Comparative Proteomics of Inner Membrane Fraction from Carbapenem-Resistant Acinetobacter baumannii with a Reference Strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A. baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A. baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A. baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A. baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A. baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A. baumannii to carbapenem

High prevalence of oxacillinases in clinical multidrug-resistant Acinetobacter baumannii isolates from the Tshwane region, South Africa – an update

BACKGROUND : Acinetobacter baumannii is an important hospital-acquired pathogen in healthcare facilities that frequently causes bacteraemia and ventilator-associated pneumonia in intensive care units. Acinetobacter baumannii can be isolated from various sites in the hospital environment like medical equipment, bed linen, medical personnel and indwelling catheters. It is difficult to treat A. baumannii infections because of their highly resistant antimicrobial profiles. The purpose of this study was to determine the prevalence of β-lactamase genes in multidrug-resistant (MDR) clinical A. baumannii isolates using Multiplex-PCR (M-PCR) assays. METHODS : One hundred MDR A. baumannii isolates were collected from the diagnostic division of the Department of Medical Microbiology after routine analysis of the submitted specimens. All collected isolates were identified and tested for susceptibility using the VITEK 2® system (bioMérieux, France). Six isolates were excluded from this study because the isolates were incorrectly identified as A. baumannii with the VITEK 2® system (bioMérieux, France). Molecular tests, namely M-PCR assays, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed. MLST analyses were performed on representative isolates from the four major pulsotypes (≥5 isolates with 80 % similarity) and selective isolates from each minor pulsotype. RESULTS : All the A. baumannii isolates showed 100 % resistance to ampicillin, amoxicillin, cefuroxime, cefuroximine axetil, cefoxitin, cefotaxime and nitrofurantoin. Seven percent of the isolates were resistant to amikacin. Two percent of the isolates were classified as having intermediate susceptibility to tigecycline. A. baumannii isolates showed an antibiotic resistance profile of 67 % and higher to antibiotics, such as ceftazidime, cefepime, imipenem, meropenem, gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole. None of the isolates were resistant to colistin. The M-PCR assays showed that 99 % of the isolates contained the OXA-51 gene and 77 % contained the OXA-23 gene. None of the isolates contained the GES, GIM, IMP, KPC, NDM, OXA-24, OXA-58, PER, SIM, SPM, VEB and VIM genes. Representative A. baumannii isolates were grouped into five existing sequence types (ST): ST106, ST258, ST339, ST502, ST758 and ST848. Isolates belonging to the pan-European clonal lineages I and II (EUI and EUII) were identified. CONCLUSION : The high prevalence of MDR A. baumannii isolates has a severe impact on available treatment choices and this in return impacts on treatment outcomes in the studied healthcare facilities. The most dominant ST among the collected isolates was ST758, member of the EUI group. The presence of the OXA-23 gene was not restricted to a specific ST. Continuous research and surveillance is necessary to monitor the circulating β-lactamase genes in clinical settings to guide infection control policies in order to try and curb the spread of this bacterium.ML was supported by a National Research Foundation (NRF) grant. The MALDI-TOF analysis is based on research supported in part by the National Research Foundation (NRF) of South Africa (Grant specific unique reference number (UID) 74426).http://www.biomedcentral.com/bmcinfectdis/am201

Antitumor Effect of the Human Immunodeficiency Virus Protease Inhibitor Ritonavir: Induction of Tumor-Cell Apoptosis Associated with Perturbation of Proteasomal Proteolysis

Ritonavir is an HIV protease inhibitor used in the therapy of HIV infection. Ritonavir has also been shown to inhibit the chymotrypsin-like activity of isolated 20S proteasomes. Here, we demonstrate that ritonavir, like classical proteasome inhibitors, has antitumoral activities. In vitro, ritonavir strongly reduced the rate of proliferation of several tumor cell lines and induced their apoptosis. Nontransformed cell lines and terminally differentiated bone-marrow macrophages were comparatively resistant to the apoptosis-inducing effect. In vivo, ritonavir, administered p.o. for a week at doses of 6-8.8 mg/mouse/day, caused significant growth inhibition (76-79% after 7 days of treatment) of established EL4-T cell thymomas growing s.c. in syngeneic C57L/6 mice. Unexpectedly, we found that ritonavir activates the chymotrypsin-like activity of isolated 26S proteasomes, in strong contrast to its effect on isolated 20S proteasomes. The net effect of low micromolar concentrations of ritonavir on the chymotrypsin-like activity in cells and cell lysates was a weak inhibition, consistent with marginal alterations of polyubiquitinated proteins, marginal alterations in acid-soluble proteolytic peptide levels, and a small accumulation of the tumor suppressor protein p53, in cells treated with ritonavir. In contrast, we found a relatively strong accumulation of the cyclin-dependent kinase inhibitor p21WAF₋₁, a sign of deregulation of cell-cycle progression typical for apoptosis induction in transformed cells by classical proteasome inhibitors. We demonstrate that p21 accumulation in the presence of ritonavir is attributable to the inhibition of proteolytic degradation. Accumulation of p21 most likely reflects a selective inhibition of proteasomes, in line with the atypical degradation of p21, which does not require ubiquitination. These findings suggest that selective perturbation of proteasomal protein degradation may play a role in the antitumoral activities of ritonavir