Multidrug-resistant Acinetobacter baumannii strains have increasingly resulted in nosocomial outbreaks worldwide, leaving limited options for treatment. To date, little has been reported on the antimicrobial susceptibilities and genomic profiles of A. baumannii strains from hospital outbreaks in the Greater Los Angeles area. In this study, we examined the susceptibilities and genetic profiles of 20 nonduplicate isolates of A. baumannii from nosocomial outbreaks in Los Angeles County (LAC) and determined their mechanisms of fluoroquinolone resistance. Antibiotic susceptibility testing indicated that the majority of these LAC isolates were not susceptible to 14 of the 17 antibiotics tested, with the exception of doxycycline, minocycline, and tigecycline. In particular, all isolates were found to be resistant to ciprofloxacin. Genomic DNA analysis revealed eight epidemiologically distinct groups among these 20 A. baumannii isolates, consistent with antibiotic susceptibility profiles. Sequencing analysis confirmed that concurrent GyrA and ParC amino acid substitutions in the "hot spots" of their respective quinolone resistance-determining regions were primarily responsible for the high-level ciprofloxacin resistance of these isolates. Antibiotic susceptibility testing using two efflux pump inhibitors suggested that the presence of efflux pumps was only a secondary contributor to ciprofloxacin resistance for some of the isolates. In summary, the present study has revealed good correlation between the antibiotic susceptibility profiles and genetic fingerprints of 20 clinical isolates from nosocomial outbreaks in Los Angeles County and has determined their mechanisms of fluoroquinolone resistance, providing an important foundation for continued surveillance and epidemiological analyses of emerging A. baumannii isolates in Los Angeles County hospitals.

With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Overexpression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17-fold and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli. From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens.

Teicoplanin (