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A total of 272 methicillin-resistant Staphylococcus aureus (MRSA) from equine infections originating from 17 equine hospitals and 39 veterinary practices in Germany as well as 67 isolates from personnel working at equine clinics were subjected to molecular typing. The majority of isolates from horses was attributed to clonal complex (CC) 398 (82.7%). Within CC398, 66% of isolates belonged to a subpopulation (clade) of CC398, which is associated with equine clinics.MRSA attributed to CC8 (ST254, t009, t036, SCCmecIV; ST8, t064, SCCmecIV) were less frequent (16.5%). Single isolates were attributed to ST1, CC22, ST130, and ST1660. The emergence of MRSA CC22 and ST130 in horses was not reported so far. Nasal MRSA colonization was found in 19.5% of veterinary personnel with occupational exposure to horses. The typing characteristics of these isolates corresponded to isolates from equine infections.Comparing typing characteristics of equine isolates with those of a substantial number of isolates from human infections typed at the German Reference Center for Staphylococci and Enterococci (2006–2014; n = 10864) yielded that the proportion of isolates exhibiting characteristics of MRSA from equine medicine is very low (< 0.5%). As this low proportion was also found among MRSA originating from nasal screenings of human carriers not suffering from a staphylococcal infection (n = 5546) transmission of MRSA from equine clinics to the community seems to be rare so far.

1 vancomycin-resistant Enterococcus faecium causing sequential 2 outbreaks over three years in a tertiary care hospital 3 4 Abstract 24 Vancomycin-resistant Enterococcus faecium (VREfm) emerged as an important cause 25 of nosocomial infections worldwide. Previous studies based on molecular typing 26 revealed that VREfm outbreaks are mainly associated with a particular genetic lineage, 27 namely clonal complex 17 (CC17), which harbours either vanA or vanB gene cluster. 28 The University Hospital of Lausanne faced several VREfm episodes of transmissions 29 between 2014 and 2017. In this study, we used whole-genome sequencing (WGS) to 30 investigate the relatedness of 183 VREfm isolates collected from 156 patients. 31Sequence type (ST) 17, ST80 and ST117 were the most predominant clones. Based 32 on epidemiological data, 10 outbreaks were identified, which were caused by at least 33 13 distinct genotypes. The majority of isolates involved in outbreaks (91%) differed by 34 only 0 to 3 SNPs. Four outbreaks involved more than one genotype and half of the 35 cases considered as sporadic were possibly linked to an outbreak. By sequencing all 36 isolates, we were able to better understand our local epidemiology of VREfm. The 37 polyclonal structure observed between the different outbreaks strains, the high level of 38 recombination detected in isolates, the time elapsed between admission and the first 39 VREfm detection and the negative screening at admission support the hypothesis of 40 the emergence of new VREfm clones within the hospitalized population. 41 42 VRE_Manuscript_Revised2.docx 3

In the early 2000s, a particular MRSA clonal complex (CC398) was found mainly in pigs and pig farmers in Europe. Since then, CC398 has been detected among a wide variety of animal species worldwide. We investigated the population structure of CC398 through mutation discovery at 97 genetic housekeeping loci, which are distributed along the CC398 chromosome within 195 CC398 isolates, collected from various countries and host species, including humans. Most of the isolates in this collection were received from collaborating microbiologists, who had preserved them over years. We discovered 96 bi-allelic polymorphisms, and phylogenetic analyses revealed that an epidemic sub-clone within CC398 (dubbed ‘clade (C)’) has spread within and between equine hospitals, where it causes nosocomial infections in horses and colonises the personnel. While clade (C) was strongly associated with S. aureus from horses in veterinary-care settings (p = 2×10−7), it remained extremely rare among S. aureus isolates from human infections.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that fall into two main categories: Crohn’s disease (CD) and ulcerative colitis (UC). The gastrointestinal tract extends from the mouth to the anus and harbors diverse bacterial communities. Several sequencing-based studies have identified an intestinal enrichment of oral-associated bacteria and demonstrated their ability to induce intestinal inflammation in mice, suggesting that intestinal pathobionts originate from the oral cavity, particularly members of the genus Streptococcus. This study aimed to investigate the composition of the salivary and fecal microbiome of IBD patients (n = 14) compared to healthy controls (n = 12) and to determine the abundance of common bacterial taxa in both niches. Metagenomic DNA was extracted from saliva and fecal samples, and the 16S rRNA gene was targeted for sequencing. Our results revealed that the overall microbial composition of saliva was significantly altered in the IBD patients compared to the control subjects (p = 0.038). At the genus level, Veillonella and Prevotella were highly abundant in IBD (median: 25.4% and 22.2%, respectively) compared to the control group (17.9% and 13.4%, respectively). In contrast, Neisseria, Streptococcus, Haemophilus, and Fusobacterium were associated with a healthy gut state. Regarding the fecal microbiome, the IBD group had a significantly higher abundance of Clostridium sensu stricto 1 and Escherichia-Shigella (both comprising pathogenic bacteria) compared with the control group. Members of both bacterial groups have previously been shown to positively correlate with intestinal inflammation and high expression of pro-inflammatory cytokines that disrupt intestinal barrier integrity. In addition, we demonstrate that the increased abundance of Clostridium sensu stricto 1 and Escherichia-Shigella has also been associated with significant upregulation of certain metabolic pathways in the feces of the IBD group, including bacterial invasion of epithelial cells. Streptococcus was the only common genus detected in both the salivary and fecal microbiome and represented the oral-gut axis in our study. Using culture-based methods, we isolated 57 and 91 Streptococcus strains from saliva as well as 40 and 31 strains from fecal samples of the controls and IBD patients, respectively. The phylogenetic tree of streptococci based on sodA sequences revealed several patient-specific clusters comprising salivary and fecal streptococcal isolates from the same patient and belonging to the same species, suggesting that the oral cavity is an endogenous reservoir for intestinal strains.

Periodontitis can result in tooth loss and the associated chronic inflammation can provoke several severe systemic health risks. Adjunctive to mechanical treatment of periodontitis and as alternatives to antibiotics, the use of probiotic bacteria was suggested. In this study, the inhibitory effect of the probiotic Streptococcus salivarius subsp. salivarius strains M18 and K12, Streptococcus oralis subsp. dentisani 7746, and Lactobacillus reuteri ATCC PTA 5289 on anaerobic periodontal bacteria and Aggregatibacter actinomycetemcomitans was tested. Rarely included in other studies, we also quantified the inverse effect of pathogens on probiotic growth. Probiotics and periodontal pathogens were co-incubated anaerobically in a mixture of autoclaved saliva and brain heart infusion broth. The resulting genome numbers of the pathogens and of the probiotics were measured by quantitative real-time PCR. Mixtures of the streptococcal probiotics were also used to determine their synergistic, additive, or antagonistic effects. The overall best inhibitor of the periodontal pathogens was L. reuteri ATCC PTA 5289, but the effect is coenzyme B12-, anaerobiosis-, as well as glycerol-dependent, and further modulated by L. reuteri strain DSM 17938. Notably, in absence of glycerol, the pathogen-inhibitory effect could even turn into a growth spurt. Among the streptococci tested, S. salivarius M18 had the most constant inhibitory potential against all pathogens, followed by K12 and S. dentisani 7746, with the latter still having significant inhibitory effects on P. intermedia and A. actinomycetemcomitans. Overall, mixtures of the streptococcal probiotics did inhibit the growth of the pathogens equally or–in the case of A. actinomycetemcomitans- better than the individual strains. P. gingivalis and F. nucleatum were best inhibited by pure cultures of S. salivarius K12 or S. salivarius M18, respectively. Testing inverse effects, the growth of S. salivarius M18 was enhanced when incubated with the periodontal pathogens minus/plus other probiotics. In contrast, S. oralis subsp. dentisani 7746 was not much influenced by the pathogens. Instead, it was significantly inhibited by the presence of other streptococcal probiotics. In conclusion, despite some natural limits such as persistence, the full potential for probiotic treatment is by far not utilized yet. Especially, further exploring concerted activity by combining synergistic strains, together with the application of oral prebiotics and essential supplements and conditions, is mandatory.

Streptococcus oralis subspecies dentisani is explored as an anti-cariogenic probiotic. Here, subjecting freshly stimulated saliva samples of 35 healthy volunteers, six epidemiologically unrelated and two related strains were isolated (prevalence around 20%) applying a newly developed three-step procedure. Furthermore, the probiotic strain S. dentisani 7746 (AB-Dentisanium®) was tested under a variety of environmental conditions for its inhibitory effect on six S. mutans , two S. sobrinus , 15 other oral or intestinal streptococci, 15 S. dentisani strains, and six representatives of other species including periodontopathogens. All except one of the S. mutans strains were inhibited by 7746 colonies or culture supernatant concentrate but only if either the test cell number was low or the producer or its bacteriocin concentration, respectively, was high. S. sanguinis OMI 332, S. salivarius OMI 315, S. parasanguinis OMI 335, S. vestibularis OMI 238, and the intestinal S. dysgalactiae OMI 339 were not inhibited, while the other 10 streptococcal strains (especially S. oralis OMI 334 and intestinal S. gallolyticus OMI 326) showed a certain degree of inhibition. From the panel of other bacterial species only Aggregatibacter actinomycetemcomitans was slightly inhibited. With the exception of OMI 285 and OMI 291 that possessed a 7746 bacteriocin-like gene cluster, all S. dentisani strains and especially type strain 7747 T were strongly inhibited by 7746. In conclusion, probiotic strain 7746 might antagonize the initiation and progression of dental caries by reducing S. mutans if not too abundant. S. dentisani strains inhibit each other, but strains with similar bacteriocin-related gene clusters, including immunity genes, are able to co-exist due to cross-resistance. In addition, development of resistance and adaptation to 7746-bacteriocins was observed during our study and needs attention. Hence, mechanisms underlying such processes need to be further investigated using omics-approaches. On the manufacturing level, probiotic strains should be continuously tested for function. Further clinical studies investigating inhibition of S. mutans by AB-Dentisanium® are required that should also monitor the impact on the oral microbiome composition including resident S. dentisani strains.

Pseudomonas aeruginosa is one of the main pathogens responsible for nosocomial infections, particularly in Intensive Care Units (ICUs). Due to the complexity of P. aeruginosa ecology, only powerful typing methods can efficiently allow its surveillance and the detection during expanding outbreaks. An increase in P. aeruginosa incidence was observed in the ICUs of the Lausanne University Hospital between 2010 and 2014. All clinical and environmental isolates retrieved during this period were typed with Double locus sequence typing (DLST), which detected the presence of three major genotypes: DLST 1-18, DLST 1-21, and DLST 6-7. DLST 1-18 (ST1076) isolates were previously associated with an epidemiologically well-described outbreak in the burn unit. Nevertheless, DLST 1-21 (ST253) and DLST 6-7 (ST17) showed sporadic occurrence with only few cases of possible transmission between patients. Whole genome sequencing (WGS) was used to further investigate the epidemiology of these three major P. aeruginosa genotypes in the ICUs. WGS was able to differentiate between outbreak and non-outbreak isolates and confirm suspected epidemiological links. Additionally, whole-genome single nucleotide polymorphisms (SNPs) results considered isolates as closely related for which no epidemiological links were suspected, expanding the epidemiological investigation to unsuspected links. The combination of a first-line molecular typing tool (DLST) with a more discriminatory method (WGS) proved to be an accurate and cost-efficient typing strategy for the investigation of P. aeruginosa epidemiology in the ICUs.