Whole genome sequence analysis of a transmissible multidrug-resistance plasmid captured without cultivation from poultry litter

Use of antibiotics in the agricultural industry introduces selective pressure and, consequently, could increase the presence of antibiotic resistant organisms in surrounding environments. One such environment is litter (manure and bedding) produced during large-scale poultry production in the Shenandoah Valley. Litter, with its microorganisms, is commonly applied to fields within the Shenandoah River watershed. Antibiotic resistance (AR) and virulence genes are potentially transmissible between organisms through horizontal gene transfer of genetic mobile elements, for which poultry litter could be a reservoir. The typical, culture-based approach to detecting and analyzing AR plasmids and other mobile genetic elements is limited due to the inability to culture, isolate, and analyze all bacteria in nearly all environments. In addition, the expense and time of extracting and sequencing plasmids from culturable isolates is great. The goals of this study were (i) to use a non-culture-dependent plasmid isolation method to isolate AR plasmids directly from poultry litter, (ii) to sequence and assemble the whole genome of the plasmid capture strain E. coli LA61RifR, and (iii) use a combination of short- and long-read sequencing and computational methods to assemble and annotate one of the captured plasmids. It was also wished to determine the antibiotic susceptibility of the captured plasmids. An exogenous plasmid capture method was used to isolate tetracycline-resistance plasmids EH1-12, some of which conferred phenotypic resistance to a range of late-generation, clinically-significant antibiotics. Of the 12 transconjugants, 11 conferred resistance to more than one antibiotic (excluding tetracycline), the most common were resistances to piperacillin and piperacillin/tazobactam. Perhaps most striking was the resistance conferred by plasmid EH11 to aztreonam, a monobactam antibiotic effective against gram negative aerobic organisms, which has rarely been observed. Other surprising resistance phenotypes included ceftazidime and ciproflaxocin which are members of the cephalosporin and quinolone drug classes, respectively. The whole genomes of both the plasmid capture strain LA61RifR and one of the multidrug resistant transconjugants, LA61RifR::pEH11, were sequenced. SPAdes and Canu were used to assemble the genomes of LA61RifR and of LA61RifR::pEH11, respectively. Ninety-seven contigs assembled from short-read sequencing data comprised the LA61RifR genome and 5 contigs assembled from long-read data comprised the LA61RifR::pEH11 genome. One contig of LA61RifR::pEH11was identified as plasmid EH11. Genes encoding antibiotic resistance, bacteriocins, and aerobactin siderophore systems were annotated with ARGannot, RAST, and Prokka . Eight repeat regions, 47 transposase genes, and two regions responsible for plasmid replication and transfer were also identified. Overall this study, through phenotypic and genotypic analyses, demonstrated that poultry litter can act as reservoir for transmissible multidrug-resistant plasmids. Genome analysis also demonstrated the potential to transfer genes that contribute to a host’s virulence. Such resistances and virulence genes, encoded on transmissible plasmids, provide advantages to infectious agents and enable their survival in poultry litter and other environments, thus possibly complicating treatment of resulting infections

Development and optimization of synergistic antimicrobial combinations against a variety of non-tuberculous mycobacteria of clinical importance

Non-tuberculous mycobacteria (NTM) are an increasing threat to the health of immunocompromised and immunocompetent individuals. Mycobacterium abscessus (MAB) and M. avium subspecies paratuberculosis (MAP) are two species of NTM that threaten respiratory and gut health, respectively. These two organisms have demonstrated both intrinsic and acquired resistance to many antibiotics. Of the two, MAB is associated with drug resistance due to genomic mutations, whereas MAP is known to adapt to an anaerobic state during which antibiotic targets are inactive. In this study, broth microdilution was used for both MIC and synergy-based assays; for MAP, the Wayne model was used for anaerobic susceptibility testing. Once MICs were established, a sub-inhibitory concentration of clofazimine was added to varying concentrations of 14 antibiotics to identify synergistic combinations. Aerobic synergy was defined as a ≥4-fold decrease in the MIC for an individual drug versus the paired MIC alone. For MAB, synergy was demonstrated between clofazimine and 12/14 antibiotics. The greatest synergy against MAB occurred with clofazimine and doxycycline, where the fold decrease in the latter was 211.20. For MAP, 4 strains were adapted to anaerobiosis and treated with 16 drugs or combinations. The threshold for successful inhibitory activity was defined as a 1-log10 reduction in viable counts. Success followed exposure to 4-drug combinations containing bedaquiline, clofazimine, and metronidazole with rifaximin or clarithromycin, or alternatively, the combination of gentamicin, rifaximin, clarithromycin, and metronidazole. No single, 2-, or 3-drug combinations achieved the targeted 1-log10 decrease. The most active drug combination against anaerobically-adapted MAP was bedaquiline, clofazimine, metronidazole, and rifaximin. These studies demonstrate that synergy testing of drug resistant NTM species should be considered as an alternative to standard susceptibility testing using single drugs, particularly in settings in which few treatment options are available. In addition, adaptation of MAP and possibly other NTMs to anaerobiosis raises questions regarding the ability of aerobically active antibiotics to eradicate such organisms in vivo. Future studies are needed to help correlate in vitro susceptibility results with in vivo efficacy, especially given the rising tide of multidrug-resistant NTM infections, including those caused by MAB and MAP

Development and optimization of synergistic antimicrobial combinations against a variety of non-tuberculous mycobacteria of clinical importance

Non-tuberculous mycobacteria (NTM) are an increasing threat to the health of immunocompromised and immunocompetent individuals. Mycobacterium abscessus (MAB) and M. avium subspecies paratuberculosis (MAP) are two species of NTM that threaten respiratory and gut health, respectively. These two organisms have demonstrated both intrinsic and acquired resistance to many antibiotics. Of the two, MAB is associated with drug resistance due to genomic mutations, whereas MAP is known to adapt to an anaerobic state during which antibiotic targets are inactive. In this study, broth microdilution was used for both MIC and synergy-based assays; for MAP, the Wayne model was used for anaerobic susceptibility testing. Once MICs were established, a sub-inhibitory concentration of clofazimine was added to varying concentrations of 14 antibiotics to identify synergistic combinations. Aerobic synergy was defined as a ≥4-fold decrease in the MIC for an individual drug versus the paired MIC alone. For MAB, synergy was demonstrated between clofazimine and 12/14 antibiotics. The greatest synergy against MAB occurred with clofazimine and doxycycline, where the fold decrease in the latter was 211.20. For MAP, 4 strains were adapted to anaerobiosis and treated with 16 drugs or combinations. The threshold for successful inhibitory activity was defined as a 1-log10 reduction in viable counts. Success followed exposure to 4-drug combinations containing bedaquiline, clofazimine, and metronidazole with rifaximin or clarithromycin, or alternatively, the combination of gentamicin, rifaximin, clarithromycin, and metronidazole. No single, 2-, or 3-drug combinations achieved the targeted 1-log10 decrease. The most active drug combination against anaerobically-adapted MAP was bedaquiline, clofazimine, metronidazole, and rifaximin. These studies demonstrate that synergy testing of drug resistant NTM species should be considered as an alternative to standard susceptibility testing using single drugs, particularly in settings in which few treatment options are available. In addition, adaptation of MAP and possibly other NTMs to anaerobiosis raises questions regarding the ability of aerobically active antibiotics to eradicate such organisms in vivo. Future studies are needed to help correlate in vitro susceptibility results with in vivo efficacy, especially given the rising tide of multidrug-resistant NTM infections, including those caused by MAB and MAP

MEK5 and ERK5 are mediators of the pro-myogenic actions of IGF-2

During the differentiation of muscle satellite cells, committed myoblasts respond to specific signalling cues by exiting the cell cycle, migrating, aligning, expressing muscle-specific genes and finally fusing to form multinucleated myotubes. The predominant foetal growth factor, IGF-2, initiates important signals in myogenesis. The aim of this study was to investigate whether ERK5 and its upstream MKK activator, MEK5, were important in the pro-myogenic actions of IGF-2. ERK5 protein levels, specific phosphorylation and kinase activity increased in differentiating C2 myoblasts. ERK5-GFP translocated from the cytoplasm to the nucleus after activation by upstream MEK5, whereas phospho-acceptor site mutated (dominant-negative) ERK5AEF-GFP remained cytoplasmic. Exogenous IGF-2 increased MHC levels, myogenic E box promoter-reporter activity, ERK5 phosphorylation and kinase activity, and rapidly induced nuclear localisation of ERK5. Transfection with antisense Igf2 decreased markers of myogenesis, and reduced ERK5 phosphorylation, kinase and transactivation activity. These negative effects of antisense Igf2 were rescued by constitutively active MEK5, whereas transfection of myoblasts with dominant-negative MEK5 blocked the pro-myogenic action of IGF-2. Our findings suggest that the MEK5-ERK5 pathway is a novel key mediator of IGF-2 action in myoblast differentiation