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t infections. Many clinical isolates have acquired multiple antibiotic resistance and can be more pan-resistant than even methicillinresistant Staphylococcus aureus . Furthermore, infections caused by Acinetobacter are not restricted to the clinical setting, and reports have emerged describing cases involving otherwise healthy individuals of varying ages, occurring in community settings, during wars, and following natural disasters. To date there has been no comparative testing for potential virulence and toxic effects of Acinetobacter species/strains isolated from clinical and environmental sources, which should be necessary prior to any intended biotechnology application. Our research objective is to expand the repertoire of useful endpoints required to predict and rank pathogenicity potential of environmental Acinetobacter strains with the aim of reducing the use of more costly and laborious in vivo test methods. In previous work, we developed a set of pathogenicity-toxicity parameters that could differentiate between potentially harmful and non-toxic strains of Bacillus species. Here we describe a side-by-side comparison of strains from seven Acinetobacter strains which were selected from both clinical and environmental sources. We used our previous test parameters, as well as others developed to study clinical Acinetobacter strains. The latter include assays which Virulence Potential of Acinetobacter Strains assess capacity PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189346 to bind or adhere to mammalian cells, 6-Methoxy-2-benzoxazolinone disrupt mammalian cell interactions , produce haemolytic or cytolytic activities, and/or infect by way of proliferation in mammalian cell culture medium. Also included is an assessment of exposure-induced immune system responses such as release of pro-inflammatory cytokines and chemoattractants -1b, IL-6, IL-8, and tumour necrosis factor-a ), as observed in vitro with laryngeal epithelial cells and cultured mouse splenocytes. Materials and Methods Bacterial Culture and Monitoring were purchased from the Sigma Chemical Company and Invitrogen. Using 96-multi-well plates, 26104 cfu of bacteria were added to each microwell containing an antibiotic dilution series in trypticase soy broth . The final concentration of each antibiotic in the series was 24, 12, 6, 3, 1.5, 0.75, 0.38 and zero mg/ml. Since growth in TSB varied between strains, plates were incubated at 28uC or 37uC for 24 h, 48 h or 96 h to generate enough bacterial growth to evaluate antibiotic susceptibility. The bacterial metabolic status of each sample was determined by adding MTT -2,5-diphenyl tetrazolium bromide) to a final concentration of one mg/ml/well. Plates were then incubated at 28uC or 37uC for two hours and examined for purple crystals. The MIC was defined as the minimum antibiotic concentration resulting in no detectable MTT bioreduction, which indicates that no metabolic activity and proliferation of bacterial cells has occurred. Haemolytic activity produced by various Acinetobacter strains was compared to that produced by the positive control. Sterile, defibrinated sheep blood was washed three times with PBS by repeated centrifugation and resuspension in 0.9% saline, before resuspension at a final concentration of 5% with cooled, autoclaved agar ) containing 1.4% pancreatic digest of casein, 0.5% NaCl, 0.45% peptone and 0.45% yeast extract. After agar solidification, each strain was deposited in a grid pattern onto the surface, and incubated at 37uC for up to 6 days. Photographs were taken daily to mon

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