Friends and Foes: Microbial Interactions and Infections

Friends and Foes: Microbial Interactions and Infection

If one were to write a recipe for bacterial infection, it would seem relatively straightforward: add a pathogen to a host and voilĂ ! However, infection is not just a product of interactions between pathogens and their hosts, but also between pathogens and other microbes within and on the host.

Investigating these microbe-microbe interactions is key to understanding mechanisms of bacterial pathogenesis, as well as facilitating the development of microbe-based strategies for combatting disease. Ultimately, “we can treat infection through the lens of ecology,” said Joseph Zackular, Ph.D., Assistant Professor of Pathology and Laboratory Medicine at the University of Pennsylvania. “You don't have to go just for the really tough bug. If you [target] its friend or its partner, you might get an advantage.”

A group of microbes connected to one another with lines, with a large bacterial cell in the center.
During infection, bacterial pathogens interact with diverse types of microbes, which, in turn, interact with each other. This web of interactions influences the course and outcomes of infection.
Source: American Society for Microbiology

Friend or Foe? It Depends.

According to Zackular, most infections are, at some level, polymicrobial. The success of pathogens is intricately linked to all other microbes at an infection site. Some of these microbes may promote growth of a pathogen, while others prevent it. Whether the pathogen prevails depends on the form and function of the broader microbial community at a given time, which itself depends on numerous other factors (e.g., medications, diet, host responses and more). Zackular pointed to the toxin-producing, gastrointestinal pathogen Clostridioides difficile—a common cause of antibiotic-associated diarrhea—as a key example.

Interactions with the gut microbiome play a central role in the pathogenesis of C. difficile. Listen to Vincent Young, Ph.D., discuss the importance of these interactions in modulating C. difficile infection susceptibility and outcomes.

The gut microbiota (i.e., the consortium of microbes inhabiting the gut) normally "does a really good job of reshaping the environment to create a hostile place for C. difficile,” Zackular said, mainly through nutrient restriction and production of metabolites that hamper C. difficile’s growth. If the microbiota is perturbed (such as after antibiotic treatment, which kills many resident gut bacteria that defend against pathogens), C. difficile can proliferate and cause disease. In fact, recent work from Zackular’s lab suggests that, after antibiotic treatment, remaining members of the microbiota may facilitate C. difficile growth, highlighting the context-dependent role of the microbiota during infection.

Zackular’s lab’s findings center around Enterococcus, a group of common intestinal bacteria often increased in the guts of people with C. difficile infection (CDI), in part because of Enterococcus’ intrinsic antibiotic resistance. Through in vitro and in vivo experiments, Zackular’s team discovered that Enterococcus promotes C. difficile growth by metabolizing the amino acid arginine to produce ornithine, a compound C. difficile readily breaks down for food. However, in the absence of arginine, C. difficile starts producing more toxin, which damages the host’s intestine.

Enterococcus is doing 2 things at the same time,” explained Zackular, who presented in the Host-Microbe Biology (HMB) track session “Commensal-Pathogen Interactions” at ASM Microbe 2023. That is, it is providing C. difficile with a food source (ornithine) while, at the same time, restricting a metabolic signal (arginine), which consequently tells C. difficile to produce toxin. Enterococcus may, in turn, “take advantage of all the chaos that C. difficile causes to get an additive advantage,” Zackular continued, though the details of that advantage are still under investigation.

Zackular noted that this Enterococcus-C. difficile interaction is just one of many relationships that modulate C. difficile’s pathogenesis. However, it is “proof of principle that one of the clinically relevant things we notice is, we think, pretty consequential.” Still, exploring other “microbial correlates” of CDI in diverse host environmental contexts (e.g., the guts of people who are susceptible to infection in the absence of antibiotic treatment, like those with inflammatory bowel disease) will be important for gaining a nuanced understanding of the ecological underpinnings of disease.

Spacing Out During Infection

A confocal microscopy image of P. aeruginosa and S. aureus.Microscopy image of fluorescently labeled Pseudomonas aeruginosa (green), Staphylococcus aureus (red) and host cells (blue) interacting 4 days post-infection in a murine chronic wound model. The organisms are often found together in chronic infections.
Source: Juan Barraza, Ph.D.
With that in mind, Carolyn Ibberson, Ph.D., Assistant Professor of Microbiology and Plant Biology at the University of Oklahoma, underscored that understanding the ecology of infection depends not just on profiling the microbial constituents of the host environment, but also on examining their physical associations. In other words, how closely bacteria live with one another is an important, often underappreciated, aspect of microbial interactions and infection.

"We are thinking about the infection environment as its own ecosystem,” said Ibberson, a presenter in the Molecular Biology & Physiology (MBP) track session, “Influencing Your Neighbor: the Effect of Polymicrobial Interactions on Bacterial Behaviors,” at ASM Microbe 2023. “In an ecosystem, you have a biological community of interacting organisms and their environment. We could think about the microbes that live in our bodies in the same terms. So, you need to know who's there; you need to know what they're doing [and] you need to know how they're organized with each other.”

The spatial organization of microbes can dictate if and how they interact. For example, Staphylococcus aureus and Pseudomonas aeruginosa are routinely found together in chronic infections (e.g., cystic fibrosis and chronic wounds), even though P. aeruginosa generally kills S. aureus if they are mixed in vitro. Ibberson, who studies chronic, polymicrobial infections, wanted to understand why. “We started thinking that maybe [the bacteria] are spacing themselves in a way that they can benefit from each other, because they do have beneficial interactions that have been shown, like increasing each other's tolerance or virulence expression, while also not inhibiting and killing each other,” she explained.

Using a mouse model of chronic wounds, Ibberson and team showed that each species did, in fact, exist in distinct aggregates about 30 microns apart—they were in the wound bed together but maintained some personal space. The separation relied on an antimicrobial compound produced by P. aeruginosa; in the absence of this compound, the bacteria intermixed. Notably, S. aureus became less tolerant to aminoglycoside antibiotics when mixed with P. aeruginosa. Though the mechanisms behind this observation are unclear, the findings indicate that the metabolic networks between bacteria, and their clinical implications, depend on their spatial orientation. To that end, Zackular’s group also showed that C. difficile and Enterococcus form mixed biofilms, which likely facilitate metabolic exchange between the organisms.

Ibberson thinks that incorporating spatial analyses into infection research, along with analyses of microbial community composition and function, is critical for taking a holistic approach to studying microbial pathogenesis. On a broader scale, she emphasized the necessity of investigating microbial interactions in their natural host environments and infection contexts, where all factors of the ecosystem are at play. This ability has become increasingly possible with the development of tools (e.g., next-generation sequencing, machine learning and more) that allow scientists to, as stated by Zackular, “embrace the complexity and study systems with so many variables.” By understanding all facets of the infection landscape, researchers are better positioned to develop tactics for preventing or treating disease.

Looking Beyond Bacteria

Confocal microscopy image of C. albicans stained blue and green.Interactions between bacterial pathogens and diverse types of microbes, including fungi, play important roles in infection outcomes. This image depicts Candida albicans, a fungus that commonly contributes to polymicrobial infections.
Source: Allison D.L., et al./Microbiology Spectrum, 2016
Adding to the complexity is the fact that the host is home to more than a few (thousand) bacterial species. For example, in the gut, “bacteria have the biggest biomass, [which] is why most research focuses on bacteria. But in terms of number, there are a lot of other nonbacterial components,” said Judith Behnsen, Ph.D., Assistant Professor of Microbiology & Immunology at the University of Illinois at Chicago College of Medicine and convener of the ASM Microbe 2023 session, “Beyond Bacteria: Trans-Kingdom Modulation of Microbial Pathogenesis.” She highlighted that viruses, archaea, fungi, protists and parasites are also members of the host ecosystem, not just in the gut, but throughout the body. These other microbes can influence bacterial pathogenesis to dictate the course and outcome of infection.

Behnsen’s research, for instance, focuses on interactions between fungi and Salmonella enterica serovar Typhimurium, a major cause of gastroenteritis. Her lab recently showed that commensal fungi in the gut, as well as in the diet, are a source of siderophores, small molecules that scavenge for iron from the host environment for S. Typhimurium. Iron is a critical nutrient for bacteria that is limited during infection. Experiments in vitro and in mice revealed that these fungal siderophores provide S. Typhimurium with a growth advantage, suggesting a potential role for “inter-kingdom cross-feeding” during infection. Behnsen noted there are also examples of fungi inhibiting bacterial pathogens (and vice versa), as well as competitive and cooperative interactions between pathogens and other types of microbes, like viruses.

However, for the most part, scientists are still in the dark when it comes to these relationships. “We do know more about what's there,” Behnsen said, reiterating how advancements in technology have made it easier to study the composition of host-associated and environmental microbial communities. “But what do [those microbes] do? That [knowledge] is still lagging quite a bit. The good thing is that more and more people are starting to look.” And continuing to look will be important. Knowledge of how these diverse members of the host ecosystem modulate disease could lead to novel strategies for thwarting bacterial pathogens.

Clinically Capitalizing on Microbial Interactions: The Case of Phage Therapy

In fact, ecological insights into bacterial pathogenesis can inform—and have already informed—methods to combat infections. Some of these methods are broad in their microbial scope (e.g., fecal microbiota transplants for treating CDI) while others weaponize specific and, in some cases, nonbacterial interactions to obliterate pathogens. Phage therapy offers an excellent example.

Bacteriophages (phages) are viruses that bind to and hijack bacterial cells to replicate themselves, often killing the cell in the process. The bactericidal capabilities of phages have piqued interest in phage therapy for treating bacterial infections, whereby a selection of phages that specifically attack the infecting bacterium are administered to a patient. “We can use the enemy of the enemy as our friend,” said Jyot Antani, Ph.D., a post-doctoral researcher in the laboratory of Paul Turner, Ph.D., at Yale University. Recently, scientists have focused not just on how phages attack bacteria, but also on bacterial responses to phages.

Swimming assay illustrating differences in motility between wild-type E. coli and E. coli evolved in the presence of flagellotropic phage.
Source: Jyot Antani, Ph.D.

Bacteria have been locked in an evolutionary arms race with phages for eons. As a result, they have developed mechanisms to resist phages at various points in the replication cycle. “One such mechanism involves altering receptors on the cell surface,” explained Antani, a presenter in the ASM Microbe MBP track session “Anti-Phage Defenses: The Battle Between Prokaryotes and Their Viruses.” Often, phage resistance comes with a price. When Antani and his colleagues experimentally evolved populations of Escherichia coli with a flagellotropic (i.e., flagellum-binding) phage in vitro, some bacteria evolved mutations in motility-regulator genes that rendered them nonmotile. Other bacteria evolved mutations in the gene encoding the flagellar filament. These flagellar mutants were still motile but less so than the ancestral strain. In both cases, bacteria “traded” motility for phage resistance. Notably, phage-resistant bacteria can also exhibit reduced virulence or increased susceptibility to antibiotics.

Antani emphasized the potential of “evolutionary medicine,” in which we can use such bacterial trade-offs to our advantage. For instance, bacterial pathogens are often motile during infection, and based on Antani’s findings, scientists could potentially use flagellotropic phages to hinder their motility. “If there are only a few motile bacteria remaining, then it gets easier to use other ways, in combination with the [flagellotropic] phage, to stop those bacteria from spreading,” Antani said. He went on to explain that this method could make the infectious population more manageable. Ultimately, insights into bacteria-phage interactions and defenses could further inform the rational design of phage therapeutics.

The Bottom Line

Infection is complicated. Understanding the process requires delineating how bacterial pathogens organize and engage with all aspects of the host ecosystem, especially its many microbes. Doing so will allow scientists to exploit these interactions to foster human health. “Microbes are not in a vacuum,” Zackular said. “The way that they interact with their environment, their ecosystem…is ultimately the thing that matters the most for [understanding] how they impact us as humans, and how they impact society.”

Author: Madeline Barron

Madeline Barron
Madeline Barron, Ph.D., is the Science Communications Specialist at ASM. She obtained her Ph.D. from the University of Michigan in the Department of Microbiology and Immunology.