Gram-Negative Bacterial Wound 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. IMPORTANCE The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials. Received 27 March 2014 Accepted 10 April 2014 Published 27 May 2014 Citation Jacobs AC, Thompson MG, Black CC, Kessler JL, Clark LP, McQueary CN, Gancz HY, Corey BW, Moon JK, Si Y, Owen MT, Hallock JD, Kwak YI, Summers A, Li CZ, Rasko DA, Penwell WF, Honnold CL, Wise MC, Waterman PE, Lesho EP, Stewart RL, Actis LA, Palys TJ, Craft DW, Zurawski DV. 2014. AB5075, a highly virulent isolate of Acinetobacter baumannii, as a model strain for the evaluation of pathogenesis and antimicrobial treatments. mBio 5(3):e01076-14. doi:10.1128/mBio.01076-14. Editor Howard Shuman, University of Chicago Copyright © 2014 Jacobs et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Daniel V. Zurawski, [email protected]. Acinetobacter baumannii is an opportunistic, Gram-negative pathogen that thrives in clinical settings and is often multidrug resistant (MDR), factors which earn it a place among the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens of clinical importance (1). Some recent isolates are resistant to all typically used antibiotics except colistin and tigecycline and thus are called extensively or extremely drug-resistant (XDR) A. baumannii (2). MDR/XDR A. baumannii strains are a worldwide problem for clinicians and caregivers in the hospital setting, particularly in the intensive care unit (ICU) (3). A. baumannii is also often isolated from infections of severe wounds sustained in military combat. These infections are responsible for increased morbidity, with prolonged wound RESEARCH ARTICLE May/June 2014 Volume 5 Issue 3 e01076-14 ® mbio.asm.org 1 m b.asm .rg on July 5, 2014 P ubished by m b.asm .rg D ow nladed fom healing and amputations of extremities when limbs cannot be salvaged (4, 5). A. baumannii was a predominant isolate from wounded soldiers serving in Iraq (4, 5) and was associated with wartime polytrauma injuries in the past (6). Additionally, there may be a link between A. baumannii and crush injuries, as A. baumannii infections were also prevalent after the recent large earthquakes in Haiti (7) and China (8). Another disturbing development that has increased the clinical importance of A. baumannii infections is that many strains have become highly antibiotic resistant. For example, in previous decades, A. baumannii isolates obtained from both military and civilian settings were often carbapenem sensitive. Now, the majority of U.S. military isolates are carbapenem resistant (9). This trend has also been mirrored in civilian hospitals around the world (10). Recently, even colistin-resistant strains have emerged in the military health care system (11). The latter development is deeply troubling, as colistin is considered the last line of defense against these MDR isolates. Exacerbating this problem is the lack of new treatments in the pharmaceutical pipeline (12); therefore, research on A. baumannii virulence factors is urgently needed, as they could constitute potentially novel targets for future antimicrobials. While previous studies attempted to examine the virulence of different clinical A. baumannii strains utilizing in vivo model systems (13, 14), the majority of A. baumannii researchers still use two American Type Culture Collection (ATCC) strains, ATCC 19606T and ATCC 17978, which were isolated more than 50 years ago and are not significantly antibiotic resistant. These strains are certainly more amenable to genetic manipulation than most clinical isolates (15, 16) and share considerable genome homology ( 90%) to current A. baumannii isolates (17), but they are not representative of contemporary isolates of this rapidly evolving pathogen. Some researchers, recognizing that the ATCC isolates are dated, have performed studies with more recent clinical isolates; however, genetic manipulation of such isolates has depended on susceptibility to aminoglycosides (18–20), which is often not found in clinical strains (21). Therefore, our goal was to carry out a systematic study of our own contemporary clinical strains isolated from patients in the U.S. military health care system to identify a strain that is more representative of current clinical isolates, that is highly virulent in established model infections, and that can be genetically manipulated without a potential sacrifice with respect to virulence and antibiotic resistance. Not only does identifying such a strain account for more recent clinical outcomes, but the increased virulence in animal models allows greater statistical power in screening new therapeutics. Moreover, the ability to manipulate the genome allows the study of virulence factors, some of which may be responsible for the emergence of this pathogen in more recent years. RESULTS Defining genetic characteristics of A. baumannii isolates. In order to identify potential reference strains, a diverse set of 33 A. baumannii isolates was chosen based on genetic, isolation site, and antibiotic resistance differences from more than 200 A. baumannii strains isolated between 2004 and 2010 from patients in the U.S. military health care system. AB0057, first isolated in 2004 at Walter Reed Army Medical Center, was also included as a comparator because this strain is well characterized, and its genome was previously sequenced (22). The diversity set of A. baumannii isolates was determined via pulsed-field gel electrophoresis (PFGE) analysis and a multiplex PCR assay previously developed to identify the international clonal complexes (ICC). Separately, antibiograms were determined using two different automated bacterial identification systems. The majority of strains were found to be multidrug resistant, typical of current clinical strains (see Table S1 in the supplemental material). As shown in Fig. 1, the genetic similarity of the strains, as determined by PFGE, ranged from 45 to 100%. PFGE types were considered to represent the same clones when their genetic similarity was 80% (23); based on this cutoff, the 33 strains represent 19 unique clones. When the genetic relatedness of these 19 clones was compared, it was found that the majority of them clustered into three groups, which generally aligned with the ICC designations determined by multiplex PCR (Fig. 1 and Table 1) (24). Exceptions were the isolates AB3560, AB4456, and AB4857, which were determined to be ICC III by the multiplex assay but appeared to be ICC I via PFGE. In this case, we relied on the multiplex data (Table 1) to be definitive. These data were used to select four representative strains for genome sequencing and evaluation in animal models. Three of the strains chosen each represented one of the three ICC groups, AB5075 (ICC I), AB5711 (ICC II), and AB4857 (ICC III). The fourth strain, AB5256 was an outlier, as the OXA-51 allele from this strain was amplified with group 1 primers (24), while the csuE allele was not. The isolates were sequenced (25) and compared to previously sequenced A. baumannii genomes using the BLAST score ratio (BSR) approach (26). This method compares putative peptides encoded in each genome based on the ratio of BLAST scores to determine if they are conserved (BSR value 0.8), divergent (0.8 BSR 0.4), or unique (BSR 0.4). The majority of the proteomes were similar among strains, meaning they had a BSR of 0.4; however, each isolate also had a set of unique proteins (see Table S2 in the supplemental material). These results are similar to what has been found previously with MDR A. baumannii clinical isolates (17), suggesting that the strains used in this study are not genetic outliers. Virulence assessed in the Galleria mellonella model. Strains were first tested in a Galleria mellonella infection model, as this model is well established to assess virulence and novel therapeutics for bacterial pathogens, including A. baumannii (27, 28). G. mellonella larvae were infected with an approximate dose of 1.0 105 CFU with each of the four sequenced A. baumannii isolates, AB4857, AB5075, AB5256, and AB5711, as well as control strain AB0057. Worms were observed for 6 days, and death was recorded. Within 24 h postinfection, approximately 25% of AB5075-infected worms remained, while the other four strains had survival rates of 70% or higher (Fig. 2). By the end of the 6-day study, AB5075-infected worms had a survival rate of 16%; strains AB4857 and AB5256 were considered moderately pathogenic in this model, with survival rates of 50% and 35%, respectively. The least lethal strains were AB5711 and AB0057, with survival rates of 85% and 83%, respectively. Phosphate-buffered saline (PBS)injected control worms displayed 100% survival through the course of the study. Based on these data, it was hypothesized that AB5075 was more virulent than the other four strains tested. Using the Mantel-Cox test with Bonferroni correction for multiple comparisons, Kaplan-Meier curves were compared, and AB5075 was found statistically to be more lethal than AB4857, AB5711, and AB0057 (all P values 0.0125). While AB5256 had a higher Jacobs et al. 2 ® mbio.asm.org May/June 2014 Volume 5 Issue 3 e01076-14 m b.asm .rg on July 5, 2014 P ubished by m b.asm .rg D ow nladed fom