Enterococcus faecium is a commensal of the human gut microbiota that can become an opportunistic pathogen, particularly in immunocompromised individuals. Small RNAs (sRNA) are thought to contribute to this shift by enabling rapid bacterial adaptation to environmental changes. Despite this, knowledge of sRNA in E. faecium remains limited. Ern0160, in particular, has attracted interest for its involvement in antibiotic and biocide responses, as well as its role in intestinal colonization in a murine model. In this study, we investigated the functions of Ern0160 in E. faecium Aus0004 reference strain and sought to identify its mRNA targets. Transcriptomic and in silico analyses revealed potential regulatory targets, including two homologous genes encoding LysM-containing domain proteins (EFAU004_01059 and EFAU004_01150), both associated with enterococcal pathogenicity. Experimental validation confirmed that increased expression of Ern0160 led to repression of these genes. We further demonstrated direct and specific interactions between Ern0160 and the two homologous target mRNAs. Functional assays in the Galleria mellonella larvae infection model showed that deletion of Ern0160 resulted in increased host mortality, whereas deletion of its targets genes resulted in decreased mortality. These results are consistent with previous findings linking these genes to E. faecium virulence in murine model of systemic and urinary tract infections. Our findings suggest that Ern0160 contributes to a regulatory network that modulates E. faecium colonization and infection by targeting genes involved in antimicrobial response and virulence. This study highlights the potential of regulatory RNAs such as Ern0160 to shape the pathogenic behavior of a multi-drug resistant and clinically significant bacterium.

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA(+)B(+)CDT(+), 22 ToxA(+)B(+)CDT(+) Hv(+) and 105 ToxA(+)B(+)CDT(-)) and 50 non-toxigenic (ToxA(-)B(-)) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA(-)B(-) strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA(+)B(+)CDT(-) strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA(+)B(+)CDT(+) strains. The classifiers' performances for Hv strain detection were linked to high specificity (>= 0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

In 2016-2019, hospital A's haematology ward experienced an outbreak of OXA-48-producing ST-22 Citrobacter freundii strains, with toilets identified as source of transmission. Between 2020 and 2022, 28 strains of OXA-48-producing ST-22 C. freundii were isolated on other wards. This study aimed to determine whether all OXA-48-producing ST-22 C. freundii strains belonged to the same clone and to investigate the persistence of this clone using whole genome sequencing. OXA-48-producing ST-22 C. freundii strains collected from patients (n = 33) and from the hospital environment (n = 20) of seven wards were sequenced using Illumina technology and clonal relationships were determined using single nucleotide polymorphism (SNP). Phylogenetic analyses were performed on 53 strains from hospital A and on 240 epidemiologically unrelated carbapenem-resistant ST-22 C. freundii isolated from elsewhere in France. SNP analysis suggested long-lasting persistence of the same clone for more than 6 years. Phylogenetic analysis showed that 52 of 53 strains isolated in hospital A belonged to the same cluster and were different from the 240 epidemiologically unrelated C. freundii ST-22. Our data suggest that this clone can persist in hospital environments for years, representing a risk for hospital-acquired infections and outbreaks. Reservoir management is essential to prevent further transmission.

Carbapenemase-producing Enterobacterales (CPE) represent a rapidly growing threat to global health. Accumulating evidence has shown the role of hospital wastewater environment as reservoirs for CPE, but also as a source of infection in some outbreaks [ 1 Park S.C. Parikh H. Vegesana K. Stoesser N. Barry K.E. Kotay S.M. et al. Risk factors associated with carbapenemase-producing Enterobacterales (CPE) positivity in the hospital wastewater environment. Appl Environ Microbiol. 2020; 86: e01715-e01720 Crossref PubMed Google Scholar , 2 Regev-Yochay G. Smollan G. Tal I. Pinas Zade N. Haviv Y. Nudelman V. et al. Sink traps as the source of transmission of OXA-48-producing Serratia marcescens in an intensive care unit. Infect Control Hosp Epidemiol. 2018; 39: 1307-1315 Crossref PubMed Scopus (41) Google Scholar , 3 Jolivet S, Couturier J, Vuillemin X, Gouot C, Nesa D, Adam M, et al. Outbreak of OXA-48-producing Enterobacterales in a haematological ward associated with an uncommon environmental reservoir, France, 2016 to 2019. Available at: https://pubmed.ncbi.nlm.nih.gov/34047273/ [last accessed December 2023]. Google Scholar ]. We conducted a study to determine the prevalence of CPE contamination in water-related environment in our 670-bed acute care hospital, outside any epidemic context or associations with known CPE carriers.

BACKGROUND:The duration of extensively drug-resistant bacteria (XDR) carriage depends on several factors for which the information can be difficult to recover. AIM:To determine whether past screening and clinical results of patients can predict the results of subsequent screening. METHODS:In total, 256 patients were retrospectively included from 10 healthcare centres in France from January 2014 to January 2022. We created a predictive clearance score, ranging from -5 to +7, that included the number of XDR species and the type of resistance detected in the sample, as well as the time from the last positive sample, the number of previous consecutive negative samples, and obtaining at least one negative PCR result in the collection. This score could be used for the upcoming rectal screening of a patient carrying an XDR as soon as the last screening sample was negative. FINDINGS:The negative predictive value was >99% for score ≤0. The median time to achieve XDR clearance was significantly shorter for a score of 0 (443 days (259-705)) than that based on previously published criteria. CONCLUSION:This predictive score shows high performance for the assessment of XDR clearance. Relative to previous guidelines, it could help to lift specific infection prevention and control measures earlier. Nevertheless, the decision should be made according to other factors, such as antimicrobial use and adherence to hand hygiene.

We describe a case of healthcare-associated bloodstream infection due to Mycobacterium fortuitum . Whole-genome sequencing showed that the same strain was isolated from the shared shower water of the unit. Nontuberculous mycobacteria frequently contaminate hospital water networks. Preventative actions are needed to reduce the exposure risk for immunocompromised patients.

Background: Extended-spectrum beta-lactamase-producing Enterobacterales (eESBL) have high prevalence in hospitals, but real-time monitoring of nosocomial acquisition through conventional typing methods is challenging. Moreover, patient-to-patient transmission varies between the main species, namely Escherichia coli and Klebsiella pneumoniae, which draws into question the relevance of applying identical preventive measures.Aim: To detect eESBL cross-transmission events (CTEs) using the quantitative antibiogram (QA) method in combination with epidemiological data (combined QA method), and to determine the effectiveness of standard or contact precautions for eESBL species. Methods: First, a validation set was used to compare the relevance of the combined QA method with a combination of pulsed-field gel electrophoresis and epidemiological data (combined PFGE method). Next, a 4-year retrospective analysis was conducted to detect eESBL CTEs in hospitalized patients. Two species were screened - ESBL E. coli (ESBL-Ec) and ESBL K. pneumoniae (ESBL-Kp). During the study, standard precautions alone were applied to patients with ESBL-Ec, whereas contact precautions were applied for patients with ESBL-Kp. Findings: As a proof of concept, use of the combined QA method and the combined PFGE method for the detection of CTEs gave identical results for E. coli, and similar results for at least 75% of K. pneumoniae. Overall, 722 patients with ESBL-Ec isolates and 280 patients with ESBL-Kp isolates were included in the retrospective analysis. Nine and 23 CTEs were identified for E. coli and K. pneumoniae, respectively, involving 20 (2.7%) and 36 (12.8%) patients, respectively.Conclusion: The combined QA method constitutes a rapid tool for epidemiological surveillance to detect CTEs. In the study hospital, standard precautions are sufficient to prevent acquisition of ESBL-Ec, whereas contact precautions must be implemented to prevent acquisition of ESBL-Kp.(c) 2023 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

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Clostridioides difficile is a major nosocomial pathogen and has a considerable burden on healthcare systems. Our objective was to determine the transmission patterns of C. difficile in a non-epidemic setting using whole-genome multi-locus sequence typing (wgMLST) and core-genome single-nucleotide polymorphism (cgSNP) analyses. A retrospective study was conducted in a 650-bed university hospital between January 2016 and February 2017. In total, 191 strains isolated from 169 symptomatic C. difficile infection (CDI) patients were analyzed by WGS. Sequences were compared using wgMLST and cgSNP analyses. Genetic data and ward movements were then combined to identify the transmission rate and the type of transmission. The transmission rate varied from 55/169 (19.5%) (wgMLST) to 33/169 (32.5%) (cgSNP). Most transmission was considered cryptic, irrespective of the genetic analysis (38/55 [69.1%] by wgMLST to 25/33 [75.8%] by cgSNP). No transmission within the same ward was observed. In a non-epidemic setting, most C. difficile transmission occurs from sources other than symptomatic CDI patients.

A set of putative novel small RNAs was recently identified as expressed in Enterococcus faecium , a major opportunistic pathogen involved in numerous healthcare-associated infections and hospital outbreaks. The aim of this study was to characterize the first functional analysis of one of them, srn0030 , by phenotypic, genomic and transcriptomic approaches. By genomic analysis and RACE mapping, we revealed the presence of this RNA (previously designated as P tet ) within the 5’-untrasnlated region (UTR) of tet (M), a gene conferring tetracycline resistance through ribosomal protection. The regulatory mechanism has previously been described as transcriptional attenuation, but has actually been poorly characterized. Hence, we provide original additional data, especially the presence of three upstream transcripts of ~100, ~150 and ~230 nt within the 5’-UTR of tet (M), suggesting an alternative regulatory mechanism. The total deletion of these three transcripts causes an unexpected decreasing of tetracycline resistance in E. faecium . The attenuation mechanism was investigated, and we confirmed that the transcriptional read-through and tet (M) overexpression induced by tetracycline addition but the function of putative peptide leader on attenuation mechanism was not supported by our data. We report here new phenotypic and transcriptomic observations in E. faecium demonstrating an alternative regulatory mechanism of tet (M) gene expression.

CLOSTRIDIOIDES DIFFICILE:UPDATED RECOMMENDATIONS Clostridioides difficile is a spore-forming anaerobic enteropathogen responsible for a wide spectrum of clinical features ranging from mild uncomplicated diarrhoea to severe debilitating disease, toxic megacolon, or even perforation and sometimes death. Risk factors for CDI include age >65 years, previous hospitalization and recent antibiotic therapy. Main virulence factors of C. difficile are toxins A (TcdA) and B (TcdB). The emergence and dissemination of a new hypervirulent strain (027/NAP/BI) in 2005 has stimulated clinical and basic research on C. difficile. Major advances have been made regarding the CDI epidemiology (better recognition of community acquired CDI), diagnosis (molecular tests) and therapy (new drugs such as fidaxomicin, bezlotoxumab, and fecal microbiota transplantation) aspects. These advances have allowed the updating of management recommendations, under the aegis of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Antibiotic treatment of CDI depends on both the severity of the infection, and the risk/number of recurrences. Prevention of CDI requires an antimicrobial stewardship policy and the implementation of contact precautions for the infected patients.

We investigated the frequency, distribution, and risk factors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) environmental contamination around infected patients during the first and third wave of the coronavirus disease 2019 pandemic. The shedding of SARS-CoV-2 in rooms of infected patients was limited in our hospital setting.

The aim of the study was to characterize the antimicrobial activity of clinically-relevant biocides (chlorhexidine digluconate, benzalkonium chloride, PVP-iodine and triclosan) and to determine the sRNA expression profiles under biocide exposure in two major bacterial opportunistic pathogens, Enterococcus faecium and Staphylococcus aureus. In vitro activities were evaluated against S. aureus HG003 and E. faecium Aus0004. We determined MIC, MBC, sub-inhibitory concentrations (SIC) and growth curves under SIC conditions. sRNA expression study under SIC exposure of biocides was performed by RT-qPCR on 3 sRNAs expressed in S. aureus (RNAIII, SprD and SprX) and the first 9 sRNAs identified as expressed in E. faecium. MICs were higher against E. faecium than for S. aureus. Growth curves under increasing biocide concentrations highlighted two types of bactericidal activity: "on/off" effect for chlorhexidine, benzalkonium chloride, PVP-iodine and a "concentration-dependent" activity for triclosan. Exposure to biocide SICs led to an alteration of several sRNA expression profiles, mostly repressed. The distinct biocide activity profiles must be evaluated with other compounds and bacterial species to enrich the prediction of resistance risks associated with biocide usage. Biocide exposure induces various sRNA-mediated responses in both S. aureus and E. faecium, and further investigations are needed to decipher sRNA-driven regulatory networks.

Objective: We aimed to report a community outbreak of an uncommon methicillin-resistant Staphylococcus aureus (MRSA) originating in a maternity ward. Patients and methods: Cases were defined by epidemiological, clinical, and microbiological investigations. Microbiological investigations included phenotypic analysis, molecular typing, and whole-genome sequencing. To control the outbreak, we applied both national recommendations to prevent in-hospital transmission and the French High Council for Public Health guidelines on the management of community-acquired MRSA infections. Results: Between March and July 2016, seven patients with MRSA infections were identified: six skin and soft tissue infections and one pulmonary infection, including six microbiologically confirmed infections. Infections occurred in community settings, but a link with the same maternity ward was found for all patients. All MRSA strains had a t690 spa type, were tetracycline-resistant, and produced Panton-Valentine leukocidin. All isolates belonged to the sequence type 88 (ST88). Conclusion: This outbreak highlights the largely underestimated risk of healthcare-associated infections in maternity wards. Healthcare workers should be aware of the importance of standard hygiene precautions and use of alcohol-based hand sanitizers for neonates and mothers.

The aim of this study was to evaluate the role of the regulatory small RNA (sRNA) Ern0160 in gastrointestinal tract (GIT) colonization by Enterococcus faecium. For this purpose, four strains of E. faecium were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Δ0160), a trans-complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). Strains were studied both in vitro and in vivo, alone and in competitive assays. In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured single while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. Then, in vivo experiments were performed using a GIT colonization mouse model. Several existing models of GIT colonization were compared while a novel one, combining ceftriaxone and amoxicillin, was developed. A GIT colonization was performed with each strain alone, and no significant difference was noticed. By contrast, significant results were obtained with co-colonization experiments. With WT + Δ0160 suspension, a significant advantage for the WT strain was observed from day 5 to the end of the protocol, suggesting the involvement of ern0160 in GIT colonization. With Δ0160_0160 + Δ0160_pAT29 suspension, the strain with the empty vector took the advantage from day 3 to the end of the protocol, suggesting a deleterious effect of ern0160 overexpression. Altogether, these findings demonstrate the potential implication of Ern0160 in GIT colonization of E. faecium. Further investigations are needed for the identification of sRNA target(s) in order to decipher underlying molecular mechanisms.

Pendant la crise sanitaire liée au SARS-CoV-2, le Centre d'appui pour la Prévention des infections associées aux soins (CPias) a fortement été sollicité par les différents secteurs de l'offre de soins. Un outil informatique commun a été créé en 2019 pour tracer les demandes d'appui du CPias. L'objectif est d'analyser les thèmes abordés, les demandeurs et les réponses émises. La saisie de ces données se fait à partir d'un formulaire en ligne. L'analyse des données est réalisée avec le logiciel statistique SAS®. Après un contrôle de la qualité des données, une analyse descriptive des conseils donnés a été réalisée. Ainsi, les variables recensées sont : la date, la fonction des appelants, le statut et le type de leurs établissements (sanitaire, ESMS, secteur libéral…) et les thèmes des demandes. Une fois la base de données exportée et les demandes étiquetées « COVID-19 » filtrées, une seconde analyse qualitative des sous-thèmes a été faite. Entre le 1er février et le 31 mai 2020, le CPias a répondu à 886 sollicitations sur le thème du COVID-19 contre 640 sur toute l'année 2019 (tous thèmes confondus), avec un pic visible autour de la semaine 12. Ainsi, 64 % (466) des demandes émanent d'établissements médicosociaux, 26 % (189) d'établissements sanitaires, 7 % (48) de tutelles et 3 % (22) de la médecine ambulatoire. Quarante-trois pour cent (363) de ces demandes sont faites par des professionnels de santé (à parts égales entre professionnels médicaux et paramédicaux), 26 % (217) par des personnels non soignants (dont 80 % (174) de directeurs de structures médicosociales), 21 % (175) par des hygiénistes (praticiens et infirmiers) et 10 % (90) d'autres professionnels. Les thèmes les plus souvent abordés concernent pour 30 % (264) les équipements de protection individuelle (conseils pour leur bonne utilisation, gestion de la pénurie), pour 23 % (206) l'environnement (entretien des locaux, gestion du risque environnemental) et pour 21 % (185) la conduite à tenir face à des patients/résidents suspects, positifs ou revenant d'hospitalisation. Viennent ensuite des questions sur la conduite à tenir pour les professionnels soit 14 % (123) (suspects/contact, ceux travaillant en ville ou à domicile) et 14 % (120) sur la maladie elle-même (contagiosité, symptômes, prélèvement, unité dédiée aux patients COVID ou suspects). Parfois ces questions ont été relayées aux infectiologues selon le niveau d'expertise requis. Enfin, 11 % (98) des demandes sont d'ordre organisationnel autour des flux internes et de l'organisation des consultations. Le CPias a été très investi dans ses missions régionales de prévention et contrôle de l'infection pendant cette crise sanitaire. Nous en tirons que de nombreux acteurs en santé avaient besoin d'une adaptation ou d'une interprétation pratique des doctrines nationales et/ou d'être confortés dans les conduites à tenir. L'analyse montre également la diversité des appelants sortants du cadre habituel de nos interlocuteurs témoignant de l'importance de l'épidémie.