Prevalence, Antimicrobial Resistance and Toxin-Encoding Genes of Clostridioides difficile from Environmental Sources Contaminated by Feces

Clostridioides difficile (C. difficile) is the most common pathogen causing antibiotic-associated intestinal diseases in humans and some animal species, but it can also be present in various environments outside hospitals. Thus, the objective of this study was to investigate the presence and the characteristics of toxin-encoding genes and antimicrobial resistance of C. difficile isolates from different environmental sources. C. difficile was found in 32 out of 81 samples (39.50%) after selective enrichment of spore-forming bacteria and in 45 samples (55.56%) using a TaqMan-based qPCR assay. A total of 169 C. difficile isolates were recovered from those 32 C. difficile-positive environmental samples. The majority of environmental C. difficile isolates were toxigenic, with many (88.75%) positive for tcdA and tcdB. Seventy-four isolates (43.78%) were positive for binary toxins, cdtA and cdtB, and 19 isolates were non-toxigenic. All the environmental C. difficile isolates were susceptible to vancomycin and metronidazole, and most isolates were resistant to ciprofloxacin (66.86%) and clindamycin (46.15%), followed by moxifloxacin (13.02%) and tetracycline (4.73%). Seventy-five isolates (44.38%) showed resistance to at least two of the tested antimicrobials. C. difficile strains are commonly present in various environmental sources contaminated by feces and could be a potential source of community-associated C. difficile infections.


Introduction
Clostridioides (Clostridium) difficile is an obligate anaerobic, spore-forming, Grampositive rod-shaped, and toxin-producing bacterium. C. difficile is among the most common nosocomial pathogens that cause antibiotic-associated diarrhea and pseudomembranous colitis worldwide [1][2][3]. The occurrence of C. difficile was well documented in hospitalized patients with C. difficile infection (CDI) but is also emerging in various environmental sources outside healthcare institutions. Little is known about environmental C. difficile isolates, and few studies were conducted on the prevalence, antimicrobial resistance, and toxin-encoding genes of environmental C. difficile in environmental sources contaminated with feces (e.g., biogas plants, digested sludge-amended soil, soil, animal feces, manure and in wastewater, raw sludge, and anaerobically digested sewage sludge). The ecology of C. difficile outside clinical settings is not fully understood, but the evolution of environmental pathogenic strains could occur in their zoonotic and environmental reservoirs. Therefore, optimization methods for the isolation and detection of C. difficile are required to elucidate the role of non-clinical sources as transmission routes of human infection.
C. difficile has several virulence factors, including toxins A and B, which are encoded by tcdA and tcdB genes, respectively, that are localized on a 19 kb Pathogenicity Locus (PaLoc) [4]. In addition, the C. difficile toxin CDT (cdtA and cdtB), which belongs to a family of binary toxins, was identified in toxigenic C. difficile strains [5].

Prevalence and Isolation of C. difficile from Fecally Contaminated Environmental Samples
The environmental C. difficile strains were isolated from 32 out of 81 (39.50%) fecally contaminated environmental samples (feces of calves (n = 10), biogas plant (n = 2), soil (n = 1), WWTP samples (n = 12), digested sludge-amended soils (n = 3), thermophilic digesters of biowaste or sewage sludge (n = 2), and anaerobic lab-scale bioreactors for the thermophilic digestion of sewage sludge (n = 2)), after the selective enrichment culture from spores in a C. difficile selective broth, supplemented with 0.1% sodium taurocholate for spore germination, 16 mg/L norfloxacin, and 32 mg/L moxalactam. The results of the presence of C. difficile in different fecal environmental samples are summarized in Table 1. Most C. difficile strains were isolated from digested sludge-amended soils and biogas plant samples, followed by WWTP samples, samples from the thermophilic digesters of biowaste or sewage sludge, soil, and the feces of calves at 100%, 75%, 66.67%, 50%, and 31.25%, respectively. However, C. difficile was not at all detected in adult cow feces, mixed storage cattle manure, treated sewage (effluent), grass and maize silages, and horse feces. A total of 169 environmental C. difficile isolates (WWTP samples (n = 69), calf feces (n = 40), digested sludge-amended soils (n = 21), anaerobic lab-scale bioreactors for the thermophilic digestion of sewage sludge (n = 17), thermophilic digesters (n = 16), biogas plant (n = 5), and soil (n = 1)) were isolated from those 32 C. difficile-positive samples, after purification by re-streaking them on an appropriate media, as described in Materials and Methods. Then, the isolates were confirmed as C. difficile via a latex agglutination C. difficile test and the amplification of the triose phosphate isomerase (tpi) gene by PCR. Table 2 illustrates the characteristics of toxin genes and antimicrobial resistance profiles of the environmental C. difficile isolates.    Fifty percent of the farm samples were positive for C. difficile in cattle feces and a correlation between the age of the cattle/calves and the detection of C. difficile in feces could be observed. The occurrence of C. difficile in calf feces and the antimicrobial prescriptions on farms are shown in Table 1. C. difficile was found in the feces of calves that were treated with paromomycin, amoxicillin-colistin (farm 6), or spectinomycin-lincomycin (6/6, 100%), (3/6, 50%), or (1/3, 33.33%), respectively, while it was not observed in the analyzed calf feces treated with sulphanilamide-neomycin.

Toxin-Encoding Genes of Environmental C. difficile Strains
Environmental C. difficile isolates were screened for toxin genes (tcdA and tcdB) and binary toxins (cdtA and cdtB) via a multiplex PCR assay. Almost all isolates were toxigenic, with 88.76% positive samples for both toxin A (tcdA) and B (tcdB). There were 75 isolates (44.38%) positive for both binary toxins CDT (cdtA and cdtB). All those isolates were positive for both toxins A and B. Nineteen isolates (11.24%) were non-toxigenic ( Table 2). The highest number of C. difficile toxigenic isolates was recovered from a WWTP (55, 32.54%) and from calf feces (40, 23.67%).

Antimicrobial Resistance of Environmental C. difficile Strains
The susceptibility of environmental C. difficile isolates to six antimicrobials was determined by the disc diffusion method and the minimum inhibitory concentrations (MICs) by using an E-test. All environmental C. difficile isolates (n = 169) were susceptible to the antimicrobials vancomycin and metronidazole ( Table 2). Most isolates (66.86%, n = 113) were resistant to ciprofloxacin, followed by clindamycin, moxifloxacin, and tetracycline, with 46.15% (n = 78), 13.02% (n = 22), and 4.73% (n = 8), respectively. Seventy-five (44.38%) out of one-hundred-sixty-nine isolates displayed resistance to at least two of the antimicrobials. Ninety-four (62.67%) and sixty-two (41.33%) out of one-hundred-fifty toxigenic isolates were resistant to ciprofloxacin and clindamycin, respectively. Furthermore, all non-toxigenic isolates were resistant to ciprofloxacin, while 15 isolates were resistant to clindamycin, 1 isolate was resistant to moxifloxacin, and 1 was resistant to tetracycline.

Standard Curves, Limit of Detection, and Detection Accuracy of qPCR for the 16S rRNA Gene
Serial dilutions of C. difficile DSM strain 1296 (CD) from 10 −1 to 10 −7 (from 3.4 × 10 6 to 0.34 CD cells) were spiked in CD-negative feces, and three standard curves were performed as described in Materials and Methods. Moreover, the standard curve was performed with C. difficile DSM 1296 pure culture. The quantification cycles (Cq) in 7 log dilutions ranged from 17.80 to 37.94 for three standards with R 2 of 0.9967, 0.9939, 0.9915, while Cq for the analytical standard of the pure culture of C. difficile ranged from 13.87 to 35.64 with R 2 of 0.9989 ( Figure 1). The two analytical standard curves were performed to evaluate the quantitative detection accuracy and the limit of detection between the pure culture of C. difficile DSM strain 1296 and CD-spiked feces. The analytical curves of the pure culture of C. difficile DSM strain 1296 and CD-spiked feces for the 16S rRNA gene had almost equal slopes. These results confirmed that the TaqMan-based qPCR method was capable of detecting the target "C. difficile" in pure culture and in CD-spiked feces with high accuracy. These results also indicated that a TaqMan-based qPCR assay was qualified to quantify the C. difficile in feces with a low detection limit of 22.66 cells/g of feces.
Antibiotics 2023, 12, x FOR PEER REVIEW 7 of 19 evaluate the quantitative detection accuracy and the limit of detection between the pure culture of C. difficile DSM strain 1296 and CD-spiked feces. The analytical curves of the pure culture of C. difficile DSM strain 1296 and CD-spiked feces for the 16S rRNA gene had almost equal slopes. These results confirmed that the TaqMan-based qPCR method was capable of detecting the target "C. difficile" in pure culture and in CD-spiked feces with high accuracy. These results also indicated that a TaqMan-based qPCR assay was qualified to quantify the C. difficile in feces with a low detection limit of 22.66 cells/g of feces.

Quantification of Environmental C. difficile in Fecal Environmental Samples
A load of C. difficile cells was estimated by TaqMan-based PCR assay for 16S rRNA gene with DNA extracted from the 81 fecally contaminated environmental samples, as described in Table 1. In total, 45 out of 81 samples (55.56%) were positive for the C. difficile 16S rRNA gene, with counts ranging from 0.044 to 1561.62 cells per g or mL ( Table 3, Table  The intra-assay CVs of the three standards were between 0.11% and 5%, 0.11% and 5.28%, and 0.11% and 5.69%, whereas the inter-assay CVs of the three standards ranged between 2.25% and 5.76, 3.05% and 5.36%, and 3.19% and 5.31%.

Quantification of Environmental C. difficile in Fecal Environmental Samples
A load of C. difficile cells was estimated by TaqMan-based PCR assay for 16S rRNA gene with DNA extracted from the 81 fecally contaminated environmental samples, as described in Table 1. In total, 45 out of 81 samples (55.56%) were positive for the C. difficile 16S rRNA gene, with counts ranging from 0.044 to 1561.62 cells per g or mL ( Table 3, Table  S1 in Supplementary Materials), and 36 samples were negative or under the detection limit (44.44%). C. difficile was mainly detected in 14 samples derived from the feces of calves and WWTP samples, and it was also detected in the samples of soil, digested sludge-amended soils, digested raw sewage sludge, and horse feces. Results of C. difficile detection in fecally contaminated environmental samples by TaqMan-based qPCR assay were compared with those derived by C. difficile selective enrichment culture (CSEC). A total of 81 environmental samples were examined with both methods. Environmental C. difficile was detected in 45 of the 81 samples (55.56%) by qPCR, whereas C. difficile was isolated by CSEC from 32 samples (39.50%) ( Figure 2, Table S2 in Supplementary Materials). C. difficile was positive for both qPCR and CSEC in 24 samples (75%), while it was negative in 28 samples (57.14%) ( Table 4). Results of C. difficile detection in fecally contaminated environmental samples by TaqMan-based qPCR assay were compared with those derived by C. difficile selective enrichment culture (CSEC). A total of 81 environmental samples were examined with both methods. Environmental C. difficile was detected in 45 of the 81 samples (55.56%) by qPCR, whereas C. difficile was isolated by CSEC from 32 samples (39.50%) ( Figure 2, Table S2 in Supplementary Materials). C. difficile was positive for both qPCR and CSEC in 24 samples (75%), while it was negative in 28 samples (57.14%) ( Table 4).  Eight confirmed enrichment culture-positive samples appeared to be negative by qPCR (Table 4), which could be explained by the lower target concentrations, meaning that the detection limit consists of less than 10 copies of the target DNA per PCR reaction [31][32][33]. Moreover, this might be related to the DNA extraction method and the increase in PCR inhibitors in those fecal samples. In addition, the DNA extraction efficiency from spores was approximately 1000 times lower than the efficiency when DNA was extracted Eight confirmed enrichment culture-positive samples appeared to be negative by qPCR (Table 4), which could be explained by the lower target concentrations, meaning that the detection limit consists of less than 10 copies of the target DNA per PCR reaction [31][32][33]. Moreover, this might be related to the DNA extraction method and the increase in PCR inhibitors in those fecal samples. In addition, the DNA extraction efficiency from spores was approximately 1000 times lower than the efficiency when DNA was extracted from vegetative cells [28]. Additionally, among the 49 CSEC-negative samples that were not in concordance, 21 (42.86%) samples were positive with qPCR but not with an enrichment culture.
In those fecal samples, environmental C. difficile was not found with selective enrichment culture. This could be referred to as the used selective medium containing antimicrobial agents, the size of the sample, and other supplements. However, from the 49 CSEC-negative samples, only 28 (57.14%) samples gave the same result in the qPCR assay (Table 4).

Discussion
C. difficile is responsible for antibiotic-associated diarrhea in humans, and it was suggested that environmental sources outside healthcare institutions, such as animal feces, manure, wastewater, and sewage sludge from WWTPs [9,[20][21][22][23]34], play a crucial role as a reservoir of community-associated C. difficile infections. The prevalence of C. difficile was found in different environments, such as animal farms [9,20,[35][36][37][38], anaerobically digested sewage sludge from WWTP [20,39], animal manure and compost [21], soil [20], and vegetables, lawn and compost [40,41]. To the best of our knowledge, this is the first study that represents the prevalence, antimicrobial susceptibility patterns, toxin-encoding genes, and quantitative numbers of environmental C. difficile in various environmental samples contaminated by feces in a limited geographical region in Germany.
In this study, the frequency of the detection of toxigenic strains was high (88.76%), especially in isolates that recovered from WWTP samples and the feces of claves and, in consequence, must be considered completely virulent and able to cause antibiotic-associated diarrhea and pseudomembranous colitis in humans. The toxigenic strains of C. difficile were previously isolated from animal manure and compost [21], poultry manure, soil, dust [22], the feces of calves [9,36], and WWTP samples [39]. The present study and some previous studies confirmed that those sources also carry both toxigenic and antimicrobial-resistant C. difficile isolates. In our study, toxigenic C. difficile isolates were resistant to ciprofloxacin and clindamycin by 62.67% and 41.33%, respectively.
Interestingly, the presence of environmental C. difficile was observed in the feces of calves that were treated with antimicrobials such as paromomycin (belonging to the aminoglycoside class) or combined antimicrobials (amoxicillin (belonging to penicillins) and colistin (belonging to polymyxins) as well as spectinomycin (belonging to the aminocyclitol class) and lincomycin (belonging to lincosamide class)) on farms with positive results of C. difficile in 100%, 50%, and 33.33% of the samples, respectively. C. difficile was not observed in calf feces treated with combined antimicrobials (sulphadiazine (belonging to sulfonamides) and neomycin (belonging to the aminoglycoside class)), probably due to their low utilization on farms. It should be noted that the administration of antimicrobials to individual calves and the fecal shedding of C. difficile from the same calf could be directly linked. It was also observed that C. difficile could be detected in feces after penicillin prescriptions on the farm [9]. In humans, penicillins were reported as being associated with C. difficile infections [8]. Moreover, it could be identified that prior antimicrobial treatment increases the frequency of C. difficile fecal shedding from calves.
In the present study, the number of ciprofloxacin resistance (2nd generation of fluoroquinolones) in environmental C. difficile isolates obtained from various environmental samples was 66.86%. The number of moxifloxacin resistance (3rd generation of fluoroquinolones) in these isolates was 13.02%. Recently, a large number of C. difficile isolates were found that expressed a higher resistance to the 2nd generation of fluoroquinolones (ciprofloxacin) than to the 3rd generation of fluoroquinolones (moxifloxacin) [11,12,40]. Fluoroquinolone resistance in C. difficile strains occurs via mutations in the quinolone resistancedetermining region (QRDR) of DNA gyrase subunits A (gyrA) and/or B (gyrB), resulting in several amino acid substitutions that confer resistance to fluoroquinolones [11,50].
Clindamycin belongs to the lincosamide class. Clindamycin resistance was discovered in 46.15% of all environmental C. difficile isolates in this study. Clindamycin resistance was reported in C. difficile isolates from different environmental sources, such as the feces of dairy calves (76.5%) [9], manure and compost samples (53.45%) [21], vegetables, lawn, and compost (33.6%) [40], swine and dairy feces (79.5 %) [37], and puddle water and soils (28.6%) [19]. Nineteen and fifteen non-toxigenic strains, classified as non-virulent, were resistant to ciprofloxacin and clindamycin, respectively. One isolate was resistant to tetracycline, and another one was resistant to moxifloxacin. These multiple antimicrobial resistances in non-toxigenic environmental C. difficile strains might serve as reservoirs of antimicrobial resistance determinants, which may be horizontally transferred to toxigenic strains, as well as into other pathogenic bacterial species via horizontal gene transfer (HGT).
The environmental C. difficile isolates recovered from raw sewage, calf feces, anaerobically digested sludge, and digested sludge-amended soils were resistant to tetracycline by 4.73%, which is comparable to the already published studies of clinical and environmental C. difficile isolates such as C. difficile isolates from soil and water (8.6%) [19] and vegetables, lawn, and compost (2.9%) [40]. In C. difficile, resistance to tetracycline is encoded by tetracy-cline (tet) resistance genes. The most widespread tet gene is tetM, usually associated with conjugative transposons Tn916/Tn916-like family and Tn5397. These elements are found to be able to transfer the tet genes among C. difficile strains and between unrelated species of bacteria present in the clinical setting, community, and in the environment, including animal reservoirs, food sources, soil, and water [7].
C. difficile resistance to antimicrobial agents (i.e., fluoroquinolones, macrolidelincosamides-streptogramin B (MLS B ), tetracyclines, or beta-lactams) could be a result of the presence of antimicrobial resistance genes (ARGs) via the transfer of mobile genetic elements (e.g., plasmids, conjugative transposons, prophages), occurrences of gene mutations, and changes in the antimicrobial targets and/or metabolic pathway of C. difficile and via biofilm formation [7,11,12,50,51]. HGT plays a key role in the spread of ARGs among toxigenic and non-toxigenic C. difficile strains and between other gut microbiota [52,53].
Culture-independent approaches with targets on the bacterial 16S rRNA gene have come into prominence for the detection and quantification of anaerobic fecal bacterial species, practically those present in relatively small numbers, such as C. difficile and C. perfringens, compared to the dominant gastrointestinal bacterial flora in animals and humans. In such cases, a selective enrichment culture is necessary, but it is time-and lab-consuming. Therefore, a TaqMan real-time qPCR assay for the rapid detection of the 16S rRNA gene of environmental C. difficile was used directly with the DNA extracted from the diverse fecally contaminated environmental samples, and a comparison with the results of the C. difficile selective enrichment culture method was performed.
To our knowledge, this is the first study that quantitatively evaluated numbers of environmental C. difficile in different environmental sources contaminated by feces and compared these with results from C. difficile selective enrichment cultures. Several studies, however, used qPCR to qualitatively and quantitatively determine the occurrence of C. difficile in clinical samples [24,28,29,54]. The fecally contaminated environmental sources outside healthcare institutions (i.e., WWTP samples, cattle feces, soil, digestion of raw sewage sludge, horse feces) could directly or indirectly spread C. difficile in the community [9,21,[35][36][37]39] and may be a potential health risk.
In our examinations, the reliable detection of C. difficile in different fecally associated samples and the comparison of results obtained between CSEC and qPCR methods (Table S1) supports the validity of TaqMan qPCR as a sensitive method to detect C. difficile in fecal environmental samples. C. difficile was detected in 45 out of 81 samples (55.56%) via qPCR, whereas it was detected in 32 samples (39.50%) by selective enrichment culture. Brown et al. [27] reported that the C. difficile 16S rRNA gene was detected in 64.6% and 43.8% of environmental surface area by qPCR and enrichment culture, respectively. However, the results obtained with qPCR correlate with the selective enrichment cultures in 24 (75%) samples, but qPCR was more sensitive and able to detect C. difficile in 21 enrichment culture-negative cases. Eight enrichment culture-positive samples were qPCR negative (Table 4). This might relate to the number of C. difficile cells or spores in fecal samples. In addition, the used DNA extraction method may reduce the target concentration, meaning that the sample consists of less than 10 copies of the target DNA [31][32][33]. In our study, the DNA concentration ranged between 1.08 and 384 ng/µL, and the DNA template was subjected to qPCR with or without dilution. In general, the DNA extraction from fecal samples and the resuspension in smaller amounts of elution buffer could not only give highly concentrated DNA but also increase fecal-derived PCR inhibitors and decrease the efficiency of amplification [28,58]. Kubota et al. [28] also reported that the qPCR assay mainly detected vegetative cells because the DNA extraction efficiency from spores was approximately 1000 times lower than the efficiency from vegetative cells. The expected reasons for not detecting C. difficile via 16S rRNA gene qPCR or in enrichment cultures are summarized in Table 5. Among the 49 CSEC-negative samples, 21 samples were qPCR-positive. The discrepant result between the selective enrichment culture and TaqMan qPCR methods in the 21 samples may reflect that the selective enrichment culture method detects only living cells; qPCR detects both living and dead cells, which could result in a higher detection frequency of C. difficile by a TaqMan qPCR assay ( Table 5). The quantification of the 16S rRNA gene by real-time qPCR in antibiotic-associated diarrhea patients was correlated with the culture, but qPCR was more sensitive and able to detect C. difficile in some culture-negative samples [54]. The low detection limit in C. difficile-spiked human stool samples by traditional PCR was 10-fold higher than the LOD from the culture method [33]. Moreover, used medium type, sample size, and selective supplemented agents (e.g., antimicrobials) might contribute to the apparent variation in C. difficile prevalence in those samples by using the enrichment culture method. The higher sensitivity found by qPCR was expected due to the detection of non-cultivable cells or spores. Additionally, the 16S rRNA gene qPCR and selective enrichment culture methods are all acceptable techniques for the detection and quantification of environmental C. difficile, but the qPCR assay is more sensitive than the selective enrichment culture.

Fecal Environmental Samples Collection
Eighty-one fecally contaminated environmental samples were collected from March 2021 to June 2022, including cattle feces, soil, digested sludge-amended soils, mixed storage cattle manure, horse feces, thermophilic digesters of biowaste or sewage sludge, biogas plant, anaerobic lab-scale bioreactors for thermophilic digestion of sewage sludge, and samples from a WWTP, located in northwestern Germany, including raw sewage (influent), treated sewage (effluent), activated sewage sludge, raw sewage sludge (mixture of activated sewage sludge and access of secondary sedimentation), and digested sewage sludge. The fecal environmental samples are summarized in Table 1.
For raw sewage (influent), activated sewage sludge, and treated sewage (effluent), 100 or 300 mL of the sewage-derived samples were centrifuged at 10,000× g for 10 min at 4 • C, the supernatant was discarded, and the pellet was resuspended in one milliliter of CD broth. Afterward, the mixture was inoculated into supplemented CD broths and incubated as described above. For soil samples, soil was processed as described previously by Janezic et al. [19] with some modifications. Briefly, 25 g of soil was resuspended in 90 mL of sterile distilled water. In order to remove the majority of soil particles, 50 mL of soil suspension was centrifuged at 50× g for 2 min. Of soil suspension, 40 mL was transferred into a new 50 mL sterile centrifugation tube and centrifuged again at 50× g for 2 min. Of the supernatant, 30 mL was centrifuged at 10,000× g for 10 min, the supernatant was discarded, and the pellet was inoculated in 9 mL of supplemented CD broth. All inoculated broths were incubated as described above.
For grass and maize silage and horse feces, five grams of each sample were vortexed in 15 mL of 1× phosphate-buffered saline (PBS) for 1 min, three times. The collected suspensions were centrifuged at 10,000× g for 10 min at 4 • C, the supernatant was discarded, and the pellets were inoculated in 9 mL supplemented CD broths and incubated as mentioned above.
Following incubation, 2 mL of each incubated CD broth was mixed with an equal amount of absolute alcohol (1:1) and incubated at room temperature for 50-60 min. The mixtures were then centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The pellet was resuspended in 200 µL 1× PBS. All resuspended liquid or at least 100 µL was plated on Clostridium difficile agar basis (CDA, Fisher Scientific GmbH, Schwerte, Germany) supplemented with 7% defibrinated horse blood (Fisher Scientific GmbH, Schwerte, Germany), (12 mg/L) norfloxacin, (32 mg/L) moxalactam, and 0.1% sodium taurocholate. All plates were incubated anaerobically in anaerobic jars (Schuett-Biotec GmbH, Göttingen, Germany) at 37 • C for two days and, if negative, re-incubated three days more. Of each plate suspected of being C. difficile, 5-10 colonies (based on morphology, grey with irregular edges) were carefully picked and streaked onto CDA or blood agar supplemented with 5% horse blood and incubated anaerobically at 37 • C for 48 h. The identity of the pure culture was evaluated on the basis of morphology and confirmed via the Oxoid C. difficile latex test (Fisher Scientific GmbH, Schwerte, Germany) and finally by analyzing the tpi gene (see below in Section 4.4). Stock cultures of confirmed C. difficile isolates were stored in brain heart infusion (BHI) broth (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) with 20% glycerol at −20 • C.

Genomic DNA Extraction from Bacterial Cells (Pure Cultures)
C. difficile colonies were transferred to 150-200 µL of 5% Chelex 100 in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8), pre-heated at 56 • C for 30 min. Afterwards, samples were boiled at 95 • C for 15 min with gentle vortexing every 5 min under continuous shaking at 300 rpm. The tube was centrifuged at high speed at 12,000× g for 3 min to pellet the Chelex. The supernatant (approximately 130-180 µL) containing the eluted genomic DNA was transferred to a new 1.5 mL Eppendorf tube. The eluted genomic DNA was centrifuged again for 3 min as described above, and 100-150 µL was removed and transferred to a final 1.5 mL Eppendorf tube. The genomic DNA was stored at −20 • C for further analysis. The genomic DNA was diluted 1:10 in MQ water, and five microliters (DNA concentration ranged between 1 and 1.5 ng/µL) were used directly in PCRs as DNA templates.

Molecular Identification of Environmental C. difficile Isolates via PCR
PCR amplification of a specific housekeeping gene, triose phosphate isomerase (tpi) was performed as previously described by Leeme et al. [59]. The PCR was performed with tpi-specific primers (tpi-F: AAAGAAGCTACTAAGGGTACAAA) and (tpi-R: CATAATAT-TGGGTCTATTCCTAC), with an amplicon size of 230 bp. The C. difficile DSM (Leibniz Institute, German Collection of Microorganisms, Braunschweig, Germany) strain 1296 was used as a positive control. PCR products were run under standard conditions on a 1% agarose gel and stained with a DNA stain (SERVA Electrophoresis GmbH, Heidelberg, Germany), and visualized under UV light.

Profiling of Toxin-Encoding Genes of Environmental C. difficile Isolates by Multiplex PCR
Amplification of toxin genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) were detected using a multiplex PCR, as described previously by Perrson et al. [60]. The primers are listed in Table 6. C. difficile DSM 1296 was used as a positive control for toxin genes, tcdA and tcdB, but negative for binary toxin genes, cdtA and cdtB. In addition, one of our C. difficile strain was sequenced with an Illumina MiSeq in order to confirm the presence of the respective toxin genes which used as a positive control for those genes. PCR products were analyzed by electrophoresis on a 1.5% agarose gel.

Antimicrobial Susceptibility Testing
Environmental C. difficile isolates were subjected to antimicrobial susceptibility testing by the disc diffusion method for the antimicrobials clindamycin, ciprofloxacin, and tetracycline (Fisher Scientific GmbH, Schwerte, Germany). The minimum inhibitory concentrations (MICs) were determined by using an E-test of the antimicrobials vancomycin, metronidazole, and moxifloxacin (bioMe'rieux Deutschland GmbH, Nürtingen, Germany). The moxifloxacin's concentration tested was 0.002-32 µg/mL. For vancomycin and metronidazole, the range tested was 0.016-256 µg/mL. The environmental C. difficile isolates were streaked on blood agar plates and were incubated anaerobically at 37 • C for 24 h. The inoculum was prepared by picking a few colonies and mixing them in two milliliters of physiological saline (0.85% NaCl). A bacterial suspension equivalent to 4 MacFarland units [61] was spread on Brucella agar plates using a sterile cotton swab (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) supplemented with hemin and vitamin K, according to Clinical and Laboratory Standards Institute (CLSI) [62] for the testing of anaerobes. Antimicrobial discs and E-test strips were placed onto agar plates. The plates were incubated anaerobically for 24-48 h at 37 • C. For the disks, the diameter of the inhibition zone was measured. For the E-test, the MIC value was read from the scale in terms of µg/mL where the ellipse edge intersects the strip. The breakpoint/epidemiological cut-off of the E-test was interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [63] guideline for vancomycin. The breakpoints of metronidazole and moxifloxacin were interpreted according to CLSI guidelines [62]. The inhibition zone diameter breakpoints of clindamycin were interpreted according to the members of the SFM Antibiogram Committee [64], while ciprofloxacin and tetracycline were interpreted according to Kouassi et al. [65].

Preparation and DNA Extraction from Fecal Environmental Samples
100-400 mg of fecal and soil samples were used for DNA extraction. Of raw and digested sewage sludge, thermophilic digesters content, storage mixed cow manure, and biogas plant digestate, 4 mL were centrifuged at 12,000× g for 5 min, the supernatant was discarded, and the pellet was used for DNA extraction as described above. For raw and treated sewage and activated sewage sludge, 35 to 300 mL of each was centrifuged at 10,000× g for 10 min at 4 • C, the supernatant was discarded, and the pellets were resuspended in provided buffer for DNA extraction. Grass and maize silage and horse feces were pre-treated, as described above in Section 4.1. 100-400 mg was weighted from the pellets, or the pellet was resuspended in provided buffer for DNA extraction. The DNA was extracted from fecal environmental samples by using Allprep ® PowerViral ® DNA/RNA Kit (Qiagen, Hilden, Germany) or Quick-DNA™ Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the respective protocols. The extracted DNA was stored at −20 • C until further analysis. The DNA concentration was quantified via Qubit 3.0 Fluorometer.

Preparation of Standard Analytic Curves of C. difficile-Spiked Feces and Pure Culture for qPCR
The standard analytic curves of C. difficile (CD) were performed as described previously by Bandelj et al. [25]. Briefly, the strain C. difficile DSM 1296 was cultured on brain heart infusion (BHI) agar plates. The plates were incubated anaerobically at 37 • C for 24 h. Afterward, the pure culture of CD was harvested from BHI agar plates into one milliliter of 1× PBS. 10-fold serial dilutions of CD stock suspension were prepared in 1× PBS (10 −1 to 10 −7 ). The number of C. difficile DSM 1296 cells was quantified by counting the cells with a microscope (Axioscope, Carl Zeiss Microscopy GmbH, Jena, Germany) using a Neubauer chamber (Marienfeld-Superior™ GmbH & Co.KG, Lauda-Königshofen, Germany). The number of CD cells per milliliter was calculated for dilutions, 10 −1 to 10 −4 , according to the following equation: Cells per mL = average count per square (from four squares) × dilution factor × 10 4 Of the serial dilutions of the pure culture ranging from 3.4 × 10 7 CD cells per mL to approximately 3.4 CD cells per milliliter, 100 µL was spiked in cattle feces. All serial dilutions were spiked in 150 mg cattle feces in triplicate that were previously confirmed by 16S rRNA gene-specific assay to be negative for C. difficile in genomic DNA directly extracted or after selective enrichment of cattle feces as well as by plating enrichment culture of cattle feces on C. difficile selective agar plates as described above in Section 4.1. The genomic DNA was isolated from the CD-negative feces spiked with a known number of CD cells and further tested in triplicate using the CD 16S rRNA gene TaqMan-based qPCR to generate a standard analytical curve. The genomic DNA was extracted from the CD-negative spiked feces by using Quick-DNA™ Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA). The extracted genomic DNA was stored at −20 • C until further analysis.
Genomic DNA was extracted from 3.4 × 10 7 cells of pure culture of C. difficile DSM 1296 with a Qiagen genomic DNA extraction kit (Qiagen, Hilden, Germany) using a silicabased kit (silica bead DNA extraction kit; Thermo Scientific, St. Leon-Rot, Germany). Its serial dilutions were applied to generate a standard analytical curve of the C. difficile DSM 1296 pure culture cells. The two standard analytical curves were compared and used to evaluate the lower detection limit and detection accuracy of this TaqMan-based qPCR assay. The DNA was used to generate CD genome qualification standards and to determine the amplification efficiency (Figure 1).

Quantification of Environmental C. difficile in Fecally Contaminated Environmental Samples by TaqMan-Based qPCR Assay
Purified DNA from fecal samples is used to establish an appropriate standard curve to enumerate a load of C. difficile in the fecal samples. Fecal samples were analyzed in duplicate by qPCR from undiluted or diluted DNA, as mentioned above. The amount of DNA measured by qPCR was converted to cell numbers. This was accomplished by using the standard curve that was generated by plotting the Cq value against CD cell numbers corresponding to each DNA dilution (Figure 1). The intra-and inter-assay coefficient of variations (CVs) of the qPCR assay, PCR efficiency, and low detectable limits (LOD) were determined. The LOD was defined as the smallest CD cell number in each standard curve.

Conclusions
The environmental C. difficile strains are commonly present in various non-clinical sources, which could serve as a potential source of community-associated CDI. The specified TaqMan-based qPCR assay showed acceptable results with respect to detection limits, which makes this assay especially suitable for the rapid detection of C. difficile not only in patients and clinical environments but also in environmental sources outside healthcare institutions. The whole genome sequences of those environmental C. difficile strains are required to characterize virulence-associated factors or the genotypically antimicrobial resistance often located on mobile genetic elements (e.g., plasmids, conjugative transposons, prophages, insertion sequences). In addition, the epidemiological relatedness between clinical strains and those from non-clinical environments and animals needs further investigation.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics12010162/s1, Table S1: Quantification of C. difficile from fecally contaminated environmental samples; Table S2: Comparison of detection results of Environmental C. difficile between qPCR and C. difficile selective enrichment culture (CSEC). Funding: This research was funded by German Research Foundation (DFG, Deutsche Forschungsgemeinschaft) within the project SUPERsafe "Survival and pathogenicity of Clostridioides difficile in sewage, sewage sludge, surface water, animal manure, fodder, crops and silage -Treatment requirements to minimize health risks", grant number (GA 546/13-1).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.