A US-based national surveillance study for the susceptibility and epidemiology of Clostridioides difficile isolates with special reference to ridinilazole: 2020–2021

ABSTRACT We have previously reported on the susceptibility and epidemiology of Clostridioides difficile isolates from six geographically dispersed medical centers in the United States. This current survey was conducted with isolates collected in 2020–2021 from six geographically dispersed medical centers in the United States, with specific attention to susceptibility to ridinilazole as well as nine comparators. C. difficile isolates or stools from patients with C. difficile antibiotic-associated diarrhea were collected and referred to a central laboratory. After species confirmation of 300 isolates at the central laboratory, antibiotic susceptibilities were determined by the agar dilution method [M11-A9, Clinical and Laboratory Standards Institute (CLSI)] against the 10 agents. Ribotyping was performed by PCR capillary gel electrophoresis on all isolates. Ridinilazole had a minimum inhibitory concentration (MIC) 90 of 0.25 mcg/mL, and no isolate had an MIC greater than 0.5 mcg/mL. In comparison, fidaxomicin had an MIC 90 of 0.5 mcg/mL. The vancomycin MIC 90 was 2 mcg/mL with a 0.7% resistance rate [both CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria]. The metronidazole MIC 90 was 1 mcg/mL, with none resistant by CLSI criteria, and a 0.3% resistance rate by EUCAST criteria. Among the 50 different ribotypes isolated in the survey, the most common ribotype was 014–020 (14.0%) followed by 106 (10.3%), 027 (10%), 002 (8%), and 078–126 (4.3%). Ridinilazole maintained activity against all ribotypes and all strains resistant to any other agent tested. Ridinilazole showed excellent in vitro activity against C. difficile isolates collected between 2020 and 2021 in the United States, independent of ribotype.

Ridinilazole is under development for the treatment of CDI (7,8).Ridinilazole is a more-narrow spectrum antibiotic than some antibiotics currently used to treat CDI, namely vancomycin and metronidazole.Clinical studies have also shown that ridinilazole is associated with less disturbance to the gut microbiota in patients with CDI compared to the standard of care, vancomycin (9).Vancomycin has a profound impact on the microbiota in the gut causing prolonged and more significant dysbiosis and loss of secondary bile acids, features known to be associated with higher relapse rates (9,10).Furthermore, based on one small study, ridinilazole may preserve the gut micro biota better than fidaxomicin, both of which have less impact on gut microbiota than vancomycin (9,11,12).
We have previously been involved with national surveillance of C. difficile susceptibil ity to various panels of agents, along with REA typing and toxin gene profiling since 2011 (13,14).As part of a surveillance program in 2020-2021, we undertook a 300-isolate study of C. difficile from six different geographically dispersed medical centers in the United States.In vitro, antimicrobial susceptibility test of these 300 contemporary C. difficile isolates to ridinilazole and comparators and their molecular typing using PCR ribotyping was performed.

RESULTS
Ridinilazole had an excellent activity against the 300 isolates tested (Table 1).The MIC 90 was 0.25 mcg/mL.In comparison, fidaxomicin had an MIC 90 of 0.5 mcg/mL.Vancomycin had an MIC 90 of 2 mcg/mL.Vancomycin resistance was seen with 0.7% of isolates based on both M11-A9 Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria.The metronidazole MIC 90 was 1 mcg/mL with none resistant by CLSI criteria, and with very little resistance noted (0.3%) by EUCAST criteria.For other notable antibiotics, significant resistance was seen.For clindamycin, the MIC 90 was greater than 32 mcg/mL with 26% resistance according to CLSI breakpoints.The moxifloxacin MIC 90 was 16 mcg/mL with 14.7% resistance noted.Imipenem had an MIC 90 of 8 mcg/mL and accompanying resistance of 5%.Rifampin and rifaximin had very low MIC 90's (Table 1).Tigecycline had an MIC 90 of 0.12 mcg/mL with an ECOFF resistance rate of 0.7%.
When one examines the ribotype distribution in comparison to 2016, we saw a change in distribution with an increase in 014-020 from 11.8% to 14.0%, whereas we saw a 50% decrease in ribotype 106 (from 15.0% to 10.3 %) and a 30% decrease in ribotype 027 (from 13.1% to 10.0%) (17).The activity of ridinilazole was maintained across all ribotypes including the hypervirulent ribotypes 027 and 078-126 (Table 2).The distribution of MIC values is shown in Fig. 2 for ridinilazole, fidaxomicin, vancomycin, and metronidazole.Only 25 isolates (8.3%) showed a ridinilazole MIC value of 0.5 mcg/mL, that is, higher than ridinilazole MIC 90, which included nine hypervirulent ribotypes (6 RT 078-126 and 3 RT 027).There were two isolates resistant to vancomycin with an MIC of 4 mcg/mL, one was ribotype 027 and the other was ribotype 053-163.
Examination of activity against isolates resistant to any of the agents tested demon strated that ridinilazole maintained its activity (Table 3).Ridinilazole retained activ ity against isolates resistant to moxifloxacin, clindamycin, imipenem, tigecycline, and vancomycin based on CLSI breakpoints or ECOFF values for EUCAST.

DISCUSSION
Ridinilazole showed an excellent in vitro activity against contemporary C. difficile isolates obtained between 2020 and 2021 from six geographically dispersed medical centers in the United States.No isolate had an MIC greater than 0.5 mcg/mL.Ridinilazole showed more in vitro potency than vancomycin or fidaxomicin, currently the preferred agent in the treatment of CDI (18).Clinical development of ridinilazole is on hold due to the Phase 3 Ri-CoDIFy study results.In this study, ridinilazole treatment achieved a higher sustained clinical response compared to vancomycin; however, the study did not pass the statistical threshold for superiority and, as such, the primary endpoint was not achieved (19).
Furthermore, we demonstrated that ridinilazole maintained activity against hypervirulent ribotypes as well as those that were resistant to one or more antimicro bial agents, either used to treat C. difficile-associated diarrhea or those used to treat anaerobic infections.Among these hypervirulent strains, many isolates clustered around the vancomycin EUCAST breakpoint of 2 mcg/mL.Most notably, Ribotype 078-126 appears to have the largest dispersion of MICs compared to the other ribotypes.RT078 represents a more diverse group than most other ribotypes, like RT027.These isolates are commonly observed in the environment and agricultural animals.Our results may reflect a trend toward increased resistance or it may also reflect a higher probability of resistance simply due to increased diversity (20)(21)(22).
When one looks at the trends in ribotype distribution, compared to 2016, there has been a change in ribotype distribution in the United States with a decline in the hypervirulent strains.This has been noted as well by others (23).
In conclusion based on the activity profile, ridinilazole has maintained its activity against C. difficile over the past few years of development and is a potential additional agent in the armamentarium to treat C. difficile-associated diarrhea.

Medical centers
Stools from patients diagnosed with CDI, or isolates recovered from stools of patients diagnosed with CDI, were referred to the Special Studies Laboratory at Tufts Medi cal Center by six medical centers for strain isolation and speciation (stools) or speci ation (isolates) and subsequent susceptibility testing (Table 4).The medical centers were as follows: Hines VA Hospital, Chicago, IL; Mayo Clinic, Rochester, MN; Johns Hopkins University School of Medicine, Baltimore MD; Tufts Medical Center, Boston, MA; Kindred Health System, Rancho Cucamonga, CA; and the University of Houston College of Pharmacy, Houston, TX.Each center has both excellent anaerobic bacteriology laboratories and investigators willing to collaborate.

Bacterial isolates
Convenience samples from patients with stools that tested positive for C. difficile toxin were obtained from Johns Hopkins, Tufts Medical Center, the University of Houston College of Pharmacy, and Hines VA hospital.These centers performed C. difficile stool culture following their standard procedures to obtain isolates and maintained the isolates stocked at −80°C.Isolates were cultured into pre-reduced anaerobically sterilized chopped meat broth (Anaerobe Systems, Morgan Hill, CA, USA) or other appropriate media for transport prior to shipment.Shipment of isolates was at ambient temperature.The isolates were kept at room temperature in the transport media as received by Tufts Medical Center until tested.Two centers, namely Mayo Clinic (Minnesota) and Kindred Healthcare (California), provided frozen stool samples from patients who had C. difficile toxin-positive samples.De-identified toxin-positive stool samples were stored at −80°C on site until being shipped overnight on dry ice to the Special Studies Laboratory at Tufts Medical Center.Upon receipt by Tufts the samples were stored at −80°C until processing.
Each institution that performed isolation of C. difficile was instructed to send an average of 50 isolates collected throughout the study period, July 2020-August 2021 while those providing stool were asked to send approximately 55 samples.Due to resource allocation during the COVID-19 pandemic, not all centers were able to provide the requisite number of samples.Additional isolates from Tufts Medical Center obtained at the appropriate time interval were included to complete a total of 300 isolates to be tested.

Processing of samples and identification of isolates
At the central laboratory, both the confirmation of the isolate as C. difficile and the culture of stool were accomplished by plating on C. difficile selective medium (cycloserine-cefox itin-fructose agar with taurocholate; Anaerobe Systems, Morgan Hill, CA, USA) and observing for characteristic colonial morphology (24,25).Thawed stool samples were ethanol shocked prior to being plated (25).A proline disc test (Remel Products, Lenexa, KS, USA) and gram stain were performed.This was followed by using the identifying method API20A ® (BioMerieux Inc., Durham, NC, USA).Speciated isolates were stored by using a cell paste swabbed from fresh plates that were suspended directly into skim milk and frozen at −80°C for future testing and reference (26).

Antimicrobial susceptibility testing
The MICs of the isolates were determined in singlicate using the CLSI-recommended agar dilution methodology against the panel of antibiotics shown in Table 5 (26,27).The medium was brucella agar (BD BBL, Franklin Lakes, NJ, USA) supplemented with 5 mg hemin, 1 mg Vitamin K 1 per liter, and 5% (vol/vol) lysed sheep blood.The antibi otic-containing plates were prepared freshly on the morning of the test.The inocula were prepared using direct suspension to achieve turbidity equivalent to a 0.5 McFarland standard and distributed accordingly into a Steers replicating block that deposited the inocula onto the test plates.The inocula density on the agar surface was ~10 4 CFU/spot.The plates were incubated for 44-48 hours at 35°C−37°C in an anaerobic chamber with an atmosphere of 85% N 2 , 5% CO 2 , and 10% H 2 .After incubation, the plates were examined against a dark, non-reflecting background and the MIC read at the concentration where a marked reduction occurred in the appearance of growth on the test plate as compared to that of growth on the control plate.Tests were to be repeated if the MICs of the control organisms were outside of the CLSI-specified range (27).The control and reference organisms used in this study were C. difficile ATCC 700057, C. difficile ATCC 43255, Bacteroides thetaiotaomicron ATCC 29741, Bacteroides fragilis ATCC 25285, and Staphylococcus aureus ATCC 29213.Isolates with high MICs to ridinilazole (≥0.5 mcg/mL), fidaxomicin (≥2 mcg/mL), vancomycin (≥4 mcg/mL), metronidazole (≥4 mcg/mL), or tigecycline (≥1 mcg/mL) were retested twice on separate days of testing.If the results were not identical, the isolate was tested a third time.There were no isolates with high MIC' to fidaxomicin or ridinilazole that met these criteria for re-testing.
The rates of resistance of the antimicrobial agents were determined using currently accepted CLSI breakpoints for anaerobes (27).For agents that did not have CLSI recommendations, FDA recommendations or the manufacturer's proposed breakpoint(s) were used (Table 5) (28).We also looked at rates of resistance using EUCAST breakpoints, based on epidemiologic cutoff values (ECOFF), which have been established for C. difficile (29).

Ribotyping
All isolates were sent to the Walk Laboratory, Montana State University, for amplicon preparation and analysis.A template for amplicon generation was obtained by growing C. difficile isolates overnight under anaerobic conditions in pre-reduced brain heart infusion broth supplemented with 0.1% cysteine.PCR was conducted directly on 10-fold diluted cultures (in sterile H 2 O).Fluorescently labeled amplicons were generated using PCR directly on 10-fold diluted cultures (in sterile H 2 O) (Promega MasterMix, M7502, Promega, Madison, WI, USA) with forward and reverse primers at 10 pmol/mcl.The forward primer was 5-GTGCGGCTGGATCACCTCCT-3 and reverse primer was 5-6-FAM/ CCCTGCACCCTTAATAACTTGACC.Thermocycling conditions included 95°C for 10 min followed by 35 cycles of 95°C for 0.5 min, 55°C for 0.5 min, 72°C for 1.5 min, and a final elongation step of 72°C for 10 min.Amplicons were stored at −20°C until fragment analysis using a Promega Spectrum Compact CE System (Promega).For CE analysis, amplicons were diluted in sterile DNase-/RNase-free water, Hi-DI Formamide (Life Technologies, Rockville, MD, USA) and ROX1000 size standard (Bioventure Inc, Murfrees boro, TN, USA) and added to a CE loading plate.Resulting .fsafiles were analyzed using the Walk Lab CdiffFragR pipeline (https://github.com/nvpinkham/CdiffFragR)against the in-house database of ribotyping profiles (version F-Ribotyping_Pes.lite.15),as previously described (16,17,30).

FIG 1
FIG 1 Distribution of ribotypes in the survey of 2020-2021.

TABLE 1
Activities of the antimicrobial agents against 300 C. difficile isolates b TheCLSI or EUCAST, as applicable, ECOFF value was applied, in the absence of a clinical breakpoint.c For tigecycline, the breakpoint for resistance recommended for anaerobes by the FDA was used.

TABLE 4
Participating study centers

TABLE 5
Antimicrobial agents tested, ranges, and breakpoints for susceptibility NA, not applicable.CLSI or EUCAST recommended breakpoint for resistance not available.bTheCLSI or EUCAST, as applicable, ECOFF value was applied, in the absence of a clinical breakpoint.c For tigecycline, the breakpoint for resistance recommended for anaerobes by the FDA was used. a