Role of Outer Membrane Vesicles in the pathogenicity of Acinetobacter baumannii infection under various antibiotic treatment and novel therapeutic options for CHDL-related carbapenem-resistant Acinetobacter baumannii infection

• Main Authors: Chun-Hsiang Chiu, 邱俊翔
• Other Authors: Chang-Phone Fung
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/4955bn

博士 === 國立陽明大學 === 臨床醫學研究所 === 107 === The genus Acinetobacter has emerged as one of the most troublesome pathogens for health care institutions globally over the past 2 decades due to the increasing prevalence and rapid development of drug resistance. Imipenem and meropenem were traditionally the most effective antimicrobials against A. baumannii. However, carbapenem-resistant A. baumannii (CRAb) are now increasingly encountered worldwide, especially in the Asia-Pacific region. There are few treatment options for CRAb, and the development of new agents remains a priority to ensure the availability of efficacious therapies. Our study focused on pathogenicity of drug resistant A. baumannii and try to develop novel therapeutic options for drug resistant A. baumannii infection. Part 1: In a previous study of 252 patients with monomicrobial A. baumannii bacteremia, we found that, despite comparable percentages of inappropriate antimicrobial therapy (56% vs. 51.4%) and Acute Physiology and Chronic Health Evaluation (APACHE II) scores (24.5 vs. 25), patients treated with antipseudomonal cephalosporins had a higher 14 day mortality rate than those treated with antipseudomonal carbapenems (42% vs. 25%, P < 0.05). This result suggested that factors other than the use of appropriate antimicrobial therapy and disease severity contribute to the differences in the mortality rates between the two groups. In a different study, we also demonstrated that treatment with sub-inhibitory concentrations of ceftazidime and imipenem could enhance the release of outer membrane vesicles (OMVs) from CRAb. Thus, we hypothesized that the difference in the mortality rates between the groups was due to the different OMV components released from A. baumannii under the different antibiotic treatments. In the first part of this study, we wanted to explore the role of OMVs in the pathogenicity of A. baumannii infection under sub-inhibitory concentrations of ceftazidime and imipenem treatment. The CRAb transformant, ATCC17978 pOXA-58-2, was treated with ceftazidime, an anti-pseudomonal cephalosporin, or imipenem, an anti-pseudomonal carbapenem, at half the minimum inhibitory concentration. The resultant OMV induced cytokine expression in RAW264.7 macrophages was then measured by ELISA or western blotting. The virulence of the OMVs in vivo was assessed in mice. OMVs and OMV-carried lipopolysaccharides were measured by nanoparticle tracking analysis and western blotting, respectively. Liquid chromatography tandem-mass spectrometry was used to identify proteins within the OMVs. We found that OMVs derived from ceftazidime treated cells led to maximum expression levels of iNOS, IL-1β, and TNF-α in the RAW264.7 cell line and increased mortality in mice. Nanoparticle tracking analysis and western blot showed that OMV amount and OMV-carried LPS increased in ceftazidime treated A. baumannii. We also found that polymyxin B diminished almost all of the OMV mediated IL-1β and TNF-α stimulation in RAW264.7. These results suggest that OMV-carried LPS is essential for the induction of IL-1β and TNF-α. In the animal study, when adjusted for the same amount of LPS, we found that ceftazidime treatment derived OMV also resulted in higher mortality compared to imipenem treatment. We therefore concluded that components other than LPS of the OMVs derived from ceftazidime treatment were also more virulent than OMVs derived from imipenem treatment. Proteomic analysis revealed that there were differences in the proteins identified in OMVs derived from different antibiotic treatment. We conclude that treatment of A. baumannii with a sub-inhibitory concentration of ceftazidime enhanced the release of OMVs which demonstrated increased virulence compared to imipenem treatment induced OMVs. Part 2: A study we conducted in 2015 revealed that OXA-58 translocates to the periplasm via a Sec pathway and is then released from bacteria within OMVs. We were interested in understanding whether or not other common carbapenem hydrolyzing class D β-lactamases (CHDLs) in A. baumannii, including the OXA-23, OXA-40, and OXA-51 families, were also released from the Sec-dependent pathway and whether secA inhibitors can also reduce the carbapenem resistance of CRAb by blocking the release of CHDLs. To determine whether the CHDL is secreted via the Sec pathway, cell fractionation and western blotting analyses were performed to detect the periplasmic His-tagged CHDLs. To evaluate the synergistic effect of a combination therapy with Sec A inhibitors and carbapenem, checkerboard analysis with a pairwise combination of carbapenems (imipenem or meropenem) and Sec A inhibitors (Rose Bengal or sodium azide) was performed with six clinical CRAb isolates harboring different CHDL genes. The results were interpreted using fractional inhibitory concentration index (FICI). The combination with the lowest FICI was subjected to a time-kill analysis. In the second part of this study, using in silico analysis, we found that OXA-23, OXA-40, and OXA-51 were preferentially translocated via the Sec system. Application of a SecA inhibitor, Rose Bengal, decreased the periplasmic translocation of His-tagged OXA-23 and OXA-83 (belonging to the OXA-51 family), but not that of OXA-72 (belonging to the OXA-40 family), from ATCC 15151 transformants. A combination of imipenem or meropenem with Rose Bengal showed synergistic effects (FICI ≤ 0.5) for 6 and 4 clinical isolates, respectively. Combination treatment using imipenem or meropenem with sodium azide exhibited no interactions (FIC index, >0.5–4) against all clinical isolates. Combination treatment using imipenem and Rose Bengal had the lowest FICI and showed synergy at 24 h in the time-kill assay. In conclusion, the combination of a SecA inhibitor and carbapenem could have synergistic effects against CRAb. Improving the potency of SecA inhibitors or targeting other proteins involved in the Sec pathway might be effective approaches against CRAb.