IncHI2 ST3 plasmids are known carriers of multiple antimicrobial resistance genes. Complete plasmid sequences from multiple drug resistant Escherichia coli circulating in Australian swine is however limited. Here we sequenced two related IncHI2 ST3 plasmids, pSDE-SvHI2, and pSDC-F2_12BHI2, from phylogenetically unrelated multiple-drug resistant Escherichia coli strains SvETEC (CC23:O157:H19) and F2_12B (ST93:O7:H4) from geographically disparate pig production operations in New South Wales, Australia. Unicycler was used to co-assemble short read (Illumina) and long read (PacBio SMRT) nucleotide sequence data. The plasmids encoded three drug-resistance loci, two of which carried class 1 integrons. One integron, hosting drfA12-orfF-aadA2, was within a hybrid Tn1721/Tn21, with the second residing within a copper/silver resistance transposon, comprising part of an atypical sul3-associated structure. The third resistance locus was flanked by IS15DI and encoded neomycin resistance (neoR). An oqx-encoding transposon (quinolone resistance), similar in structure to Tn6010, was identified only in pSDC-F2_12BHI2. Both plasmids showed high sequence identity to plasmid pSTM6-275, recently described in Salmonella enterica serotype 1,4,[5],12:i:-that has risen to prominence and become endemic in Australia. IncHI2 ST3 plasmids circulating in commensal and pathogenic E. coli from Australian swine belong to a lineage of plasmids often in association with sul3 and host multiple complex antibiotic and metal resistance structures, formed in part by IS26.
Chronic wound infections are a major burden to both society and the health care industry. Bacterial biofilms are the major cause of chronic wound infections and are notoriously recalcitrant to treatments with antibiotics, making them difficult to eradicate. Thus, new approaches are required to combat biofilms in chronic wounds. One possible approach is to use drug combination therapies. Manuka honey has potent broad-spectrum antibacterial activity and has previously shown synergistic activity in combination with antibiotics against common wound pathogens, including Staphylococcus aureus. In addition, manuka honey exhibits anti-biofilm activity, thereby warranting the investigation of its potential as a combination therapy with antibiotics for the topical treatment of biofilm-related infections. Here we report the first use of MacSynergy II to investigate the response of established S. aureus (strain NCTC 8325) biofilms to treatment by combinations of Medihoney (medical grade manuka honey) and conventional antibiotics that are used for preventing or treating infections: rifampicin, oxacillin, fusidic acid, clindamycin, and gentamicin. Using checkerboard microdilution assays, viability assays and MacSynergy II analysis we show that the Medihoney-rifampicin combination was more effective than combinations using the other antibiotics against established staphylococcal biofilms. Medihoney and rifampicin were strongly synergistic in their ability to reduce both biofilm biomass and the viability of embedded S. aureus cells at a level that is likely to be significant in vivo. Other combinations of Medihoney and antibiotic produced an interesting array of effects: Medihoney-fusidic acid treatment showed minor synergistic activity, and Medihoney-clindamycin, -gentamicin, and -oxacillin combinations showed overall antagonistic effects when the honey was used at sub-inhibitory concentration, due to enhanced biofilm formation at these concentrations which could not be counteracted by the antibiotics. However, these combinations were not antagonistic when honey was used at the inhibitory concentration. Confocal scanning laser microscopy confirmed that different honey-antibiotic combination treatments could eradicate biofilms. Our results suggest that honey has potential as an adjunct treatment with rifampicin for chronic wounds infected with staphylococcal biofilms. We also show that MacSynergy II allows a comprehensive examination of the synergistic effects of honey-antibiotic combinations, and can help to identify doses for clinical use.
BackgroundProfiling of microbial communities via metagenomic shotgun sequencing has enabled researches to gain unprecedented insight into microbial community structure and the functional roles of community members. This study describes a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq) protocol for reduced representation metagenome profiling.MethodsThis technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containing DNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection. We assessed the performance of the metagenomic ddRADseq approach by applying the full method to human stool samples and generating sequence data.ResultsThe ddRADseq data yields a similar estimate of community taxonomic profile as obtained from shotgun metagenome sequencing of the same human stool samples. No obvious bias with respect to genomic G + C content and the estimated relative species abundance was detected.DiscussionAlthough ddRADseq does introduce some bias in taxonomic representation, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies.
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