Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

Cross-protection and cross-feeding between Klebsiella pneumoniae and Acinetobacter baumannii promotes their co-existence.

Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae against the cephalosporin, cefotaxime. Our study demonstrates distinct syntrophic interactions occur between A. baumannii and K. pneumoniae, helping to elucidate the basis for their co-existence in polymicrobial infections.

Delving into defence: identifying the Pseudomonas protegens Pf-5 gene suite involved in defence against secreted products of fungal, oomycete and bacterial rhizosphere competitors.

Competitive behaviours of plant growth promoting rhizobacteria (PGPR) are integral to their ability to colonize and persist on plant roots and outcompete phytopathogenic fungi, oomycetes and bacteria. PGPR engage in a range of antagonistic behaviours that have been studied in detail, such as the production and secretion of compounds inhibitory to other microbes. In contrast, their defensive activities that enable them to tolerate exposure to inhibitory compounds produced by their neighbours are less well understood. In this study, the genes involved in the Pseudomonas protegens Pf-5 response to metabolites from eight diverse rhizosphere competitor organisms, Fusarium oxysporum, Rhizoctonia solani, Gaeumannomyces graminis var. tritici, Pythium spinosum, Bacillus subtilis QST713, Pseudomonas sp. Q2-87, Streptomyces griseus and Streptomyces bikiniensis subspecies bikiniensi, were examined. Proximity induced excreted metabolite responses were confirmed for Pf-5 with all partner organisms through HPLC before culturing a dense Pf-5 transposon mutant library adjacent to each of these microbes. This was followed by transposon-directed insertion site sequencing (TraDIS), which identified genes that influence Pf-5 fitness during these competitive interactions. A set of 148 genes was identified that were associated with increased fitness during competition, including cell surface modification, electron transport, nucleotide metabolism, as well as regulatory genes. In addition, 51 genes were identified for which loss of function resulted in fitness gains during competition. These included genes involved in flagella biosynthesis and cell division. Considerable overlap was observed in the set of genes observed to provide a fitness benefit during competition with all eight test organisms, indicating commonalities in the competitive response to phylogenetically diverse micro-organisms and providing new insight into competitive processes likely to take place in the rhizosphere.

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD.

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.