Co-evolutionary adaptations of Acinetobacter baumannii and a clinical carbapenemase-encoding plasmid during carbapenem exposure.

OXA-23 is the predominant carbapenemase in carbapenem-resistant Acinetobacter baumannii. The co-evolutionary dynamics of A. baumannii and OXA-23-encoding plasmids are poorly understood. Here, we transformed A. baumannii ATCC 17978 with pAZJ221, a bla OXA-23-containing plasmid from clinical A. baumannii isolate A221, and subjected the transformant to experimental evolution in the presence of a sub-inhibitory concentration of imipenem for nearly 400 generations. We used population sequencing to track genetic changes at six time points and evaluated phenotypic changes. Increased fitness of evolving populations, temporary duplication of bla OXA-23 in pAZJ221, interfering allele dynamics, and chromosomal locus-level parallelism were observed. To characterize genotype-to-phenotype associations, we focused on six mutations in parallel targets predicted to affect small RNAs and a cyclic dimeric (3' → 5') GMP-metabolizing protein. Six isogenic mutants with or without pAZJ221 were engineered to test for the effects of these mutations on fitness costs and plasmid kinetics, and the evolved plasmid containing two copies of bla OXA-23 was transferred to ancestral ATCC 17978. Five of the six mutations contributed to improved fitness in the presence of pAZJ221 under imipenem pressure, and all but one of them impaired plasmid conjugation ability. The duplication of bla OXA-23 increased host fitness under carbapenem pressure but imposed a burden on the host in antibiotic-free media relative to the ancestral pAZJ221. Overall, our study provides a framework for the co-evolution of A. baumannii and a clinical bla OXA-23-containing plasmid in the presence of imipenem, involving early bla OXA-23 duplication followed by chromosomal adaptations that improved the fitness of plasmid-carrying cells.

In vitro and in vivo activity of Manuka honey against NDM-1-producing Klebsiella pneumoniae ST11.

AIM: To determine the therapeutic potential of Manuka honey against New Delhi metallo-ß-lactamase-1-producing Klebsiella pneumoniae ST11 in vitro and in vivo. MATERIALS & METHODS: Carbapenamases and metallo-ß-lactamases-producing K. pneumoniae ST11 isolated from blood culture was confirmed by VITEK-2® system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing, followed by determination of minimum inhibitory concentration (µg/ml) using VITEK-2 system. Genetic analysis of bla NDM-1 was done by PCR, pulsed-field gel electrophoresis and DNA hybridization. In vitro and in vivo efficacy of Manuka honey was performed by microbroth dilution assay and BALB/c mice model respectively. RESULTS: K. pneumoniae ST11 displayed resistance to commonly used antibiotics. bla NDM-1 was located on 150 and 270kb plasmids. Minimum inhibitory concentration and minimum bactericidal concentration of Manuka honey was 30% (v/v) and substantial reduction of bacterial mean log value (>1 log) was observed in mice. Histological analysis of mice liver and kidneys demonstrated mild to moderate inflammation. CONCLUSION: Manuka honey can be used as an alternate therapeutic approach for management of New Delhi metallo-ß-lactamase-producing pathogens.