracted using the PureLink™ Genomic DNA Mini-Kit (ThermoFisher Scientific, Waltham, MA, United States) following the manufacturer's instructions and stored at -20°C.Genomic DNA was used for library preparation using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (NEB, France) according to the manufacturer's instructions.Whole genome sequencing was performed on an Illumina NextSeq 500 instrument (Illumina).After sequencing, raw data were assembled de novo using the CLC genomics 10.2 program (Qiagen, Les Ulis, France), and the genomes were analyzed online using software available at the Center for Genomic Epidemiology-CGE. 3 The latter included multilocus sequence typing (MLST) with the CGE MLST 2.0 software to determine sequence types (ST), and acquired resistance gene determinations using ResFinder 4.1 (25)(26)(27).Similarly, plasmid replicon types and virulence genes were identified using PlasmidFinder 2.0 (26,28) and VirulenceFinder 2.0., respectively (26,29,30).Reference plasmids sequence were retrieved from the NCBI database, using a local BLAST algorithm.Reads and/or Contigs carrying carbapenemase genes were mapped to reference plasmids, using the CLC genomics 10.2 program (Qiagen).

Plasmids from CR-E isolates
Plasmids were extracted from the carbapenem-resistant isolates by the Kieser method as previously described (31).Transfer of plasmid-borne resistance markers was assessed by electroporation of the plasmids into electro-competent E. coli TOP10 (Invitrogen, Saint-Aubin, France).Transformants were selected on Trypticase soy agar (TSA) supplemented with ticarcillin (100 μg/mL).Transformants were PCR screened for the carbapenemase genes likely transferred to the recipient E. coli.Plasmids were visualized using electrophoresis on 0.7% agarose.E. coli NCTC 50192, which harbors four plasmids (7, 48, 66, and 154 kb) was used as size markers during electrophoresis (31).

Typing of the CR-E isolates using infrared spectrometry
The CR-E isolates were typed using the Bruker IR Biotyper spectrometer (IRBT, Bruker, Hamburg, Germany).An amount of 1 μL of bacterial colonies selected from the confluent part of the culture was re-suspended in 50 μL of 70% ethanol solution in an IR Biotyper suspension vial.After vortexing, 50 μL of deionized water was added, and the solution was mixed by pipetting.The bacterial suspensions (15 μL) were spotted in three technical replicates onto the 96-spot silicon IR Biotyper target and let dry for 15-20 min at 35°C ± 2°C.In each run, prior to sample spectra acquisition, quality control was performed with the Infrared Test Standards (IRTS 1 and 2) of the IR Biotyper kit.IRTS 1 and IRTS 2 were re-suspended in 90 μL deionized water and 90 μL of absolute ethanol was added and mixed.Subsequently, 12 μL of the suspension was spotted onto the IR Biotyper target and let dry as previously described.The relationships between the isolates were analyzed using the Bruker IR Biotyper Software (version 2.1.0.195,Bruker) (32).An online tool 4 was used to assess the quantitative data of discriminatory power and concordance of the typing methods.Simpson's index of diversity (SID) was used to evaluate the discriminatory power of the typing method, calculating the probability that two unrelated isolates from the test strain set will be clustered into different typing groups.Adjusted Rand index (ARI) with 95% confidence intervals was used to evaluate the concordance of IRBT typing results (33).

Carbapenemase detection and susceptibility testing of CR-E isolates
The Carba NP hydrolysis test and the NG-Test ® CARBA-5, are displayed in Table 1 and Table S2.Among the 33 CR-E isolates, 23 were positive using the Carba NP test, while the remaining isolates were repeatedly negative.Using the NG-Test ® CARBA-5 ICA, all 33 isolates were CPs: 17 isolates (51.5%) were positive for NDM and 16 (48.5%)for OXA-48-like enzymes.Out of the 26 E. coli isolates different AMR phenotypes were observed (Figure S1).NDM (n = 14) and OXA-48-like (n = 12) were the only carbapenemases detected in these isolates.Three K. pneumoniae isolates were positive for OXA-48like, and two for NDM.The two E. cloacae complex isolates produced either an OXA-48-like or an NDM.
Furthermore, alignment of the ftsI gene sequences with that of a wild-type gene (E. coli NCTC 9022, accession number LR134237) revealed a four amino-acid insertion in PBP3 after residue 333 in 13 of 14 NDM-5-producing E. coli isolates.Two types of insertions were detected: YRIN (n = 7) and YRIK (n = 6).YRIN insertion was found among isolates with ST167 and ST361, and YRIK insertion was present in isolates with ST648 and ST405.ST90 isolate contained neither YRIN nor YRIK insertions.These 4 AA insertions could be correlated with increased MICs to aztreonam/avibactam (4, 8, and 16 mg/L) and cefiderocol among bla NDM-5 producing E. coli isolates, as compared to similar isolates lacking a 4 AA insertion (0.06 mg/mL).

Virulence determinants and plasmids in the CR-Escherichia coli isolates
Virulence factors (VFs) in the CR-E. coli isolates were identified using the CGE VirulenceFinder 2.0.TraT protein, previously shown to mediate resistance to bacterial killing by serum, was detected in 14 E. coli isolates, including a bla OXA-48 -positive isolate belonging to ST69.Genes encoding adhesins (ipfA, fimH, afaA, afaC, afaD, afaE) were found in five E. coli that were positive for bla OXA-244 (Table 3).Additionally, iss encoding an outer membrane lipoprotein that enhances serum resistance, was detected only in one bla OXA-244 -positive E. coli isolate.The capsular genes, kpsE and kpsM, were both detected in eight isolates, of which three were bla NDM-5 -positive (ST648), and 5 were bla OXA-244 -positive (ST69).Most VFs were detected in two isolates belonging to ST69.

Genetic relatedness as revealed by IR Biotyper in comparison to WGS
Sixteen CR-E. coli and the five CR-K.pneumoniae isolates positive with the Carba NP test were assessed with the IR Biotyper for strain typing and the results were compared with those obtained by WGS analysis (Figures S2, S3).Overall IRBT results corroborated WGS for the typing of the five CR K. pneumoniae, which were classified into four IR types (Figure S2).However, IRBT differentiated 16 E. coli isolates that belonged to 7 STs into 11 IR types, with 8 IR types comprising only a single isolate (Table 5).The Simpson Index of Diversity (SID) was used to determine the discriminatory power of the typing methods.WGS had the highest discriminatory power (0.967) followed by IRBT (0.933), while the lowest SID was for MLST (0.875).Main discrepancies were observed between IRBT and WGS with E. coli ST-648 isolates (O85D6, O85E2, O85G1, and O85F3), which clustered into IR type 7 (Figure S3), but differed from each other by 53, 400, and 1,360 SNIPs.

Discussion
The increase in ESCR-and CR-E isolates has been observed in hospitals worldwide.In Lebanon, two nationwide hospital-based retrospective studies documented a high prevalence of MDR pathogens, including ESBL-producing Enterobacterales (34% in 2016) (5).Recently, a community-based study revealed that approximately half of the population carries ESCR-E.coli (14), with an important dynamic of acquisition and loss of MDR strains and limited plasmid spread.The occurrence of Enterobacterales with decreased susceptibility to carbapenems, raised from 0.4% in 2008-2010 to 1.6% in 2012 and 3.3% in 2019 in hospitalized patients (12,35).Despite the concerning increase and reports, there is a lack of molecular data on the epidemiology of ESBL-and CR-E in Lebanon.
Our results highlighted the predominance of the bla CTX-M-15 gene (52/57, 91.2%) among ESBL-producing isolates regardless of the co-existence of other ß-lactam resistance determinants, as observed globally (36).CTX-M-15-producing Enterobacterales clones have been widely reported in Lebanon among human, animal, and environmental sources (14,15,(37)(38)(39).The finding that one single E. coli isolate may carry two CTX-M (−15 and − 27) variants capable   (12,35).OXA-48-like enzymes were the most prevalent carbapenemases among Enterobacterales in Lebanon over the last decade while NDM-5 was uncommon in hospital and community settings (12,40).In our study, the bla NDM-5 gene has become the predominant carbapenemase gene (14 of 26 E. coli isolates).The bla OXA-244 gene (10/26) came in second place, while bla OXA-48 (1/33), and bla OXA-181 (1/33) genes are rare.OXA-244 producing E. coli isolates are increasingly described worldwide (41), and represents a threat to public health because of the difficulties in their detection using classical screening media based on carbapenems and temocillin (42).Indeed, our results confirmed that OXA-244-producing isolates have lower MICs to temocillin and carbapenems as compared to other CP-E, which results in the absence of growth on screening media and thus an underestimation and silent spread.Furthermore, these isolates were negative for the Carba NP test, and only the use of an ICA (here the NG-Test Carba5) revealed a positive signal for OXA-48-like carbapenemase, suggesting that both tests should be used together to maximize the chances of detecting all OXA-48-like carbapenemases.Additionally, the CR K. pneumoniae isolates harbored bla OXA-48 (3/5) and bla NDM-1 (2/5), while the E. cloacae and E. hormaechei isolates harbored bla OXA-48 and bla NDM-1 , respectively.
MLST results demonstrated several distinct genetic backgrounds for the CR-E isolates, suggesting horizontal gene transfer of the carbapenemase gene carrying plasmids, particularly those carrying the bla NDM-5 gene, rather than a clonal spread of a single clone.Interestingly, we found that the carbapenemase genes were plasmidborne (except in eight bla OXA-244 -producing E. coli) and on different plasmid types (Table 4).Of the NDM-5-producing E. coli isolates, six and seven had the insertion of YRIN or YRIK in PBP3, respectively, which resulted in a significant increase of MICs to aztreonam/ avibactam and cefiderocol as well as to other PBP3-targeting ß-lactams (34).Surprisingly, the bla OXA-244 gene was chromosomally encoded in 8 isolates, while it was plasmid-mediated in only two isolates belonging to ST69 and ST10.The chromosomal location of the bla OXA- 244 gene was previously reported in ST38 and ST69 in France (43).Taken together, the occurrence of different resistance markers in diverse genetic backgrounds as well as plasmid types constitute a risk to patients, potentially highlighting a more severe problem and highlighting an urgent need to monitor and control the spread of resistance in hospitals in Lebanon.
The study identified various MLST types, including ST648, ST167, ST361, and ST405, in bla NDM-5 -producing E. coli.These STs are recognized as high-risk global clones that contribute to the widespread dissemination of drug resistance determinants among Enterobacterales (44).For instance, ST648 is recognized as a major global ESBL-producing E. coli clone (45,46), particularly associated with bla CTX-M-15 , in humans, birds, and companion animals (47).Additionally, ST648 was identified in Lebanon among clinical CRE (48), including bla NDM-5-producing E. coli (49).Besides, ST167 has been linked to the global spread of bla NDM in humans, animals, and food (50).ST405 has been detected in several countries, including the United States (51), Japan (52), and Lebanon (48), allowing the transmission of bla CTX-M-15 and aac(6′)-Ib-cr genes (53).Notably, both ST361 and ST648 have been reported among Syrian refugees in Lebanon (54), and ST90 was found in the effluent of Al-Qaa refugee camp (55).Additionally, we identified ST38 among bla OXA-244producing E. coli, previously described in estuary water in Lebanon (56).Several European countries have reported increased dissemination of the bla OXA-244 gene (57,58), with ST38 being the most common sequence type among bla OXA-244 -producing E. coli isolates (59).Moreover, ST38 bla OXA-48 -producing E. coli has also been found in fowls in Lebanon (60).As observed in our study, ST940 E. coli carrying the bla OXA-181 gene was previously reported at the American University of Beirut Medical Center (61).Among the K. pneumoniae isolates, there were five isolates belonging to four different sequence types.One of them, K. quasipneumoniae subsp.similipneumoniae ST1770, has been previously reported in hospital wastewater effluents in Japan (62), but had not been reported in Lebanon.Additionally, clinical K. pneumoniae isolates ST35 and ST45 have been documented in Lebanon (63,64).ST37 has also been closely associated with ESBLs (65).Furthermore, the ST182 bla NDM-1 E. hormaechei isolate, previously reported in Lebanon (66), has been frequently isolated from clinical specimens in China, Mexico, the Czech Republic, and the United States (67).
Although there is a paramount need to monitor the spread of critical AMR strains in hospitals in Lebanon, these efforts are complicated by the unavailability of resources.Molecular typing methods such as whole genome sequencing are relatively timeconsuming and expensive (32).Therefore, we evaluated the Bruker IR Biotyper for reliable detection of the relatedness and discrimination between strains.Our findings showed that Fourier Transform Infrared (FT-IR) technology is a powerful tool for strain typing, showing slightly better results to MLST and comparable results to WGS among CR-E.coli and K. pneumoniae isolates.Its advantages are summed up in the simple preparation of samples, ease of use, and low running costs.Together with its relatively high discriminatory power, the FT-IR seems to be a good tool for outbreaks real-time surveillance and infection control in clinical settings.
In conclusion, our study showed that in-depth studies are crucial to better understand the emergence and dissemination of drugresistant determinants within and across healthcare institutions.Specifically, we highlighted an unprecedented diversity of ESBL-and CR-E determinants compared to other studies in Lebanon.Furthermore, the MLST and the associated plasmid types suggested that these determinants were circulating in diverse strains, complicating the control efforts and suggesting the need for evidencebased antimicrobial stewardship programs.In the distressing situation of Lebanon, the accessibility to novel antibiotic molecules remains a major concern, impacting public health.The economic crisis had a profound impact leading to significant challenges in antibiotics procurement and even more so to new molecules, which are unavailable and not yet routinely tested.Furthermore, our data support that the fight against MDR bacteria in LMICs, such as Lebanon, requires a comprehensive One Health approach because of the diffuse sources and factors that affect the spread of resistance in hospitals and the community.The latter corroborates our previous research that showed a wide reliance on and sometimes indiscriminate use of critically important antibiotics in healthcare settings, agriculture, and the community in Lebanon (8,9).This approach and science-based interventions are urgently needed to control the spread

TABLE 2
(18)biotic susceptibility patterns of selected carbapenemase-producing Escherichia coli isolates for antibiotics considered last resort molecules using broth microdilution test (Sensititre), and interpreted according to the EUCAST guidelines(18).

TABLE 3
Virulence genes of the 26 carbapenemase-producing E. coli clinical isolates.

TABLE 4
Carbapenemase gene location was determined by plasmid extraction using Kieser technique and by applying a BLASTN algorithm for carbapenemase-genes-carrying contigs and then these contigs were mapped to reference plasmids using CLC genomics.

TABLE 5
Comparison of different typing methods for carbapenemaseproducing Escherichia coli clinical isolates.Plasmid incompatibility groups present in the 33 clinical carbapenemase-producing Enterobacterales isolates, were identified using PlasmidFinder 2.1 online tool available at the Center for Genomic Epidemiology -CGE (http://www.genomicepidemiology.org/) using

TABLE 4
Our findings further support the shift from bla OXA-48-like variants toward bla NDM-5 among E. coli in hospital settings in Lebanon 10.3389/fpubh.2023.1290912Frontiers in Public Health 12 frontiersin.org of AMR in Lebanon, which represents an essential threat nationally and globally as AMR is known to spill across international borders.