Antibiotic Modulation of Capsular Exopolysaccharide and Virulence in Acinetobacter baumannii

February Antibiotic Modulation of Capsular Exopolysaccharide and Virulence in Acinetobacter baumannii Edward Geisinger 0 2 3 Ralph R. Isberg 0 2 3 0 1 Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 2 Howard Hughes Medical Institute , Boston, Massachusetts , United States of America 1 F32AI098358. RRI is an Investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Funding: This work was supported by Howard Hughes Medical Institute. EG was supported by a postdoctoral fellowship from National Institute of Allergy and Infectious Diseases 3 Editor: David Weiss, Emory University School of Medicine, UNITED STATES Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen. - Competing Interests: The authors have declared that no competing interests exist. Acinetobacter baumannii has gained notoriety as a cause of hospital-acquired infections that are difficult to treat due to extensive antibiotic resistance. While the microorganism rarely causes disease in the community, it commonly infects patients receiving antibiotics. The factors intrinsic to the bacterium that enable growth in the presence of antibiotics are not well characterized. Furthermore, the consequences of subinhibitory antibiotic concentrations on A. baumannii disease are unknown. Here we examined the K locus, a bacterial disease determinant responsible for the production of protective surface polysaccharides, and asked whether this determinant also contributes to antibiotic resistance. We found that K locus polysaccharides facilitate resistance to multiple antibiotics, and, unexpectedly, that the bacterium responds to certain antibiotics at subinhibitory concentrations by increasing production of capsule, the principal K locus polysaccharide. This augmented production of capsule, which is mediated by upregulation of K locus gene expression, increased the ability of the bacterium to overcome attack by the complement system, an important anti-pathogen host defense, and result in lethal disease during experimental bloodstream infection in mice. Our studies indicate that A. baumannii increases its disease-causing potential in the setting of inadequate antibiotic treatment, which may promote the development of opportunistic infections. Hospital-acquired infections with multidrug resistant (MDR) bacteria pose increasingly difficult challenges for patient care. These diseases are often intransigent to initial empiric broadspectrum antibiotic therapy, delaying effective treatment and resulting in significant morbidity and mortality in already vulnerable patient populations. An emerging cause of such troublesome infections is Acinetobacter baumannii. This organism is responsible for a spectrum of diseases in susceptible individuals, including hospital-acquired pneumonia, sepsis, and wound infections [1]. A. baumannii is especially problematic in intensive care units (ICUs), where it is now among the 5 most common pathogens associated with ventilator-associated pneumonia in US hospitals [25]. Moreover, these infections are associated with alarming increases in drug resistance rates. A recent survey reported that most hospital-acquired A. baumannii infections are MDR [5], and strains resistant to all clinically useful antibiotics are emerging [68]. Observations such as these have led the Infectious Diseases Society of America and Food and Drug Administration to designate A. baumannii a high priority target for new antibiotic development [9,10]. Novel approaches to treat A. baumannii are urgently needed. An understanding of the pathobiology of A. baumannii infections would facilitate the development of novel control strategies. These infections typically target critically ill, hospitalized patients with indwelling devices [1], in whom they can be found as colonizers before the onset of disease [11,12]. In addition, multivariate analyses have consistently identified prior or inappropriate antibiotic treatment as an independent risk factor for A. baumannii nosocomial diseases [1315]. How antibiotics modulate host susceptibility to infection is not well understood, although several mechanisms are possible, including indirect effects on the host, such as reduction in competitive, drug-susceptible populations within the patient microbiota and/or modulation of innate immune defenses, as well as direct effects on A. baumannii physiology and virulence potential. Regarding bacterial factors that contribute to pathogenicity, the A. baumannii envelope is associated with many of the determinants of virulence in mammalian disease models [1622]. Among these, capsular exopolysaccharide has emerged as a universal virulence factor owing to several observations. In a study of greater than 40 A. baumannii patient isolates, almost al (...truncated)