Decade of Carbapenemase-Producing Enterobacterales in New Caledonia: Integrative Surveillance Though Genomic, Phenotypic and Clinical approaches

Decade of Carbapenemase-Producing Enterobacterales in New Caledonia: Integrative Surveillance Though Genomic, Phenotypic and Clinical approaches | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Decade of Carbapenemase-Producing Enterobacterales in New Caledonia: Integrative Surveillance Though Genomic, Phenotypic and Clinical approaches Julien Colot, Alexandre Bourles, Léo Cousin, Gauthier Delvallez, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5067243/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Carbapenemase-producing Enterobacterales (CPE) have been identified by the World Health Organization as global priority pathogens. The dissemination of these bacteria and outbreaks within healthcare facilities are of serious concern. This study investigated the transmission patterns and genetic characteristics of CPE isolated in New Caledonia from 2013 to 2022. The isolates were identified and characterized both phenotypically and whole-genome sequencing (WGS). In total 214 CPE were isolated: 199 non duplicate clinical isolates from 164 patients and 15 from hospital environmental surface. The most common genera in clinical samples were Enterobacter (34%) and Klebsiella (25%), with 194 isolates (98%) carrying IMP-type carbapenemase. WGS of 89 isolates revealed the dominance of the bla IMP-4 carbapenemase gene, found in 82 isolates. The bla IMP-4 was primarily predicted to be carried by IncL/M-type plasmid, found in 69% of the sequenced isolates. Our work revealed the circulation of 12 bacterial clusters with 61 strains involved in outbreaks or persistent over time. Genomic, phenotypic and clinical approaches identified 12 distinct outbreaks involving IMP producers. These results highlight the importance of studying plasmid transmission to better prevent silent spread of CPE. Ultimately, this study provides new guidelines for limiting the clinical spread of CPE in New Caledonia. Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Biological sciences/Genetics/Genomics/Medical genomics Antibiotic resistance blaIMP-4 Clinical Outbreak Surveillance Hygiene Investigations New Caledonia Plasmid Whole Genome Sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The World Health Organization (WHO) has recognized antimicrobial resistance (AMR) as a major threat to global health 1 . Among mechanisms of antibiotic resistance, carbapenem resistance is particularly concerning, and carbapenemase-producing Enterobacterales (CPE) are categorized into critical groups to guide research and development and public health interventions into the 2024 WHO Bacterial Priority Pathogens List 2 . The advent and availability of genomic approaches have led to substantial advancements in comprehending antibiotic resistance genes dissemination 3 , 4 . ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance are developed for clinical and public health microbiology 5 . Genomic data provide additional information, specifically on the identification of Antibiotic Resistance Genes (ARGs) and plasmids carrying these genes, complementing to the phenotypic approaches typically used in medical laboratories. In Enterobacterales, carbapenem resistance can be driven by the acquisition of carbapenemase-encoding genes, including Ambler class A serine β-lactamases ( bla KPC , bla GES ), metallo β-lactamases ( bla NDM , bla VIM , bla IMP ) and carbapenem-hydrolysing class D β-lactamases ( bla OXA−48−like) 6 . These genes are often carried on plasmids, which can be horizontally transmissible between Enterobacterales driving the rapid spread of resistance 7 . In addition, these plasmids often carry other resistance genes, making the bacteria extremely drug resistant and limiting treatment options. New Caledonia (NC), a French archipelago located in the tropical southwest Pacific Ocean, is not spared by diffusion of CPE. Notably, since the implementation of CPE surveillance on the territory (2004, two major CPE outbreaks have been identified using epidemiological investigations in the Centre Hospitalier Territorial (CHT), the main hospital in NC: one in the Intensive Care Unit (ICU) in 2017 and another one in the Neonatal Intensive Care Unit (NICU) in 2018 8 . Although NC’s population is relatively isolated, there are links notably with Australia, the main site of many medical evacuations (Med Evac), and mainland France, where many population transfers occur. In this archipelago, IMP-type carbapenemase-producing isolates were the first CPE identified in 2013. Since then, the number of IMP-type carbapenemase) carriers increased from 2013 (4 isolates) to 2018 (33 isolates found) 8 . This carbapenemase, encoded here by the bla IMP−4 gene, is prevalent in the Asia-Pacific region 9 – 11 and has become particularly dominant in Australia 12 – 15 . In this study, we combined epidemiological and genomic analyses of all CPE cases detected in NC between July 2013 and December 2022 to provide a comprehensive overview of the local epidemiology of CPE. Clinical and epidemiological data, along with screening test results, were collected for each patient. Suspected outbreaks were first identified through epidemiological investigations: hygiene control investigations and routine microbial analyses. Then, whole-genome sequencing (WGS) was performed to identify ARGs, as well as associated plasmids and integrons, and to confirm suspected outbreaks. Results 1. Patients’ characteristics and corresponding isolates As shown in Table 1 , out of the 164 patients, 57 (34%) of isolates came from clinical samples from patients with symptoms, whereas 107 (66%) where obtained from screening samples (contacts). Figure 1a presents this proportion along the years. Among the clinical samples, 14 (9%) were from blood cultures, 22 (13%) from urine samples, 5 (3%) from respiratory samples, and 16 (10%) from other sources, mainly abscesses. Ninety-nine patients (60%) were males and 65 (40%) were females. One hundred twenty-five patients (78%) were from the CHT and 30 (18%) from other external laboratories. Among the 125 patients from CHT, 51 were admitted in ICU (31%), 18 (11%) in the cohorting unit 7 (4%) in pediatrics, 14 (9%) in NICU, 11 in nephrology and 24 in other departments (Fig. 1b). Following a significant increase observed in 2017 and 2018 (Fig. 1a and 1b), CPE incidence between 2019 and 2022 was relatively stable (13.30 for 100,000 hospitalization-days and 12.63 for 100,000 hospitalization-days respectively). The clinical data, collected for 118 out of 125 patients admitted in CHT are summarized in Table 2 . The median hospital stay before CPE isolation was 19.5 days [interquartile:10–38]. Fifty three percent of patients had been hospitalised for an infectious condition, 78% had been in the ICU, and 87% had received antibiotics prior CPE detection. Table 1 Characteristics of samples from 164 patients Characteristics No.(%) Sex Male 99(60.3) Female 65(39.6) Age 60 85(51.8) Ward ICU* 51(31.1) External laboratory 30(18.2) Other * 24(14.6) Cohorting unit* 18(10.9) NICU* 14(8.5) Nephrology* 11(6.7) Pediatrics* 7(4.2) CHN 5(3.0) CHS 4(2.4) Origin Fecal swab 107(65.2) Urines 22(13.4) Other 16(9.7) Blood 14(8.5) Respiratory 5(3.0) Ward suspicion Yes 72(43.9) No 97(56.1) Spread events 2017-ICU 17(23.6) 2018-NICU 10(13.8) 2018-ICU 6(8.3) 2018-Pediatrics 6(8.3) Other clusters in ICU 17(23.6) Other clusters outside of ICU 16(22.2) Services marked with a star (*) are associated with CHT. Services without a star are from other hospitals (CHN, CHS) or external laboratories. Table 2 Characteristics of the 118 clinical data recovered hospitalized at the CHT No(%) Lenght of stay (days) 30 37(31,36) Medical history Surgery 58(49.1) None 39(33.0) Diabetes 33(27.9) Chronic obstructive Pulmonary Disease 14(11.8) Tumor 13(11.0) Med Evac 13(11.0) Travel 10(8.4) Chemotherapy 5(4.2) ICU Yes 92(77.9) No 26(22.0) Lenght of stay in ICU (days) 30 15(16.3) Risk factor Antibiotics 103(87.2) Catheter 79(66.9) Mechanical ventilation 62(52.5) Multi-drug resistant bacteria carrier 38(32.0) None 9(7.6) Death Yes 20(16.9) No 98(83.0) 2. Phenotypic and genomic profiles of CPE isolates A total of 199 CPE strains were collected from the 164 patients between July 2013 and December 2022. The predominant genera identified using MALDI-TOF were Enterobacter (34%), Klebsiella (25%), Citrobacter (18%), Escherichia (12%) and Serratia (8%; Fig. 1c). Among these isolates, 194 strains (98%) were identified as possessing an IMP-type carbapenemase (Fig. 1d). Additionally, we included 15 IMP-carrying isolates from hospital environmental surfaces, belonging to the genera Enterobacter (n = 7), Serratia (n = 7) and Klebsiella (n = 1). These isolates were collected during three suspected large outbreaks (2017 in ICU, 2018 in both NICU, and in Pediatrics), and two suspected small outbreaks (2020 in Nephrology 1 and 2). AST was conducted on 130 CPE isolates (119 from patients and 11 from hospital environmental surface) with clinical isolates results detailed in Fig. 2. All clinical isolates were resistant to the tested third generation cephalosporins (3GCs; cefotaxime, ceftazidime) except for one Citrobacter (CLIN139, isolated from a blood culture) and one Klebsiella producing an OXA-48 carbapenemase (CLIN189, isolated from a cutaneous wound). Notably, 25/119 isolates (21%) showed susceptibility to at least one carbapenem (ertapenem or imipenem). In addition, 57 out of 119 clinical isolates (48%) were susceptible to ciprofloxacin and 71/119 (60%) were susceptible to trimethoprim-sulfamethoxazole. Susceptibility to the piperacillin-tazobactam combination was evaluated in 112 clinical CPE isolates, with 27 isolates (25%) showing susceptibility. This finding was confirmed for all 14 isolates tested using the Etest®. Notably, all clinical Serratia isolates tested were susceptible to piperacillin-tazobactam (n = 11). WGS performed on the selection of 89 representative CPE isolates throughout the study period allowed us to precisely identify 19 species using TYGS (Supplementary Table 1). The resistance gene annotation revealed the dominance of the IMP-4 producers (82/89). For two old isolates (CLIN02 and CLIN04) and CLIN61, the bla IMP−4 was not identified in our sequencing, but these strains had been confirmed using immunochromatographic assays during routine analysis Moreover, across all isolates, bla IMP−4 gene was consistently associated with a class 1 integron structure (Supplementary Table 1). This class 1 integron (with bla IMP−4 carbapenemase-encoding gene) was predicted mainly on an IncL/M type plasmid associated with the AA002 MOB_typer primary cluster (61/89 isolates, 69%) (Table 3 , Figs. 3 and 4a). Interestingly, this IncL/M plasmid replicon was also found in 6 isolates in which the bla IMP−4 gene was predicted on other plasmids. This plasmid was identified in isolates from the following genera: Enterobacter (n = 29), Serratia (n = 18), Klebsiella (n = 15), Escherichia (n = 4), Citrobacter (n = 3). Additionally, 10 of these bla IMP−4 /IncL/M CPE were isolated from the hospital environmental surface (Table 3 , Fig. 3). Other ARGs such as bla TEM−1B , aac(3)IId, catB3, mph(A), qnrB and sul1 were predicted to co-occur on this plasmid (Fig. 4a). The bla IMP−4 carbapenemase gene was also predicted for 9 CPE to be carried on an IncC type plasmid (AA860 MOB_typer primary cluster) found in Citrobacter (n = 1), Enterobacte r (n = 4) and Klebsiella (n = 4) with two CPE isolated from the hospital environmental surface (Table 3 , Fig. 3). The same ARGs described above were predicted on this plasmid for these CPE (Fig. 4a). In addition, according to MOB_type prediction, 3 CPE harboured the bla IMP−4 gene on an IncHI2A-type plasmid (AA739 MOB_typer primary cluster) (Table 3 , Fig. 3). Finally, bla NDM−1 was detected in two Escherichia coli isolates (CLIN01 and CLIN116) predicted to be carried on a IncC-type plasmid (AA860 MOB_typer primary cluster) within a class 1 resistance integron structure and in two Providencia rettgerii isolates (CLIN111 and CLIN114) on an undefined incompatibility plasmid (AA855 MOB_typer primary cluster) also within a class 1 resistance integron structure. Notably, in CLIN114 isolate, bla IMP−4 and bla NDM−1 were predicted to be collocated on the same IncC type plasmid (AA860 MOB_typer primary cluster). Table 3 Characteristics of plasmids found in sequenced isolated MOB_typer primary cluster Plasmid Type predicted_mobility Carbapenemase predicted Genera Sample Implicated in suspected spread event AA002 (n = 68) IncL/M conjugative except non mobilizable for CLIN05 ; CLIN102 ; CLIN32 ; CLIN61 ; CLIN95 bla IMP−4 (n = 61) Citrobacter (n = 3) Enterobacter (n = 29) Escherichia (n = 3) Klebsiella (n = 15) Serratia (n = 18) Infectious sample (n = 23) Screening sample (n = 35) Hospital Environmental sample (n = 10) 2017-ICU (n = 10) 2018-Digestive Surgery (n = 1) 2018-NICU (n = 11) 2018-Pediatrics (n = 10) 2018-ICU (n = 6) 2019-ICU 1 (n = 2) 2019-ICU 3 (n = 2) 2020-Nephrology1 (n = 2) 2020-Nephrology2 (n = 1) 2021-Cohorting unit (n = 1) AA855 (n = 2) Rep_cluster_1506: CLIN114 Rep_cluster_1220: CLIN11 conjugative: CLIN 114 non mobilizable: CLIN 111 bla IMP−4 (n = 1) bla NDM−1 (n = 2) Providencia (n = 2) Screening sample (n = 2) 2022-ICU (n = 2) AA627 (n = 7) InC mobilizable bla IMP−4 (n = 1) Citrobacter (n = 7) Infectious sample (n = 3) Screening sample (n = 4) 2021-Cohorting unit (n = 3) AA275 (n = 3) IncFIB, IncFII, rep_cluster_2183 conjugative bla IMP−4 (n = 3) Klebsiella (n = 3) Infectious sample (n = 2) Screening sample (n = 1) 2017-ICU (n = 2) 2019-ICU (n = 1) AA860 (n = 11) IncC conjugative bla IMP−4 (n = 9) bla NDM−1 (n = 2) Citrobacter (n = 1) Enterobacte r (n = 4) Escherichia (n = 2) Klebsiella (n = 4) Infectious sample (n = 5) Screening sample (n = 4) Hospital Environmental sample (n = 2) 2018-Digestive Surgery (n = 1) 2019-ICU 1 (n = 2) 2019-ICU 3 (n = 1) 2020-Nephrology 1 (n = 1) 2020-Nephrology 2 (n = 1) AA739 (n = 9) IncHI2A conjugative bla IMP−4 (n = 3) Enterobacter (n = 8) Escherichia (n = 1) Infectious sample (n = 3) Screening sample (n = 4) Hospital Environmental sample (n = 2) 2018-Pediatrics (n = 2) AB130 (n = 5) Col(VCM04) conjugative bla IMP−4 (n = 3) Citrobacter (n = 5) Infectious sample (n = 2) Screening sample (n = 3) 2021-Cohorting unit (n = 2) The relationships of the sequenced isolates are detailed in Fig. 3 and core genome SNP analysis are reported in Supplementary Table 2. Clonal relationships between strains (core genome SNP threshold < 21) are reported in several STs (ST98, ST254, ST104, ST125, ST536) on in each Serratia groups. Unfortunately, not all strains within each ST have SNPs below 21. Using plasmid content and AST profiles (considering the random errors of the VITEK and clonal relationships based on WGS, we considered identical ASTs if they differed by no more than one result) and hygiene investigations (link between patients), (Fig. 4) we were able to regroup strains between clusters. We identified 12 clusters (core genome SNP threshold < 50) encompassing 64 CPE isolates (72%; Supplementary Table 2). Among them, the three main clusters were associated with the genera Serratia and Enterobacter . The Serratia cluster group III (mean SNPs: 16.75, max: 33, min: 1) identified as the recently described S. sarumanii 16 associated with also IncL/M type plasmid. The latter group includes 10 CPE isolates from outbreak at the NICU in 2018. The Enterobacter ST104 cluster (mean SNPs: 23.86; max: 33; min: 11), identified as E. hoffmanii according to the latest nomenclature 17 , includes 9 resistant strains harbouring the IncL/M type plasmid, isolated during two outbreaks at the ICU in 2017 and 2018. The Citrobacter ST98 cluster (mean SNPs: 19.16; max: 32; min: 1); identified as Citrobacter freundii with however several plasmids (IncL/M, IncC, Col (VCM04)) and found at several time and ward during the study. 3. CPE clustering Based on hygiene investigations, 4 large outbreaks (n ≥ 6) and 13 small outbreaks (n < 6) were initially suspected over the study period. The 13 small outbreaks between 2018 and 2022 involved 2 to 5 CPE isolates each. Six of these episodes occurred in the ICU, and two in the Nephrology ward, where CPE were also isolated from the hospital environmental surface (Table 3 ). The remaining episodes occurred sporadically across other wards. Taken together, WGS, AST and epidemiological investigations confirmed the 4 large outbreaks identified on epidemiological grounds and 5 of the 13 small outbreaks (Fig. 5). Two suspected small outbreaks were not confirmed and six could not be investigated because the isolates were not preserved. In addition, 1 isolate initially labelled as isolated cases (CLIN104) was assigned to one small outbreak (Cohorting unit 2021). These analyses also uncovered 3 small outbreaks that had not been previously suspected (Fig. 5). Interestingly, E. hoffmanii ST104 was found in the two large outbreaks in ICU (Figs. 4 and 5) with one strain isolated from the hospital environmental surface in 2017. Moreover, IncL/M plasmid type was implicated in the four large outbreaks (Fig. 4). For example, during the large outbreak in Pediatrics in 2018, four genera ( Citrobacter, Enterobacter, Klebsiella and Serratia ) are implicated harbouring IncL/M type plasmid predicted as carrier of bla IMP−4 (Fig. 5). Discussion Since the initial detection of the first CPE in NC ( Klebsiella pneumoniae IMP on a catheter in a patient admitted to the CHT’s pneumology unit) in July 2013, the number of patients carrying CPE has increased, peaking in 2018 with outbreaks observed in the ICU and the NICU 8 . This increase is also observed in Asia Pacific region 18 . However, since these spread events, we observed a stabilisation in the situation and even a reduction in the number of cases. This situation can be partially explained by the COVID-19 pandemic with a zero-covid strategy applied in New Caledonia with a rapid border closure in New Caledonia, reinforcement of hygiene measures and reduction of patients number in the hospital 19 . All the measures put in place within the hospital, such as the introduction of a cohorting unit, systematic screening of patients before and after MedEvac from Australia (2015), and automated email alerts when the admission of an identified CPE carrier occurs (2017), could limit a potential increase of CPE in New Caledonia 8 . Although the incidence has stabilised over the last four years in NC, the risk of CPE spreading should not be overlooked, hence the need for increased surveillance to prevent new outbreaks. Indeed, our findings indicate that increase of CPE was associated with spread events involving multiple strains, as well as the circulation of dominant carbapenemase-encoding plasmids among various species and genera. In the Western Pacific region, implementing pathogen genomics surveillance is an evidence 20 . Regarding the circulation of several strains, in NC we identified through WGS the persistence of K. pneumoniae ST16, also reported in Fiji 21 which is globally recognized as a CPE sublineage with critical public health implications 22 . Additionally, we isolated Citrobacter freundii ST98 between 2014 and 2021. This ST is also known to include carbapenemase producers and to be present in several countries 23 . In NC, the first isolates of these two lineages were detected in samples from external laboratories before outbreaks at the CHT (ICU 2017; Cohorting unit 2021). We might hypothesize that these STs were circulating within the community prior to their identification at the hospital. Even if we were as exhaustive as possible (the CHT is the reference centre for CPE in New Caledonia), very few community strains were collected. It would therefore be interesting to extend this study outside hospitals to get a more accurate picture of the circulation of CPE in NC. In addition to the characterization major STs, the WGS approach enabled to establish connections between several outbreaks. In our study a threshold at 50 core genome SNPs, was proposed to discriminate hospital outbreak. This is in agreement with the current literature where thresholds range from 21 24 to 80 core genome SNPs 25 to discriminate hospital outbreaks depending on the studied bacterial genus. We found that E. hoffmanii ST104 and related strains were implicated in ICU outbreaks in 2017 and 2018. The presence of an isolate in the hospital environmental surface (CLIN37) suggests a single outbreak. E. hoffmanii ST254 were implicated in outbreaks in ICU and Pediatrics in 2018 suggesting link between these two events. Interestingly, this ST was previously reported to be implicated in the widespread of transmission of bla IMP−4 in Australia 26 . In the collection, our results showed that the carbapenemase-encoding genes are primarily associated with an IncL/M type flanking structure. This replicon was implicated in four large and small outbreaks and isolated cases highlighting its significance in New Caledonia. The putative IncL/M plasmid reconstructed with MOB-suite software was predicted to be conjugative and found in 5 different bacterial genera in our collection, emphasizing its notable diffusion potential 27 . It is therefore essential to enhance monitoring and control of this plasmid, as limiting its spread will help to prevent further CPE outbreaks in NC. Although our short read sequencing data do not allow us to determine whether the bla I MP−4 gene was localized on a particular plasmid when multiple plasmid structures are found within the same strain, our results showed that in all cases the bla IMP−4 gene was located on a class 1 resistance integron. This structure is also widely recognised in Australia for harbouring the bla IMP−4 gene 28 . Some CPE contain several entities of the plasmids described in Table 3 , which are likely to harbour the bla IMP−4 gene. However, short-read sequencing often results in the integron being found in small contigs, making it difficult to pinpoint the exact bla IMP−4 -carrying plasmid. Long reads sequencing is planned to study the genetic environment around these integrons and to confirm the position of the ARGs on a given plasmid. Our results highlight the value of studying plasmids, particularly during dissemination episodes involving different strains but the same plasmid (Pediatry − 2018). Long reads sequencing is currently being implemented at the CHT. Given the results of genomics in this work (dominance of IncL/M) and the speed with which results can be obtained (few hours in Madueno et al., (2024) 29 ), its implementation appears to be essential to limit the spread of CPE in New Caledonia, particularly if an outbreak is suspected. With plasmids, this time could be further reduced, for example by displaying plasmid profiles on an electrophoresis gel before sequencing. This plasmid gel migration technique could be implemented within the hospital to provide additional, less precise but more timely information for rapid decision-making. This method may demonstrate intra- and interspecies horizontal transmission and thus help hygiene operational teams. Also, once the plasmids have been circularised, RFLP (restriction fragment length polymorphism) approaches combined with separation by gel electrophoresis or plasmid-specific PCR could be used in the future. We conducted a retrospective epidemiologic and genomic study, confirming the dominance of IMP-type carbapenemase in NC. This carbapenemase, encoded here by the bla IMP−4 gene, is prevalent in the Asia-Pacific region 9 – 11 and has become particularly dominant in Australia 12 – 15 . Our study revealed that in over 68% of sequenced isolates, bla IMP−4 was predicted to be carried on an IncL/M type plasmid (AA002 MOB_typer primary cluster). This plasmid type has also been identified in Australian outbreaks described in Sydney 14 , 28 , 30 . Interestingly, using MOB-typer prediction, the nearest IncL/M type plasmid (AA002 MOB_typer primary cluster) plasmids obtained using mash distance are reported in strains isolated from The Alfred Hospital in Sydney 14 . This hospital is particularly associated with medical evacuations of patients from NC. Of the 13 patients who tested positive upon return from these medical evacuations, seven isolates were sequenced. Six of these were found to carry on a IncL/M plasmid, and two of them were linked to local outbreaks (ICU-2017 and Pediatrics-2018). Several clones, such as C. freundii ST98 with AA860 MOB_typer primary cluster 31 , K. pneumoniae ST20 with AA02 MOB_typer primary cluster 14 , with AA739 MOB_typer primary cluster or AA860 MOB_typer primary cluster 5 have been previously reported in Australia. These findings suggest potential epidemiological links between Australia and NC as already reported for Vancomycin resistant Enteroccocus 8 or Corynebacterium diphtheriae 32 . In addition, one patient repatriated from Australia was identified with an Enterobacter xiangfangensis (CLIN71; formerly named E . hormaechei subsp. xiangfangensis ) with an IncHI2A type plasmid (AA739 MOB_typer primary cluster), which has been previously reported in this country 17 , 33 . Although our WGS panel included a wide variety of bacterial strains, those belonging to the genus Enterobacter were predominant. The bla IMP−4 gene is particularly prevalent in this genus in Australia 28 , 34 . Notably, E. xiangfangensis , the main species in our dataset, has been involved in an outbreak 33 . In contrast, Enterobacter chengduensis isolates are rarely described as carbapenemase producer in the literature. Apart from CLIN07 (ST414), the only identified bla IMP−4 carriers have also been reported in neighbouring Australia 35 . Further genomic analysis and collaboration are essential to confirm the microbial and antibiotic resistance connections between our two territories, but these suspected links with Australia at various points underscore the importance of screening patients returning from medical evacuations. The data presented in this study underscores the importance of using screening agar to effectively detect CPE strains carrying the bla IMP− 4 gene. Goire et al . (2016) demonstrated in Australia that screening media such as Brilliance™ CRE Agar are insufficiently sensitive for detecting bla IMP− 4 , unlike other CPE types 15 . For bla IMP− 4 endemic situation, the authors recommended combining ESBL screening media with the CarbaNP test and in-house bla IMP− 4 real-time PCR. Here, we also confirmed the low level of resistance to carbapenems in strains carrying the bla IMP− 4 gene 9 , 33 , which are however well captured when using ESBL agar plate. Our study reported a 15% fatality in infected patients, lower than the rates reported in the literature 36 , 37 . This low fatality rate might be attributed to the limited number of associated resistances, with retained susceptibility to cotrimoxazole (59%) and fluoroquinolones (47%). Additionally, the low level of resistance to carbapenems conferred by bla IMP− 4 allowed the use of certain carbapenems in combination with cotrimoxazole or fluoroquinolones 38 , 39 . The introduction of the cohorting unit to isolate patient seems beneficial in NC, as the number of CPE cases has stabilised, and no further outbreaks have occurred in CHT since its establishment. However, our WGS data are more concerning, as we detected Citrobacter freundii ST98 in several patients associated with this unit, raising questions about the efficacy of the cohorting system 40 . Many clones detected from patients were also found in environmental isolates, suggesting an important role of the hospital environment in their spread and persistence. In our study, most of Serratia isolates originated from the hospital environment, a finding consistent with previous reports highlighting the relevance of environmental reservoirs in ICU 41 or NICU 42 in the endemicity of opportunistic pathogens such as S. marcescens . Based on these results and litterarure 43 , a more in-depth analysis of the hospital environmental surface and notably cohorting unit environment should be considered. To summarise, we integrated phenotypic, genomic and epidemiological analyses to investigate the transmission pathways of CPE in NC. Our findings revealed that multiple clones were responsible for outbreaks in ICU and NICU and identified an IncL/M plasmid as probable vehicle for the dissemination of bla IMP−4 in NC since 2013. These results highlight the importance of studying plasmid transmission to better prevent the spread of CPE and support the concept that AMR is a silent pandemic. Methods We conducted a retrospective study to analyse all CPE isolates collected in NC from July 2013 (first isolate detected) to December 2022. NC’s healthcare infrastructure includes three public hospitals and one private clinic for patients’ hospitalization. These facilities operate under the authority of the Department of Health and Social Affairs (DASS) of New Caledonia Government, which has decision-making powers in health-related matters, a feature that is consistent with the administrative structure in France. Study was conducted in the CHT, the main and reference hospital of the archipelago. It comprises 645 beds, 12 operating rooms, medical units, surgery units and intensive care units. The number of hospitalizations approximates 40,000 a year. Moreover, CHT laboratory is the reference laboratory for CPE in NC, where all suspected CPE isolates are sent for expertise. In order to prevent spread of CPE, patients with a CPE at the CHT are transferred in a cohorting unit by a dedicated medical team 8 . 1. Workflow for CPE isolation at CHT All patients admitted to the ICU, and those returning from a foreign hospital, or known previous carriers of CPE were systematically screened for CPE carriage. When a CPE is isolated in a patient, all contacts within the department were also screened. CPE screening involves using a faecal swab culture on ChromID® ESBL (bioMérieux, Marcy-l'Étoile, France), with results available 24 to 48 h after incubation at 37°C. All colony morphologies are identified using MALDI-TOF mass spectrometry (Microflex LT-MS, Bruker Daltonics). Subsequently, Enterobacterales were plated on Mueller-Hinton agar with different antibiotic discs: ertapenem (10µg), temocillin (30µg) and ceftolozane/tazobactam (30/10µg) inspired by the 2022 European Committee on Antimicrobial Susceptibility Testing 44 . Confirmation was performed using NG-CARBA-5® immunochromatographic assays (NG Biotech Laboratories, Guipry, France), which detects the major carbapenemase types (VIM, IMP, NDM, KPC, and OXA-48-like) 45 , 46 . Antimicrobial agent Susceptibility Testing (AST) was systematically conducted for clinical strains on VITEK-2® system (bioMérieux, Marcy l'Etoile, France) with AST N-234 or N-372 cards. Finally, carbapenem minimum inhibitory concentrations (MICs) can be confirmed using the Etest® method (bioMérieux, Marcy l'Etoile, France). This approach was applied to all samples collected during the study and retrospectively to all CPE isolates available in our collection. The study included CPE strains from both carrier and infected patients, irrespective of the type of specimen collected, as well as environmental isolates (recovered from table surface and sink in the lavatory in patient rooms, or healthcare trolley) identified during a suspected outbreak for comparison. Furthermore, to ensure the accuracy and reliability of the results, all ASTs were interpreted using the EUCAST guidelines as updated in 2022 44 . 2. Collection of clinical data We systematically recorded the origin of each sample, specifying the hospital ward or name of the external laboratory, and categorized the sample types into five mains groups: rectal swab, blood culture, urine, respiratory, and other. For all patients hospitalized at CHT, we collected clinical data relevant to the study and recognized as risk factors for CPE acquisition in other studies 47 . Finally, we recorded whether the isolate was likely associated with a spread event. Suspected outbreaks were characterised by the incidental discovery leading to the detection of a secondary case, or the detection of several CPE in the same department at short period (less than one month). In this study, an outbreak was considered as a large outbreak if there were at least five secondary cases, otherwise, if there are fewer than 5 secondary cases, we considered this event as a small outbreak. 3. Whole-genome sequencing A batch of 64 CPE (corresponding to first isolates in NC or suspected of belonging to outbreaks) isolated from 2013 to 2019 was submitted to WGS by the French National Reference Center of Antimicrobial Resistance using Pasteur P2M core facility. Sequencing was conducted using a NextSeq500 instrument (Illumina, San Diego, CA; 2 × 150-nucleotide paired-end protocol). Additionally, WGS was performed on 25 CPE (in order to complete the study period until 2022) collected between 2020 and 2022 by the Beijing Genomics Institute (BGI) Tech Solutions Hong Kong Co., Limited. Isolates were inoculated in Luria Broth medium and incubated overnight at 37°C. Genomic DNA was extracted from 1.5 ml of LB culture using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's instructions. DNA purity was determined using Nanodrop (ThermoFisher Scientific, Waltham, MA, USA) and agarose gel deposition. Paired-end libraries were prepared with an insert size of approximately 300 bp and sequenced on DNB-SEQ PE150 according to the supplier’s protocol. a. Genome assembly and quality assessment Paired-end reads were de novo assembled using the fq2dna workflow V21.06, which also includes various steps such as trimming, error correction, contaminant removal, and polishing. ( https://gitlab.pasteur.fr/GIPhy/fq2dna ). For completeness and contamination analysis, CheckM (V1.1.3, taxonomy_wf at genus level) and QUAST version 5.2.0 48 . were employed. b. In-silico species identification and genomic screening Multi-locus sequence typing (MLST) was conducted using MLST software V2.19.0 ( https://github.com/tseemann/mlst ) and associated profiles provided by PubMLST 49 or BIGSdb-Pasteur ( Klebsiella pneumoniae species complex). To evaluate ARGs, Abricate software V1.0.1 was employed ( https://github.com/tseemann/abricate ; --minid 70 --mincov 90 --db resfinder) 50 . The prediction of genomic location of ARGs, plasmid identification and typing were performed using MOB-suite V3.0.1 27 . Furthermore, the identification of integrons carrying ARGs was conducted using IntegronFinder V2.0.2 51 . c. Phylogeny and relationship between isolates The initial phylogenetic relationships between isolates from the different compartments and sample sites were examined using the k-mer-based tool JolyTree V2.1 52 . The associated tree was midpoint-rooted and annotated with iTOL V6 53 . Species identification was achieved for each assembly through the utilisation of a digital DNA:DNA hybridization approach (dDDH cut-off ≥ 70.0% formula d 4 ) as facilitated by the Type Strain Genome Server (TYGS) 54 . Regarding Enterobacter genus, an additional analysis was performed using the approach and taxonomy proposed by Wu et al. with FastANI V1.33 (ANI cut-off ≥ 96.0%) 17 . Due to the significant genetic diversity of the sample and the need for precise comparison within clinical isolates, we conducted core genome-based phylogenetic analyses on isolates of the same sequence type (ST) with more than two assemblies. For each ST group, a good-quality reference genome was identified by using the online tool SimilarGenomeFinder against all public genomes in the BV-BRC resource center (V3.30.19a;) 55 , 56 . Mapping was performed using Snippy V4.6.0 against these selected references. The resulting core-SNP alignment was used to infer maximum-likelihood phylogenetic trees for each ST group using IQ-tree V2.1.4 57 , and recombination elements were removed with ClonalFrameML V1.12 58 . Then, specific SNP differences matrixes were generated with snp-dists V0.8.2. As proposed by David et al. , (2019) 59 , we used 21 core genome SNP as threshold for discrimination of clonal diffusion. Declarations Funding source This work was supported by the Centre Hospitalier Territorial de Nouvelle-Calédonie, French National Reference Center of antibiotic resistance, Government of New Caledonia [Postdoctoral contract, 2020-2023] and Institut Pasteur de Nouvelle-Calédonie. Author contributions A.B. and J.C. designed the study, L.C., G.D, P.S. and J.C. delivered clinical data, A.B., M.K. and T.O. performed the microbiology experiments, S.B., R.A.B. and L.D performed the WGS experiments, A.B., F.P. and M.P. performed the bioinformatics analyses, P.S performed statistical analyses, A.B., S.B., R.A.B., L.D., F.V and J.C. purchased the funds, A.B., L.C and J.C. wrote the original manuscript, A.B.,G.D., A.Bi., T.R., R.A.B., L.D., S.B., P.S., M.P., F.V., C.G and J.C revised the manuscript. Every authors read and approved the final manuscript. Data availibility Access to the assembly data from this study is available on GenBank, under the BioProject ID PRJNA1146064. Competing interests The authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical approval This work was approved by the CHT ethics committee (Avis N°3 2024). Acknowledgments We would like to acknowledge the contribution of the microbiology laboratory from the Centre Hospitalier Territorial Gaston Bourret and P2M core facility (Institut Pasteur, Paris) for sequencing some isolates. We also thank students of Institut Pasteur de Nouvelle-Calédonie: M. Zami and L. Nemia for the technical support. References World Health Organization. Antimicrobial Resistance: Global Report on Surveillance . (World Health Organization, 2014). WHO. WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance. https://www.who.int/publications-detail-redirect/9789240093461 (2024). 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legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5067243/v1/96f9ed375c94f3d30e1496e7.png"},{"id":70955516,"identity":"b6e4a973-27c8-4dce-90e8-ed069992bf82","added_by":"auto","created_at":"2024-12-09 14:19:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":147212,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5067243/v1/395842ab863e4208867105de.png"},{"id":70956590,"identity":"d79339aa-b4d9-40bd-bb8b-6e745981b670","added_by":"auto","created_at":"2024-12-09 14:27:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2250358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5067243/v1/fea464fd-cb0e-466c-89f5-4be88483395d.pdf"},{"id":70955259,"identity":"1701b298-b9e9-440e-b984-04cd5820f88d","added_by":"auto","created_at":"2024-12-09 14:11:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":106592,"visible":true,"origin":"","legend":"Supplemmentary Table 1","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5067243/v1/a4a1abcc420d062eaa6707dc.xlsx"},{"id":70955258,"identity":"53463f5b-5220-495a-84e4-4426091ef9f1","added_by":"auto","created_at":"2024-12-09 14:11:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40291,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5067243/v1/ae7ed0b7d49d16e24bfe7abe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Decade of Carbapenemase-Producing Enterobacterales in New Caledonia: Integrative Surveillance Though Genomic, Phenotypic and Clinical approaches","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe World Health Organization (WHO) has recognized antimicrobial resistance (AMR) as a major threat to global health \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among mechanisms of antibiotic resistance, carbapenem resistance is particularly concerning, and carbapenemase-producing Enterobacterales (CPE) are categorized into critical groups to guide research and development and public health interventions into the 2024 WHO Bacterial Priority Pathogens List \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe advent and availability of genomic approaches have led to substantial advancements in comprehending antibiotic resistance genes dissemination \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance are developed for clinical and public health microbiology \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Genomic data provide additional information, specifically on the identification of Antibiotic Resistance Genes (ARGs) and plasmids carrying these genes, complementing to the phenotypic approaches typically used in medical laboratories. In Enterobacterales, carbapenem resistance can be driven by the acquisition of carbapenemase-encoding genes, including Ambler class A serine β-lactamases (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eGES\u003c/sub\u003e), metallo β-lactamases (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e) and carbapenem-hydrolysing class D β-lactamases (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u0026minus;like)\u003c/sub\u003e \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These genes are often carried on plasmids, which can be horizontally transmissible between Enterobacterales driving the rapid spread of resistance \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In addition, these plasmids often carry other resistance genes, making the bacteria extremely drug resistant and limiting treatment options.\u003c/p\u003e \u003cp\u003eNew Caledonia (NC), a French archipelago located in the tropical southwest Pacific Ocean, is not spared by diffusion of CPE. Notably, since the implementation of CPE surveillance on the territory (2004, two major CPE outbreaks have been identified using epidemiological investigations in the Centre Hospitalier Territorial (CHT), the main hospital in NC: one in the Intensive Care Unit (ICU) in 2017 and another one in the Neonatal Intensive Care Unit (NICU) in 2018 \u003csup\u003e8\u003c/sup\u003e. Although NC\u0026rsquo;s population is relatively isolated, there are links notably with Australia, the main site of many medical evacuations (Med Evac), and mainland France, where many population transfers occur. In this archipelago, IMP-type carbapenemase-producing isolates were the first CPE identified in 2013. Since then, the number of IMP-type carbapenemase) carriers increased from 2013 (4 isolates) to 2018 (33 isolates found) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This carbapenemase, encoded here by the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene, is prevalent in the Asia-Pacific region \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and has become particularly dominant in Australia \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e In this study, we combined epidemiological and genomic analyses of all CPE cases detected in NC between July 2013 and December 2022 to provide a comprehensive overview of the local epidemiology of CPE. Clinical and epidemiological data, along with screening test results, were collected for each patient. Suspected outbreaks were first identified through epidemiological investigations: hygiene control investigations and routine microbial analyses. Then, whole-genome sequencing (WGS) was performed to identify ARGs, as well as associated plasmids and integrons, and to confirm suspected outbreaks.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Patients\u0026rsquo; characteristics and corresponding isolates\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, out of the 164 patients, 57 (34%) of isolates came from clinical samples from patients with symptoms, whereas 107 (66%) where obtained from screening samples (contacts). Figure\u0026nbsp;1a presents this proportion along the years. Among the clinical samples, 14 (9%) were from blood cultures, 22 (13%) from urine samples, 5 (3%) from respiratory samples, and 16 (10%) from other sources, mainly abscesses. Ninety-nine patients (60%) were males and 65 (40%) were females. One hundred twenty-five patients (78%) were from the CHT and 30 (18%) from other external laboratories. Among the 125 patients from CHT, 51 were admitted in ICU (31%), 18 (11%) in the cohorting unit 7 (4%) in pediatrics, 14 (9%) in NICU, 11 in nephrology and 24 in other departments (Fig.\u0026nbsp;1b). Following a significant increase observed in 2017 and 2018 (Fig.\u0026nbsp;1a and 1b), CPE incidence between 2019 and 2022 was relatively stable (13.30 for 100,000 hospitalization-days and 12.63 for 100,000 hospitalization-days respectively). The clinical data, collected for 118 out of 125 patients admitted in CHT are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The median hospital stay before CPE isolation was 19.5 days [interquartile:10\u0026ndash;38]. Fifty three percent of patients had been hospitalised for an infectious condition, 78% had been in the ICU, and 87% had received antibiotics prior CPE detection.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of samples from 164 patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCharacteristics\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNo.(%)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSex\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99(60.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65(39.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32(19.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u0026ndash;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47(28.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e85(51.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWard\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eICU*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51(31.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExternal laboratory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30(18.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther\u0026nbsp;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24(14.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCohorting unit*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18(10.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNICU*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14(8.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNephrology*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11(6.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePediatrics*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7(4.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCHN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5(3.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCHS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4(2.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOrigin\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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char=\".\" colname=\"c3\"\u003e \u003cp\u003e14(8.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRespiratory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5(3.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWard suspicion\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72(43.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97(56.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSpread events\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2017-ICU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17(23.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2018-NICU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10(13.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2018-ICU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6(8.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2018-Pediatrics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6(8.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther clusters in ICU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17(23.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther clusters outside of\u0026nbsp;ICU\u0026nbsp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16(22.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eServices marked with a star (*) are associated with CHT. Services without a star are from other hospitals (CHN, CHS) or external laboratories.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the 118 clinical data recovered hospitalized at the CHT\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNo(%)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLenght of stay (days)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21(17,80)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60(50,85)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37(31,36)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedical history\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58(49.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39(33.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiabetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33(27.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChronic obstructive Pulmonary Disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14(11.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13(11.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMed Evac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13(11.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTravel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10(8.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChemotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(4.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eICU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92(77.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26(22.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLenght of stay in ICU\u0026nbsp; (days)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25(27.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52(56.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15(16.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRisk factor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103(87.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatheter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79(66.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMechanical ventilation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62(52.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMulti-drug resistant bacteria carrier\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38(32.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9(7.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDeath\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20(16.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98(83.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2. Phenotypic and genomic profiles of CPE isolates\u003c/h2\u003e \u003cp\u003eA total of 199 CPE strains were collected from the 164 patients between July 2013 and December 2022. The predominant genera identified using MALDI-TOF were \u003cem\u003eEnterobacter\u003c/em\u003e (34%), \u003cem\u003eKlebsiella\u003c/em\u003e (25%), \u003cem\u003eCitrobacter\u003c/em\u003e (18%), \u003cem\u003eEscherichia\u003c/em\u003e (12%) and \u003cem\u003eSerratia\u003c/em\u003e (8%; Fig.\u0026nbsp;1c). Among these isolates, 194 strains (98%) were identified as possessing an IMP-type carbapenemase (Fig.\u0026nbsp;1d). Additionally, we included 15 IMP-carrying isolates from hospital environmental surfaces, belonging to the genera \u003cem\u003eEnterobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7), \u003cem\u003eSerratia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7) and \u003cem\u003eKlebsiella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1). These isolates were collected during three suspected large outbreaks (2017 in ICU, 2018 in both NICU, and in Pediatrics), and two suspected small outbreaks (2020 in Nephrology 1 and 2). AST was conducted on 130 CPE isolates (119 from patients and 11 from hospital environmental surface) with clinical isolates results detailed in Fig.\u0026nbsp;2. All clinical isolates were resistant to the tested third generation cephalosporins (3GCs; cefotaxime, ceftazidime) except for one \u003cem\u003eCitrobacter\u003c/em\u003e (CLIN139, isolated from a blood culture) and one \u003cem\u003eKlebsiella\u003c/em\u003e producing an OXA-48 carbapenemase (CLIN189, isolated from a cutaneous wound). Notably, 25/119 isolates (21%) showed susceptibility to at least one carbapenem (ertapenem or imipenem). In addition, 57 out of 119 clinical isolates (48%) were susceptible to ciprofloxacin and 71/119 (60%) were susceptible to trimethoprim-sulfamethoxazole. Susceptibility to the piperacillin-tazobactam combination was evaluated in 112 clinical CPE isolates, with 27 isolates (25%) showing susceptibility. This finding was confirmed for all 14 isolates tested using the Etest\u0026reg;. Notably, all clinical \u003cem\u003eSerratia\u003c/em\u003e isolates tested were susceptible to piperacillin-tazobactam (n\u0026thinsp;=\u0026thinsp;11).\u003c/p\u003e \u003cp\u003eWGS performed on the selection of 89 representative CPE isolates throughout the study period allowed us to precisely identify 19 species using TYGS (Supplementary Table\u0026nbsp;1). The resistance gene annotation revealed the dominance of the IMP-4 producers (82/89). For two old isolates (CLIN02 and CLIN04) and CLIN61, the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e was not identified in our sequencing, but these strains had been confirmed using immunochromatographic assays during routine analysis Moreover, across all isolates, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene was consistently associated with a class 1 integron structure (Supplementary Table\u0026nbsp;1). This class 1 integron (with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e carbapenemase-encoding gene) was predicted mainly on an IncL/M type plasmid associated with the AA002 MOB_typer primary cluster (61/89 isolates, 69%) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Figs.\u0026nbsp;3 and 4a). Interestingly, this IncL/M plasmid replicon was also found in 6 isolates in which the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene was predicted on other plasmids. This plasmid was identified in isolates from the following genera: \u003cem\u003eEnterobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;29), \u003cem\u003eSerratia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;18), \u003cem\u003eKlebsiella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;15), \u003cem\u003eEscherichia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4), \u003cem\u003eCitrobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3). Additionally, 10 of these \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e/IncL/M CPE were isolated from the hospital environmental surface (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;3). Other ARGs such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1B\u003c/sub\u003e, \u003cem\u003eaac(3)IId, catB3, mph(A), qnrB\u003c/em\u003e and \u003cem\u003esul1\u003c/em\u003e were predicted to co-occur on this plasmid (Fig.\u0026nbsp;4a). The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e carbapenemase gene was also predicted for 9 CPE to be carried on an IncC type plasmid (AA860 MOB_typer primary cluster) found in \u003cem\u003eCitrobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eEnterobacte\u003c/em\u003er (n\u0026thinsp;=\u0026thinsp;4) and \u003cem\u003eKlebsiella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4) with two CPE isolated from the hospital environmental surface (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;3). The same ARGs described above were predicted on this plasmid for these CPE (Fig.\u0026nbsp;4a). In addition, according to MOB_type prediction, 3 CPE harboured the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene on an IncHI2A-type plasmid (AA739 MOB_typer primary cluster) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;3). Finally, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e was detected in two \u003cem\u003eEscherichia coli\u003c/em\u003e isolates (CLIN01 and CLIN116) predicted to be carried on a IncC-type plasmid (AA860 MOB_typer primary cluster) within a class 1 resistance integron structure and in two \u003cem\u003eProvidencia rettgerii\u003c/em\u003e isolates (CLIN111 and CLIN114) on an undefined incompatibility plasmid (AA855 MOB_typer primary cluster) also within a class 1 resistance integron structure. Notably, in CLIN114 isolate, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e were predicted to be collocated on the same IncC type plasmid (AA860 MOB_typer primary cluster).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of plasmids found in sequenced isolated\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOB_typer primary cluster\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasmid Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epredicted_mobility\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarbapenemase predicted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenera\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eImplicated in suspected spread event\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA002 (n\u0026thinsp;=\u0026thinsp;68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncL/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003econjugative\u003c/p\u003e \u003cp\u003eexcept non mobilizable for CLIN05\u0026nbsp;; CLIN102\u0026nbsp;; CLIN32\u0026nbsp;; CLIN61\u0026nbsp;; CLIN95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCitrobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003cp\u003e\u003cem\u003eEnterobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;29)\u003c/p\u003e \u003cp\u003e\u003cem\u003eEscherichia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003cp\u003e\u003cem\u003eSerratia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfectious sample (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;35) Hospital Environmental sample (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2017-ICU (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003cp\u003e2018-Digestive Surgery (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e2018-NICU (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003cp\u003e2018-Pediatrics (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003cp\u003e2018-ICU (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003cp\u003e2019-ICU 1 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003e2019-ICU 3 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003e2020-Nephrology1 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003e2020-Nephrology2 (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e2021-Cohorting unit (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA855 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRep_cluster_1506: CLIN114\u003c/p\u003e \u003cp\u003eRep_cluster_1220: CLIN11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003econjugative: CLIN 114\u003c/p\u003e \u003cp\u003enon mobilizable: CLIN 111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eProvidencia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2022-ICU (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA627 (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emobilizable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCitrobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfectious sample (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2021-Cohorting unit (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA275 (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncFIB, IncFII, rep_cluster_2183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003econjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfectious sample (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2017-ICU (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003e2019-ICU (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA860 (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003econjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCitrobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e\u003cem\u003eEnterobacte\u003c/em\u003er (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003cp\u003e\u003cem\u003eEscherichia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfectious sample (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003cp\u003eHospital Environmental sample (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2018-Digestive Surgery (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e2019-ICU 1 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003e2019-ICU 3 (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e2020-Nephrology 1 (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e2020-Nephrology 2 (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA739 (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncHI2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003econjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEnterobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003cp\u003e\u003cem\u003eEscherichia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfectious sample (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003cp\u003eHospital Environmental sample (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2018-Pediatrics (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAB130 (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCol(VCM04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003econjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCitrobacter\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInfectious sample (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003cp\u003eScreening sample (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2021-Cohorting unit (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe relationships of the sequenced isolates are detailed in Fig.\u0026nbsp;3 and core genome SNP analysis are reported in Supplementary Table\u0026nbsp;2. Clonal relationships between strains (core genome SNP threshold\u0026thinsp;\u0026lt;\u0026thinsp;21) are reported in several STs (ST98, ST254, ST104, ST125, ST536) on in each \u003cem\u003eSerratia\u003c/em\u003e groups. Unfortunately, not all strains within each ST have SNPs below 21. Using plasmid content and AST profiles (considering the random errors of the VITEK and clonal relationships based on WGS, we considered identical ASTs if they differed by no more than one result) and hygiene investigations (link between patients), (Fig.\u0026nbsp;4) we were able to regroup strains between clusters. We identified 12 clusters (core genome SNP threshold\u0026thinsp;\u0026lt;\u0026thinsp;50) encompassing 64 CPE isolates (72%; Supplementary Table\u0026nbsp;2). Among them, the three main clusters were associated with the genera \u003cem\u003eSerratia\u003c/em\u003e and \u003cem\u003eEnterobacter\u003c/em\u003e. The \u003cem\u003eSerratia\u003c/em\u003e cluster group III (mean SNPs: 16.75, max: 33, min: 1) identified as the recently described \u003cem\u003eS. sarumanii\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e associated with also IncL/M type plasmid. The latter group includes 10 CPE isolates from outbreak at the NICU in 2018. The \u003cem\u003eEnterobacter\u003c/em\u003e ST104 cluster (mean SNPs: 23.86; max: 33; min: 11), identified as \u003cem\u003eE. hoffmanii\u003c/em\u003e according to the latest nomenclature \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, includes 9 resistant strains harbouring the IncL/M type plasmid, isolated during two outbreaks at the ICU in 2017 and 2018. The \u003cem\u003eCitrobacter\u003c/em\u003e ST98 cluster (mean SNPs: 19.16; max: 32; min: 1); identified as \u003cem\u003eCitrobacter freundii\u003c/em\u003e with however several plasmids (IncL/M, IncC, Col (VCM04)) and found at several time and ward during the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3. CPE clustering\u003c/h2\u003e \u003cp\u003eBased on hygiene investigations, 4 large outbreaks (n\u0026thinsp;\u0026ge;\u0026thinsp;6) and 13 small outbreaks (n\u0026thinsp;\u0026lt;\u0026thinsp;6) were initially suspected over the study period. The 13 small outbreaks between 2018 and 2022 involved 2 to 5 CPE isolates each. Six of these episodes occurred in the ICU, and two in the Nephrology ward, where CPE were also isolated from the hospital environmental surface (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The remaining episodes occurred sporadically across other wards. Taken together, WGS, AST and epidemiological investigations confirmed the 4 large outbreaks identified on epidemiological grounds and 5 of the 13 small outbreaks (Fig.\u0026nbsp;5). Two suspected small outbreaks were not confirmed and six could not be investigated because the isolates were not preserved. In addition, 1 isolate initially labelled as isolated cases (CLIN104) was assigned to one small outbreak (Cohorting unit 2021). These analyses also uncovered 3 small outbreaks that had not been previously suspected (Fig.\u0026nbsp;5). Interestingly, \u003cem\u003eE. hoffmanii\u003c/em\u003e ST104 was found in the two large outbreaks in ICU (Figs.\u0026nbsp;4 and 5) with one strain isolated from the hospital environmental surface in 2017. Moreover, IncL/M plasmid type was implicated in the four large outbreaks (Fig.\u0026nbsp;4). For example, during the large outbreak in Pediatrics in 2018, four genera (\u003cem\u003eCitrobacter, Enterobacter, Klebsiella\u003c/em\u003e and \u003cem\u003eSerratia\u003c/em\u003e) are implicated harbouring IncL/M type plasmid predicted as carrier of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e (Fig.\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince the initial detection of the first CPE in NC (\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e IMP on a catheter in a patient admitted to the CHT\u0026rsquo;s pneumology unit) in July 2013, the number of patients carrying CPE has increased, peaking in 2018 with outbreaks observed in the ICU and the NICU \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This increase is also observed in Asia Pacific region \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, since these spread events, we observed a stabilisation in the situation and even a reduction in the number of cases. This situation can be partially explained by the COVID-19 pandemic with a zero-covid strategy applied in New Caledonia with a rapid border closure in New Caledonia, reinforcement of hygiene measures and reduction of patients number in the hospital \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. All the measures put in place within the hospital, such as the introduction of a cohorting unit, systematic screening of patients before and after MedEvac from Australia (2015), and automated email alerts when the admission of an identified CPE carrier occurs (2017), could limit a potential increase of CPE in New Caledonia \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although the incidence has stabilised over the last four years in NC, the risk of CPE spreading should not be overlooked, hence the need for increased surveillance to prevent new outbreaks. Indeed, our findings indicate that increase of CPE was associated with spread events involving multiple strains, as well as the circulation of dominant carbapenemase-encoding plasmids among various species and genera.\u003c/p\u003e \u003cp\u003eIn the Western Pacific region, implementing pathogen genomics surveillance is an evidence \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Regarding the circulation of several strains, in NC we identified through WGS the persistence of \u003cem\u003eK. pneumoniae\u003c/em\u003e ST16, also reported in Fiji \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e which is globally recognized as a CPE sublineage with critical public health implications \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Additionally, we isolated \u003cem\u003eCitrobacter freundii\u003c/em\u003e ST98 between 2014 and 2021. This ST is also known to include carbapenemase producers and to be present in several countries \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In NC, the first isolates of these two lineages were detected in samples from external laboratories before outbreaks at the CHT (ICU 2017; Cohorting unit 2021). We might hypothesize that these STs were circulating within the community prior to their identification at the hospital. Even if we were as exhaustive as possible (the CHT is the reference centre for CPE in New Caledonia), very few community strains were collected. It would therefore be interesting to extend this study outside hospitals to get a more accurate picture of the circulation of CPE in NC.\u003c/p\u003e \u003cp\u003eIn addition to the characterization major STs, the WGS approach enabled to establish connections between several outbreaks. In our study a threshold at 50 core genome SNPs, was proposed to discriminate hospital outbreak. This is in agreement with the current literature where thresholds range from 21 \u003csup\u003e24\u003c/sup\u003e to 80 core genome SNPs \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e to discriminate hospital outbreaks depending on the studied bacterial genus.\u003c/p\u003e \u003cp\u003eWe found that \u003cem\u003eE. hoffmanii\u003c/em\u003e ST104 and related strains were implicated in ICU outbreaks in 2017 and 2018. The presence of an isolate in the hospital environmental surface (CLIN37) suggests a single outbreak. \u003cem\u003eE. hoffmanii\u003c/em\u003e ST254 were implicated in outbreaks in ICU and Pediatrics in 2018 suggesting link between these two events. Interestingly, this ST was previously reported to be implicated in the widespread of transmission of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e in Australia \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In the collection, our results showed that the carbapenemase-encoding genes are primarily associated with an IncL/M type flanking structure. This replicon was implicated in four large and small outbreaks and isolated cases highlighting its significance in New Caledonia. The putative IncL/M plasmid reconstructed with MOB-suite software was predicted to be conjugative and found in 5 different bacterial genera in our collection, emphasizing its notable diffusion potential \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It is therefore essential to enhance monitoring and control of this plasmid, as limiting its spread will help to prevent further CPE outbreaks in NC. Although our short read sequencing data do not allow us to determine whether the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eI\u003c/em\u003eMP\u0026minus;4\u003c/sub\u003e gene was localized on a particular plasmid when multiple plasmid structures are found within the same strain, our results showed that in all cases the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene was located on a class 1 resistance integron. This structure is also widely recognised in Australia for harbouring the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Some CPE contain several entities of the plasmids described in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, which are likely to harbour the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene. However, short-read sequencing often results in the integron being found in small contigs, making it difficult to pinpoint the exact \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e-carrying plasmid. Long reads sequencing is planned to study the genetic environment around these integrons and to confirm the position of the ARGs on a given plasmid. Our results highlight the value of studying plasmids, particularly during dissemination episodes involving different strains but the same plasmid (Pediatry \u0026minus;\u0026thinsp;2018).\u003c/p\u003e \u003cp\u003eLong reads sequencing is currently being implemented at the CHT. Given the results of genomics in this work (dominance of IncL/M) and the speed with which results can be obtained (few hours in Madueno et al., (2024)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e), its implementation appears to be essential to limit the spread of CPE in New Caledonia, particularly if an outbreak is suspected. With plasmids, this time could be further reduced, for example by displaying plasmid profiles on an electrophoresis gel before sequencing. This plasmid gel migration technique could be implemented within the hospital to provide additional, less precise but more timely information for rapid decision-making. This method may demonstrate intra- and interspecies horizontal transmission and thus help hygiene operational teams. Also, once the plasmids have been circularised, RFLP (restriction fragment length polymorphism) approaches combined with separation by gel electrophoresis or plasmid-specific PCR could be used in the future.\u003c/p\u003e \u003cp\u003eWe conducted a retrospective epidemiologic and genomic study, confirming the dominance of IMP-type carbapenemase in NC. This carbapenemase, encoded here by the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene, is prevalent in the Asia-Pacific region \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and has become particularly dominant in Australia \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our study revealed that in over 68% of sequenced isolates, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e was predicted to be carried on an IncL/M type plasmid (AA002 MOB_typer primary cluster). This plasmid type has also been identified in Australian outbreaks described in Sydney \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Interestingly, using MOB-typer prediction, the nearest IncL/M type plasmid (AA002 MOB_typer primary cluster) plasmids obtained using mash distance are reported in strains isolated from The Alfred Hospital in Sydney\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This hospital is particularly associated with medical evacuations of patients from NC. Of the 13 patients who tested positive upon return from these medical evacuations, seven isolates were sequenced. Six of these were found to carry on a IncL/M plasmid, and two of them were linked to local outbreaks (ICU-2017 and Pediatrics-2018). Several clones, such as \u003cem\u003eC. freundii\u003c/em\u003e ST98 with AA860 MOB_typer primary cluster\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e ST20 with AA02 MOB_typer primary cluster \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, with AA739 MOB_typer primary cluster or AA860 MOB_typer primary cluster \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e have been previously reported in Australia. These findings suggest potential epidemiological links between Australia and NC as already reported for Vancomycin resistant \u003cem\u003eEnteroccocus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e or \u003cem\u003eCorynebacterium diphtheriae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition, one patient repatriated from Australia was identified with an \u003cem\u003eEnterobacter xiangfangensis\u003c/em\u003e (CLIN71; formerly named \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ehormaechei\u003c/em\u003e subsp. \u003cem\u003exiangfangensis\u003c/em\u003e) with an IncHI2A type plasmid (AA739 MOB_typer primary cluster), which has been previously reported in this country \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Although our WGS panel included a wide variety of bacterial strains, those belonging to the genus \u003cem\u003eEnterobacter\u003c/em\u003e were predominant. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene is particularly prevalent in this genus in Australia \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eE. xiangfangensis\u003c/em\u003e, the main species in our dataset, has been involved in an outbreak \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eEnterobacter chengduensis\u003c/em\u003e isolates are rarely described as carbapenemase producer in the literature. Apart from CLIN07 (ST414), the only identified \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e carriers have also been reported in neighbouring Australia \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Further genomic analysis and collaboration are essential to confirm the microbial and antibiotic resistance connections between our two territories, but these suspected links with Australia at various points underscore the importance of screening patients returning from medical evacuations.\u003c/p\u003e \u003cp\u003eThe data presented in this study underscores the importance of using screening agar to effectively detect CPE strains carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;\u0026thinsp;4\u003c/sub\u003e gene. Goire \u003cem\u003eet al\u003c/em\u003e. (2016) demonstrated in Australia that screening media such as Brilliance\u0026trade; CRE Agar are insufficiently sensitive for detecting \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;\u0026thinsp;4\u003c/sub\u003e, unlike other CPE types \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;\u0026thinsp;4\u003c/sub\u003e endemic situation, the authors recommended combining ESBL screening media with the CarbaNP test and in-house \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;\u0026thinsp;4\u003c/sub\u003e real-time PCR. Here, we also confirmed the low level of resistance to carbapenems in strains carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;\u0026thinsp;4\u003c/sub\u003e gene \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, which are however well captured when using ESBL agar plate. Our study reported a 15% fatality in infected patients, lower than the rates reported in the literature \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This low fatality rate might be attributed to the limited number of associated resistances, with retained susceptibility to cotrimoxazole (59%) and fluoroquinolones (47%). Additionally, the low level of resistance to carbapenems conferred by \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;\u0026thinsp;4\u003c/sub\u003e allowed the use of certain carbapenems in combination with cotrimoxazole or fluoroquinolones \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe introduction of the cohorting unit to isolate patient seems beneficial in NC, as the number of CPE cases has stabilised, and no further outbreaks have occurred in CHT since its establishment. However, our WGS data are more concerning, as we detected \u003cem\u003eCitrobacter freundii\u003c/em\u003e ST98 in several patients associated with this unit, raising questions about the efficacy of the cohorting system \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Many clones detected from patients were also found in environmental isolates, suggesting an important role of the hospital environment in their spread and persistence. In our study, most of \u003cem\u003eSerratia\u003c/em\u003e isolates originated from the hospital environment, a finding consistent with previous reports highlighting the relevance of environmental reservoirs in ICU \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e or NICU \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e in the endemicity of opportunistic pathogens such as \u003cem\u003eS. marcescens\u003c/em\u003e. Based on these results and litterarure\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, a more in-depth analysis of the hospital environmental surface and notably cohorting unit environment should be considered.\u003c/p\u003e \u003cp\u003eTo summarise, we integrated phenotypic, genomic and epidemiological analyses to investigate the transmission pathways of CPE in NC. Our findings revealed that multiple clones were responsible for outbreaks in ICU and NICU and identified an IncL/M plasmid as probable vehicle for the dissemination of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e in NC since 2013. These results highlight the importance of studying plasmid transmission to better prevent the spread of CPE and support the concept that AMR is a silent pandemic.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eWe conducted a retrospective study to analyse all CPE isolates collected in NC from July 2013 (first isolate detected) to December 2022. NC\u0026rsquo;s healthcare infrastructure includes three public hospitals and one private clinic for patients\u0026rsquo; hospitalization. These facilities operate under the authority of the Department of Health and Social Affairs (DASS) of New Caledonia Government, which has decision-making powers in health-related matters, a feature that is consistent with the administrative structure in France. Study was conducted in the CHT, the main and reference hospital of the archipelago. It comprises 645 beds, 12 operating rooms, medical units, surgery units and intensive care units. The number of hospitalizations approximates 40,000 a year. Moreover, CHT laboratory is the reference laboratory for CPE in NC, where all suspected CPE isolates are sent for expertise. In order to prevent spread of CPE, patients with a CPE at the CHT are transferred in a cohorting unit by a dedicated medical team\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e1. Workflow for CPE isolation at CHT\u003c/h2\u003e\n \u003cp\u003eAll patients admitted to the ICU, and those returning from a foreign hospital, or known previous carriers of CPE were systematically screened for CPE carriage. When a CPE is isolated in a patient, all contacts within the department were also screened. CPE screening involves using a faecal swab culture on ChromID\u0026reg; ESBL (bioM\u0026eacute;rieux, Marcy-l\u0026apos;\u0026Eacute;toile, France), with results available 24 to 48 h after incubation at 37\u0026deg;C. All colony morphologies are identified using MALDI-TOF mass spectrometry (Microflex LT-MS, Bruker Daltonics). Subsequently, Enterobacterales were plated on Mueller-Hinton agar with different antibiotic discs: ertapenem (10\u0026micro;g), temocillin (30\u0026micro;g) and ceftolozane/tazobactam (30/10\u0026micro;g) inspired by the 2022 European Committee on Antimicrobial Susceptibility Testing \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Confirmation was performed using NG-CARBA-5\u0026reg; immunochromatographic assays (NG Biotech Laboratories, Guipry, France), which detects the major carbapenemase types (VIM, IMP, NDM, KPC, and OXA-48-like) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Antimicrobial agent Susceptibility Testing (AST) was systematically conducted for clinical strains on VITEK-2\u0026reg; system (bioM\u0026eacute;rieux, Marcy l\u0026apos;Etoile, France) with AST N-234 or N-372 cards. Finally, carbapenem minimum inhibitory concentrations (MICs) can be confirmed using the Etest\u0026reg; method (bioM\u0026eacute;rieux, Marcy l\u0026apos;Etoile, France).\u003c/p\u003e\n \u003cp\u003eThis approach was applied to all samples collected during the study and retrospectively to all CPE isolates available in our collection. The study included CPE strains from both carrier and infected patients, irrespective of the type of specimen collected, as well as environmental isolates (recovered from table surface and sink in the lavatory in patient rooms, or healthcare trolley) identified during a suspected outbreak for comparison. Furthermore, to ensure the accuracy and reliability of the results, all ASTs were interpreted using the EUCAST guidelines as updated in 2022 \u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2. Collection of clinical data\u003c/h2\u003e\n \u003cp\u003eWe systematically recorded the origin of each sample, specifying the hospital ward or name of the external laboratory, and categorized the sample types into five mains groups: rectal swab, blood culture, urine, respiratory, and other. For all patients hospitalized at CHT, we collected clinical data relevant to the study and recognized as risk factors for CPE acquisition in other studies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Finally, we recorded whether the isolate was likely associated with a spread event. Suspected outbreaks were characterised by the incidental discovery leading to the detection of a secondary case, or the detection of several CPE in the same department at short period (less than one month). In this study, an outbreak was considered as a large outbreak if there were at least five secondary cases, otherwise, if there are fewer than 5 secondary cases, we considered this event as a small outbreak.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3. Whole-genome sequencing\u003c/h2\u003e\n \u003cp\u003eA batch of 64 CPE (corresponding to first isolates in NC or suspected of belonging to outbreaks) isolated from 2013 to 2019 was submitted to WGS by the French National Reference Center of Antimicrobial Resistance using Pasteur P2M core facility. Sequencing was conducted using a NextSeq500 instrument (Illumina, San Diego, CA; 2 \u0026times; 150-nucleotide paired-end protocol). Additionally, WGS was performed on 25 CPE (in order to complete the study period until 2022) collected between 2020 and 2022 by the Beijing Genomics Institute (BGI) Tech Solutions Hong Kong Co., Limited. Isolates were inoculated in Luria Broth medium and incubated overnight at 37\u0026deg;C. Genomic DNA was extracted from 1.5 ml of LB culture using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer\u0026apos;s instructions. DNA purity was determined using Nanodrop (ThermoFisher Scientific, Waltham, MA, USA) and agarose gel deposition. Paired-end libraries were prepared with an insert size of approximately 300 bp and sequenced on DNB-SEQ PE150 according to the supplier\u0026rsquo;s protocol.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003ea. Genome assembly and quality assessment\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePaired-end reads were \u003cem\u003ede novo\u003c/em\u003e assembled using the fq2dna workflow V21.06, which also includes various steps such as trimming, error correction, contaminant removal, and polishing. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gitlab.pasteur.fr/GIPhy/fq2dna\u003c/span\u003e\u003c/span\u003e). For completeness and contamination analysis, CheckM (V1.1.3, taxonomy_wf at genus level) and QUAST version 5.2.0 \u003csup\u003e48\u003c/sup\u003e. were employed.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eb. In-silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;species identification and genomic screening\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMulti-locus sequence typing (MLST) was conducted using MLST software V2.19.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/mlst\u003c/span\u003e\u003c/span\u003e) and associated profiles provided by PubMLST \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e or BIGSdb-Pasteur (\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e species complex). To evaluate ARGs, Abricate software V1.0.1 was employed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/abricate\u003c/span\u003e\u003c/span\u003e; --minid 70 --mincov 90 --db resfinder) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The prediction of genomic location of ARGs, plasmid identification and typing were performed using MOB-suite V3.0.1 \u003csup\u003e27\u003c/sup\u003e. Furthermore, the identification of integrons carrying ARGs was conducted using IntegronFinder V2.0.2 \u003csup\u003e51\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003ec. Phylogeny and relationship between isolates\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe initial phylogenetic relationships between isolates from the different compartments and sample sites were examined using the k-mer-based tool JolyTree V2.1 \u003csup\u003e52\u003c/sup\u003e. The associated tree was midpoint-rooted and annotated with iTOL V6 \u003csup\u003e53\u003c/sup\u003e. Species identification was achieved for each assembly through the utilisation of a digital DNA:DNA hybridization approach (dDDH cut-off \u0026ge;\u0026thinsp;70.0% \u003cem\u003eformula d\u003c/em\u003e\u003csub\u003e\u003cem\u003e4\u003c/em\u003e\u003c/sub\u003e) as facilitated by the Type Strain Genome Server (TYGS) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Regarding \u003cem\u003eEnterobacter\u003c/em\u003e genus, an additional analysis was performed using the approach and taxonomy proposed by Wu \u003cem\u003eet al.\u003c/em\u003e with FastANI V1.33 (ANI cut-off \u0026ge;\u0026thinsp;96.0%) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Due to the significant genetic diversity of the sample and the need for precise comparison within clinical isolates, we conducted core genome-based phylogenetic analyses on isolates of the same sequence type (ST) with more than two assemblies. For each ST group, a good-quality reference genome was identified by using the online tool SimilarGenomeFinder against all public genomes in the BV-BRC resource center (V3.30.19a;) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Mapping was performed using Snippy V4.6.0 against these selected references. The resulting core-SNP alignment was used to infer maximum-likelihood phylogenetic trees for each ST group using IQ-tree V2.1.4 \u003csup\u003e57\u003c/sup\u003e, and recombination elements were removed with ClonalFrameML V1.12 \u003csup\u003e58\u003c/sup\u003e. Then, specific SNP differences matrixes were generated with snp-dists V0.8.2. As proposed by David \u003cem\u003eet al.\u003c/em\u003e, (2019)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, we used 21 core genome SNP as threshold for discrimination of clonal diffusion.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Centre Hospitalier Territorial de Nouvelle-Calédonie, French National Reference Center of antibiotic resistance, Government of New Caledonia [Postdoctoral contract, 2020-2023] and Institut Pasteur de Nouvelle-Calédonie.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.B. and J.C. designed the study, L.C., G.D, P.S. and J.C. delivered clinical data, A.B., M.K. and T.O. performed the microbiology experiments, S.B., R.A.B. and L.D performed the WGS experiments, A.B., F.P. and M.P. performed the bioinformatics analyses, P.S performed statistical analyses, A.B., S.B., R.A.B., L.D., F.V and J.C. purchased the funds, A.B., L.C and J.C. wrote the original manuscript, A.B.,G.D., A.Bi., T.R., R.A.B., L.D., S.B., P.S., M.P., F.V., C.G and J.C revised the manuscript. Every authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccess to the assembly data from this study is available on GenBank, under the BioProject ID PRJNA1146064.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was approved by the CHT ethics committee (Avis N°3 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the contribution of the microbiology laboratory from the Centre Hospitalier Territorial Gaston Bourret and P2M core facility (Institut Pasteur, Paris) for sequencing some isolates. We also thank students of Institut Pasteur de Nouvelle-Calédonie: M. Zami and L. Nemia for the technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWorld Health Organization. \u003cem\u003eAntimicrobial Resistance: Global Report on Surveillance\u003c/em\u003e. (World Health Organization, 2014).\u003c/li\u003e\n \u003cli\u003eWHO. WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance. https://www.who.int/publications-detail-redirect/9789240093461 (2024).\u003c/li\u003e\n \u003cli\u003eStruelens, M. J. \u003cem\u003eet al.\u003c/em\u003e Real-time genomic surveillance for enhanced control of infectious diseases and antimicrobial resistance. \u003cem\u003eFront. Sci.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 1298248 (2024).\u003c/li\u003e\n \u003cli\u003eBaker, K. 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Microbiol.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1919\u0026ndash;1929 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Antibiotic resistance; blaIMP-4, Clinical Outbreak Surveillance, Hygiene Investigations, New Caledonia, Plasmid, Whole Genome Sequencing","lastPublishedDoi":"10.21203/rs.3.rs-5067243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5067243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbapenemase-producing Enterobacterales (CPE) have been identified by the World Health Organization as global priority pathogens. The dissemination of these bacteria and outbreaks within healthcare facilities are of serious concern. This study investigated the transmission patterns and genetic characteristics of CPE isolated in New Caledonia from 2013 to 2022. The isolates were identified and characterized both phenotypically and whole-genome sequencing (WGS). In total 214 CPE were isolated: 199 non duplicate clinical isolates from 164 patients and 15 from hospital environmental surface. The most common genera in clinical samples were \u003cem\u003eEnterobacter\u003c/em\u003e (34%) and \u003cem\u003eKlebsiella \u003c/em\u003e(25%), with 194 isolates (98%) carrying IMP-type carbapenemase. WGS of 89 isolates revealed the dominance of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-4\u003c/sub\u003e carbapenemase gene, found in 82 isolates. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-4\u003c/sub\u003e was primarily predicted to be carried by IncL/M-type plasmid, found in 69% of the sequenced isolates. Our work revealed the circulation of 12 bacterial clusters with 61 strains involved in outbreaks or persistent over time. Genomic, phenotypic and clinical approaches identified 12 distinct outbreaks involving IMP producers. These results highlight the importance of studying plasmid transmission to better prevent silent spread of CPE. Ultimately, this study provides new guidelines for limiting the clinical spread of CPE in New Caledonia.\u003c/p\u003e","manuscriptTitle":"Decade of Carbapenemase-Producing Enterobacterales in New Caledonia: Integrative Surveillance Though Genomic, Phenotypic and Clinical approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 14:11:11","doi":"10.21203/rs.3.rs-5067243/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d52bec02-7b53-44d7-a5b2-342bf53940f9","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":37627689,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":37627690,"name":"Biological sciences/Genetics/Genomics/Medical genomics"}],"tags":[],"updatedAt":"2024-12-09T14:11:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-09 14:11:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5067243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5067243","identity":"rs-5067243","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}