Assessment of sustained efficacy and resistance emergence under human-simulated exposure of cefiderocol against Acinetobacter baumannii using in vitro chemostat and in vivo murine infection models.

Objectives: This study evaluated the sustained kill and potential for resistance development of Acinetobacter baumannii exposed to human-simulated exposure of cefiderocol over 72 h in in vitro and in vivo infection models. Methods: Seven A. baumannii isolates with cefiderocol MICs of 0.12-2 mg/L were tested. The sustained bactericidal activity compared with the initial inoculum and the resistance appearance over 72 h treatment were evaluated in both an in vitro chemostat and an in vivo murine thigh infection model under the human-simulated exposure of cefiderocol (2 g every 8 h as 3 h infusion). Results: In the in vitro model, regrowth was observed against all seven tested isolates and resistance emergence (>2 dilution MIC increase) was observed in five test isolates. Conversely, sustained killing over 72 h and no resistance emergence were observed in six of seven tested isolates in vivo. The mechanism of one resistant isolate that appeared only in the in vitro chemostat studies was a mutation in the tonB-exbB-exbD region, which contributes to the energy transduction on the iron transporters. The resistance acquisition mechanisms of other isolates have not been identified. Conclusions: The discrepancy in the sustained efficacy and resistance emergence between in vitro and in vivo models was observed for A. baumannii. Although the resistance mechanisms in vitro have not been fully identified, sustained efficacy without resistance emergence was observed in vivo for six of seven isolates. These studies reveal the in vivo bactericidal activity and the low potential for development of resistance among A. baumannii evaluated under human-simulated exposures.

Reduced susceptibility mechanism to cefiderocol, a siderophore cephalosporin, among clinical isolates from a global surveillance programme (SIDERO-WT-2014).

OBJECTIVE: To investigate possible mechanistic factors to explain cefiderocol (CFDC) non-susceptibility, we characterized 38 clinical isolates with a CFDC minimum inhibitory concentration (MIC) of >4µg/mL from a multi-national surveillance study. METHODS: The MIC measurement in the presence of ß-lactamase inhibitors and whole genome sequencing were performed. RESULTS: The MIC decrease of CFDC by ß-lactamase inhibitors was observed against all of the test isolates. Among the 38 isolates, NDM and PER genes were observed in 5 and 25 isolates, respectively. No other ß-lactamases responsible for high MIC were identified in the other eight isolates. The MIC of CDFC against Escherichia coli isogenic strains introduced with NDM and PER ß-lactamase increased by ≥16-fold, suggesting the contribution of NDM and PER to the non-susceptibility to CFDC. Against NDM producers, a ≥8-fold MIC increase was observed only when both serine- and metallo-type ß-lactamase inhibitors were added. In addition, many of the PER or NDM producers remained susceptible to CFDC. These results suggested that the presence of only NDM or PER would not lead to non-susceptibility to CFDC and that multiple factors would be related to CFDC resistance. CONCLUSION: Multiple factors including NDM and PER could be related to reduced susceptibility to CFDC.

Escherichia coli strains possessing a four amino acid YRIN insertion in PBP3 identified as part of the SIDERO-WT-2014 surveillance study.

BACKGROUND: In addition to carbapenemases, dissemination of recently reported Escherichia coli lineages possessing a four amino acid insertion in PBP3 (encoded by ftsI) that confers reduced susceptibility to PBP3-targeted ß-lactams, such as ceftazidime, can pose a threat of antimicrobial resistance. OBJECTIVES: To evaluate genotypic and phenotypic characteristics of E. coli possessing the mutated PBP3 collected during SIDERO-WT-2014 surveillance. METHODS: A subset of 65 E. coli clinical isolates with MICs ≥2 mg/L for ceftazidime/avibactam, ceftolozane/tazobactam or cefiderocol, among a total of 1529 isolates from the multinational surveillance study, were subjected to gene analysis and antimicrobial susceptibility testing. Isogenic PBP3 mutants were constructed to confirm experimentally an impact on antimicrobial susceptibility. RESULTS: Eleven strains possessing a YRIN-inserted PBP3 were identified, consisting of nine strains collected from the same hospital in Turkey (ST1284) and one each from the USA and Italy (ST361). Strains associated with each ST lineage possessed similar genetic backgrounds including ß-lactamase genotypes; all nine strains from Turkey carried CMY-42, OXA-1 and the OXA-181 carbapenemase (five strains additionally carried CTX-M-15 ESBL), whereas the two other strains carried CMY-42 and TEM-1, indicating dissemination driven by selective pressure. The presence of the YRIN insertion contributed to reduced susceptibility to aztreonam, ceftazidime, cefepime and ceftolozane/tazobactam, although the strains remained susceptible to ceftazidime/avibactam despite relatively high MICs. CONCLUSIONS: E. coli strains of both ST1284 and ST361 lineages, possessing YRIN-inserted PBP3, are disseminating in several regions. The YRIN insertion in PBP3 occurred with multiple ß-lactamases, which indicates frequent cross-resistance to other ß-lactams.

A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import.

Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.

Optimization of enrichment conditions on TiO2 chromatography using glycerol as an additive reagent for effective phosphoproteomic analysis.

Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.