Osteomyelitis (OM) from multidrug-resistant (MDR) has emerged in >30% of combat-related Velcade injuries in Iraq and Afghanistan. PCR for the specific gene confirmed a 100% infection rate with 75% of the MDR strains and testing confirmed that all strains were sensitive to colistin. We also developed a real-time quantitative PCR (RTQ-PCR) assay that could detect as few as 10 copies of in a sample. To demonstrate the efficacy of colistin prophylaxis in this model mice were treated with either parenteral colistin (0.2mg colistinmethate i.m. for 7 days) local colistin (PMMA bead impregnated with 1.0mg colistin sulfate) or an unloaded PMMA bead control. While the parenteral colistin failed to demonstrate any significant effects vs. the placebo the colistin PMMA bead significantly reduced the infection rate such that only 29.2% of the mice had detectable levels of at 19 days (p<0.05 vs. i.m. colistin and placebo). species which can be difficult to cure in some settings.9-11 Surprisingly this pathogen has been reported in less than 2% of nosocomial infections within the United States but has emerged in over 30% of admitted deployed soldiers.10 In contrast to complex (ABC) are Gram-negative non-fermentative non-spore forming strictly aerobic oxidase-negative coccobacillary organisms. Additionally infections caused by appear to be hospital-acquired and not from an initial colonization of the injury. 13 Thus one critical question involving is whether or not they can produce osteolytic OM on their own or if they are only present in super-infections with other microorganisms. Another important question is whether or not the MDR OM can be effectively prevented with parenteral or local antibiotic therapy at the time of initial surgery. In support of this a clinical study has demonstrated that most MDR Acinetobacter strains are sensitive to colistin 10 and that colistin heteroresistance primarily occurs in patients treated with colistin.14 Thus we aimed to test the hypotheses that: i) pure clinical isolates of MDR can induce implant-associated OM and ii) prophylactic colistin prevents these orthopaedic infections. To the end we used a quantitative murine style of implant-associated OM orginally created for can stimulate OM in the lack of Velcade additional pathogens nevertheless these lesions are blastic instead of osteolytic and don’t contain obvious biofilms in necrotic bone tissue. Moreover regional however not parenteral pharmacological doses of colistin are capable of preventing the establishment of MDR Acinetobacter implant-associated OM. Methods Bacterial Velcade strains Four clinical isolates of with confirmed resistance to amikacin ampicillin aztraeonam ceftriaxone ciprofloxacin gentamicin imipenem tobramycin and vancomycin were obtained from wounded soldiers treated at the Brooke Army Medical Center at Fort Sam Houston San Antonio under Institutional Review Board approved protocols. Bacterial strains were grown overnight at 250rpm and 37°C in Tryptic Soy broth (Sigma-Aldrich St. Louis MO). Strain sensitivity Rabbit polyclonal to AP1S1. to colistin was determined by streaking overnight cultures on Trypic Soy agar plates containing 10μg/ml of colistinmethate (Paddock Laboratories Inc Minneapolis MN). DNA extraction PCR cloning of parC and RTQ-PCR of Acinetobacter DNA DNA was extracted from all four strains using a DNeasy Blood and Velcade Tissue Kit (Qiagen Valencia CA). DNA primers (forward 5’-AAAAATCAGCGCGTACAGTG-3’ and reverse 5’-CGAGAGTTTGGCTTCGGTAT-3’) specific for the topoisomerase gene gene was amplified and sequenced Velcade using an i-cycle PCR machine (Bio-Rad Hercules CA). The amplification protocol was as follows: 95°C pre-melt for 15min followed by 40 cycles of 95°C for 30s 54 for 30s and 72°C for 30s. To generate a standard curve for RTQ-PCR the 196bp fragment was cloned into the pTOPO2.1 cloning vector (Invitrogen Carlsbad CA) and 10-fold serial dilutions were used to perform Syber Green (Thermo Scientific) RTQ-PCR (Corbett Research Sydney AU). A similar curve was generated using the mouse β-primers (forward 5’-AGATGTGAATCAGCAAGCAG-3’ and reverse 5’-GCGCAAGTTAGGTTTTGTCA-3’) to control for sample integrity as we have previously described.15 In order to calculate the gene copies in a tibia sample we first generated a standard curve with genomic DNA purified directly from an overnight culture. The standard curve was generated with 10-fold dilutions of the TOPO plasmid with insert. The mean of the 3.