Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B . pseudomallei virulence and dormancy. The peptidoglycan remodeling enzymes, lytic transglycosylases (Ltgs) are potential targets for such new antibiotics. Ltgs cleave the glycosidic bonds within bacterial peptidoglycan allowing for the insertion of peptidoglycan precursors during cell growth and division, and cell membrane spanning structures such as flagella and secretion systems. Using bioinformatic analysis we have identified 8 putative Ltgs in B . pseudomallei K96243. We aimed to investigate one of these Ltgs, LtgG (BPSL3046) through the generation of deletion mutants and biochemical analysis. We have shown that LtgG is a key contributor to cellular morphology, division, motility and virulence in BALB/c mice. We have determined the crystal structure of LtgG and have identified various amino acids likely to be important in peptidoglycan binding and catalytic activity. Recombinant protein assays and complementation studies using LtgG containing a site directed mutation in aspartate 343, confirmed the essentiality of this amino acid in the function of LtgG.

IntroductionThe degree to which bacteria in the human respiratory tract are aerosolised by individuals is not established. Building on our experience sampling bacteria exhaled by individuals with pulmonary tuberculosis using face masks, we hypothesised that patients with conditions frequently treated with antimicrobials, such as chronic obstructive pulmonary disease (COPD), might exhale significant numbers of bacteria carrying antimicrobial resistance (AMR) genes and that this may constitute a previously undefined risk for the transmission of AMR.MethodsFifteen-minute mask samples were taken from 13 patients with COPD (five paired with contemporaneous sputum samples) and 10 healthy controls. DNA was extracted from cell pellets derived from gelatine filters mounted within the mask. Quantitative PCR analyses directed to the AMR encoding genes: blaTEM (β-lactamase), ErmB (target methylation), mefA (macrolide efflux pump) and tetM (tetracycline ribosomal protection protein) and six additional targets were investigated. Positive signals above control samples were obtained for all the listed genes; however, background signals from the gelatine precluded analysis of the additional targets.Results9 patients with COPD (69%), aerosolised cells containing, in order of prevalence, mefA, tetM, ErmB and blaTEM, while three healthy controls (30%) gave weak positive signals including all targets except blaTEM. Maximum estimated copy numbers of AMR genes aerosolised per minute were mefA: 3010, tetM: 486, ErmB: 92 and blaTEM: 24. The profile of positive signals found in sputum was not concordant with that in aerosol in multiple instances.DiscussionWe identified aerosolised AMR genes in patients repeatedly exposed to antimicrobials and in healthy volunteers at lower frequencies and levels. The discrepancies between paired samples add weight to the view that sputum content does not define aerosol content. Mask sampling is a simple approach yielding samples from all subjects and information distinct from sputum analysis. Our results raise the possibility that patient-generated aerosols may be a significant means of AMR dissemination that should be assessed further and that consideration be given to related control measures.

Bacterial pathogens exhibit physiologically distinct forms that enable their survival in an infected host, the environment and following exposure to antimicrobial agents. B. pseudomallei causes the disease melioidosis, which has a high mortality rate and is difficult to treat with antibiotics. The bacterium is endemic to several countries and detected in high abundance in the environment.

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