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Title: Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions}
Article:
Abstract:
While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis.
Introduction:
The diversity of bacterial-host interactions is extraordinary and ranges from symbioses critical for health, to infections that rapidly kill. Even more remarkable is the fact that the same species of bacteria can produce markedly different disease manifestations, depending upon the clinical setting. The opportunistic pathogen Pseudomonas aeruginosa is a prime example. In some patients, like those with pneumonia or sepsis, it produces acute infections that can be rapidly lethal[1]. In other patients, like those with chronic wounds, sinus disease, or cystic fibrosis( CF), large numbers of organisms can remain localized at infection sites for years without systemic spread[1]. Staphylococcus aureus, Escherichia coli, Streptococcus sp. and others also produce life-threatening acute infections in some settings and smoldering chronic infections in others[2]. While knowledge of acute infection pathogenesis has advanced, understanding of chronic infections has lagged. An emerging model suggests chronic infection may involve a two-step process, with different functions mediating initial and established phases of infection[3–5]. Early-on, acute virulence functions are postulated to be active, enabling bacteria to establish a foothold within the host[3, 4]. Once infection is established, virulence factor activity may diminish, and biofilm formation is induced[3–5]. Biofilms are surface-attached and matrix-encased communities that are resistant to killing by antibiotics and immune responses. The net effect of these mechanisms could enable infection initiation, but yet produce the non-invasive and persistent phenotype of chronic infections. While this model is compelling, the evidence supporting phased activity of virulence and biofilm formation is inconclusive. For example, work using a murine surgical wound model found many genes which influence virulence such as motility, LPS, and quorum sensing were not essential for survival in chronic wound[6]. The only virulence factor which was both upregulated and important for fitness in this chronic wound model was the type 3 secretion system[6]. Studies of CF isolates has shown that bacterial genes mediating acute virulence are frequently inactivated by mutations acquired as infecting bacteria evolve in vivo[3, 7, 8]. This finding is consistent with the idea that some virulence factors could be dispensable, and indeed disadvantageous for infecting bacteria once chronic infection is established. However, it does not provide information about their activity during the initiation or early phases of infection. The importance of biofilm formation functions in chronic infections is also unsettled. For example, examination of infected specimens suggests that many bacteria in infected wounds and CF lungs are living in biofilm-like bacterial aggregates[9–11]. Furthermore, biofilm-mediated antibiotic tolerance is consistent with the treatment-resistant phenotype of chronic infections[11–13]. However, isolates taken from chronic CF infections often exhibit in vitro biofilm defects[14–16], and genes encoding a key biofilm-mediated polysaccharide was found to be repressed in murine chronic wounds[6]. In addition, recent work indicates that environmental conditions at infection sites( such as viscous mucus[17], proteases that inactivate flagella[17], and /or polymer-rich secretions[18]) can force bacteria to grow as aggregates. Thus, it is not known if aggregates form in vivo via the activity of biofilm functions, or if host conditions are responsible. Here we performed studies to test the importance of virulence and biofilm formation functions in wounds, and better understand wound infection pathogenesis. We studied human wound infection isolates, and performed genomic-level analysis in P. aeruginosa to identify bacterial functions that mediate bacterial fitness in murine chronic wounds. We also attempted to model the selective environment in wounds using ex vivo conditions. We focused on chronic wounds as they are a major and understudied problem, and P. aeruginosa is a predominant wound pathogen.
Discussion:
This study advances understanding of the P. aeruginosa functions needed to initiate and maintain chronic wound infections and the stress conditions that affect bacterial fitness in wounds. A key finding was that many virulence functions were inactivated in human wound infection isolates, and only one of the virulence functions we tested affected bacterial fitness in murine chronic wounds. Type three secretion was the exception as our mutant by mutant wound experiments found that inactivation of type three secretion compromised bacterial fitness in wounds, consistent with previous work[6]. Our finding that many P. aeruginosa virulence factors could be inactivated without reducing bacterial fitness in wounds, along with consistent findings from other studies[3, 6–8], suggest that chronic wounds sites may be relatively permissive environments in which bacterial nutrient acquisition and growth may not depend upon the invasive or toxic functions. A permissive host environment could also explain why low virulence “commensal” organisms commonly inhabit wounds[38, 39]. It is notable that isolates from chronic CF infections have been shown to exhibit defects in many of the same virulence functions. However, our experiments do not indicate that wound environments are stress-free. We found that genes mediating resistance to anaerobic and membrane stress, and certain metabolic pathways were critical in infection. Independent experiments investigating the phenotypes of mutants with high and low wound fitness corroborated the importance of anaerobic, oxidative, and membrane stress in wounds. Previous work using Tn-seq and gene expression analysis in wounds also found nutritional functions to be important( including those mediating purine and amino acid biosynthesis)[6]. Our experiments using colonies and stationary phase cultures suggested that the selective environment in wounds could be approximated by high-density growth conditions in the absence of a host. This finding is consistent with work showing that the P. aeruginosa gene expression profile in murine wounds resembles that in high-cell density colonies[25]. Taken together, these findings raise the possibility that the predominant stresses on organisms in wounds may be applied by the bacteria themselves, rather than the host. It is also possible that overlapping functions counter bacterial and host-applied stresses. Our data also sheds light on the role of biofilms in chronic infections. Our finding that isolates that successfully infect human wounds are often biofilm-deficient in vitro is similar to results using CF isolates[14]. We also found that genetic manipulations that markedly changed the capacity of P. aeruginosa to form biofilms did not produce corresponding effects in murine wounds. These results argue against the importance of biofilm formation functions, that are defined using laboratory systems, in these chronic infections. This is consistent with a previous report that expression of the predominant exopolysaccharide in PAO1, psl, is not increased in the chronic murine wound environment[6] despite the appearance of the bacteria in multi-cellular aggregates[9]. Biofilm formation functions may be dispensable in CF and wounds because the non-invasive and resistant bacterial phenotypes characteristic of chronic infection are a consequence of host conditions[40]. For example, cell aggregates may be a default growth mode in thick polymer-rich exudates present in CF airway and wounds[17, 18]. It is also possible that active mechanisms of aggregation are needed in vivo, but these functions do not overlap with biofilm formation functions identified in vitro. Nevertheless, the correlation between the fitness of mutants in wounds and high-density growth conditions suggest that the physiological stresses inherent to biofilm growth may be relevant in wounds, even if biofilm formation functions are not. Our study had several limitations. First, the clinical isolates we studied from acute infections all came from different patients, so we were unable to determine if isolate populations from individual subjects exhibited diversity in biofilm formation or virulence factor expression as wound isolates did. Second, limited clinical information was available about subjects from whom wound isolates were collected. Third, while the Tn-seq experiments comprehensively examined the effect of most non-essential genes, we did not test all identified virulence factors in mutant-by-mutant wound infection experiments. Thus, it is possible that some virulence functions( in addition to type three secretion) affect bacterial fitness in murine wounds. Fourth, the in vivo Tn-seq screening and verification experiments involved competitions between strains to identify fitness defects. This is a limitation, as some defects can be compensated for by the wild type in competitions. However, we found that all of the isolates from two wounds were protease-negative and all of the isolates from three wounds were rhamnolipid negative. Thus the subpopulations expressing these functions are very small, if they exist. Furthermore, the strain lacking both quorum sensing systems, and thus many secreted virulence factors, exhibited no significant defect in murine wounds in pure culture. These findings argue against the interpretation that subpopulations of bacteria producing virulence factors generally compensated for defective mutants in our experiments. Fifth, we note that while the biofilm and virulence gene mutants we studied were engineered deletions, we also used transposon mutants in some experiments to verify the Tn-seq results. Thus, it is possible that in some cases the transposon, or its insertion location, may have affected results. However, control experiments showed that the transposon did not generally affect in vivo fitness. Furthermore, the fitness defects of genes verified to be important for infection were replicated by many different mutants in each gene. These included the many independent mutants( in each gene) generated in the Tn-seq libraries( see Fig 4B and S7 Fig) and the independent sequence-defined transposon mutants[30, 31] used in verification experiments. Sixth, we used a well-defined laboratory strain, PAO1, which likely differs both in genomic content and gene expression from clinical wound isolates. However, despite these differences the virulence and biofilm phenotypes of many clinical wound isolates were generally corroborated by our experiments with PAO1 in the murine wound model. Finally, we note that the murine infection model does not represent all relevant conditions of human wounds. In particular, our murine infection model was used primarily to examine the necessity of genes early in chronic infection( within the first five days) which is shorter than the six weeks of infection which can be sustained by this model or the months to years of infection observed in human wounds. Our study also has had strengths that increased confidence in the results. We verified the fitness defects identified by Tn-seq with individual mutants. Such verifications are critical, as we found that ~1/3 of the defects identified by Tn-seq were not reproducible in mutant-by-mutant assays. This is consistant with other studies which have shown similar levels of reproducibility from Tn-seq screens[32–34]. In addition, results with clinical isolates and experimental murine infections gave generally concordant results regarding the importance of virulence and biofilm formation functions in wounds. Likewise, tests with wound-defective mutants and independent Tn-seq experiments both suggested that bacterial fitness in high-density growth conditions and wounds were strongly correlated. Finally, the wound model had key strengths. Diabetic mice were used, and diabetes is a key factor in human wound pathogenesis[41]. Furthermore, P. aeruginosa inhibited wound-healing in the model, which directly links infection to disease. In addition to increasing understanding of wound infection pathogenesis, this study highlights challenges and opportunities in therapeutics development. One novel approach seeks to target infection pathogenesis functions, rather than trying to kill or inhibit bacteria[42]. However, our data suggest that targeting biofilm formation and many virulence functions may not be useful in chronic infections. Our finding that the functions needed in wounds and high-density cultures overlap suggests that separating basic growth and chronic infection pathogenesis functions may be difficult. Our findings also point toward opportunities. The fact that stationary phase cultures and colonies generally represented the selective environment of wounds raises the possibility that these systems could be used as an initial screen for potential therapeutics, or therapeutic targets. Such approaches could offer “first-pass” efficiency and throughput advantages as compared to animal or cell-based models. Finally, our work suggests parallels in the pathogenic mechanisms of two of the most significant chronic bacterial infections: those afflicting people with chronic wounds and cystic fibrosis. This is important because animal models that faithfully represent most chronic bacterial infections are lacking, and progress may be accelerated if therapeutic targets relevant for multiple chronic infections could be identified.
Chronic infections are poorly understood , hard to model , and treatment resistant . Future progress depends upon understanding infection pathogenesis . Our study suggests that many bacterial virulence and biofilm formation functions are not required for fitness of infecting bacteria in chronic wounds . Instead , we found that functions that mitigate stresses associated with high-density bacterial growth are critical for infection . These findings suggest new approaches to model and target chronic bacterial infections .