By Avery Young

Introduction

The evolutionary arms race between humans and the pathogens that call them home has been raging since our ancestral lineages diverged many millions of years ago. For every deadly adaptation a pathogen has acquired, humans have evolved some strategic resistance in response. Lauded as a major medical discovery, Sir Alexander Fleming’s 1928 discovery of penicillin afforded humankind an arsenal upgrade against our microscopic foes. Unfortunately, microbes were quick to cultivate antibiotic resistance, and the competition to discover newer and more effective treatments has been contested ever since. Rising virulence in the human intestinal pathogen known as Clostridioides difficile (C. difficile) has made research about this rapidly evolving bacterium a top priority.

THE EVOLUTIONARY ARMS RACE BETWEEN HUMANS AND PATHOGENS... HAS BEEN RAGING SINCE OUR ANCESTRAL LINEAGES DIVERGED MANY MILLIONS OF YEARS AGO...

General Physiology and Metabolism

C. difficile is a strict anaerobe, meaning that it cannot grow in the presence of oxygen. It has gained notoriety as a hospital-acquired infection due to its highly contagious nature, toxin production, and antibiotic resistance. In some areas of North America, C. difficile infections (CDI) have surpassed methicillin-resistant Staphylococcus aureus (MRSA) as the number one hospital-acquired infection.¹ C. difficile begins cycles of infection in the colon and travels between hosts via fecal-oral transmission routes. Clinical manifestations of CDI often occur in cycles and range in severity from asymptomatic carrier states to fatal colonic inflammation.² Typical signs and symptoms of infection include watery diarrhea, food refusal, nausea, and painful cramping.

Patients presenting with the highest risk for contracting C. difficile infections include the elderly, newborns, and anyone with recent antibiotic exposure. Broad spectrum antibiotic treatments, often prescribed for preexisting infections, disrupt the naturally healthy and protective microbiome of the colon. This makes treatment of CDI precariously difficult because the medications prescribed to treat other illnesses are often the footholds that provide C. difficile the perfect opportunity for colonization.

While many aspects of the C. difficile infection cycle remain poorly understood, it is well established that the synthesis of toxins is a large proponent of disease symptoms.³ Toxins are secreted by C. difficile into the host colon and affect the biology of host colonic cells at the cytoskeletal level.⁴ Cytoskeletal disturbance results in a loss of the cell’s structural integrity and apoptosis (cell suicide).⁵ Apoptosis and intestinal inflammation subsequently manifest as the hallmark symptom of infection: diarrhea.

To ensure survival and the successful propagation of disease, C. difficile cells will form metabolically dormant and dehydrated endospores in the face of environmental stressors. Researchers and healthcare professionals pay significant attention to spores because of their role in the transmission cycle. Through the process known as sporulation, a vegetative C. difficile cell can respond to nutrient deprivation, immune system attacks, or antibiotic therapy by encasing a complete copy of its genome in many layers of thick, protective protein, as depicted in Figure 1.

These spores present with remarkable resistance properties and are able to persist in hostile environments. Spores even demonstrate an extraordinary adherence to stainless steel, a material often used in hospitals for appliances, furnishings, and other items with which patients and physicians come into daily contact.⁷ C. difficile endospores can withstand exposure to extreme heat, oxygen (C. difficile is a strict anaerobe), radiation, antibiotics, and chemicals.⁷ This even includes the standard disinfectants utilized by custodial staff. Once shed in the feces of hosts, these infectious and microscopic particles saturate the environment and can remain viable for years until ingestion completes the cycle of transmission.

Each C. difficile spore core contains a complete copy of the genome, as well as all proteins necessary for outgrowth upon germination. Once in the small intestine, the presence of specific bile acids will trigger spores to undergo germination, meaning they will transform once again into vegetative cells capable of disease. The specificity of bile acids necessary for germination indicates that a large portion of the spores’ fates depends on the secretion activity of the liver and gallbladder. Bile acids are small cholesterol-derived acids that are necessary for the healthy absorption of fats and cholesterol during digestion. Bile acids are divided into two families: cholic acids, which stimulate C. difficile spore germination, and chenodeoxycholic acid derivatives (CDCA), which inhibit C. difficile spore germination.⁸

FIGURE 1. The main structural layers of a bacterial endospore, not drawn to scale. Figure adapted from Paredes-Sabja.6

Objectives

The focus of this research was to characterize a recently identified C. difficile ribotype 106 clinical isolate (LC5624). Ribotypes are used by research laboratories to classify strains of the same species; a molecular analysis of rRNA allows bacteria of the same genus and species to be further characterized into what is roughly analogous to a subspecies. Ribotyping as a technique has further classified the 180 validly described C. difficile species into 6 phylogenetic clades, with pathogenic strains dispersed throughout each clade. Notably, instances of CDI caused by strains of ribotype 106 have overtaken the historically prevalent ribotype 027 in cases of active circulation in healthcare communities across the globe. Using a well-studied ribotype 027 strain as a standard of comparison, investigations into a ribotype 106 clinical isolate were conducted to better understand the current evolutionary state of this pathogen. As a snapshot of this pathogen’s community progression, this research highlights trends of evolution between current and historical strains and potentially serves to curtail any further advancements in pathogenicity. Through assessments of sporulation and germination assays in the presence of germinants and inhibitors, I compiled a detailed physiological profile of C. difficile LC5624. Bridging this molecular understanding of laboratory physiology to a range of clinical severities aids scientific research aimed at eliminating this human pathogen.

Methodology

In order to identify the novel germination phenotype of clinical isolate LC5624 (ribotype 106), the thoroughly studied C. difficile strain R20291 (ribotype 027) was used as a control and standard of comparison throughout all experimentation. Optical density and dipicolinic acid (DPA) release assays were used to study the effective rates of growth and germination in C. difficile strains in the presence of known germinants (taurocholate and a glycine cogerminant) and inhibitors (chenodeoxycholic acid) at various concentrations.

PATIENTS PRESENTING WITH THE HIGHEST RISK FOR CONTRACTING C. DIFFICILE INFECTIONS INCLUDE THE ELDERLY, NEWBORNS, AND ANYONE WITH RECENT ANTIBIOTIC EXPOSURE... 

Principle Behind Optical Density Assays

Upon germination, an endospore will rehydrate its core and shed its thick, protective spore coat and exosporium (Figure 1), culminating in the outgrowth of a living cell. This transition from a dense spore into a vegetative cell is monitored through changes in optical density at 600 nm over time and is commonly seen as a transition from a bright to a dark phase under contrast microscopy, as seen in Figure 2. An ideal graphical representation of optical density as a function of time is shown. The optical density rate of change as a function of time varies with the amount and identity of germinant or inhibitor present in solution.

Principle Behind Terbium Assays

A large component of spore resistance is attributable to a DPA chelate found packaged in the spore core. The DPA chelate is a complex layer of calcium ions coordinately bound to the ligand DPA and is thought to be primarily responsible for the endospore’s core dehydration and impressive heat tolerance. During germination, this DPA chelate is released to allow the spore core to rehydrate and develop into a vegetative cell. In the laboratory, DPA release can be monitored in real time because DPA readily binds to terbium ions added to the solution and results in a fluorescent signal. An ideal graphical representation of fluorescence as a function of time is shown in Figure 3.

Figure 2. (A) Ungerminated, phase bright C. difficile spores suspended in buffer. (B) Germinated, phase dark spores after exposure to 10mM TA and 30 mM glycine. All images were taken under the 100X objective in phase contrast microscopy by Ritu Shrestha.

Figure 3. Dehydrated spore cores are rich in a DPA chelate that is released upon initiation of germination. Upon this release, DPA can form a complex with terbium ions that is excitable at 270nm.

Quantifying Kinetic of the Multi-enzyme Germination Process

As mentioned previously, taurocholate (TA) and glycine are potent activators of C. difficile spore germination and CDCA is a potent inhibitor. The concentration of a compound that results in the half-maximum germination rate is known as the effective concentration 50%, or EC50. From the EC50, the interaction of an inhibitor of germination can be quantified as Ki. Concentration values for glycine and TA are reported as EC50, and CDCA is reported as Ki. Spores with a decreased sensitivity for germinants or inhibitors present with higher EC50 or Ki values respectively. The kinetics of the DPA release across a range of TA concentrations (0 mM−50 mM) and a constant concentration of glycine were used to calculate TA EC50 values for both strains. Similarly, a range of glycine concentrations (0−30 mM), and a constant concentration of TA were used to determine a glycine EC50 value for both strains. To calculate CDCA Ki values, a range of TA concentrations (0−50 mM) and a constant concentration of CDCA were tested in terbium assays. EC50 and Ki experiments were designed to include constant concentrations of a co-germinant or inhibitor to ensure the range of TA or glycine concentrations was the sole independent variable.

THE MOST NOTABLE ASCPECT OF THE C. DIFFICILE... IS THE FACT THAT THE SPORES SHOW A SENSITIVITY TO BOTH BILE ACID GERMINANTS AND INHIBITORS.

Results

In the calculations reported in Table 1, a lower EC50 value for LC5624 indicates a more potent interaction with substrate and, subsequently, more efficient germination. Wildtype R20291 spores require greater than 9 times the concentration of TA and 2.2 times the concentration of glycine than do C. difficile LC5624 spores to induce half maximal germination rates (Table 1). In regard to inhibitor CDCA, C. difficile R20291 spores were inhibited at approximately 1.6 times greater concentrations of CDCA than were C. difficile LC5624 spores (Table 1).

Arguably the most notable aspect of the C. difficile LC5624 germination profile is the fact that LC5624 spores show a significantly heightened sensitivity to both bile acid germinants and inhibitors. Based on a comparison of calculated EC50 values, LC5624 spores are more sensitive to the bile acid germinant taurocholate (TA) and amino acid co-germinant glycine than are R20291 spores. This aspect of the germination profile may be misleading if not viewed in the context of a typical transmission cycle. Inside a host colon, a typical endospore will naturally be exposed to TA, glycine, and CDCA. For R20291 spores, the Ki of the CDCA inhibitor is less than that of the EC50 of both primary germinant TA and co-germinant glycine. This means lesser concentrations of CDCA are required to inhibit germination in R20291, and greater concentrations of TA and glycine are necessary to initiate germination. In contrast, C. difficile LC5624 spores require lower concentrations of TA and glycine to initiate germination, and greater concentrations of CDCA to inhibit germination. Such variance in spore germination sensitivities carries impactful consequences in regard to infection; a spore derived from C. difficile strain LC5624 may be more likely to germinate in the presence of physiological concentrations of bile acids than are spores of C. difficile R20291. At the same concentration of TA, glycine, and even CDCA, C. difficile LC5624 spores appear to germinate into viable, disease-causing organisms at faster rates than C. difficile R20291 spores.

Table 1. Comparison of EC50and Ki values for known germinants and inhibitors between C. difficile strain R20291 and clinical isolate LC5624.

Discussion

C. difficile R20291 (ribotype 027) was isolated during an epidemic outbreak of CDI in the early 2000s; LC5624 (ribotype 106) was a clinical isolate identified in 2017. In little more than a decade, ribotype 106 evolved with rates of germination that are more than double that of historical strains. This increased sensitivity to the presence of natural, host-derived germinants may have contributed to the spread of ribotype 106 within hospital and healthcare communities.

The clear advance in the spread of this strain also raises questions pertaining to other active strains of the same genetic ribotype. Are heightened sensitivities to germinants conserved among all ribotype 106 strains? Have the minimum concentrations for germination inhibitors risen in all strains? While it is not appropriate to suggest this trend is pervasive in all actively circulating strains of ribotype 106, there is no evidence to indicate such an increase would not also be modeled in other strains. Further investigations into other strains would certainly prove beneficial in this regard and would better guide the physicians who are providing treatment to patients afflicted with CDI.

The characterization of phenotypes seen in C. difficile clinical isolates allows for a deeper understanding of the pathogen faced by physicians and patients in healthcare today. A clear picture of the pathogen in its current evolutionary state could curtail potential increases in virulence through better informed treatments of active cases. Further investigations into clinical isolates, their unique phenotypes, and levels of disease severity allow for a greater understanding of the pathogen in its community evolution and provide physicians with an edge over the evolutionary rival found in C. difficile.

Acknowledgments

I would like to thank Dr. Joseph Sorg of the Texas A&M University Department of Biology for all his mentorship and guidance throughout the research. I would also like to acknowledge PhD candidates Ritu Shrestha and Kathleen McAllister for all their patience, love, and encouragement during the process. Special thanks also to Dr. Larry Kociolek of the division of Pediatric Infectious Diseases at Feinberg School of Medicine at Northwestern University (Chicago, IL) for generously sharing the C. difficile LC5624 clinical isolate.

Avery Young ‘19

Avery Young is a junior molecular and cell biology major from Richardson, Texas. Avery completed the work for this article while participating in the 2017–2018 class of the Undergraduate Research Scholars under the guidance of Dr. Joseph Sorg. Avery plans to attend graduate school in history beginning in the fall of 2019 after which she hopes to work for organizations that specialize in combating infectious diseases.

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