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C Diff Research Papers

In this study, we sought to identify species-level abundances of C. difficile in 16S rRNA gene sequence datasets from different patient populations using a validated algorithm (Resphera Insight). Similar to previous studies of Listeria monocytogenes18 and Salmonella enterica,19,20 validation using a high-resolution taxonomic assignment method from 804 novel C. difficile isolates established an overall sensitivity of 99.9% with a marginal false positive rate less than 0.1%, suggesting that C. difficile could be distinguished from other related microbiota members.

Compared to the microbiota of healthy individuals, we observed a higher presence and relative abundance of C. difficile in microbiota data collected from two CDI patient cohorts. 8.5% of healthy individuals were positive for C. difficile using our approach, supporting previous epidemiological assessments of asymptomatic carriage rates.25,26,27,28 Although analysis of CDI datasets revealed a wide distribution of C. difficile relative abundances (ranging from virtually undetectable to above 50% of total sequences), the relative abundance of detected C. difficile in relation to other members of the microbiota was significantly lower in healthy individuals than that of CDI patients. The ability to assess C. difficile levels as part of the microbiota community is potentially more important within population surveys compared to diagnosis using traditional PCR or GDH/EIA tests that merely account for the presence of C. difficile using toxin B or GDH as a proxy.

While detection of C. difficile from 16S rRNA gene sequence data is limited by sequencing depth, our results suggest that C. difficile does not generally reside in healthy adults. In contrast, we did not detect C. difficile in all patients with CDI. The relative presence of C. difficile in these patients is likely below the detection limit given the available sequencing depth, however some of the samples collected from patients in the Seekatz dataset were collected during antibiotic treatment, thus potentially limiting growth of C. difficile during those time points. Indeed, Seekatz et al. report that they were unable to retrieve C. difficile strains from all patient time points via anaerobic cultivation, generally the gold standard for C. difficile detection and diagnosis.

In a third cohort of 14 recurrent CDI patients receiving fecal microbiota transplantation from nine healthy donors (FMT; Table S2, Fig. 3), C. difficile was less frequently detected than the Seekatz and Khanna index CDI patient groups. Only 4 of 14 FMT patients had any detectable levels of C. difficile before treatment, and 3 of 14 had observations of C. difficile post-FMT. Notably, Resphera Insight detected C. difficile presence in both patients who went on to develop symptomatic CDI post-FMT (recipient IDs 005 and 006).9 Prior to FMT, all patients were treated with vancomycin (125 mg 4× per day) for at least 4 days before and the day of transplantation. Thus, we attribute the reduced detection of C. difficile in this cohort to differences in patient treatment before sampling.

Applying our approach to a longitudinal dataset of 38 premature infants in a single NICU, we identified C. difficile in two-thirds of this patient cohort. Asymptomatic carriage of C. difficile among infants has been observed to be higher than for adults, and it remains unknown whether infant cases of CDI represent true disease.29,30 While CDI testing of infants is not recommended,30 recent epidemiological studies indicate 26% of children hospitalized with CDI are infants under 12 months of age, and 5% are neonates.31 In one study of 753 pediatric patients 0 to 12 years of age, 2.9% of CDI outpatients, 4.6% of CDI inpatients, and 6.6% of healthy controls were positive for C. difficile toxin B.32 Another recent study of C. difficile in 338 healthy infants (<2 yrs) in the United Kingdom found 10% were colonized at enrollment with a toxigenic strain, and 49% became colonized with a toxigenic strain post-enrollment.33 Symptomatic Clostridium difficile infections are believed not to occur in infants due to the expected lack of specific toxin receptors and under-developed signaling pathways in the gut; however, these proposed mechanisms have not been rigorously evaluated in studies of humans.34,35,36 Multiple case studies have argued that CDI can occur in this patient population,36 and there is ongoing debate about the appropriate policy for treatment of symptomatic children who test positive for C. difficile.37,38

Our analysis of an infant case study of asymptomatic colonization during the first 18 months of life identified a reduction in C. difficile relative abundance after abrupt transition from human milk to cow’s milk. Yet in a large longitudinal study by Stoesser and colleagues, multivariate analysis demonstrated that breastfeeding (mixed with formula or exclusively) was protective against asymptomatic C. difficile colonization.33 As noted by Davis and colleagues,16C. difficile does not carry the functional capacity for cleaving monosaccharides from oligosaccharide side chains and thus depends on the generation of monomeric glucose by other commensal members of the gut microbiome.39 Additionally, C. difficile relies on sialic acid as a carbon source for expansion made available by other commensals such as Bifidobacterium species.40 Therefore, the reduction of C. difficile after transition to cow’s milk is potentially the result not of milk source alone, but shifting microbial community composition and the presence of substrates by which C. difficile may thrive.

We were also able to identify a significant negative correlation between the abundance of C. difficile and C. scindens in one of the CDI cohorts, confirming similar trends reported by Buffie et al.15C. scindens, a secondary bile acid producer of deoxycholic acid which has been shown to protect against CDI, may have important translational implications.13,41 New and consistent negative correlations were also identified between C. difficile and multiple species within the Blautia genus including B. faecis, B. luti, B. schinkii, and B. wexlerae. Notably, some members of the Blautia genus are known for 7α-dehydroxylating activity of primary bile acids,42,43,44 however this remains to be evaluated for the species we identified in this study. These data suggest that species other than C. scindens may provide relevant functional capabilities in the context of CDI and prove to be informative in the development of future microbial-based therapeutics. One exception to these findings was the lack of negative correlations identified within the NICU infant cohort, which can be attributed to the very limited observations of these Blautia species and C. scindens in the overall dataset (Table S3). Indeed, among the 322 NICU infant samples analyzed, only B. luti and B. wexlerae were observed at all, and only in 5 (1.6%) and 2 (0.6%) samples, respectively, which precluded their evaluation with the CCREPE method.

While microbiome profiling through 16S rRNA gene sequencing is unlikely to replace existing methods for routine diagnosis of CDI, sequence-based assessment of C. difficile levels in the context of microbiota profiling rather than presence alone may prove valuable in surveillance of C. difficile in patient populations, prediction of disease outcome, or the development of new therapies for CDI. Although our study is limited to 16S rRNA gene-based identification of C. difficile and cannot predict whether a strain produces toxin or carries a functional pathogenicity locus,45 consideration for accurate identification of C. difficile and related members may be useful in assessing clinical outcomes of new microbial therapies that rely on 16S rRNA gene sequencing to validate recovery of the microbiota.

Abstract

The initial period of studies on Clostridium difficile (published during 1978–1980) appeared to provide a nearly complete portfolio of criteria for diagnosing and treating C. difficile infection (CDI). The putative pathogenic role of C. difficile was established using Koch's postulates, risk factors were well-defined, use of a cell cytotoxicity assay as the diagnostic test provided accurate results, and treatment with oral vancomycin was highly effective and rapidly incorporated into practice. During the next 10 years, enzyme immunoassays (EIAs) were introduced as diagnostic tests and became the standard for most laboratories. This was not because EIAs were as good as the cell cytotoxicity assay; rather, EIAs were inexpensive and yielded results quickly. Similarly, metronidazole became the favored treatment because it was less expensive and quelled fears of colonization with vancomycin-resistant organisms, not because it was better than vancomycin therapy. Cephalosporins replaced clindamycin as the major inducers of CDI because they were so extensively used, rather than because they incurred the same risk. Some serious issues remained unresolved during this period: the major challenges were to determine ways to treat seriously ill patients for whom it was not possible to get vancomycin into the colon and to find methods that stop persistent relapses. These concerns persist today.

Antibiotic-associated colitis due to Clostridium difficile has been under intense investigation since 1974. This article reviews studies on C. difficile infection (CDI) that were published between 1974 and the mid-1990s and set the stage for more-recent work.

Early Studies

Three separate lines of study fostered initial knowledge of C. difficile and CDI: initial work on the organism, investigation of antibiotic-associated typhlitis in rodents, and anatomic studies of pseudomembranous colitis (PMC).

Initial work. C. difficile was originally described by Hall and O'Toole [1] in 1935 as a component of the normal intestinal flora of newborn infants. These investigators also showed that this organism produced a toxin that was highly lethal to mice. In fact, the toxin was only 10–100-fold less toxic than botulinum toxin, but subsequent studies by the famous authority on clostridia, L. D. S. Smith, showed that C. difficile was not biologically important in extraintestinal infections. A review by Smith and King [2] indicated that C. difficile infection had no unique features that suggested a histotoxic clostridial syndrome.

The rodent model. Work on the rodent model was first performed by Hambre et al. [3] during World War II to investigate the potential benefit of penicillin for treatment of gas gangrene. They found that penicillin caused typhlitis, which actually proved to be more lethal than Clostridium perfringens–induced gas gangrene. Subsequent studies showed that multiple rodent species were susceptible to the development of typhlitis and that many types of antibiotics were associated with this complication; however, the cause was elusive. Relevance of this work to CDI in humans lies in the fact that the hamster model eventually proved to be the source of nearly all of the clinically important early data on this complication. Furthermore, Green et al. [4], who were studying penicillin-induced death in guinea pigs, found that stool specimens contained cytopathic changes, which they attributed to the activity of a latent virus. This, in retrospect, appears to be the first identification of C. difficile cytotoxin.

Anatomic studies of PMC. J. M. T. Finney performed the first anatomic studies of PMC. In 1893, Finney [5] reported pseudomembranous changes in the intestinal tract of a 22-year-old postoperative patient being treated by William Osler. PMC became a commonly recognized complication of antibiotic use in the early 1950s and was primarily encountered by surgeons, who reported rates as high as 14%–27% among postoperative patients [6, 7]. Staphylococcus aureus was the suspected pathogen, and vancomycin given orally became standard treatment for this condition [8].

The “C. difficile era” began in 1974, when Tedesco et al. [9] reported high rates of PMC among patients at Barnes Hospital (St. Louis, MO) who were receiving clindamycin. This study was the first in which endoscopy was a routine diagnostic procedure for patients with antibiotic-associated diarrhea. Of 200 patients given clindamycin, diarrhea developed in 42 (21%). Twenty clindamycin recipients (10%) had PMC at the time of endoscopy. Although not stated in the article, stool cultures were negative for S. aureus, despite the ease of growing this organism on selective media. This article crystallized interest in this adverse drug reaction and spawned studies to define the cause, pathophysiological characteristics, and management of “clindamycin colitis.”

Detection ofC. difficile.The early work on C. difficile detection involved clinical specimens and hamster models and was performed primarily by Keighley et al. [10] at a hospital in Birmingham, England, where C. difficile was endemic on a surgical ward; by the group headed by Bob Fekety and Joe Silva in Ann Arbor, Michigan [11]; and by my group in Boston, Massachusetts [12]. In most of the early studies, the hamster model turned out to be pivotal in establishing methods for detection of C. difficile [13], confirming the diagnostic role of the “cell cytotoxicity assay” [13], detecting toxin B produced by C. difficile [14], confirming that antibiotics (including oral vancomycin and metronidazole) are inducers of PMC [15, 16], and confirming the efficacy of oral vancomycin for treatment of CDI [17].

The initial work on the cell cytotoxicity assay was by Te-Wen Chang, who demonstrated that stool specimens from hamsters with antibiotic-associated typhlitis and from patients with PMC contained a potent cytopathic toxin that was neutralized with Clostridium sordellii antitoxin [18]. However, because cultures of stool specimens from hamsters did not yield C. sordellii, my colleagues and I [12] analyzed other clostridial species recovered from hamster stool specimens to determine which species could produce the cytopathic toxin that was neutralized by C. sordellii antitoxin. C. difficile satisfied this criterion. C. difficile antitoxin was not available, so the standard test for detection of C. difficile was to demonstrate the presence of a cytotoxin neutralized by C. sordellii antitoxin. The first person with a positive test result was a postoperative patient in California with cephalothin-induced “clindamycin colitis.” This patient had a cytotoxin titer of 106, which remains the highest titer I have measured in a patient [13]. (Of note, Tedesco sent some stool specimens to Upjohn, which sponsored the study at Barnes Hospital [9]. In 1978, I received 6 stool specimens from Upjohn with a note indicating that they had “been stored in unspecified conditions for 4 to 5 years”; all of the specimens were positive for C. difficile cytotoxin.)

Studies from the Late 1970s through the Early 1990s

Pathologic characteristics of PMC. Like most enteric bacterial pathogens, C. difficile causes disease with a wide spectrum of severity, ranging from mild “nuisance” diarrhea with a normal colonic mucosa to PMC, the most characteristic and severe form of CDI. PMC lesions are nearly always limited to the through 1992 only 7 cases involving the small bowel had been reported [19]. The prior reports of S. aureus enterocolitis showed that the small bowel was commonly involved, suggesting a distinction between “S. aureus enterocolitis” and “C. difficile colitis” [6, 7]; recent work has supported this distinction [20]. Of practical importance, oral vancomycin would be appropriate treatment for both conditions, but metronidazole would not.

PMC in the great majority of patients seen since 1978 has been caused by C. difficile, and a subset of patients have histologic lesions most characteristic of PMC. Anatomic studies of these lesions revealed that the pseudomembrane is composed of fibrin, mucin, sloughed mucosal epithelial cells, and acute inflammatory cells. There are various differences between lesions in different persons, but lesions in the same person appear to be uniform [21, 22]. The initial lesion has focal necrosis and inflammation, as well as the characteristic “summit” (figure figure 1A and figure 1B). The most advanced disease involves complete structural necrosis with extensive involvement of the lamina propria, which is overlaid by a thick, confluent pseudomembrane. There are multiple other causes of PMC, including intestinal obstruction, colon cancer, leukemia, severe burns, shock, uremia, heavy metal poisoning, hemolytic-uremic syndrome, Crohn disease, shigellosis, neonatal necrotizing enterocolitis, ischemic colitis, and Hirschsprung disease. Nevertheless, the vast majority of PMC cases seen since 1978 have been attributed to C. difficile.

Figure 1

Typical summit lesions associated with pseudomembranous colitis. A, Pseudomembranous colitis with typical raised lesions (diameter, 0.2–1 cm). The appearance is light yellow against a hyperemic bowel mucosa. B, Microscopic pathologic characteristics of a mushroom-shaped pseudomembrane.

Figure 1

Typical summit lesions associated with pseudomembranous colitis. A, Pseudomembranous colitis with typical raised lesions (diameter, 0.2–1 cm). The appearance is light yellow against a hyperemic bowel mucosa. B, Microscopic pathologic characteristics of a mushroom-shaped pseudomembrane.

Anatomic lesions are best detected by colonoscopy; in 20%–30% of cases, PMC is limited to the proximal colon and therefore may be missed by sigmoidoscopy [23, 24]. Characteristic lesions may also be detected by CT scan [25]. Nevertheless, because of stool toxin assays, demonstration of anatomic findings is now seldom necessary, although imaging or endoscopy may sometimes be performed for other reasons.

Risk factors for CDI. The major risk factors for CDI are hospitalization, older age (i.e., β65 years), and antibiotic exposure. Use of nearly any antibiotic with a spectrum of antibacterial activity has been implicated as a risk factor in people and hamsters. The initial attention was on clindamycin. The connection between this agent and PMC was the source of concern in the 1970s and was the subject of the report by Tedesco et al. [9], the findings of which showed that “clindamycin colitis” is synonymous with “antibiotic-associated colitis.” However, most of the studies done in the 1980s showed that cephalosporins had become by far the most frequently implicated agents [26–29]. Broad-spectrum penicillins, including amoxicillin, were the second-most frequently implicated drugs.

Care in the interpretation of these data is important. Clindamycin was associated with the greatest risk of CDI; cephalosporins and broad-spectrum penicillins were associated with the greatest number of CDI cases because of their extensive use. Macrolides, trimethoprim-sulfamethoxazole, metronidazole, rifampin, and tetracyclines were far less commonly associated with CDI. Ciprofloxacin was the first fluoroquinolone created and was approved for human use in 1987, but fluoroquinolones did not seem to figure prominently in cases of CDI until recently [30]. The only other class of drugs recognized to induce CDI comprised antineoplastic agents, primarily methotrexate [31, 32]. There were additional cases of CDI that were not associated with antibiotic exposure, but these were described in anecdotal case reports. Attempts to identify C. difficile in other diseases, such as neonatal necrotizing enterocolitis, sudden infant death syndrome, Crohn disease, ulcerative colitis, and chronic enigmatic diarrhea, were uniformly unsuccessful (table 1) [33].