Contribution of MALDI-TOF mass spectrometry and machine learning including deep learning techniques for the detection of virulence factors of Clostridioides difficile strains.

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.

Investigation of a Mycobacterium fortuitum catheter-related bloodstream infection in an oncology unit.

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.

Environmental severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contamination in hospital rooms during the first and third coronavirus disease 2019 (COVID-19) waves.

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.

[Clostridioides difficile: updated recommendations]. / Clostridioides difficile : des recommandations actualisées.

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.

CLOSTRIDIOIDES DIFFICILE: DES RECOMMANDATIONS ACTUALISÉES Clostridioides difficile est un bacille à Gram positif anaérobie sporulé responsable d'un large spectre d'infections digestives : de la diarrhée simple spontanément résolutive à la colite pseudomembraneuse qui peut se compliquer de mégacôlon toxique, de perforation et entraîner le décès du patient. Les principaux facteurs de risque d'infections à C. difficile (ICD) sont l'âge supérieur à 65 ans, l'administration d'antibiotiques et les antécédents d'hospitalisation. La virulence des souches est liée à la production de deux toxines, TcdA et TcdB. L'émergence et la dissémination rapide d'un clone dit « hypervirulent ¼ (027/ NAP/BI) en 2005 a stimulé la recherche clinique et fondamentale. Des avancées majeures ont été réalisées tant sur le plan épidémiologique (meilleure reconnaissance des formes communautaires d'ICD), diagnostique (nouveaux tests moléculaires) que thérapeutique (nouveaux traitements comme la fidaxomicine, le bezlotoxumab, ou la transplantation de microbiote fécal) ; ces progrès ont permis la mise à jour des recommandations de prise à charge, sous l'égide de la Société européenne de microbiologie clinique et des maladies infectieuses (ESCMID). Le traitement antibiotique des ICD dépend à la fois de la sévérité de l'infection, du risque et du nombre de récidives. Le contrôle de l'infection requiert, d'une part, la maîtrise de la consommation d'antibiotiques et, d'autre part, la mise en œuvre de précautions « contact ¼ vis-à-vis des patients infectés.

Small RNA-mediated regulation of the tet(M) resistance gene expression in Enterococcus faecium.

We investigated the role of a novel small RNA expressed in Enterococcus faecium (named Ern0030). We revealed that ern0030 was encoded within the 5'untranslated region of tet(M), a gene conferring tetracycline resistance through ribosomal protection. By RACE mapping, we accurately determined the boundaries of ern0030, which corresponded to Ptet. This upstream sequence of tet(M), Ptet, was previously described within transcriptional attenuation mechanism. Here, Northern blot analyses revealed three transcripts of different lengths (ca. 230, 150 and 100 nucleotides) expressed from Ptet. Phenotypically, the total deletion of ern0030 conferred a decrease in tetracycline MICs that was consistent with gene expression data showing no significant tet(M) induction under tetracycline SIC in ern0030-deleted mutant as opposed to a 10-fold increase of tet(M) expression in the wild-type strain. We investigated the transcriptional attenuation mechanism by toeprint assay. Whereas the expected tet(M) ribosome-binding site (RBS) was detected, the RBS of the putative leader peptide was not highlighted by toeprint assay, suggesting the transcriptional attenuation was unlikely. Here, we demonstrate that Ern0030 has a role in regulation of tet(M) expression and propose a novel model of tet(M) regulation alternative or complementary to transcriptional attenuation.

The Regulatory RNA ern0160 Confers a Potential Selective Advantage to Enterococcus faecium for Intestinal Colonization.

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.

Distinct expression profiles of regulatory RNAs in the response to biocides in Staphylococcus aureus and Enterococcus faecium.

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.