Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.

Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe–microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species.

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Bacteria in the human oral cavity often grow in an attached multispecies biofilm community. Members of this community display defined interactions that have an impact on the physiology of the individual and the group. Here, we show that during coculture growth with streptococci, the oral pathogen Aggregatibacter actinomycetemcomitans displays enhanced resistance to killing by host innate immunity. The mechanism of resistance involves sensing of the streptococcal metabolite hydrogen peroxide by A. actinomycetemcomitans, which stimulates a genetic program resulting in enhanced expression of the complement resistance protein ApiA. The oxidative stress response regulator OxyR mediates induction of apiA transcription, and this induction is required for coculture resistance to killing by human serum. These findings provide evidence that interaction between community members mediates prokaryotic resistance to host innate immunity and reinforce the need to understand how polymicrobial growth affects interaction with the host immune system.Aggregatibacter ͉ ApiA ͉ complement ͉ peroxide ͉ Streptococcus gordonii

Quantitative detection of hydrogen peroxide in solution above a Streptococcus gordonii (Sg) bacterial biofilm was studied in real time by scanning electrochemical microscopy (SECM). The concentration of hydrogen peroxide was determined to be 0.7 mM to 1.6 mM in the presence of 10 mM glucose over a period of 2 to 8 h. The hydrogen peroxide production measured was higher near the biofilm surface in comparison to Sg grown planktonically. Differential hydrogen peroxide production was observed both by fluorometric as well as by SECM measurements. The interaction between two different species in a bacterial biofilm of Sg and Aggregatibacter actinomycetemcomitans (Aa) in terms of hydrogen peroxide production was also studied by SECM. One-directional y-scan SECM measurements showed the unique spatial mapping of hydrogen peroxide concentration across a mixed species biofilm and revealed that hydrogen peroxide concentration varies greatly dependent upon local species composition.real-time (detection) | metabolite efflux | local concentration | oral flora | Au UME S treptococcus gordonii (Sg) is a member of the viridans group streptococci-Gram-positive oral microbes that are known to ferment sugars into lactic acid and produce hydrogen peroxide in the presence of oxygen (1). The presence of these beneficial oral streptococci has been shown to improve oral health, by either competition with pathogens for nutrients in the oral cavity or by the production of inhibitory concentrations of hydrogen peroxide. Populations of viridans group streptococci negatively correlate with the presence of many notable oral pathogens (2, 3). However, recent work has demonstrated that in vitro Sg can grow in coculture with the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans (Aa) (4). In co-culture Aa preferentially utilizes Sg-produced lactic acid (5) and detoxifies Sg-produced hydrogen peroxide using the KatA enzyme (6). Recent work has demonstrated that hydrogen peroxide induces katA expression as well as apiA, which encodes an immunoprotective factor that renders Aa more resistant to killing by host innate immunity (5). These studies demonstrated induction of gene expression in mixed species biofilms by Sg-produced hydrogen peroxide. Because hydrogen peroxide is rapidly degraded by catalase and can also react with other biological materials, we sought to quantify local hydrogen peroxide concentrations in real time to be utilized for future polymicrobial experiments between Sg, Aa, and other oral bacteria.Previous measurements of hydrogen peroxide have been performed using fluorescence, spectroscopy and other methods (1, 7-10). However, current techniques lack the ability to quantify local hydrogen peroxide concentrations at the surface of a biofilm. In this study, scanning electrochemical microscopy (SECM) was used to address this problem. SECM has the unique ability to set the exact distance from a sensing tip [an ultramicroelectrode (UME) of size ∼10 to 25 μm diameter] to a substrate through a feedback approach curve (11)...