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Objectives: This study aimed to provide detailed genetic characterization of Tn6636, a multidrug-resistant and composite mobile element, in clinical isolates of Staphylococcus aureus. Methods: A total of 112 ermB-positive methicillin-susceptible S. aureus (MSSA) and 224 ermB-positive methicillin-resistant S. aureus (MRSA) isolates collected from 2000 to 2015 were tested for the presence of Tn6636. Detection of the plasmids harboring Tn6636 was performed by S1 nuclease digestion pulsed-field gel electrophoresis (PFGE) analysis, conjugation test, and whole genome sequencing (WGS). Results: Prevalence of Tn6636 in MSSA is higher than that in MRSA. Ten MSSA isolates and 10 MRSA isolates carried Tn6636. The 10 MSSA isolates belonged to three sequence types (ST), including ST7 (n = 6), ST5 (n = 3), and ST59 (n = 1). The 10 MRSA isolates belonged to ST188 (n = 8) and ST965 (n = 2). Analysis of plasmid sequences revealed that Tn6636 was harbored by six different mosaic plasmids. In addition to resistance genes, some plasmids also harbored toxin genes. Conclusion: The presence of multi-resistant Tn6636 in plasmids of both MSSA and MRSA with various STs suggests its broad dissemination. Results indicate that Tn6636 has existed for at least 16 years in Taiwan. The mosaic plasmids harboring Tn6636 can be transferred by conjugation. Ongoing surveillance of Tn6636 is essential to avoid continued spreading of resistant plasmids.

ABSTRACT: Objectives: This study aimed to clarify the characteristics of Tn7-derivatives transposons in MDR Proteus mirabilis strains isolated from anal swabs of chicken and swine in China from 2015–2020. Methods: The Tn7 tnsA gene was screened in 207 P. mirabilis isolates by polymerase chain reaction (PCR). These strains were subjected to antimicrobial susceptibility testing. Illumina Hiseq (200 × coverage) was used for genome sequencing. Transposon maps were completed by PCR and Sanger sequencing and analysed by BLAST. Results: The Tn7 tnsA gene was detected in 21 strains by PCR. Eight novel Tn7-derivatives, named Tn6667, Tn6668, Tn6669, Tn6670, Tn7095, Tn7096, Tn7097 and Tn7098, were characterised. Three types of hybrid class 2/1 integrons were found at the right end of Tn7 derivatives. A novel Tn7-like transposon Tn6666 with an active integrase gene intI2, whose transposition module shows 93% nucleotide identity to the corresponding region of Tn7, was characterised in three strains. Tn6666 is also found next to Tn7097 or Tn7098 in the chromosomes of two clonally related P. mirabilis strains. The number of resistance genes carried by the novel transposons varied from 1 to 18. A novel variant of class A extended-spectrum beta-lactamase gene, blaPER-16, with eight base substitutions compared with blaPER-12, was harboured by Tn7098. Conclusion: Our study characterised diverse novel Tn7-derivatives and a new Tn7-like transposon in P. mirabilis. An active integrase gene intI2 might promote the diversification of Tn7-like transposons. More attention should be paid to the prevalence and evolution of Tn7-derivatives and Tn7-like transposons and antimicrobial resistance genes they carry.

IntroductionThe continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action.MethodsUtilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin.ResultsA proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2.DiscussionOur study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.

Abstract High temperature causes ubiquitous environmental stress to microorganisms, but studies have not fully explained whether and to what extent heat shock would affect genome stability. Hence, this study explored heat-shock-induced genomic alterations in the yeast Saccharomyces cerevisiae. Using genetic screening systems and customized single nucleotide polymorphism (SNP) microarrays, we found that heat shock (52 °C) for several minutes could heighten mitotic recombination by at least one order of magnitude. More than half of heat-shock-induced mitotic recombinations were likely to be initiated by DNA breaks in the S/G2 phase of the cell cycle. Chromosomal aberration, mainly trisomy, was elevated hundreds of times in heat-shock-treated cells than in untreated cells. Distinct chromosomal instability patterns were also observed between heat-treated and carbendazim-treated yeast cells. Finally, we demonstrated that heat shock stimulates fast phenotypic evolutions (such as tolerance to ethanol, vanillin, fluconazole, and tunicamycin) in the yeast population. This study not only provided novel insights into the effect of temperature fluctuations on genomic integrity but also developed a simple protocol to generate an aneuploidy mutant of yeast.