Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and responsible for causing life-threatening infections. The emergence of hypervirulent and multidrug-resistant (MDR) S. aureus strains led to challenging issues in antibiotic therapy. Consequently, the morbidity and mortality rates caused by S. aureus infections have a substantial impact on health concerns. The current worldwide prevalence of MRSA infections highlights the need for long-lasting preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine candidates and drug target proteins against the 16 strains of MRSA using reverse vaccinology and subtractive genomics approaches. Using the reverse vaccinology approach, 4 putative antigenic proteins were identified; among these, PrsA and EssA proteins were found to be more promising vaccine candidates. We applied a molecular docking approach of selected 8 drug target proteins with the drug-like molecules, revealing that the ZINC4235426 as potential drug molecule with favorable interactions with the target active site residues of 5 drug target proteins viz., biotin protein ligase, HPr kinase/phosphorylase, thymidylate kinase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase, and pantothenate synthetase. Thus, the identified proteins can be used for further rational drug or vaccine design to identify novel therapeutic agents for the treatment of multidrug-resistant staphylococcal infection.

Antimicrobials in wastewater promote the emergence of antibiotic resistance, facilitated by selective pressure and transfer of resistant genes. Enteric bacteria belonging to Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Citrobacter species (n = 126) from hospital effluents and proximate wastewater treatment plant were assayed for susceptibility to four antimicrobial classes. The β-lactamase encoding genes harbored in plasmids were genotyped and the plasmids were sequenced. A multidrug resistance phenotype was found in 72% (n = 58) of E. coli isolates, 70% (n = 43) of Klebsiella species isolates, and 40% (n = 25) of Enterobacter and Citrobacter species. Moreover, 86% (n = 50) of E. coli, 77% (n = 33) of Klebsiella species, and 25% (n = 4) of Citrobacter species isolates phenotypically expressed extended spectrum β-lactamase. Regarding ESBL genes, bla_CTX-M-27 and bla_TEM-1 were found in E. coli, while Klebsiella species harbored bla_CTX-M-15, bla_CTX-M-30, or bla_SHV-12. Genes coding for aminoglycoside modifying enzymes, adenylyltransferases (aadA1, aadA5), phosphotransferases (aph(6)-1d, aph(3″)-Ib), acetyltransferases (aac(3)-IIa), (aac(6)-Ib), sulfonamide/trimethoprim resistant dihydropteroate synthase (sul), dihydrofolate reductase (dfrA), and quinolone resistance protein (qnrB1) were also identified. Monitoring wastewater from human sources for acquired resistance in clinically important bacteria may provide a cheaper alternative in regions facing challenges that limit clinical surveillance.

Staphylococcus aureus is a major human and animal pathogen although the animal-associated S. aureus can be a potential risk of human zoonoses. Acquisition of phage-related genomic islands determines the S. aureus species diversity. This study characterized and compared the genome architecture, distribution nature, and evolutionary relationship of 65 complete prophages carried by human and animal-associated S. aureus strains spreading across the European regions. The analyzed prophage genomes showed mosaic architecture with extensive variation in genome size. The phylogenetic analyses generated seven clades in which prophages of the animal-associated S. aureus scattered in all the clades. The S. aureus strains with the same SCCmec type, and clonal complex favored the harboring of similar prophage sequences and suggested that the frequency of phage-mediated horizontal gene transfer is higher between them. The presence of various virulence factors in prophages of animal-associated S. aureus suggested that these prophages could have more pathogenic potential than prophages of human-associated S. aureus. This study showed that the S. aureus phages are dispersed among the several S. aureus serotypes and around the European regions. Further, understanding the phage functional genomics is necessary for the phage-host interactions and could be used for tracing the S. aureus strains transmission.

In the present study, a total of 35 S. aureus isolates collected from two different geographical locations viz., Germany and Hungary were tested for their methicillin-resistant phenotype which revealed a high incidence of methicillin-resistant S. aureus. The quantitative test for biofilm production revealed that 73.3% of isolates were biofilm producers. The isolates were further characterized using a set of biochemical and genotypic methods such as amplification and analysis of S. aureus species-specific sequence and mecA gene. The 33 mecA positive isolates were then characterized by the amplification of SCCmec and pvl toxin genes. Further, based on the biofilm-forming phenotype, 15 isolates were selected and characterized through PCR–RFLP of coa gene, polymorphism of spa gene and amplification of biofilm-associated genes. The dendrogram prepared from the results of both biochemical and genotypic analyses of the 15 isolates showed that except for the isolates SA G5 and SA H29, the rest of the isolates grouped themselves according to their locations. Thus, the two isolates were selected for further characterization through whole-genome sequencing. Comparative genome analysis revealed that SA G5 and SA H29 have 97.20% ANI values with 2344 gene clusters (core-genome) of which 16 genes were related to antibiotic resistance genes and 57 genes encode virulence factors. The highest numbers of singleton genes were found in SA H29 that encodes proteins for virulence, resistance, mobile elements, and lanthionine biosynthesis. The high-resolution phylogenetic trees generated based on shared proteins and SNPs revealed a clear difference between the two strains and can be useful in distinguishing closely related genomes. The present study demonstrated that the whole-genome sequence analysis technique is required to get a better insight into the MRSA strains which would be helpful in improving diagnostic investigations in real-time to improve patient care.

This study investigated the in vitro effect of propolis ethanolic extract (PEE) on planktonic growth and biofilm forming abilities of five commercial probiotics (Enterol, Protexin, Normaflore, BioGaia and Linex). Broth microdilution method was used to investigate the susceptibility of the microbes of five commercial probiotics to PEE. Crystal violet assay was used for the quantitative assessment of biofilm formation and mature biofilm eradication tests. Effect of PEE on autoaggregation ability and swarming motility of Normaflore microbes was determined. Planktonic forms of probiotics showed varied susceptibilities with minimal inhibitory concentration values in the range of 100–800 µg/mL of PEE. However, low PEE concentrations significantly enhanced the planktonic growth of Linex and BioGaia microbes. Biofilm studies revealed that Enterol and Protexin were non-biofilm formers, while BioGaia, Linex and Normaflore showed weak biofilms, which were inhibited by 12.5, 25, and 800 µg/mL of PEE, respectively. PEE revealed double-face effect on the biofilms of Normaflore and Linex, which were enhanced at low concentrations of PEE and inhibited at higher concentrations. Interestingly, Normaflore biofilms were shifted from weak to strong biofilms at low PEE concentrations (12.5, 25, and 50 µg/mL). In conclusion, PEE has strain dependent controversial effects on the planktonic growth and biofilm forming ability of the tested probiotics, although high concentrations have inhibitory effect on all of them, low concentrations may have strain dependent prebiotic effect.

Staphylococcus aureusis a drug-resistant pathogen, capable of colonizing diverse ecological niches and causing a broad spectrum of infections related to a community and healthcare. In this study, we choose four methicillin-resistantS. aureus(MRSA) clinical isolates from Germany and Hungary based on our previous polyphasic characterization finding. We assumed that the selected strains have a different genetic background in terms of the presence of resistance and virulence genes, prophages, plasmids, and secondary metabolite biosynthesis genes that may play a crucial role in niche adaptation and pathogenesis. To clarify these assumptions, we performed a comparative genome analysis of these strains and observed many differences in their genomic compositions. The Hungarian isolates (SA H27 and SA H32) with ST22-SCCmectype IVa have fewer genes for multiple-drug resistance, virulence, and prophages reported in Germany isolates. Germany isolate, SA G6 acquires aminoglycoside (ant(6)-Ia and aph(3’)-III) and nucleoside (sat-4) resistance genes via phage transduction and may determine its pathogenic potential. The comparative genome study allowed the segregation of isolates of geographical origin and differentiation of the clinical isolates from the commensal isolates. This study suggested that Germany and Hungarian isolates are genetically diverse and showing variation among them due to the gain or loss of mobile genetic elements (MGEs). An interesting finding is the addition of SA G6 genome responsible for the drastic decline of the core/pan-genome ratio curve and causing the pan-genome to open wider. Functional characterizations revealed thatS. aureusisolates survival are maintained by the amino acids catabolism and favor adaptation to growing in a protein-rich medium. The dispersible and singleton genes content ofS. aureusgenomes allows us to understand the genetic variation among the CC5 and CC22 groups. The strains with the same genetic background were clustered together, which suggests that these strains are highly alike; however, comparative genome analysis exposed that the acquisition of phage elements, and plasmids through the events of MGEs transfer contribute to differences in their phenotypic characters. This comparative genome analysis would improve the knowledge about the pathogenicS. aureusstrain’s characterization, and responsible for clinically important phenotypic differences among theS. aureusstrains.