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Welcome to the Microbial Jungle

In 2016, researchers overturned the long-standing belief that a person’s microbiome – the sum total of microbes living in and on our bodies – outnumbered human cells 10 to 1.1 Instead, it’s closer to a 1 to 1 ratio, they estimated. While it’s heartening to learn that we’re not outnumbered in our own skin, the tremendous ubiquity of bacteria, both on us and around us, underscores the notion that it’s really a bacterial world and we’re just living in it.

It's a bacterial world. We'rd just living in it.

Artistic rendering of BacteriaThe good news is that only about 1 percent of bacteria have the potential to be actively harmful to people or animals.2 The remaining 99% live in symbiotic relationships with us and other organisms that are often mutually beneficial. “Most microbes do not make us sick. At worst, they are hitchhikers. At best, they are invaluable parts of our bodies.”3

As our largest and most accessible organ, the skin is clearly prime real estate for bacteria looking to settle down and raise a colony or two. Not only does skin harbor a great number of bacteria, it also supports a tremendously diverse population of resident microbes that appear to be mostly unique to individuals. A recent study found that over two-thirds of the complement of bacteria detected on the skin is seemingly exclusive to an individual.4 One potentially useful reason for this microbial variety may be to create competitive pressures that keep the numbers of “bad” or pathogenic bacteria low.

But what happens when an active infection occurs, for example actively infected wounds? Does this signal a disrupted or maladapted ecosystem overpopulated with the more “nefarious” bacteria? And more perplexingly, how does microbial diversity shape and contribute to the delayed healing that is symptomatic of chronic wounds?

Despite the straightforward nature of these questions, the answers are anything but simple and comprise an emerging area of research that entwines ecology theory with microbiology. In the microbial jungle of the wound, it may not be as simple as survival of the fittest.

Lions, Tigers & Bears, Oh My!

Sketch of a Lion

Oblong bacteria drawingMicrobes exist in almost every strata of the natural environment, most often as communal and highly diverse aggregates called biofilms. Various regions of the human body also harbor biofilms, including skin, teeth, and gastrointestinal and vaginal mucosa. While not considered a “natural” biofilm, chronic wounds have been shown to contain highly diverse and abundant populations of microbes that form biofilms.5

Although comprising vast numbers of diverse microbial members, biofilms are built as a cohesive unit anchored by large, natural polymers that are secreted by the microbes into their local environment. These polymers (or extracellular polymeric substances; EPS) form a structural scaffold that provides housing for the biofilm’s microbial communities. In chronic wounds, it is thought that the EPS may also promote inherent resistance mechanisms of the biofilm to both antimicrobials and host immune response, among other functions.6

As an ecosystem, biofilms are rife with competitive forces that can drastically shape the diversity of the organisms living in it. In order to survive and thrive in this microbial jungle, bacteria have evolved a myriad of strategies that help them work with – or against – other organisms competing for the same pool of resources.7 For example, in chronic wounds, cooperative behaviors may enable microbes to work with other species in ways that promote survival and facilitate tolerance and/or resistance to antimicrobial agents.6

Driven by a constant battle for resources and space, hierarchy within microbial ecosystems is often stratified by relative abundance. Species present at high levels form the “dominant” or majority populations; overlaid onto these major groups are highly diverse, low-abundance populations described as the rare biosphere.7

Within the chronic wound biofilm, published studies have demonstrated that a great variety of common and rare aerobic and anaerobic species populate the wound ecosystem. However, no single study to date has conclusively and consistently been able to predict how microbial composition in wound biofilms may predict healing prognosis.7 Thus, the search for microbial “signatures” that denote the transition of wounds into a non-healing, stagnant state remains ongoing.

Elephant Drawing Within the chronic wound biofilm, published studies have demonstrated that a great variety of common and rare aerobic and anaerobic species populate the wound ecosystem. However, no single study to date has conclusively and consistently been able to predict how microbial composition in wound biofilms may predict healing prognosis.[7]

Bacteria with tendrils sketchWhile not strictly prognostic, the presence of pathogens in chronic wound biofilms is concerning for clinicians because of their potential risks, including antimicrobial resistance, persistence, and virulence factor production. Distribution of pathogens with the biofilm and its overall pathogenicity may also be shaped by competitive forces. For example, mutualistic interactions between microbial residents in the wound can create ecological niches that allow certain pathogenic species to flourish. A good example of this is the production of anaerobic regions in the biofilm that support growth of strictly anaerobic organisms via the activity of facultative anaerobes.6

The wound ecosystem can also be exposed to competitive forces driven by external factors, such as changes in pH, temperature, nutrient levels, type of administered wound dressing or antimicrobial and the host’s immune response.6 All of these factors can alter the wound bed in ways that may selectively increase or decrease the competitive “fitness” of one microbial species over another. This could lead to shifts in the wound ecosystem that promote growth of relatively more “fit” species at the expense of other biofilm residents.

This can become problematic if these ecological shifts cause enrichment of opportunistic pathogen species in the biofilm while suppressing nonpathogenic but competing bacteria. If the more pathogenic bacteria become the dominant species in the wound bed, there is increased likelihood of negative outcomes. For example, delayed healing, occurrence of infections and increased pathogenicity may be some of the consequences of this ecological shift.6

The Zoo ≠ The Jungle

Sketch of an Ostrich

Accurately cataloguing the biodiversity of wounds has been problematic because, until recently, most methods relied on cultivating or “growing” microbial populations in isolation prior to identifying them. The overwhelming majority of microorganisms cannot be routinely cultured in a planktonic growth state, which severely limits our ability to capture the full scale of microbial diversity in wounds.8

Furthermore, the remaining 2% of bacteria that are amenable to growth in planktonic states can be thought of as viable, but not necessarily cultivable. This means that while isolation and growth are possible, they are not necessarily easy. This is important because these organisms can still play a significant role in non-healing and infections but their presence has been historically unrecognized or underreported. Anaerobes are a good example, as they are traditionally difficult to propagate in the laboratory setting without specialized collection methods, growth media, and environmental controls; however they can constitute a primary and important component of many recalcitrant wounds.8

Petri Dish Sketch The overwhelming majority of microorganisms cannot be routinely cultured in a planktonic growth state, which severely limits our ability to capture the full scale of microbial diversity in wounds.[8]

On the other end of the spectrum, there are a handful of species that flourish in the artificial conditions of the laboratory and can out-compete other species. The problem is that these may or may not be the same species that dominate the biosphere of the wound ecosystem. Cultivation methods introduce artificial competition into the microbial ecosystem, and could potentially reorganize the microbial hierarchy by selecting for one species over another.

This selection bias could lead to loss of microbial biodiversity and ultimately, inaccurate reports of microbial composition and abundance in the wound. These limitations of cultivation-dependent methods may underscore why most studies evaluating clinical outcome and microbial composition of chronic wounds have yet to demonstrate a conclusive and consistent relationship between specific microbes and patient prognosis.

Building a Better Lion/Tiger/Bear Trap

Drawing of a Bear Trap

The advent of newer molecular techniques has redefined our understanding of the microbiology of chronic wounds and provided a powerful, sensitive tool to investigate wound biofilms and their implications to non-healing and infection. The polymerase chain reaction (PCR) technique is a staple of molecular biology and when applied to wound microbiology, can provide exquisitely sensitive microbial detection and differentiation of polymicrobial specimens in particular. Using a single copy of microbial DNA, the technique exploits the power of exponential amplification to produce billions or more copies of that specific DNA sequence. Because the amplified DNA can be unique to an organism, highly accurate and direct identification of microbes is possible, potentially to the species level.8

PCR is a staple of molecular biology and when applied to wound microbiology, can provide exquisitely sensitive microbial detection and differentiation of polymicrobial specimens in particular.

Spherical Bacteria DrawingApplication of genomic technologies has allowed microbiologists to deploy less biased approaches that do not rely on the ability to grow microbes. Instead of being restricted to only the 2% of viable, but not necessarily cultivable biosphere, microbiologists and clinicians can potentially access the full complement of microorganisms residing within wound ecosystems. Because genomic techniques can allow for (in some cases massively) parallel processing and analysis, a single specimen collected from a wound could yield comprehensive information about the microbiome in as little as 24 hours.

Data from analyses using these types of approaches have spotlighted the fact that cultures often under-represent the true microbial diversity and load of both chronic and acute wounds. We are increasingly finding that similar to natural biofilms found on the human body and elsewhere, the wound ecosystem hosts a richly diverse and intricately balanced complement of microbial life. By building a better trap, we may be able to better capture and assess the microbial jungle of the wound microbiome. In turn, this may help us find more accurate answers to the complex questions facing microbiologists and wound clinicians today.

Stay tuned. There’s more.

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References
  1. Sender R, et al. Revised estimates for the number of human and bacteria cells in the body. PLOS Biology 2016; http://www.biorxiv.org/content/early/2016/01/06/036103.
  2. MedlinePlus, US National Library of Medicine. Accessed August 9, 2017. https://medlineplus.gov/bacterialinfections.html
  3. Quote from Jeffrey Gordon, PhD. Accessed August 9, 2017. https://www.theguardian.com/science/2016/aug/25/gut-reaction-surprising-power-of-microbes
  4. Gao, Zhan et al. Molecular Analysis of Human Forearm Superficial Skin Bacterial Biota. PNAS 2007; Accessed August 9, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1815283/
  1. Rhoads DD, et al. Comparison of Culture and Molecular Identification of Bacteria in Chronic Wounds. Int. J. Mol. Sci. 2012, 13, 2535-2550; doi:10.3390/ijms13032535
  2. Percival SL, et al. Biofilms and bacterial imbalances in chronic wounds: anti-Koch. Int Wound J 2010; 7:169–175: 2927–2932.
  3. Hibbing ME, et al. Bacterial competition: surviving and thriving in the microbial jungle. Nat Rev Microbiol. 2010; 8(1): 15–25. doi:10.1038/nrmicro2259.
  4. Tatum, OL and Dowd, SE. Wound Healing Finally Enters the Age of Molecular Diagnostic Medicine. Advances in Wound Care, 2011 Vol 1, No 3.