Critical Review. Insights into Acinetobacter baumannii Pathogenicity. Gustavo M. Cerqueira 1 and Anton Y. Peleg 1,2,3

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1 IUBMB Life, 63(12): , December 2011 Critical Review Insights into Acinetobacter baumannii Pathogenicity Gustavo M. Cerqueira 1 and Anton Y. Peleg 1,2,3 1 Faculty of Medicine, Nursing and Health Sciences, Department of Microbiology, Monash University, Melbourne, Australia 2 Department of Infectious Diseases, Alfred Hospital, Melbourne, Australia 3 Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA Summary Acinetobacter spp. have justifiably received significant attention from the public, scientific, and medical communities. Over recent years, Acinetobacter, particularly Acinetobacter baumannii, has become a red-alert human pathogen, primarily because of its exceptional ability to develop resistance to all currently available antibiotics. This characteristic is compounded by its unique abilities to survive in a diverse range of environments, including those within healthcare institutions, leading to problematic outbreaks. Historically, the virulence of the organism has been questioned, but recent clinical reports suggest that Acinetobacter can cause serious, life-threatening infections. Furthermore, its metabolic adaptability gives it a selective advantage in harsh hospital environments. This review focuses on current understanding of A. baumannii pathogenesis and the model systems used to study this interesting organism. Ó 2011 IUBMB IUBMB Life, 63(12): , 2011 Keywords A. baumannii; antibiotic resistance; nosocomial infection; pathogenesis; virulence. INTRODUCTION The genus Acinetobacter comprises important opportunistic human pathogens, as well as environmental organisms (1). To date, the genus Acinetobacter comprises 23 validly named species ( and a number of DNA- DNA hybridization groups (genomic species) with provisional designations (2); however, Acinetobacter baumannii is the most prevalent clinical species worldwide. Hospital-acquired pneumonia remains the most important clinical syndrome, often Received 8 June 2011; accepted 9 June 2011 Address correspondence to: Anton Y. Peleg, Department of Microbiology, Monash University, Building 76, Melbourne, VIC 3168, Australia. Tel: anton.peleg@monash.edu affecting mechanically ventilated patients in intensive care units. Bloodstream infection is less common but potentially lifethreatening, and other infectious syndromes such as urinary tract infection, hospital-acquired meningitis, and traumatic skin, soft tissue and bone infection, also occur (1). The clinical impact of A. baumannii infection is challenging to assess, as infected patients are often unwell for many reasons, and clinical studies are often limited by their retrospective design and lack of adjustment of important confounding variables. However, it is clear that A. baumannii has a unique ability in surviving in the hospital environment and in developing resistance to antibiotics, directly leading to troublesome hospital outbreaks and therapeutic challenges for documented infections. In general, the success of A. baumannii can be attributed to several factors: (i) its ability to form biofilms and resist dessication on abiotic surfaces (i.e. medical devices and environmental surfaces (3 6); (ii) its ability to adhere to, colonize and invade human epithelial cells (7, 8); (iii) its repertoire of antibiotic resistance mechanisms that are able to be promptly up-regulated as required; and (iv) its ability to acquire foreign genetic material through lateral gene transfer to promote its own survival under antibiotic and host selection pressures (9, 10). Specific virulence mechanisms identified to date are summarized in Table 1. This review focuses on current knowledge of A. baumannii pathogenesis and the model systems used to study its virulence attributes. Nevertheless, despite recent exciting advances, knowledge regarding the molecular pathogenesis and genetics of A. baumannii is still in its infancy. BIOFILM FORMATION AND USE OF IN VITRO ABIOTIC MODELS TO STUDY A. BAUMANNII PATHOGENESIS Attachment and adherence to medical equipment and environmental surfaces appears to be important for A. baumannii pathogenesis. For example, the mean survival time of clinical isolates on glass coverslips when subjected to dehydrating ISSN print/issn online DOI: /iub.533

2 1056 CERQUEIRA AND PELEG Table 1 Summary of known A. baumannii virulence factors Designation Reference 1. CsuA/BABCDE chaperone-usher 5 pili assembly system 2. Siderophore-mediated iron acquisition system 3. Outer membrane protein A (OmpA) AbaI autoinducer synthase Biofilm-associated protein (Bap) 6 6. Two-component regulatory 19 system, BfmRS 7. Penicillin-binding protein 7/8 (PBP-7/8) PNAG-constituted biofilm Capsule Lipopolysaccharide Phospholipase D Phospholipase C 25 conditions was 27 days (26), and it has been shown that A. baumannii can survive on hospital bed rails for up to 9 days (27). Indeed, the pioneering studies on A. baumannii pathogenesis focused on the ability of A. baumannii to form biofilms on abiotic surfaces, as well as the organism s ability to survive in iron-limiting conditions (5, 11, 12). The biofilm studies were mainly performed using a 96-well polystyrene assay, as well as attachment to glass and other plastic surfaces. One of the earliest studies showed the importance of exopolysaccharide production and pilus formation to biofilm development in A. baumannii (5). Using random mutagenesis and screening of mutants for biofilm formation, csue was identified as being important in bacterial attachment and biofilm formation, as well as pili production and assembly (5). Further genetic characterization identified a csu operon, made up of six genes (csua/babcde), which encodes a chaperoneusher secretion system that is important for pilus assembly (5). Despite the importance of the csu operon in biofilm formation on abiotic surfaces, it is less important for adherence to mammalian cells (28). Subsequent work identified a two-component regulatory system for the csu operon, known as bfmrs (19). Inactivation of the bfmr response regulator, but not the bfms sensor kinase, resulted in complete abolition of csu expression, resulting in no pili production, loss of adhesive properties, and loss of biofilm formation on plastic (19). In contrast, inactivation of bfms interfered only partially with biofilm formation and had no influence on downstream target genes (19). As seen with other two-component regulatory systems, this observation suggests that BfmR can use other sensor kinases for its activation. Apart from pili formation, another surface protein was recently identified as being important for biofilm structure in A. baumannii. Loehfelm et al. (6) identified a staphylococcal homologue of biofilm-associated protein (Bap), and after generating a transposon-mediated bap mutant in a contemporary A. baumannii clinical strain ( ), it was shown that the mutant produced \50% of the biofilm thickness produced by the parent strain. Bap is a surface exposed protein and, rather than being involved in bacterial cell attachment, appears to be important in maintaining mature biofilm architecture (6). Interestingly, antibodies directed to Bap bound to 93% of the four most common strain types from a clinical outbreak, as determined by MLST, whereas they bound to only 28% of the 35 less common strain types (6). The significance of this observation is unclear, but further research into the clinical importance of Bap in A. baumannii is clearly warranted. Another outer membrane protein that has been implicated in biofilm formation is OmpA (14 17). It appears that OmpA is not essential for biofilm development, but is required for the formation of thick biofilms on polystyrene (17). The role of this protein is discussed in more detail below. Apart from cellular appendages and surface proteins, exopolysaccharides form a key component of A. baumannii biofilms, with the major polysaccharide polymer being poly-b-(1-6)-nacetylglucosamine (PNAG) (21). It appears that PNAG is not essential for biofilm formation under static conditions, but may be required for maintaining the integrity of A. baumannii biofilms in more dynamic and stressful environments (21). Furthermore, as shown in other bacteria, PNAG may play a role in cell cell adherence (29), as well as in protection against innate host defenses (30). However, these functions are yet to be confirmed in A. baumannii. Finally, the quorum-sensing molecule 3-hydroxy-C 12 -HSL encoded by the abai autoinducer synthase gene has been implicated in A. baumannii biofilm formation on abiotic surfaces (18). This gene was identified using a screen with an Agrobacterium tumefaciens trag-lacz biosensor. An abai mutant was able to adhere and form an initial biofilm, but later stages of biofilm formation were impaired (18). This defect was corrected when supernatant from the isogenic parent A. baumannii strain, which contains wild-type 3-hydroxy-C 12 -HSL, was added to the biofilm assay (18). However, despite these observed changes, the differences in biofilm formation were relatively subtle, statistical differences were not reported, and assessment in a mammalian infection model was not performed. Furthermore, no differences in killing were observed when the abai mutant and its parent strain were assessed in the Galleria mellonella (described below) infection model (31). The clinical relevance of results obtained using the in vitro biofilm assay on abiotic surfaces remains unclear. Theoretically, it seems logical that there would be a correlation between laboratory results and the clinic. However, investigations of a large set of well-described A. baumannii clinical strains (n 5 45) that differed in both epidemicity and clonality in terms of their ability to form biofilms on plastic revealed a wide variation in results, with no correlation between epidemic, outbreak or antibiotic-resistant strains and the amount of biofilm formed (32). Furthermore, great variability was observed at the strain level in

3 INSIGHTS INTO ACINETOBACTER BAUMANNII PATHOGENICITY 1057 other Acinetobacter spp. (32). This poor correlation between clinical A. baumannii strains and the results of in vitro biofilm assays on abiotic surfaces has also been shown in another study (33). These data may suggest that biofilm formation on abiotic surfaces is not critical for the clinical success of A. baumannii, or more likely, that the biofilm assays used need to be rethought to more appropriately mimic the host environment. CELLULAR CYTOTOXICITY AND USE OF IN VITRO BIOTIC MODELS TO STUDY A. BAUMANNII PATHOGENESIS Adherence to biotic surfaces is an essential step toward colonization and infection (34). A. baumannii has a predilection for causing respiratory tract infections, and therefore most published studies have focused on A. baumannii adherence, invasion, and cytotoxicity of respiratory tract cell lines. Several elegant papers have characterized this process and identified OmpA (previously Omp38) as a key virulence factor of A. baumannii. An initial study described how A. baumannii infection triggers apoptosis in epithelial cells (HeLa) through a caspase-3 dependent process (35). A subsequent study confirmed this caspase-dependent apoptosis in human laryngeal HEp-2 cells, and further described mitochondrial damage as a trigger for caspase activation and release of apoptosis-inducing factor (caspase-independent process) (14). After screening a random transposon-mediated A. baumannii mutant library, a mutant strain lacking a 38 kda outer membrane protein (Omp), had a significant decrease in cellular toxicity and apoptosis (14). This protein was termed Omp38 and is now known as OmpA. Supporting these findings, exposure of HEp-2 cells to purified OmpA produced an indistinguishable phenotype to wild-type A. baumannii (14). OmpA localized to the mitochondria, causing damage and activation of caspases and apoptosis-inducing factors (14). This work was extended to show that Omp38 binds to a range of eukaryotic cells (HEp-2, Cos-7, and macrophages), is important for A. baumannii cellular invasion, and that it targets both the nucleus and mitochondria (15, 16). Furthermore, Omp38 was shown to degrade chromosomal DNA by DNAse I- like enzymatic activity (36). Finally, in a murine pneumonia model, the OmpA 2 mutant produced less lung inflammation and tissue destruction, and a lower bacterial density in blood (16). Other studies have also implicated OmpA in pathogenesis, including toxicity toward the human fungal pathogen Candida albicans (17). Thus OmpA is one of the most well-described and important virulence factors identified in A. baumannii. It also seems that OmpA is the most abundant Omp and that it can also be secreted by A. baumannii in the form of outer membrane vesicles (37). Unlike biofilm on abiotic surfaces, pilus-like structures and the Csu chaperone-usher system were not found to be involved in adherence of A. baumannii to bronchial epithelial cells (28). Furthermore, similar amounts of inflammatory cytokines, such as interleukin (IL)-6 and IL-8 were produced by the epithelial cells in response to infection with the csue mutant and its parent strain (28). These data question further the clinical significance of in vitro biofilm assays on abiotic surfaces. However, it is plausible that the mechanisms of adherence to these diverse surfaces are different. USE OF IN VIVO NON-MAMMALIAN MODEL SYSTEMS TO STUDY A. BAUMANNII PATHOGENESIS Non-mammalian model systems or invertebrate models have emerged over the last decade as useful tools to study microbial pathogenesis. The most commonly used models include the Caenorhabditis elegans worm model, the Drosophila melanogaster fly model, the G. mellonella caterpillar model, and the Dictyostelium discoideum amoebic model. The identification of novel, as well as established virulence determinants in a wide range of human pathogenic bacteria and fungi have been described through the use of these models (38). Remarkably, many of the virulence mechanisms used by these pathogenic organisms to cause mammalian disease are also important for disease in invertebrate hosts. In general, non-mammalian model systems have significant logistical, ethical, and financial advantages over traditional mammalian models, and they allow assessment of large numbers of engineered bacterial mutant strains, which would be impossible with larger animal models. Each invertebrate model has its own unique advantages. For example, C. elegans has a short reproductive cycle (days) and has large progeny production, which facilitates rapid experimentation. Also, the worm is small (~1 mm) and transparent, providing excellent opportunity for detailed microscopy using confocal laser microscopy. Finally, the C. elegans genome is fully sequenced, and sophisticated genetic tools, such as RNA interference, are available for functional genomics. The G. mellonella caterpillar model is complimentary to C. elegans and has the advantage of being maintained at 37 8C rather than 25 8C for C. elegans. The fly model also has a fully sequenced genome, as well as a more detailed immunological response, including toll-like receptors, which were first identified in Drosophila (39). Overall, these models provide a useful balance between complexity, genetic tractability, and ease of experimentation; however, it is recognized that conclusions about virulence requires confirmation in a mammalian system. The use of non-mammalian models to study A. baumannii pathogenesis began with a study that revealed that the growth and salt tolerance of A. baumannii was augmented in the presence of Saccharomyces, and this was attributed to low concentrations of ethanol secreted by the fungus (40). To determine whether ethanol altered the virulence of A. baumannii, a C. elegans killing assay was used to measure survival of the worms after exposure to A. baumannii with and without ethanol (1%). The worms died faster in the presence of A. baumannii and ethanol, suggesting an increase in virulence (40). Relative to other human pathogens that have been studied using C. elegans, such as Pseudomonas aeruginosa and Staphylococcus

4 1058 CERQUEIRA AND PELEG aureus, the rate of killing when infected with A. baumannii was very slow (50% of the worms killed after 5 7 days), suggesting an overall low-level of virulence for A. baumannii against C. elegans. As a follow on to this work, C. elegans and D. discoideum were used to screen a transposon-generated A. baumannii mutant library (n ) for mutants with attenuated virulence in the presence of ethanol (9). Rather than using a C. elegans killing assay, this study used an assay that relies on worm proliferation, and the speed at which the worms consume a lawn of bacteria. In the presence of a virulent mutant strain and ethanol, the worms would be sick, proliferate poorly, and take longer to eat a lawn of bacteria. This assay was more amenable to screening large numbers of mutant strains. Fourteen mutants were identified that were attenuated in virulence in the presence of ethanol in both the worm and amoebic model, and whose genes were not found in the non-pathogenic Acinetobacter baylyi species (9). Mutations were identified in several genetic islands that contain genes with putative virulence function. However, all the mutants produced similar biofilms to the parent strain, and no mammalian virulence studies were performed (9). Moreover, when tested in the G. mellonella model, no difference in killing was observed for any of the mutants (personal unpublished data). These data support the fact that confirmation in a mammalian system is required before final conclusions can be made about virulence. Despite this, this important study highlighted the power of broad-based approaches to virulence gene detection. The same group recently studied global gene expression of A. baumannii using RNA-sequencing in the presence of ethanol, and were able to identify phospholipase C as being an important up-regulated gene with modest virulence function toward epithelial cells (25). A further study extended the paradigm of using C. elegans to study microbial pathogenesis by using the model system to study polymicrobial infections (41). This study investigated the interactions between C. albicans, the most common human fungal pathogen, and A. baumannii within a living host. When C. elegans are infected with C. albicans, the yeast form cells proliferate within the worm gut lumen. As the infection ensues, the yeast form cells undergo a morphological transition to form filaments, which pierce through the tissue of the worm, leading to death. Filament formation has been shown to be a key C. albicans virulence mechanism in mammals. When the worms were infected with A. baumannii and C. albicans, no fungal filaments were produced, and the worms survived for a longer period of time compared to infection with C. albicans alone (41). Thus, A. baumannii was affecting the viability and virulence of C. albicans. This work was further supported by a study showing that OmpA was involved in virulence toward C. albicans in an in vitro environment (17). Finally, a new model system has been developed to study A. baumannii pathogenesis; the G. mellonella caterpillar model (31). G. mellonella are caterpillars of the greater wax moth, and are infected by injecting the pathogen into the hemocel of the animal. A. baumannii was shown to kill G. mellonella in a dose-dependent manner, and to be more virulent than non-pathogenic species such as A. baylyi, supporting the utility of the model (31). Interestingly, antibiotics could be administered to the caterpillars after a lethal dose of bacteria, with therapeutic success being dependent on the antibiotic susceptibility profile of the infecting strain (31). This is an exciting extension to the utility of the model, and has recently been applied to study the efficacy of combination therapy against multidrug-resistant A. baumannii (42). USE OF IN VIVO MAMMALIAN MODEL SYSTEMS TO STUDY A. BAUMANNII PATHOGENESIS As mentioned above, mammalian infection model systems remain the gold standard for pathogenesis-based research, even though significant limitations may exist in extrapolating results to humans. Historically, mammalian models have been used with A. baumannii to study the efficacy of antibiotics. Given that A. baumannii most commonly causes pneumonia, lung infection models (intranasal or intra-tracheal inoculation) in small rodents have predominated. One of the earlier studies assessing A. baumannii pathogenesis focused on host responses to infection and identified the importance of the lipopolysaccharide (LPS)-induced inflammatory responses to A. baumannii infection, more specifically the requirement for CD14 and Tolllike receptor (TLR)4, which are both important in the LPSsignaling process (43). This work was supported by assessing the response of A. baumannii LPS on TLR-deficient human cell lines (44). Over recent years, several studies have utilized a previously established approach of creating a transposon-generated random mutant library, screening the library for growth in human fluid (serum or ascites), and then testing mutants that grow poorly in a mammalian model. The mammalian models and end-points have varied in complexity and strength, and have included a mouse or rat pneumonia model, as well as a rat soft-tissue model where bacteria are injected into a soft-tissue pouch and then the pouch is sampled over time for bacterial density. A pilot study assessing A. baumannii infection of bone in a rat model has also been performed (45). Virulence factors that have been identified using this approach include penicillinbinding protein 7/8 (20), a glycosyltransferase (LpsB) involved in LPS biosynthesis (23), and genes (ptk and epsa) important for capsule formation (22). Phospholipase D was shown to be important for growth in human serum and for epithelial cell invasion, but in the murine pneumonia model, similar bacterial burdens and histopathology were identified (24). However, rates of bacteremia were less at 24 H in the pld mutant, and subsequent bacterial burden in extra-pulmonary tissues (heart and liver) was also less (24). These results are similar to that observed for OmpA in a murine pneumonia model, where the mutant strain had similar lung densities to the wild-type, but lung inflammation and tissue destruction, as well as bacterial burden in the blood, were less for the mutant (16).

5 INSIGHTS INTO ACINETOBACTER BAUMANNII PATHOGENICITY 1059 CONCLUSIONS A. baumannii is a remarkable organism that causes problematic outbreaks and infections in hospitals worldwide. Relative to other pathogenic gram-negative organisms, very little is known about its virulence mechanisms and host responses to infection. Model systems have now been established to study A. baumannii pathogenesis, including in vitro abiotic and biotic models, and in vivo systems including invertebrate and mammalian models. Each of these has provided an important base for knowledge growth in A. baumannii pathogenesis. There is still much to learn with regard to the genetic tools for targeted manipulation of genes in A. baumannii, the success of which appears to be straindependent; however, random transposon-mediated mutagenesis and complementation are established. 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