Military medical facilities treating patients injured in Iraq and Afghanistan have identified a large number of multidrug-resistant (MDR)Acinetobacter baumannii isolates. In order to anticipate the impact of these pathogens on patient care, we analyzed the antibiotic resistance genes responsible for the MDR phenotype in Acinetobacter sp. isolates collected from patients at the Walter Reed Army Medical Center (WRAMC). Susceptibility testing, PCR amplification of the genetic determinants of resistance, and clonality were determined. Seventy-five unique patient isolates were included in this study: 53% were from bloodstream infections, 89% were resistant to at least three classes of antibiotics, and 15% were resistant to all nine antibiotics tested. Thirty-seven percent of the isolates were recovered from patients nosocomially infected or colonized at the WRAMC. Sixteen unique resistance genes or gene families and four mobile genetic elements were detected. In addition, this is the first report of bla OXA-58 -like and bla PER -like genes in the U.S. MDR A. baumannii isolates with at least eight identified resistance determinants were recovered from 49 of the 75 patients. Molecular typing revealed multiple clones, with eight major clonal types being nosocomially acquired and with more than 60% of the isolates being related to three pan-European types. This report gives a "snapshot" of the complex genetic background responsible for antimicrobial resistance in Acinetobacter spp. from the WRAMC. Identifying genes associated with the MDR phenotype and defining patterns of transmission serve as a starting point for devising strategies to limit the clinical impact of these serious infections.

Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability.

Our findings suggest that environmental contamination of field hospitals and infection transmission within health care facilities played a major role in this outbreak. On the basis of these findings, maintaining infection control throughout the military health care system is essential. Novel strategies may be required to prevent the transmission of pathogens in combat field hospitals.

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Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.

The activities of iron chelators (deferoxamine, deferiprone, Apo6619, and VK28) were evaluated against type strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. Deferiprone, Apo6619, and VK28 each inhibited growth in standard and RPMI tissue culture medium, while deferoxamine had no effect. Additionally, time-kill assays revealed that VK28 had a bacteriostatic effect against S. aureus. Therefore, these newly developed iron chelators might provide a nontraditional approach for treatment of bacterial infections.

Helicobacter hepaticus has been reported to induce colitis, hepatitis, and hepatocellular carcinoma in several different murine models. The aim of this study was to determine if H. hepaticus will cause colitis in monoassociated mice lacking the interleukin-10 gene (IL-10 ؊/؊ mice) and potentiate colitis in specific-pathogen-free (SPF) IL-10 ؊/؊ mice. Germfree IL-10 ؊/؊ mice on either a mixed (C57BL/6 ؋ 129/Ola) or inbred (129/SvEv) genetic background were monoassociated with H. hepaticus ATCC 51448 by oral feeding and rectal enemas. In a second experiment, germfree IL-10 ؊/؊ mice were colonized with stool from SPF mice that harbored or did not harbor endogenous H. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was isolated for histology and IL-12 secretion, and mesenteric lymph node cells were assessed for T-cell activation markers. It was found that IL-10 ؊/؊ mice monoassociated with H. hepaticus for up to 16 weeks showed almost no histologic colitis or increased IL-12 production. SPF IL-10-knockout mice had no significant difference in weight loss, mortality rate, histologic scores, colonic IL-12 secretion, or T-cell activation with or without H. hepaticus. We conclude that H. hepaticus does not induce or potentiate disease in our IL-10 ؊/؊ mice and therefore is not required to induce colitis in genetically susceptible hosts.

Acinetobacter isolates associated with casualties from the Iraq conflict from the United States were compared with those from the United Kingdom by pulsed-field gel electrophoresis and integron analysis. Representatives of the main outbreak strain associated with casualties from both countries were indistinguishable in DNA profile. Two further outbreak strains were common to both sets of isolates.