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Background: Enterococcus lactis (Elts) [former Enterococcus faecium (Efm) clade-B] has been greatly associated with human colonization, but its epidemiology is unknown since this reclassification [1]. We aimed to assess the contemporary faecal carriage of enterococci species among healthy-humans (HH) in Portugal and get novel insights about Efm/Elts differences. Methods: Fifty-one faecal samples (29-women/22-men;18-85/~45-years) from HH in Northern Portugal (February-July 2022) were processed by enrichment/selection steps with/without ampicillin, vancomycin or linezolid. Efm, Elts and other species were identified by PCR [2, 3] and antibiotic-susceptibility by disk-diffusion/broth-microdilution (EUCAST/CLSI). Representatives/sample (n=40) were characterized by Whole-Genome-Sequencing/CGE-tools, including a homemade bacteriocins(bac) database. Qualitative bacteriocin production/sensitivity was performed in sequenced Efm/Elts and selected clinical VREfm (vancomycin-resistant-Efm) and Elts (all-against-all) using the soft-agar-overlay technique. Results: All samples carried Enterococcus (n=337), with most containing Elts-73% (p, Scientific Letters, Vol. 1 No. Sup 1 (2023)

Background: Vancomycin-resistant Enterococcus faecium (VREfm) prevalence greatly varies in European countries yet continues to rise [1]. We aimed to characterize clinical-VREfm from a local hospital (800-beds) during 2009-2021. Methods: VREfm [n=175; mostly from urine (40%)] were collected from medical-47% and surgical-32% wards. Antibiotic-resistance (ABR) and occurrence of vanA/vanB, plasmids [rep-pLG1/rep-pRUM/rep-Inc18(rep1/rep2)] and virulence genes were screened by disk-diffusion/broth-microdilution (EUCAST/CLSI) and PCR, respectively. Representative VREfm/year (n=81) were selected for MLST+WGS (Illumina-NovaSeq) and analysed through CGE-tools (homemade virulence and bacteriocins(bac) databases) and cgMLST/Ridom-SeqSphere+. Results: All isolates were resistant to glycopeptides [98%-vanA;2%-vanB], ampicillin, ciprofloxacin, erythromycin [erm(B)/msr(C)], less to gentamicin (49%;aac(6’)-Ie-aph(2’’)-Ia), tetracycline [27%; tet(M)/tet(L)], quinupristin-dalfopristin (5%) and/or linezolid (2%;GT2576 mutation). VREfm were oligoclonal (until 7 STs/year), mostly clustering into ST117-31%, ST78-19% and ST80-12%. For each ST, different complex-types (CT) were detected each year, but some were identified in different wards from 3-months (ST412-CT258/ST117-CT4659) to 2-4-years (ST117-CT24/CT6602/CT6603; ST78-CT330). Sequenced VREfm showed slight overlap (ST117-CT24 and ST78-CT230) with other global Efm. Recent years (>2019) concentrated the majority of novel CTs, without common clones over time, and ST494 (associated with a German outbreak) was introduced. ABR and virulence patterns were similar throughout the years. Bac-profiles (13 patterns) were similar between common STs/CTs. One new bacteriocin was detected >2017, Bac43 increased over time and BacAS9 was exclusively detected in ST117. The typical VREfm plasmidome [RepA_N-pRUM/pLG1-like, Inc18, Rep3-pB82/pEF418, Rep_Trans-pEFNP1/pRI1] was observed across the years however, Rep-pRUM decreased. A novel RepA_N-replicase (69% nucleotide identity with pRUM) was identified in most VREfm during 2009-2021. In silico analysis showed its occurrence in ~100 Efm-genomes/GenBank from different countries after 2000. Conclusion: Current clinical VREfm display a strong oligoclonal nature. Nosocomial transmission events and/or import of VREfm from colonized patients may explain the long-term persistence of particular clones. The continuous flux between community and hospital clones may feed the emergence of new lineages/opportunities for adaption hampering classical infection control interventions., Scientific Letters, Vol. 1 No. Sup 1 (2023)

Background: Staphylococcus aureus is the leading bacterial cause of death globally [1]. Nasal carriage of S. aureus increases the risk of invasive infections, including by methicillin-resistant S. aureus (MRSA) strains, but studies including Portuguese university students (PUS) are scarce. Objective: To analyse the prevalence of methicillin-susceptible S. aureus (MSSA) and MRSA among PUS enrolled in different courses/years (1st-4th) at IUCS-CESPU, characterize their antibiotic resistance profiles, and assess the potential risk factors. Methods: Swabs collected during March-December 2022 from anterior nares of 156 volunteers (median 22-years) were processed in mannitol-salt agar and, in parallel, enriched in brain-heart broth with NaCl 6.5% further plated onto ChromID® MRSA SMART. Typical colonies were stored for species identification (MALDITOF-MS) and antibiotic susceptibility testing (disk diffusion; EUCAST/CLSI guidelines). Each student completed a questionnaire comprising demographic/clinical/social parameters. Statistical analysis was conducted in IBM-SPSS Statistics 26 using binary logistic regression applying a backward stepwise (likelihood ratio) method, with α=0.05, selecting variables using Chi-square tests and Mann-Whitney U tests for which p≤0.20, >10 occurrences, not biologically correlated [2]. Results: Prevalence of MSSA and MRSA (cefoxitin screening) were 28.8% and 1.9%, respectively. From the 45 positive samples, 9% were multidrug-resistant, 38% were resistant to penicillin, 40% to erythromycin, 40% to clindamycin (inducible), 7% to cefoxitin, 2% to tetracycline, and 2% to rifampicin. Self-reported frequent contact with animals (OR=3.44, CI 95%: 1.10–10.66) were positively associated with S. aureus, while regular sports participation presented a negative association (OR=0.36, CI 95%: 0.17–0.77). Sports participation was not correlated with self-reported excellent health (χ2=0.680, p=0.409). Conclusions: This is one of the first studies assessing MSSA/MRSA rates in PUS after the COVID-19 pandemics imposing higher self-protection/hygienization. While PUS-MSSA rates are similar to that previously observed, PUS-MRSA rates are slightly higher. Additional samples are being processed to explore future trends and other potential One Health factors influencing MSSA/MRSA colonization., Scientific Letters, Vol. 1 No. Sup 1 (2023)

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years, recently acquiring higher relevance in nosocomial infections control worldwide. Despite of this, CHX tolerance has been poorly understood amongEnterococcus faecium, one of the leading agents causing nosocomial infections. This study provides new phenotypic and molecular data for a better identification of CHX tolerantE. faeciumsubpopulations in community and clinical contexts. MICCHXdistribution of 106E. faeciumsuggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of 2CS-CHXToperon, here identified. It encodes a glucose and an amino-acid-polyamine-organocation family transporters, besides the DNA-binding-response-regulator ChtR with a P102H mutation previously described in only CHX tolerant clade A1E. faecium, and the ChtS sensor. Combined data from normal MIC distribution and 2CS-CHXToperon characterization supports a tentative epidemiological cut-off (ECOFF) of 8 mg/L to CHX, useful to detect tolerantE. faeciumpopulations in future surveillance studies. The spread of tolerantE. faeciumin diverse epidemiological backgrounds alerts for a prudent use of CHX in multiple contexts.ImportanceChlorhexidine is one of the substances included in the World Health Organization’s List of Essential Medicines which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic since the 1950s in healthcare (skin, hands, mouthwashes, eye drops), its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here we provide a comprehensive study on chlorhexidine tolerance amongEnterococcus faecium, one of the leading nosocomial agents worldwide, and identified a novel 2CS-CHXToperon as a signature of tolerant strains occurring in diverse phylogenomic groups. Finally, our data allowed to propose a tentative epidemiological cut-off of 8 mg/L, useful to detect tolerantE. faeciumpopulations in future surveillance studies in community and clinical contexts.

Wild birds have been suggested to be reservoirs of antimicrobial resistant and/or pathogenic Enterococcus faecalis (Efs) strains, but the scarcity of studies and available sequences limit our understanding of the population structure of the species in these hosts. Here, we analysed the clonal and plasmid diversity of 97 Efs isolates from wild migratory birds. We found a high diversity, with most sequence types (STs) being firstly described here, while others were found in other hosts including some predominant in poultry. We found that pheromone-responsive plasmids predominate in wild bird Efs while 35% of the isolates entirely lack plasmids. Then, to better understand the ecology of the species, the whole genome of fivestrains with known STs (ST82, ST170, ST16 and ST55) were sequenced and compared with all the Efs genomes available in public databases. Using several methods to analyse core and accessory genomes (AccNET, PLACNET, hierBAPS and PANINI), we detected differences in the accessory genome of some lineages (e.g. ST82) demonstrating specific associations with birds. Conversely, the genomes of other Efs lineages exhibited divergence in core and accessory genomes, reflecting different adaptive trajectories in various hosts. This pangenome divergence, horizontal gene transfer events and occasional epidemic peaks could explain the population structure of the species.