The colonization of patients with carbapenemase-producing Enterobacterales (CPE) has been associated with increased mortality among vulnerable individuals, particularly those admitted to intensive care units (ICUs) (Dautzenberg et al., 2015; Tischendorf et al., 2016). Preventing the spread of these multidrug-resistant bacteria in ICUs involves a multifaceted approach, including the promotion and strict adherence to hand hygiene rules, proper disinfection of reusable medical equipment, implementation of strict cleaning and disinfection protocols to reduce environmental contamination, the appropriate use of broad-spectrum antibiotics, education and training of healthcare professionals, implementation of surveillance to quickly detect CPE-associated nosocomial infections, and the quick identification and isolation of CPE carriers to prevent transmission to other patients. Complementing the survey of CPE-associated infections and aiding in the implementation of preventive measures to curb the spread of CPE within ICUs, the epidemiological monitoring of patients carrying CPE is a key-tool to document the epidemiology of these bacteria in ICUs. The ongoing epidemiological data facilitates early detection of potential outbreaks, and assists in the better selection of probabilistic treatments to manage locally acquired infections.

Comprehensive epidemiological data on patient CPE-colonization within ICUs in Mediterranean countries are rather limited, in particular prevalence studies. Prior studies predominantly focused on outbreaks associated with K. pneumoniae carrying the bla_OXA-48 gene, notably the 2011 Casablanca study (Barguigua et al., 2015), and those in 2014 involving patients from Algeria and Tunisia (Cuzon et al., 2015; Mathlouthi et al., 2016). Another significant source of clonal dissemination has been identified in K. pneumoniae carrying the bla_NDM-1 gene, previously documented in Moroccan ICUs in 2011 (Barguigua et al., 2015) and in ICUs in Cairo in 2013 and 2016 (Wassef et al., 2016; Abdulall et al., 2018). A study conducted in 2016 by Hammami and colleagues highlighted that 34.9% of ICU patients at the University Hospital of Tunis harbored CPE with the bla_OXA-48 and/or bla_NDM-1 genes in their intestinal flora (Hammami et al., 2017). Additionally, to the best of our knowledge, the colonization of ICU patients in Mediterranean countries by colistin-resistant CPE has not been extensively reported, except for an outbreak of colistin-resistant bla_OXA48 K. pneumoniae in 2015 among ICU patients at the University Hospital of Mahdia in Tunisia (Mansour et al., 2017). In this instance, the resistance to colistin was not attributed to the presence of mcr genes within the plasmids carried by this K. pneumoniae.

Our study aimed to assess the prevalence of CPE carriage among ICU patients in hospitals across diverse Mediterranean countries. This involved a multicenter prevalence study conducted just before the onset of the COVID-19 pandemic, in the first quarter of 2019 across a group of nine ICUs. Carbapenem-resistant strains, isolated from rectal or fecal samples, underwent molecular analyses to determine their susceptibility to antibiotics. Additionally, whole-genome sequencing was performed on 40 representative CPE strains to document the molecular characteristics of the predominant clones. Examination of the clinical data was also undertaken to investigate the risk factors associated with the intestinal carriage of CPE among patients in the participating ICUs.

A total of 256 ICU patients were included in the study. The clinical data were available for 226 patients (Table 1). For all but one of the participating centers, the number of patients per center was approximately 30. Due to patient refusals to participate, center C8 enrolled only 16 patients. Out of the 256 patients, 100 were women (44.0%), and 126 were men (46.0%), with ages ranging from 6 days to 102 years (mean age: 57 years). Various medical conditions were observed among the patients, with 15.5% having experienced trauma, 31.4% having diabetes mellitus, and 26.1% having malignancies. Among the patient population, 44.5% had recent hospitalization history, and 48.0% had received antibiotic treatment in the last 6 months. The most common antibiotics used included third-generation cephalosporins (28.1%) and carbapenems (10.1%). On the day of the study, 74.2% of the patients were under antibiotic treatment, which primarily consisted of third-generation cephalosporins (37.2%), carbapenems (20.4%), amoxicillin-clavulanic acid (13.3%), or fluoroquinolones (7.5%).

Our comprehensive multicenter point-prevalence survey investigated CPE carriage among 226 ICU patients in nine hospitals across Morocco, Tunisia, Egypt, and Lebanon, conducted pre-COVID-19 pandemic. To our knowledge, this study is unique in providing comprehensive prevalence data on patient colonization in Mediterranean ICUs, distinguishing it as the most recent investigation of its kind.

Firstly, the study unveiled a noteworthy prevalence of CPE strains in eight of the participating ICU centers, demonstrating CPE carriage rates ranging from 16.1 to 69.0% among ICU patients. Additionally, we observed intestinal colonization by colistin-resistant CPE strains in patients from centers 3, 5, and 6. The heightened prevalence of CPE colonization across nearly all ICUs, with the exception of one, increases the risk of subsequent infections in these vulnerable patients harboring highly resistant isolates. This poses inherent challenges in treatment. While it’s highly probable that the prevalence of CPE and colistin-resistant CPE strains has increased during the pandemic (Pascale et al., 2021), our findings are particularly concerning due to the high mortality associated with the identified clones within intensive care settings. Strengthening or set up infection control measures in these ICUs is essential to mitigate the heightened mortality risk among patients. These findings also emphasize the importance of strictly implementing isolation measures for patients originating from the Mediterranean region when transferred to other healthcare institutions.

Secondly, molecular typing of the identified CPE strains unveiled distinct scenarios across centers. In the two ICUs with more than one CPE carrier and no identified clusters (centers C2 and C8), the predominant CPE strains were mostly E. coli strains (8/12; 66.6%). WGS analysis revealed carbapenemase-producing E. coli belonging to three international high-risk clones [ST410 (Roer et al., 2018), ST167 (Roer et al., 2018; Garcia-Fernandez et al., 2020), and ST69 (Shawa et al., 2022)], known for causing life-threatening infections, particularly affecting critically ill patients with severe underlying diseases and comorbidities, and five globally recognized clones, previously reported in livestock and food products (Stolle et al., 2013; Aizawa et al., 2014; Guo et al., 2015; Wang et al., 2018; Vasconcelos et al., 2020). It appears plausible that the acquisition of these genetically diverse and unrelated E. coli strains occurred in the community setting before patients’ hospitalization. In contrast, in the six remaining centers where clusters were identified, CPE strains were predominantly K. pneumoniae strains (32/49; 65.3%). The one-day prevalence study did not afford an investigation into the mechanisms of patient acquisition of CPE strains from these clusters. Longitudinal data tracking CPE colonization over time would offer insights into trends, persistence, and factors influencing acquisition and clearance of CPE strains. However, the WGS analysis showed that the identified K. pneumoniae strains were predominantly associated with international high-risk clones known for their propensity to thrive in the ICU bedside environment and initiate outbreaks (Weterings et al., 2015; Gordon et al., 2017; Liu et al., 2018; Naha et al., 2021; da Silva et al., 2022; De Koster et al., 2022; Edward et al., 2022; Shi et al., 2022). Thus, our data tentatively suggest nosocomial acquisition of CPE K. pneumoniae by patients in the ICUs where clusters were identified. Further evidence is needed for conclusive confirmation.

Concerning the genetic determinants responsible for carbapenem and colistin resistance, our study corroborates previous findings. In alignment with recent global epidemiology of carbapenemase genes (van Duin and Doi, 2017; Pitout et al., 2019), bla_OXA-48 and bla_NDM-1 were the most frequently detected genes among the identified CPE strains. Additionally, consistent with observations in Tunisian patients, the studied colistin-resistant K. pneumoniae strains did not show the presence of mcr-1 to mcr-10 genes (Jaidane et al., 2018).

When comparing carriers and non-carriers, our study did not identify prior hospitalization or antibiotic treatment as significant risk factors for CPE colonization. The limited number of ICU patients and the high rate of hospitalization and antibiotic treatment in our sample may have influenced these results. However, our findings confirmed that ICU stays of 7 days or more significantly increased the risk of CPE colonization [1;2]. Once again, the identification of this risk factor reinforces the hypothesis of a major acquisition of CPE strains in the ICU setting during patient care, potentially exacerbated by the failure to identify and isolate CPE carriers within the participating centers

Our research conclusively establishes the presence and widespread distribution of CPE strains within Mediterranean hosipitals. Significantly, our study adds value by furnishing data on the prevalence of patient colonization and the genetic characteristics of the detected CPE strains—information notoriously challenging to procure. However, the study including a relatively small sample size of ICU patients, with variable enrollment rates across centers, the generalizability of the findings to broader populations may be limited, and the variability in enrollment rates could introduce biases. Prioritizing the awareness of healthcare professionals regarding the escalating threat of antibiotic resistance is crucial, particularly within ICUs. Replicating this study in additional Mediterranean hospitals would promote the implementation of preventive measures and the adjustment of empirical antibiotic treatments for infected ICU patients, if necessary. Finally, our findings highlight the importance of vigilance concerning patients previously hospitalized in Mediterranean facilities and undergoing medical repatriations, to prevent the introduction of CPE into downstream healthcare facilities.

The original contributions presented in the study are publicly available. This data can be found here: https://www.ebi.ac.uk/ena, PRJEB71867.

The study was conducted in collaboration with hospital directors, attending physicians, and the infection control pilot team in Tours, France, in adherence to French Healthcare recommendations for infection prevention. Ethical approvals were obtained at eight hospital centers, ensuring anonymization of patient and sample data prior to analysis. Data from center 9 were not included in the study. The study was conducted following a protocol in adherence to French Healthcare recommendations for infection prevention. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board also waived the requirement of written informed consent for participation from the participants or the participants' legal guardians/next of kin because prior verbal consent was obtained.

SS: Methodology, Writing – original draft, Writing – review & editing, Formal analysis, Investigation. SD: Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing. AB: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. KZ: Data curation, Investigation, Writing – original draft, Writing – review & editing. DG: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. RE-M: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. SE: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. IB: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. AH: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. RC: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. ZD: Data curation, Investigation, Validation, Writing – original draft, Writing – review & editing. LM: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. PF: Formal analysis, Methodology, Software, Validation, Writing – original draft, Writing – review & editing. NM-M: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing.