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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: Gardnerella spp. is often seen as evidence of vaginal pathologies, although members of this genus can also be found in the urinary and vaginal microbiota of asymptomatic women [1,2], making its role in the urogenital tract unclear. Objective: To assess phylogenomic and functional analysis of Gardnerella genus. Resistance (ARGs) and virulence genes (Vg) was also explored to unveil their role in health and urogenital disease development. Methods:Twenty-nine Gardnerella isolates from urine (U, n=22) and vaginal swabs (VSn=7) of women [24 asymptomatic, 5 with overactive bladder (OAB)] were identified by cpn60 and WGS (NovaSeq 6000; Illumina). Together with118 genomes from public databases (from U (n=35), VS (n=82) blood (n=1), and unknown samples (n=2)), we used TYGS platform and fastANI for species identification. Phylogenomic and pangenome analyses were performed using anvi’o v7.1 and Roary. Vg were annotated with COG and KEGG databases, and ARGs with AMRFinder Plus. Vg presence/absence outoputs were analyzed with R. Results: 4 species and 10 genomic species were identified, 41% of the collection being comprised by G. vaginalis. ANI and dDDH values are insufficient for distinguishing Gardnerella species. Pangenome was composed by4537 gene clusters and the core genome of by 514. Differences in carbohydrates and amino acid metabolism and absence of correlation of functions with human body sites or disease were observed. The virulome and Gardnerellaspecies/genomospecies exhibit are intertwined. ARG to aminoglycosides (aph(3')-Ia), macrolides (mefA, msrD, ermX), tetracyclines (tetM, tetL), lincosamide and streptogramins (lsaC) were detected in several isolates. Open pangenomes were observed in G. vaginalis, G. leopoldii, G. swidsinskii and GG3. Conclusions: Gardnerella comprises 4 species and 10 genomic species. Core genome analysis, ANI and dDDH are recommended for appropriate species assignment. Gardnerella species/genomospecies are associated with a particular set of VFs and metabolic functions. ARGs with clinical relevance were also observed in different Gardnerella species., 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)