A Method for generating marker-less gene deletions in multidrug-resistant Acinetobacter baumannii

10.1186/1471-2180-13-158BMC Microbiology131581-1

Clinical management of severe infections caused by carbapenem-resistant Gram-negative bacteria: a worldwide cross-sectional survey addressing the use of antibiotic combinations.

OBJECTIVES: Optimal treatment of carbapenem-resistant Gram-negative (CR-GNB) infections is uncertain due to the lack of good-quality evidence and the limited effectiveness of available antibiotics. The aim of this survey was to investigate clinicians' prescribing strategies for treating CR-GNB infections worldwide. METHODS: A 36-items-questionnaire was developed addressing the following aspects of antibiotic prescribing: respondent's background, diagnostic and therapeutic availability, preferred antibiotic strategies and rationale for selecting combination therapy. Prescribers were recruited following the snowball-sampling approach, and a post-stratification correction with inverse proportional weights was used to adjust the sample's representativeness. RESULTS: 1012 respondents from 95 countries participated in the survey. Overall, 298 (30%) of respondents had local guidelines for treating CR-GNB at their facility and 702 (71%) had access to Infectious Diseases consultation, with significant discrepancies according to country economic status: 85% (390/502) in High-Income-Countries vs 59% (194/283) in Upper-Medium-Income-Countries and 30% (118/196) in Lower-Middle-Income-Countries/Lower-Income-Countries). Targeted regimens varied widely, ranging from 40 regimens for CR-Acinetobacter spp. to more than 100 regimens for CR-Enterobacteriaceae. Although the majority of respondents acknowledged the lack of evidence behind this choice, dual combination was the preferred treatment scheme and carbapenem-polymyxin was the most prescribed regimen, irrespective of pathogen and infection source. Respondents noticeably disagreed around the meaning of 'combination therapy' with 20% (150/783) indicating the simple addition of multiple compounds, 42% (321/783) requiring the presence of in vitro activity and 38% (290/783) of in vitro-synergism. CONCLUSIONS: Management of CR-GNB infections is far from being standardized. Strategic public health focussed randomised controlled trials are urgently required to inform evidence-based treatment guidelines

Metabolomics Reveal Potential Natural Substrates of AcrB in Escherichia coli and Salmonella enterica Serovar Typhimurium.

In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors

AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity.

The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies