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The diagnosis of infective endocarditis (IE) remains a challenge. One of the rare bacterial species recently associated with biofilms and negative cultures in infective endocarditis is Aerococcus urinae. Whether the low number of reported cases might be due to lack of awareness and misidentification, mainly as streptococci, is currently being discussed. To verify the relevance and biofilm potential of Aerococcus in endocarditis, we used fluorescence in situ hybridization to visualize the microorganisms within the heart valve tissue. We designed and optimized a specific FISH probe (AURI) for in situ visualization and identification of A. urinae in sections of heart valves from two IE patients whose 16S rRNA gene sequencing had deteced A. urinae. Both patients had a history of urinary tract infections. FISH visualized impressive in vivo grown biofilms in IE, thus confirming the potential of A. urinae as a biofilm pathogen. In both cases, FISH/PCR was the only method to unequivocally identify A. urinae as the only causative pathogen for IE. The specific FISH assay for A. urinae is now available for further application in research and diagnostics. A. urinae should be considered in endocarditis patients with a history of urinary tract infections. These findings support the biofilm potential of A. urinae as a virulence factor and are meant to raise the awareness of this pathogen., Competing Interests: The authors have declared that no competing interests exist.

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.

Aerococcus viridans is a wide spread bacterium in the environment and clinically this organism is associated with different diseases in animals and humans. However, the geno- and phenotypic characterization of A. viridans associated with bovine mastitis has not yet been reported. The objectives of this study were to investigate the genetic and phenotypic diversity of A. viridans isolates using three different molecular methods including 16S rRNA gene sequencing, pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD) along with biochemical tests, including antimicrobial susceptibility test. In total, 60 A. viridans strains were cultured from dairy herds presenting with subclinical mastitis. The results of biochemical tests revealed that most of the isolates (75.0%) were accurately identified by API Rapid 20 Strep system and the majority of A. viridans strains (96.7%) were found to be catalase negative, while two (3.3%) isolates were weakly positive. All isolates were resistant to trimethoprim-sulfamethoxazole, followed by streptomycin (96.7%), tetracycline (65.0%) and clindamycin (56.7%) by minimum inhibition concentration-determining broth microdilution technique. As compared to the sequence of 16S rRNA gene, both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A. viridans. Furthermore, most of the isolates obtained from the same herd or region belonged to the same major RAPD group, which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A. viridans. This is a novel approach to use three molecular techniques and to compare their efficiency regarding the genetic diversity of A. viridans. The data suggest that A. viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.