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Additional file 2: Figure S1. PCR amplification of the zrlA gene in A. baumannii strains. Amplicons of 579 bp were detected in all A. baumannii strains tested. Figure S2. Production of recombinant ZrlA proteins. SDS-PAGE was performed to detect recombinant proteins of ca. 24 kDa (arrow).

Burkholderia cepacia is an opportunistic pathogen that infects patients with debilitating underlying diseases. This study investigated the production of outer membrane vesicles (OMVs) by B. cepacia cultured with sub-minimum inhibitory concentrations (MICs) of antibiotics and examined their pathogenic roles both in vitro and in vivo. B. cepacia ATCC 25416 produced more OMVs under antibiotic stress conditions than controls. OMVs isolated from B. cepacia cultured in Luria-Bertani (LB) broth (OMVs/LB) induced cytotoxicity and the expression of pro-inflammatory cytokine genes in A549 cells in a dose-dependent manner. Host cell cytotoxicity and pro-inflammatory responses were significantly higher in A549 cells treated with B. cepacia OMVs cultured with 1/4 MIC of ceftazidime (OMVs/CAZ) than in the cells treated with OMVs/LB, OMVs cultured with 1/4 MIC of trimethoprim/sulfamethoxazole (OMVs/SXT), or OMVs cultured with 1/4 MIC of meropenem. Intratracheal injection of B. cepacia OMVs also induced histopathology in vivo in mouse lungs. Expressions of IL-1β and TNF-α genes were significantly up-regulatedin the lungs of mice treated with OMVs/CAZ compared to mice administered other OMVs; the expression of the GRO-α gene, however, was significantly up-regulated in OMVs/SXT. In conclusion, OMVs produced by B. cepacia under different antibiotic stress conditions induce different host responses that may contribute to the pathogenesis of B. cepacia.

Shukho Kim,1 Jung Hwa Woo,1 Nayeong Kim,1 Mi Hyun Kim,1 Se Yeon Kim,1 Joo Hee Son,1 Dong Chan Moon,2 Suk-Kyung Lim,2 Minsang Shin,1 Je Chul Lee1 1Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; 2Bacterial Disease Division, Animal and Plant Quarantine Agency, Gimcheon-si, Gyeongsangbuk-do, Republic of KoreaCorrespondence: Je Chul LeeDepartment of Microbiology, School of Medicine, Kyungpook National University, 680 Gukchaebosang-ro, Jung-gu, Daegu 41944, Republic of KoreaTel +82-53-420-4844Fax +82-53-427-5664Email leejc@knu.ac.krPurpose: Colistin resistance in gram-negative bacteria from humans and livestock has been increasingly reported worldwide. The aim of this study was to investigate the underlying mechanisms of chromosome-mediated colistin resistance in Escherichia coli isolates from livestock in Korea.Materials and methods: Thirty mcr-1-negative isolates were selected from a collection of colistin-resistant E. coli isolates collected from livestock in 2005 and 2015 in Korea. Amino acid alterations in PmrAB, PhoPQ, MgrB, and PmrD were investigated. Colistin-resistant derivatives were produced by serial passage of colistin-susceptible E. coli isolates in colistin-containing media.Results: Thirty colistin-resistant mcr-negative E. coli isolates were classified into 26 sequence types. Twenty-two isolates carried diverse amino acid alterations in PmrB, PhoP, PhoQ, MgrB, and/or PmrD, whereas no mutation in any of these genes was found in the remaining eight isolates. Sixteen out of the 22 isolates shared a total of nine polymorphic positions that were found in colistin-susceptible E. coli strains. Colistin-resistant derivatives from two colistin-susceptible isolates showed the same genetic alterations that were observed in colistin-resistant clinical isolates.Conclusion: Our results suggest that the mechanism underlying chromosome-mediated colistin resistance remain to be discovered in E. coli. Selective pressure of colistin in vitro induced the same genetic mutations associated with colistin resistance in vivo. Efforts to reduce colistin consumption in livestock should be redoubled, to prevent the occurrence of colistin-resistant E. coli strains.Keywords: colistin resistance, two-component system, livestock, genetic mutation, mcr gene