The concept of NTCD inhibiting or interfering with the infectivity of a toxigenic strain of C. difficile was first reported by Wilson and Sheagren (1983) in the hamster model in 1983. They demonstrated that following cefoxitin administration, colonization with NTCD prior to challenge with toxigenic C. difficile increased hamster survival from 21% to 93%, however, durability of prevention was not determined. Simultaneous administration of the NTCD strain and a toxigenic C. difficile strain did not increase survival. Borriello and Barclay (1985) extended these observations by colonization of hamsters with three different strains of NTCD, M-1, D-1 and S-1 administered at doses ranging from 3 × 10⁶ to 3 × 10⁹ organisms followed by challenge 5 days later with a toxigenic strain B-1 at a dose of 3 × 10⁷ organisms. Results of these experiments showed at 25 days post toxigenic challenge survival of 13 of 18 (72%) vs. 0/21 (0%) hamsters not given an NTCD strain. Additional experiments showed (1) that protection was dependent upon live NTCD organisms; heat-killed NTCD did not protect hamsters, (2) that protection was dependent upon NTCD colonization; decolonization with vancomycin resulted in no protection against toxigenic challenge, and (3) that protection was specific to C. difficile; attempts to colonize with C. perfringens, C. bifermentans, and C. beijerincki, failed, and C. sporogenes colonized but failed to protect against toxigenic C. difficile challenge.

Borriello and Barclay (1985) also attempted to identify the mechanism by which NTCD protected against toxigenic C. difficile. They showed that NTCD did not degrade or alter the potency of C. difficile toxin. They investigated the mucosal association of NTCD by harvesting cecum samples from three NTCD-colonized hamsters at day 51 of colonization, washing them eight times with brain heart infusion (BHI) broth and saline, then homogenizing the tissue and doing quantitative culture of the homogenate. They found association of NTCD with the cecal mucosa ranging from 6 × 10³ to 3 × 10⁵ cfu/g tissue, suggesting there may be association of NTCD with the cecal mucosa or alternatively, they suggested competition for essential nutrients. However, in the discussion they report that after day 25 death occurred in most animals and that toxigenic C. difficile and cytotoxin could be detected in the cecum of these animals. Sequential analysis of the fecal pellets in these animals detected a change over time from only NTCD recovery to recovery of toxigenic C. difficile or both. This was of concern and the authors speculated that the presence of toxin enabled toxigenic strains of C. difficile to have a colonization advantage over NTCD. In a subsequent publication, Borriello et al. (1988) showed that a virulent toxigenic strain B-1 of C. difficile was more adherent to mucosa of small and large bowel in the hamster than NTCD strain M-1. They also showed that injection of toxin into the cecum of hamsters enhanced colonization of M-1 in the colon, but not in the small bowel or cecum when compared to toxigenic strain B-1.

Similar observations of protection against toxigenic C. difficile by NTCD were shown in gnotobiotic mice by Corthier and Muller (1988). NTCD protected against toxigenic challenge from 18h to 10 days following NTCD in 100% of gnotobiotic mice. If there was no delay in administration of the toxigenic strain following NTCD, 60% of mice survived. Survival of mice beyond 10 days was not recorded.

These early bench and animal studies were not further pursued beyond the 1980s, most likely because the protective effect in hamsters did not appear to be durable. However, these observations provide a valuable early insight into the potential utility of NTCD as a preventive strategy for CDI.

The first published report of the use of NTCD in patients was by Seal et al. (1987). Two patients with multiple recurrences of CDI were treated orally with a BHI culture of NTCD strain M-1 previously shown by Borriello and Barclay (1985) to prevent CDI in hamsters transiently. One ml of the 24h culture was diluted in 50 ml of milk (final concentration ~1 × 10⁷ cfu/ml) which was administered once daily for 3 days. Patient A, age 88 years, was treated for her first CDI episode with metronidazole and for episodes two and three with oral vancomycin. Although successfully treated with resolution of symptoms, she had persistent C. difficile in her stool for at least 3 days following the last vancomycin treatment but was negative on stool culture just prior to receiving NTCD. Following NTCD her stool cultures were positive for NTCD for 14 days with no toxigenic C. difficile detected, and she had no further CDI recurrences for at least 4 months. Patient B, age 76, was also treated for her first CDI episode with metronidazole and for episodes two and three with vancomycin. She was treated successfully for her diarrhea symptoms with vancomycin, but had high counts of toxigenic C. difficile in her stool for the first 3 days of NTCD administration (5.9–7.5 log₁₀/gm stool) with no NTCD detected. NTCD did not appear in the stool until day 6 at which time there were ~10⁷ cfu/gm stool of both NTCD and toxigenic C. difficile, but on days 9 and 10 only toxigenic C. difficile was found at a concentration of ~10⁵ cfu/gm stool. Starting on day 17 she had 2 days of self-limited diarrhea with both toxigenic C. difficile and NTCD at ~10³ cfu/gm stool and stool toxin detected. On day 28 counts of both toxigenic C. difficile and NTCD had declined to ~10² cfu/gm and on day 45 neither were detected in stool. The patient died 1 month later of an unrelated cause but had no further diarrhea. In view of the mild recurrence of diarrhea with stool toxin detected at day 17 it is likely that this patient failed preventive treatment with NTCD, whereas patient A was a success. No further patient treatments with NTCD were reported until well into the 21^st century, however, considerable clinical information on detection of NTCD in patients was obtained in the interim.

In the 1980s it became apparent that CDI was a healthcare associated infection (Gerding et al., 1986; McFarland et al., 1989), and typing systems for identification of specific C. difficile strains were developed (Kuijper et al., 1987; McFarland et al., 1989; Clabots et al., 1993) based not only on isolates from patients with CDI, but also from specimens collected from asymptomatic patients using rectal swabs (McFarland et al., 1989; Johnson et al., 1990; Clabots et al., 1992). McFarland et al. (1989) were the first to detect the presence of large numbers of patients in hospital that were asymptomatically colonized with C. difficile. They demonstrated that transmission of C. difficile was frequent in the hospital setting using immunoblot organism typing and space/time relations. They found that nearly 2/3 of patients that acquired C. difficile remained asymptomatic whereas just over 1/3 had symptomatic CDI (McFarland et al., 1989).

The use of restriction endonuclease analysis (REA) typing of C. difficile markedly enhanced the epidemiologic investigation of CDI in hospitals (Kuijper et al., 1987; Clabots et al., 1993). Early in the development of REA there were 75 distinct groups identified by letters of the alphabet, 43 cytotoxin positive groups, 28 NTCD groups, and 4 groups that included both cytotoxin positive and negative isolates. Isolates for typing were obtained from CDI patients, rectal swabs of asymptomatic patients and multiple environmental sites. In comparison with other typing systems then in use REA demonstrated greater sensitivity than immunoblot typing, bacteriophage-bacteriocin typing and ribotyping (Clabots et al., 1993). Remarkably, the most frequently isolated REA group was the NTCD group M which was found more commonly than any toxigenic groups and may be a reflection of the inclusion of large numbers of isolates collected from asymptomatic patients using rectal swabs.

Using a similar weekly rectal swab methodology used by McFarland et al. (1989) to detect C. difficile asymptomatic colonization coupled with REA typing of C. difficile (Kuijper et al., 1987; Clabots et al., 1993), Johnson et al. (1990) showed that 21% of patients hospitalized on three wards were culture-positive for C. difficile, the majority 51 of 60 (85%) were asymptomatic. All of the 9 cases of CDI were caused by two closely related toxigenic REA types, B and B2, and none occurred among the 51 asymptomatic colonized patients, 9 of whom were colonized with the B or B2 strain types but remained asymptomatic. Fifteen of the 17 patients with the B or B2 isolates were located on the same surgical ward suggesting transmission on that ward. Among the 18 different REA type C. difficile isolates found on the 3 study wards, 10 were toxigenic and 8 (44%) were non-toxigenic including 7 distinct REA groups of NTCD, groups A, C, M, P, S, T, and U. This was the first indication that NTCD strains were carried frequently by hospital patients. In addition, it was found that asymptomatic C. difficile colonization occurred in approximately the same frequency on all 3 hospital wards, ranging from 12.2 to 19.3% of patients, all of whom had relatively long hospital stays. All the patients with toxigenic C. difficile or NTCD in their stool remained asymptomatic.

A larger similar study using rectal swab cultures was conducted on a single surgical ward with a historically high rate of CDI by Clabots et al. (1992). The study was conducted over 9 months and enrolled 634 (94%) of 678 admissions to this ward, obtaining rectal swab cultures on admission and weekly thereafter. Of the 634 ward admissions, 65 (10.3%) were either asymptomatically culture-positive for C. difficile (61 patients) or had CDI (4 patients). Twelve additional asymptomatic admissions were positive for C difficile when first sampled, but were not sampled within 48 h of admission and were indeterminate as to whether they acquired C. difficile on the ward or were already colonized when admitted. The majority of admissions (355, 56%) were admitted from home and had not been hospitalized within the previous 30 days. Twenty-two (7%) of these home admissions were culture-positive for C. difficile indicating the high potential for introduction of C. difficile strains to the hospital from the community. Of the 557 admissions to the ward that were not culture-positive for C. difficile, 54 (9.7%) acquired C difficile while on the ward, but only 3 had CDI. REA typing identified 58 different distinct types during the study. Of the top seven most frequently isolated REA types, three were NTCD types and constituted 23/68 (34%) of these isolates. The most commonly isolated NTCD strain type was M3 which subsequently, together with types M23 and T7, was selected for laboratory trials in hamsters for prevention of CDI.

Low rates of CDI among patients previously asymptomatically colonized with C. difficile were observed in several trials utilizing weekly rectal swab cultures and suggested that colonization might be protective against CDI if symptoms did not develop relatively quickly following toxigenic C. difficile exposure, or if patients were colonized by NTCD. Data from four such prospective studies were combined in a random effects model by Shim et al. (1998). All four studies employed similar weekly rectal swab cultures for C. difficile in asymptomatic hospitalized patients and utilized stool cultures for C. difficile if CDI symptoms developed. Patients recovering from prior CDI were excluded as they have a high colonization rate with toxigenic C. difficile post treatment and are prone to recurrences of CDI. Asymptomatic patients were included if they had at least two consecutive weekly C. difficile rectal swab cultures and were classified as either colonized or not colonized. Colonized patients were further categorized as colonized with a toxigenic C. difficile strain or NTCD. All isolates underwent REA typing as well as toxin testing. Among the 618 non-colonized patients, 22 (3.6%) developed CDI whereas only 2 (1%) of 192 colonized patients developed CDI (pooled risk difference −2.3%, p = 0.021, Figure 1). When the analysis was confined only to patients who had received antibiotics within 14–21 days of the start of the sampling or during sampling, the pooled risk difference was −3.2%, p = 0.024. Greater exposure to antibiotics in non-colonized patients could explain the increased rate of CDI, but antibiotic exposure was lower (79%) in non-colonized patients compared to colonized patients (92%). The mechanism by which colonization prevents CDI is unknown, and toxigenic strains of C. difficile as typed by REA that caused CDI were also found to be carried asymptomatically suggesting that strain type did not explain prevention. In these 4 studies there was a very high proportion (44%) of patients that were colonized by NTCD. There were 15 different REA groups of NTCD found among the 76 NTCD isolates, but the majority of NTCD were in two REA groups, M (49%) and T (19%). No CDI occurred among NTCD colonized patients and may account at least partially for the reduced CDI rates in colonized patients.

Taken together these clinical observations documented the relatively high rate of colonization of hospitalized patients by non-toxigenic strains of C. difficile and documented a very low rate of CDI among all colonized patients. Furthermore, REA typing demonstrated the ability to group and type NTCD strains in a manner similar to toxigenic C. difficile and identified two dominant groups of NTCD, the M and T groups by REA typing.

Bedside clinical and epidemiologic observations coupled with the evolution of molecular typing of C. difficile prompted further laboratory testing of C. difficile, particularly in animal models. Of specific interest was whether strains of C. difficile responsible for hospital outbreaks were more virulent than other strains that were found to asymptomatically colonize patients more often than they caused CDI. Sambol et al. (2001) tested three toxigenic epidemic C. difficile strains, REA types B1, J9, and K14 in the hamster model and compared them to toxigenic REA type Y2 which was found to commonly colonize patients but caused little CDI. A fifth strain, toxin variant REA type CF2, which is toxin A-negative, toxin B-positive and associated with CDI outbreaks was also included. The hamster protocol was similar to that of Borriello and Barclay (Borriello and Barclay, 1985), but differed in that only spores of C. difficile were administered by gavage 5 days following a single oral clindamycin dose of 30 mg/kg. A minimum infective dose for each C. difficile strain was predetermined to be 100 spores to mimic a postulated low inoculum exposure. The epidemic strains B1, J9, and K14 infected hamsters within 24–48 h of administration and caused 100% mortality by 48 h. REA type Y2 infected 9 of 10 hamsters over a range of 1–4 days, somewhat more slowly than the epidemic strains, but eventually all 9 infected hamsters died. Toxin variant strain CF2 infected only 6 of 10 hamsters over a range of 1 to 10 days and only 3 of the 6 died; the remaining 3 hamsters were colonized through study termination at day 79 and appeared to have no symptoms. The study documented differences in virulence of C. difficile strains in the hamster model and served as a basis on which to test preventive strategies with NTCD in the hamster model.

The above pre-clinical data were used to file investigational new drug applications with agencies in the US, Canada and Europe. Manufacturing of spores of NTCD-M3 under cGMP was undertaken successfully by ViroPharma, Inc. and the spores prepared in liquid suspension for safety testing in healthy volunteers at doses of 10⁴, 10⁶, and 10⁸ spores/dose or placebo (Villano et al., 2012). NTCD-M3 was found to be safe in younger volunteers age 18–45 years at all three single doses given in the absence of any antibiotic pre-treatment. To better simulate the likely age of patients with CDI, volunteers age >60 years (age range 60–73 years) were given single doses of 10⁴, 10⁶, and 10⁸ spores or placebo safely. Volunteers >60 years were then given the highest dose, 10⁸ spores twice daily for 5 days and tolerated this safely. This was the only group in which NTCD could be detected in the stools of the volunteers in the absence of prior antibiotic administration. Stool cultures first became positive on days 2–4 and were positive in 6 of 8 volunteers at day 7 but in none at day 14 and 21 suggesting the possibility that this may have been pass-through of NTCD spores and not colonization.

Finally, groups of 12 >60 year old volunteers were pre-treated with 5 days of oral vancomycin 125 mg 4 × daily to simulate patients being treated for CDI to determine if colonization with NTCD would occur post vancomycin treatment. Each group of 9 NTCD and 3 placebo subjects was given 10⁴ or 10⁶ or 10⁸ spores of NTCD-M3 or placebo daily for 14 days beginning the day following the last vancomycin dose. In each group the 9 volunteers given NTCD-M3 had NTCD detected in their stools during the 14-day administration and in 4 to 5 volunteers on days 21 and 28 following NTCD. In the 3 volunteers receiving placebo in the 10⁴ and 10⁶ NTCD-M3 spore cohorts one volunteer in each cohort became colonized with a toxigenic strain of C. difficile but remained asymptomatic. In the cohort receiving 10⁸ spores of NTCD-M3 the 3 placebo patients all became colonized with NTCD-M3, indicating that at this highest dose of NTCD-M3 there was transmission of NTCD-M3 from the active recipient group or the environment to the placebo group who were housed on the same clinical trial facility. No safety issues were identified in the volunteers receiving NTCD-M3.

For the first time since Seal et al. (1987) reported the treatment of 2 CDI patients with NTCD in 1987, NTCD was again administered in a Phase 2 double blind, randomized, dose-ranging, placebo controlled trial of NTCD for prevention of recurrent CDI in patients who have had their first episode or first recurrence of CDI. The primary outcome of the study was safety, with fecal colonization rate, CDI recurrence rate, and optimal dosing of NTCD-M3 as exploratory secondary outcomes. The study design shown in Figure 8 compared 2 doses, 10⁴ and 10⁷ spores given orally in liquid once daily for 2 durations, 7 days (10⁷ spores) and 14 days (10⁴ and 10⁷ spores) compared to placebo for 14 days. NTCD-M3 was begun 1–2 days following completion of successful treatment of CDI with vancomycin or metronidazole (Gerding et al., 2015). A total of 168 patients were randomized and treated and 157 completed treatment. The four arms of the study were well balanced in terms of patient age (range 18–94 years) with 39% >65 years old. Most patients were having their primary CDI episode (83%), were outpatients (76%), and were treated with metronidazole (60%). Vancomycin was used for treatment in 20% of subjects and 20% were treated with both vancomycin and metronidazole.

Safety results indicated that participants who received NTCD-M3 had fewer treatment emergent adverse events and fewer serious adverse events than placebo patients with the exception of headache which occurred in 10% of NTCD-M3 recipients and 2% of placebo (Gerding et al., 2015). No serious adverse events were considered treatment related. Colonization by NTCD-M3 was defined as detection of NTCD-M3 in stool at any time following completion of treatment. Figure 9 demonstrates that colonization with toxigenic C. difficile following antibiotic treatment of CDI is common (63% of placebo subjects at week 2) whereas with administration of NTCD-M3, much of the toxigenic C. difficile colonization was replaced by NTCD-M3; 71% of participants who received 10⁷ spores and 63% who received 10⁴ spores. Colonization with NTCD-M3 was maximal in weeks 1–3 and slowly declined thereafter until last detected in week 22. A small number of participants still harbored toxigenic C. difficile at 26 weeks, the last time stools were sampled.

Prevention of recurrence of CDI is summarized in Table 1. Using the protocol definition of recurrence of CDI at 6 weeks, recurrence rate was 13 (30%) of 43 placebo subjects and 14 (11%) of 125 NTCD-M3 subjects (odds ratio [OR], 0.28; 95%CI, 0.11–0.69; P = 0.006); the lowest recurrence was in 2 of 43 (5%) patients receiving 10⁷ spores/day for 7 days (OR, 0.1; 95%CI, 0.0–0.6; P = 0.01 vs. placebo). Using an alternate definition of recurrent CDI defined as use of antibacterial treatment for CDI, the recurrence rate was 14 of 43 (33%) placebo subjects and 17 of 125 (14%) NTCD-M3 subjects (p = 0.009). NTCD-M3 also reduced all-cause diarrhea from 77% in placebo subjects to 57% of NTCD-M3 subjects (p = 0.020). There was good correlation of CDI prevention with colonization by NTCD-M3; among subjects who received NTCD-M3 and became colonized, the CDI recurrence rate was 2 of 86 (2%) vs. 12 of 39 (31%) subjects who received NTCD-M3 and were not colonized (p < 0.001). Dose-ranging indicated that 10⁷ spores were more effective than 10⁴ spores, and there was no advantage to extending treatment to 14 days vs. 7 days for the 10⁷ dose. In summary, NTCD-M3 was safe and effective in preventing CDI in patients who were experiencing their first CDI episode or first recurrence of CDI.

DG holds patents (unlicensed, no royalties) and technology for the use of NTCD for prevention of CDI and is Scientific Advisory Board Member for Rebiotix, Actelion, Merck, Summit and DaVolterra, is a consultant for Pfizer, MGB Pharma, and Sanofi Pasteur, and holds a research grant from Seres Therapeutics. SJ is Scientific Advisory Board Member for Bio-K+, Synthetic Biologics, Summit and CutisPharma. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.