Urvish Trivedi

Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection

Most infections result from colonization by more than one microbe. Within such polymicrobial infections, microbes often display synergistic interactions that result in increased disease severity. Although many clinical studies have documented the occurrence of synergy in polymicrobial infections, little is known about the underlying molecular mechanisms. A prominent pathogen in many polymicrobial infections is Pseudomonas aeruginosa, a Gram-negative bacterium that displays enhanced virulence during coculture with Gram-positive bacteria. In this study we discovered that during coinfection, P. aeruginosa uses peptidoglycan shed by Gram-positive bacteria as a cue to stimulate production of multiple extracellular factors that possess lytic activity against prokaryotic and eukaryotic cells. Consequently, P. aeruginosa displays enhanced virulence in a Drosophila model of infection when cocultured with Gram-positive bacteria. Inactivation of a gene (PA0601) required for peptidoglycan sensing mitigated this phenotype. Using Drosophila and murine models of infection, we also show that peptidoglycan sensing results in P. aeruginosa-mediated reduction in the Gram-positive flora in the infection site. Our data suggest that P. aeruginosa has evolved a mechanism to survey the microbial community and respond to Gram-positive produced peptidoglycan through production of antimicrobials and toxins that not only modify the composition of the community but also enhance host killing. Additionally, our results suggest that therapeutic strategies targeting Gram-positive bacteria might be a viable approach for reducing the severity of P. aeruginosa polymicrobial infections.

Requirements for Pseudomonas aeruginosa Acute Burn and Chronic Surgical Wound Infection

Opportunistic infections caused by Pseudomonas aeruginosa can be acute or chronic. While acute infections often spread rapidly and can cause tissue damage and sepsis with high mortality rates, chronic infections can persist for weeks, months, or years in the face of intensive clinical intervention. Remarkably, this diverse infectious capability is not accompanied by extensive variation in genomic content, suggesting that the genetic capacity to be an acute or a chronic pathogen is present in most P. aeruginosa strains. To investigate the genetic requirements for acute and chronic pathogenesis in P. aeruginosa infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in murine models of burn and non-diabetic chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for colonization and persistence in these infections. Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection. In addition, we determined that P. aeruginosa requires chemotactic flagellar motility for fitness and virulence in acute burn wound infections, but not in non-diabetic chronic wound infections. Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

Bacterial fight-and-flight responses enhance virulence in a polymicrobial infection

Significance Polymicrobial synergy occurs when infections caused by more than one species are more severe than the sum of the individual species acting alone. Here, we show that a bacterial fight-and-flight response to an antimicrobial, hydrogen peroxide (H2O2), is required for a pathogen to display synergy with a commensal bacterium in vivo. H2O2 is generated by the commensal, and in response, the pathogen either enzymatically destroys (fights) the antimicrobial or disperses away (takes flight) from the antimicrobial-producing commensal. Remarkably, both behaviors are critical for the pathogen to display synergy during coinfection. Moreover, when the pathogen is unable to disperse, the community loses spatial organization, trapping the pathogen next to the commensal. The oral pathogen Aggregatibacter actinomycetemcomitans (Aa) resides in infection sites with many microbes, including commensal streptococci such as Streptococcus gordonii (Sg). During infection, Sg promotes the virulence of Aa by producing its preferred carbon source, l-lactate, a phenomenon referred to as cross-feeding. However, as with many streptococci, Sg also produces high levels of the antimicrobial hydrogen peroxide (H2O2), leading to the question of how Aa deals with this potent antimicrobial during coinfection. Here, we show that Aa possesses two complementary responses to H2O2: a detoxification or fight response mediated by catalase (KatA) and a dispersion or flight response mediated by Dispersin B (DspB), an enzyme that dissolves Aa biofilms. Using a murine abscess infection model, we show that both of these responses are required for Sg to promote Aa virulence. Although the role of KatA is to detoxify H2O2 during coinfection, 3D spatial analysis of mixed infections revealed that DspB is required for Aa to spatially organize itself at an optimal distance (>4 µm) from Sg, which we propose allows cross-feeding but reduces exposure to inhibitory levels of H2O2. In addition, these behaviors benefit not only Aa but also Sg, suggesting that fight and flight stimulate the fitness of the community. These results reveal that an antimicrobial produced by a human commensal bacterium enhances the virulence of a pathogenic bacterium by modulating its spatial location in the infection site.

Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this hypothesis, we developed an in vivo chronic wound, diabetic mouse model to determine the ability of the opportunistic pathogen, Pseudomonas aeruginosa, to cause biofilm-associated infections. Utilizing this model, we observed that diabetic mice with P. aeruginosa-infected chronic wounds displayed impaired bacterial clearing and wound closure in comparison with their non-diabetic littermates. While treating diabetic mice with insulin improved their overall health, it did not restore their ability to resolve P. aeruginosa wound infections or speed healing. In fact, the prevalence of biofilms and the tolerance of P. aeruginosa to gentamicin treatment increased when diabetic mice were treated with insulin. Insulin treatment was observed to directly affect the ability of P. aeruginosa to form biofilms in vitro. These data demonstrate that the chronically wounded diabetic mouse appears to be a useful model to study wound healing and biofilm infection dynamics, and suggest that the diabetic wound environment may promote the formation of biofilms. Further, this model provides for the elucidation of mechanistic factors, such as the ability of insulin to influence antimicrobial effectiveness, which may be relevant to the formation of biofilms in diabetic wounds.

Kin selection, quorum sensing and virulence in pathogenic bacteria

Bacterial growth and virulence often depends upon the cooperative release of extracellular factors excreted in response to quorum sensing (QS). We carried out an in vivo selection experiment in mice to examine how QS evolves in response to variation in relatedness (strain diversity), and the consequences for virulence. We started our experiment with two bacterial strains: a wild-type that both produces and responds to QS signal molecules, and a lasR (signal-blind) mutant that does not release extracellular factors in response to signal. We found that: (i) QS leads to greater growth within hosts; (ii) high relatedness favours the QS wild-type; and (iii) low relatedness favours the lasR mutant. Relatedness matters in our experiment because, at relatively low relatedness, the lasR mutant is able to exploit the extracellular factors produced by the cells that respond to QS, and hence increase in frequency. Furthermore, our results suggest that because a higher relatedness favours cooperative QS, and hence leads to higher growth, this will also lead to a higher virulence, giving a relationship between relatedness and virulence that is in the opposite direction to that usually predicted by virulence theory.

Probing bacterial metabolism during infection using high-resolution transcriptomics

A fundamental aspect of most infectious diseases is the need for the invading microbe to proliferate in the host. However, little is known about the metabolic pathways required for pathogenic microbes to colonize and persist in their hosts. In this study, we used RNA sequencing (RNA-seq) to generate a high-resolution transcriptome of the opportunistic pathogen Aggregatibacter actinomycetemcomitans in vivo. We identified 691 A. actinomycetemcomitans transcriptional start sites and 210 noncoding RNAs during growth in vivo and as a biofilm in vitro. Compared to in vitro biofilm growth on a defined medium, ∼14% of the A. actinomycetemcomitans genes were differentially regulated in vivo. A disproportionate number of genes coding for proteins involved in metabolic pathways were differentially regulated in vivo, suggesting that A. actinomycetemcomitans in vivo metabolism is distinct from in vitro growth. Mutational analyses of differentially regulated genes revealed that formate dehydrogenase H and fumarate reductase are important A. actinomycetemcomitans fitness determinants in vivo. These results not only provide a high-resolution genomic analysis of a bacterial pathogen during in vivo growth but also provide new insight into metabolic pathways required for A. actinomycetemcomitans in vivo fitness.

Prevalence of Multiple Antibiotic Resistant Infections in Diabetic versus Nondiabetic Wounds

Diabetes mellitus (DM) affects 23.6 million people in the USA and approximately 20–25% of diabetic patients will develop foot ulceration during the course of their disease. Up to a quarter of these patients will develop infections that will necessitate amputation. Although many studies report that the rates of antibiotic resistant infections have increased dramatically in the DM population over the last decade, to our knowledge there have been no reports directly comparing the rates of antibiotic resistant infections in DM versus non-DM wounds. We performed a retrospective study comparing the wound infections of 41 DM patients to those of 74 non-DM patients to test the hypothesis that infections with multidrug resistant organisms (MDRO) were more prevalent in the DM population. We found that 63.4% of DM and 50% of non-DM patients had MDRO infections, which was not statistically different. However, 61% of the DM patients had Pseudomonas infections compared to only 18.9% of non-DM patients. Furthermore, DM patients had significantly more coinfections with both Pseudomonas and Staphylococcus aureus. Though our initial hypothesis was incorrect, we demonstrated a significant correlation between Pseudomonas and Pseudomonas/S. aureus coinfections within DM wounds.

A post-planktonic era of in vitro infectious models: issues and changes addressed by a clinically relevant wound like media

Abstract Medical science is pitted against an ever-increasing rise in antibiotic tolerant microorganisms. Concurrently, during the past decade, biofilms have garnered much attention within research and clinical practice. Although the significance of clinical biofilms is becoming very apparent, current methods for diagnostics and direction of therapy plans in many hospitals do not reflect this knowledge; with many of the present tools proving to be inadequate for accurately mimicking the biofilm phenomenon. Based on current findings, we address some of the fundamental issues overlooked by clinical labs: the paradigm shifts that need to occur in assessing chronic wounds; better simulation of physiological conditions in vitro; and the importance of incorporating polymicrobial populations into biofilm models. In addition, this review considers using a biofilm relevant in vitro model for cultivating and determining the antibiotic tolerance and susceptibility of microorganisms associated with chronic wounds. This model presents itself as a highly rapid and functional tool that can be utilized by hospitals in an aim to improve bedside treatments.