Antimicrobial resistance of Clostridioides difficile in veterinary medicine around the world: A scoping review of minimum inhibitory concentrations

Objective To provide a comprehensive characterization of Clostridioides difficile antimicrobial resistance (AMR) data in veterinary medicine based on the minimum inhibitory concentrations (MICs) of all antimicrobial agents tested in relation to the techniques used. Methods A systematic scoping review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for scoping reviews (PRISMA-ScR) and its associated checklist. The objective was to provide a synthesis of the evidence in a summarized and analyzed format. To this end, three scientific databases were consulted: Scopus, PubMed, and Web of Science, up until December 2021. Subsequently, all identified literature was subjected to screening and classification in accordance with the established study criteria, with the objective of subsequent evaluation. Study selection and data extraction A comprehensive analysis was conducted on studies regarding Clostridioides difficile antimicrobial resistance (AMR) in veterinary medicine across various animal species and related sources. The analysis included studies that presented data on antimicrobial susceptibility testing using the E-test, agar dilution, or broth microdilution techniques. The extracted data included minimum inhibitory concentration (MIC) values and a comprehensive characterization analysis. Results A total of 1582 studies were identified in scientific databases, of which only 80 were subjected to analysis. The research on Clostridioides difficile antimicrobial resistance (AMR) in veterinary medicine is most prolific in Europe and North America. The majority of isolates originate from production animals (55%) and pets (15%), with pigs, horses, and cattle being the most commonly studied species. The tested agents' minimum inhibitory concentrations (MICs) and resulting putative antimicrobial resistance profiles exhibited considerable diversity across animal species and sources of isolation. Additionally, AMR characterization has been conducted at the gene and genomic level in animal strains. The E-test was the most frequently utilized method for antimicrobial susceptibility testing (AST). Furthermore, the breakpoints for interpreting the MICs were found to be highly heterogeneous and frequently observed regardless of the geographical origin of the publication. Conclusions Antimicrobial susceptibility testing techniques and results were found to be diverse and heterogeneous. There is no evidence of an exclusive antimicrobial resistance pattern in any animal species. Despite the phenotypic and genomic data collected over the years, further interdisciplinary studies are necessary. Our findings underscore the necessity for international collaboration to establish uniform standards for C. difficile antimicrobial susceptibility testing (AST) methods and reporting. Such collaboration would facilitate a “One Health” approach to surveillance and control, which is of paramount importance.

Objective: To provide a comprehensive characterization of Clostridioides difficile antimicrobial resistance (AMR) data in veterinary medicine based on the minimum inhibitory concentrations (MICs) of all antimicrobial agents tested in relation to the techniques used.Methods: A systematic scoping review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for scoping reviews (PRISMA-ScR) and its associated checklist.The objective was to provide a synthesis of the evidence in a summarized and analyzed format.To this end, three scientific databases were consulted: Scopus, PubMed, and Web of Science, up until December 2021.Subsequently, all identified literature was subjected to screening and classification in accordance with the established study criteria, with the objective of subsequent evaluation.

Study selection and data extraction:
A comprehensive analysis was conducted on studies regarding Clostridioides difficile antimicrobial resistance (AMR) in veterinary medicine across various animal species and related sources.The analysis included studies that presented data on antimicrobial susceptibility testing using the E-test, agar dilution, or broth microdilution techniques.The extracted data included minimum inhibitory concentration (MIC) values and a comprehensive characterization analysis.Results: A total of 1582 studies were identified in scientific databases, of which only 80 were subjected to analysis.The research on Clostridioides difficile antimicrobial resistance (AMR) in veterinary medicine is most prolific in Europe and North America.The majority of isolates originate from production animals (55%) and pets (15%), with pigs, horses, and cattle being the most commonly studied species.The tested agents' minimum inhibitory concentrations (MICs) and resulting putative antimicrobial resistance profiles exhibited considerable diversity across animal species and sources of isolation.Additionally, AMR characterization has been conducted at the gene and genomic level in animal strains.The E-test was the most frequently utilized method for antimicrobial susceptibility testing (AST).Furthermore, the breakpoints for interpreting the MICs were found to be highly heterogeneous and frequently observed regardless of the geographical origin of the publication.Conclusions: Antimicrobial susceptibility testing techniques and results were found to be diverse and heterogeneous.There is no evidence of an exclusive antimicrobial resistance pattern in any animal species.Despite the phenotypic and genomic data collected over the years, further interdisciplinary studies are necessary.Our findings underscore the necessity for international collaboration to establish uniform standards for C. difficile antimicrobial susceptibility testing (AST) methods and reporting.Such collaboration would facilitate a "One Health" approach to surveillance and control, which is of paramount importance.

Introduction
Clostridioides difficile is a well-documented enteric anaerobic pathogen with a significant impact on hospital environments.Since its initial identification in 1935, the organism has been consistently documented globally [1,2].C. difficile has been identified as the primary pathogen responsible for antibiotic-associated diarrhea, which results in a significant cumulative cost to healthcare systems [3].The use of antimicrobial agents such as clindamycin, fluoroquinolones, and cephalosporins has been identified as a risk factor or inducer of C. difficile infection (CDI) in susceptible or vulnerable populations within hospital environments [3].
The successful completion of antimicrobial susceptibility testing (AST) and laboratory diagnosis of C. difficile requires the presence of highly competent staff and the establishment of a robust infrastructure [4,5].The consistent harmonization of technology and reporting style regarding minimum inhibitory concentration (MIC) data for antimicrobial agents remains a challenge in the effort for laboratory results comparability; standardized documentation for C. difficile AST and data interpretation is currently only available for human source isolates.Nevertheless, there is no consensus regarding clinical breakpoints for the majority of available antimicrobial agents [6].
The phenomenon of antimicrobial resistance (AMR) in strains of clinical origin in humans has been extensively studied [7].These profiles are characterized by reports of high resistance to various classes of agents in the studied populations [8,9].In a similar vein, although to a lesser extent, research into C. difficile AMR profiling has also been conducted in veterinary medicine [10].Although, the known reports in veterinary medicine are of an isolated nature [11].Consequently, it is necessary the integration and contextualization of this AMR data.
In recent years, there has been a growing interest in genomic assessment of strains of clinical and community origin due to the consistent and ongoing host overlapping presence of virulent C. difficile genotypes such as ribotype (RT) RT027, RT014, and RT078 [12].The epidemiology of C. difficile infection (CDI) exhibits zoonotic components; while numerous reports on antimicrobial resistance have been published, it is evident that a comprehensive approach is required from the One Health perspective.This approach should permit the evaluation of antimicrobial resistance and its interrelated determinants in the environment, as well as in animal sources and their relationship with public health [13].
The objective of this study is to present a systematic collection of animal source data that will assist in contextualizing the magnitude of antimicrobial resistance in C. difficile.The integration of this data with evidence derived from environmental and clinical settings will enable the generation of scientific research that is based on agreed-upon methodologies and comparable results.These are essential for the advancement of this research field.

Protocol and registration
A scoping review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Scoping Review Extension (PRISMA-ScR) and its checklist.The protocol stated that the aim was to investigate the patterns and profiles of C. difficile antimicrobial resistance (AMR) by means of minimal inhibitory concentration (MIC) assessment.The resulting data will assist us in understanding how AMR patterns are reported and distributed across the available literature.In this regard, the final protocol was registered at Open Science Framework on June 11, 2021 (https://osf.io/jcbs4/).

Search strategy and selection criteria
The search strategy was developed and refined in accordance with the population-exposure-outcome (PEO) search methodology approach, with input from our librarian expert.The consulted databases were PubMed (https://pubmed.ncbi.nlm.nih.gov/),Scopus (Elsevier, http s://www.scopus.com/)and Web of Science (Clarivate Analytics, https ://www.webofscience.com/wos/woscc/advanced-search) for the purpose of retrieving pertinent published literature until December 2021 in the English language.The search strategy included cross-sectional research, short communication studies, and review articles in which the abstracts described or reported C. difficile from animal or animalrelated sources.Additionally, the search included articles that presented AST data using agar dilution, broth dilution, or E-test techniques.The retrieved data was meticulously evaluated to identify only those articles that presented their findings based on a proportion or number of tested samples.A more comprehensive and detailed list of inclusion/ exclusion criteria, data charting, and our search strategy is available via our registered protocol and supplemental materials.

Data extraction
A revised, standardized tool was developed through a consensus process, and pertinent data and key findings were extracted from the screened literature.Special attention was paid to AMR data, with particular consideration given to the technique and MICs reported.In the event that the proportion or number of samples for a given minimum inhibitory concentration (MIC) category was absent from the data set, it was calculated from the available information.In instances where MIC ranges or data were derived also from human sources, particular attention was paid to MIC 90 values for animal data.

Patient and public involvement
In this review project, no animals or human patients were involved.

Results
A search across three databases (PubMed, Scopus, and Web of Science) yielded 1582 studies.A total of 501 duplicates were identified and removed.After applying the inclusion criteria, 80 studies were subjected to analysis and data extraction (Fig. 1).A comprehensive list of key findings is presented in Table 1.The objective of all included studies was to characterize C. difficile antimicrobial resistance patterns.

Clostridioides difficile AMR data distribution and characterization
The antimicrobial susceptibility testing of C. difficile in veterinary medicine commenced in 1997 with the assessment of isolates from horses [14].Since that time, numerous studies have been conducted, including investigations of isolates from a variety of animal species and sources.The findings of Jang et al. (1997) and Båverud et al. (1998) [14,15], provided the first insights into the diagnosis, management, and control of C. difficile in clinical veterinary microbiology.Subsequently, in 2003, isolates from dogs were analyzed, and it was proposed that genomic assessment, in addition to a more comprehensive phenotypic characterization, would provide further information for antibiotic treatment [16].
From the outset of the C. difficile AMR characterization, it was evident that the establishment of breakpoints for the tested antimicrobial agents would necessitate a consensus to render the data clinically relevant [6,16].Nevertheless, despite the extensive analysis of a diverse range of literature, this objective has not been successfully achieved.The comparison of results using the breakpoint interpretation criteria set by the authors became highly complicated due to the complexity and high heterogeneity of the criteria.Consequently, alternative methodologies, such as the characterization and assessment of minimum inhibitory concentrations (MICs), were employed to elucidate the prevalence of C. difficile antimicrobial resistance and its impact on global public health.
A quantitative analysis of available data revealed that Europe and North America were responsible for the majority of research and knowledge contributions regarding C. difficile in veterinary medicine, collectively accounting for 45% and 26% of total published and indexed research, respectively (see Fig. 2).The first reports from Latin America and Asia did not emerge until 2013, followed by contributions from Oceania in 2014 and the Middle East in 2021 (Table 1).
From 4834 isolates, a total of 55% (2659) of the isolates tested originated from the bovine, avian, caprine, porcine, and equine production sector on a global scale.The most frequently analyzed production farm animals were pigs, followed by bovines and equines.Fifteen percent (725) of the strains were obtained from pets (dogs, cats, reptiles, and some birds), 11% (532) from animal-related products or sources (meat, environment, food products), 10% (483) from mixed sources (more than one category, or animal samples and environmental-related samples), and 9% (435) from wildlife and sea life, including zoo collections, captive animals, and mollusks (Fig. 3).

MICs distribution and patterns
A total of 57 antimicrobial agents and 23 groups of agents were extracted from the literature for the purpose of determining their minimum inhibitory concentrations (MICs).A comprehensive and summarized overview of the minimum inhibitory concentrations (MICs) and ranges from six combined geographic regions is presented in Fig. 4. A detailed overview of MICs from individual regions is also available in Tables S1 and S2 of the supplementary materials.

Aminoglycosides
Gentamicin was tested in four studies from Europe and Oceania.In Europe, the MICs of 106 isolates that came from production animals and mixed sources ranged between 8 and > 16 μg/ml [39,43].Conversely, the MICs for 45 porcine isolates in Oceania were higher ranging between 16 and 64 μg/ml [59,69].Streptomycin MICs were found to be in a range of ≤1000 ->1000 μg/ml in European isolates [39,43].Regarding the MICs for spectinomycin and tobramycin, tested only in Oceania, concentrations reached up to 128 μg/ml for both agents [59,69].

Ansamycins
Europe has the most extensive data set on the minimum inhibitory concentrations of rifampicin/rifampin.In some of the 353 isolates from production animals and mollusks, the MICs were >256 μg/ml [15,18,37,38,43,49,55,89]; in North America, the MICs from 481

Table 1
Characterization of studies assessing Clostridioides difficile antimicrobial resistance in veterinary medicine (1997-2021).Along with the listing, a description of the sources of isolation along with number of tested isolates and key findings are provided; the resistance or susceptibility data here presented correspond to criteria used by the authors and should be considered along with the epidemiological context.A more detailed and extensive information is available in Supplemental Material Tables S1 and S2.production animal tested isolates were ≥ 128 μg/ml [14,60].Notably, the strains with highest MIC values were found in equines [14].In contrast, ten porcine isolates from Latin America exhibited MICs lower than 0.004 μg/ml [75].In Oceania, 96 isolates with a similar origin to those previously described were tested against rifaximin.MICs ranged between 0.001 and 0.015 μg/ml [62,69].
Of the third-generation cephalosporins, ceftriaxone was the most frequently tested agent, with a total of 464 isolates tested worldwide.
MICs of ≥256 μg/ml were observed in isolates from Asia, while MICs up to 512 μg/ml were reported in Slovenian isolates in Europe [52,58,68].
A total of 137 isolates from Oceania exhibited MICs between 8 and 32 μg/ml for the same agent [59,62,69,83].In Asia, MICs >512 μg/ml against cefotaxime and ceftiofur were detected among 44 strains from production animals [82]; in North America MICs against ceftiofur were > 256 μg/ml in 80 piglet strains [19].In a lesser extent, MICs of ≥64 μg/ ml were also detected for cefotaxime among 161 European isolates from production animals [71]; similarly, 49 isolates from North and Latin  Antimicrobial agents in BOLD correspond to those that have been classified by the authors as "resistant" in antimicrobial susceptibility testing reports.On the other hand, many antimicrobials do not currently have breakpoints, but high MICs have occasionally been reported, so antimicrobials that are not in bold should not be considered susceptible.4. Metronidazole heteroresistance. 5. Abbreviation NR: Not explicitly reported.

Fig. 2. Global distribution of studies assessing
Clostridioides difficile in veterinary medicine.After screening and data analysis, it was found that Europe was the highest literature contributor with 36 publications, followed by North America with 21 publications.Asia, Latin America, Oceania and Middle East contributed together with a total of 23 publications.Percentages have been adjusted by rounding.
Strains from adult dogs and puppies were also tested against ertapenem in Spain with MICs ranging between 8 to ≥32 μg/ml [56].Finally, a strain collection from a zoo in Spain yielded MICs ≥32 μg/ml for both, ertapenem and meropenem [45].

Glycopeptides
The resulting worldwide data collection of 3726 isolates for vancomycin is more complex (Table S1).The highest MICs were reported in isolates from production animals from North America, yielding values up to 16 μg/ml [60].MICs of 8 μg/ml or greater were observed in isolates from pets in Asia and Latin America [58,78,81].Teicoplanin was tested in 279 European isolates from different source categories showing MICs between 0.023 and 0.5 μg/ml [37,38,45,56,64,65,72].

Macrolides
Among the macrolides, erythromycin was the most frequently tested antimicrobial agent worldwide with 2369 isolates (Table S1).In Asia and Europe, MICs of erythromycin in production animal strains have been reported to exceed 512 μg/ml [37,68,82].In the present study, all regions, with the exception of the Middle East, have reported MICs ≥256 μg/ml for the same agent in at least one of the analyzed categories.

Polypeptides
Antimicrobial susceptibility testing was conducted on a total of 237 isolates against Bacitracin in North American horses (1997), pigs (2004), and European horses and their environment (2003).The results are presented in Table S1.The highest MICs were observed in North American equine isolates with values exceeding 1024 μg/ml [14].The MICs of the porcine isolates were found to be >256 μg/ml [19].

Tetracyclines
Tetracycline was the most prevalent antimicrobial agent in this group, with MIC reports reaching 128 μg/ml in 124 isolates from production animals in Asia [82] and North America [19].The MICs of European and Oceanian isolates were reported to be up to 32 μg/ml [67,69,76].In 26 isolates of meat from different animal species from Saudi Arabia, MICs of 0.015 to 8.0 μg/ml have been reported [90,91].The MICs for oxytetracycline were found to be >256 μg/ml in 43 isolates from Latin America [44,50].Finally, MICs up to 12 μg/ml and 0.5 μg/ml were described for doxycycline [33] and minocycline [72], respectively.

Antimicrobial agents commonly tested for assessing C. difficile AMR
A comparison of the AST results from the various geographic regions revealed that the most commonly tested agents across the analyzed literature were vancomycin, clindamycin, metronidazole, tetracycline, and moxifloxacin, with AST data entries from all regions.Furthermore, 83% (5/6) of the assessed regions had entries for erythromycin and amoxicillin/clavulanic acid.The considerable variability in the collection of AST data has demonstrated that there is no general consensus regarding the selection of antimicrobials for the assessment of AMR in C. difficile (Table S2).
As illustrated in Table 1, since the initial report of metronidazoleresistant C. difficile strains in equine isolates back in 1997 [14], reports of increased MICs up to >256 μg/ml have been published globally (Fig. 5).The strains exhibiting decreased susceptibility or resistance were isolated from a wide range of sources, including production animals and household pets [14,45,47,53,60,64,71,86]. Furthermore, to the complex timeline of high MICs for metronidazole, reports of increased MICs for vancomycin can be added, although these are quite scarce and originate from diverse sources, including pets [58,60,78,81].In addition to these two CDI therapeutic agents, high MIC values and resistance for the fluoroquinolone moxifloxacin, which is frequently tested in C. difficile AST, have been reported [28,37,48,53,55,61,64,66,67,79,82,84,89].

Genomic characterization of C. difficile AMR across the analyzed literature in veterinary medicine
In some studies, included in this scoping review, not only phenotypic AMR were investigated but also genetic AMR determinants.As the determination of genetic AMR determinants did not influence the selection of publications included in this scoping review, this paragraph can just give a little insight in genetic AMR determinants of C. difficile in veterinary medicine.

Tetracycline resistance
In some studies (Table S1), the assessment of AMR against tetracycline has been conducted by the detection of mobile genetic elements in conjuction with the phenotypic characterization of tetracycline MICs.In 2010, the genetic mobile element Tn916-like [26] was identified in all tetracycline-resistant porcine isolates.This was subsequently confirmed in 2013, when it was found in porcine isolates [42].Moreover, additional strains, predominantly isolated from pigs, have demonstrated the presence of transposons Tn6190 [51] and Tn5397-like [69].Nevertheless, the detection of genetic mobile elements has been employed not only to trace the origin for antimicrobial resistance to tetracycline, but also to characterize it.For instance, tet gene variants have been identified in toxigenic and non-toxigenic strains from veal calves [28,33], swine [34,42,69], horses [76], dogs [88], and cattle [89].

Fluoroquinolones and another agents' resistance
To a lesser extent, the resistance of fluoroquinolones has been assessed by investigating the gyrase genes across the literature.Mutations resulting in amino acid substitutions in GyrA from porcine isolates [42,61,70,82], and mutations resulting in amino acid substitutions in GyrB from equine [76], porcine [82], and avian isolates [82] have been identified.The presence of genetic resistance determinants, such as erm genes, were investigated to assess resistance to antimicrobial agents of the macrolide-lincosamide-streptogramin B (MLS B ) group, which includes erythromycin and clindamycin.The gene erm(B) was found in strains from compost [73], horses [76] and dogs [78,88].Furthermore, the genetic element cfr was identified in calves alongside erm(B) [89].Finally, other AMR determinants including lnuC, uppP2, terD1-4 or cme, and blaR, have also been fully described in strains isolated from pigs [69].

C. difficile AST methodology assessment
With regard to laboratory methodology, 69.1% (57/80) of the published studies have employed the epsilometric gradient diffusion method known as E-test (E-TEST®, M.I.C.E™) as the methodology to assess C. difficile antimicrobial resistance in AST.Agar dilution methodology, which is considered the "gold standard", was employed in 20.9% (17/81) of the studies, particularly in Oceania, Asia and Latin America.Only three studies from Slovenian researchers employed methods such as broth microdilution (Fig. 6).
The interpretative criteria and breakpoints selected were highly diverse across the analyzed data.To illustrate, despite the existence of CLSI [94] and EUCAST [95] breakpoints for metronidazole (CLSI: ≥32 μg/ml; EUCAST: >2 mg/L) and vancomycin (CLSI: No breakpoint available; EUCAST: >2 mg/L), the reported and used breakpoints for data interpretation were highly variable (Table S1).In general, modification or implementation of breakpoints derived from literature was observed as a common practice among researchers without a clear consensual rationale.Moreover, the source of interpretative criteria was frequently omitted despite the availability of standardized breakpoints provided by CLSI, EUCAST or the published literature (Table S1).
Finally, the selection and use of media, as well as the supplementation of blood-derived and specialized supplements such as vitamin K or hemin, varied according to the methodologies and incubation conditions reported.The most frequently reported incubation conditions were 24-48 h at 35-37 • C. Nevertheless, some publications omitted details regarding methodology, regardless of the region of origin (Table S1).

Discussion
After an exhaustive analysis of the literature, our objective was to provide a comprehensive characterization of antimicrobial resistance data from a clinical and categorical perspective.Indeed, after assessing the inherent complexity for comparability, we have characterized the antimicrobial resistance of C. difficile in veterinary medicine through a complete mapping of the isolate's sources and the evaluation of MICs reported against 57 antimicrobial agents (Fig. 4 and Supplemental Material).This provides a practical and consistent contextualization that, when considered alongside the global attention being paid to antimicrobial resistance reports, could contribute to methodological and interpretative harmonization of antimicrobial susceptibility testing and reporting.
C. difficile can be isolated from a wide variety of sources.The organism's presence in the environment [18,32,39,63,68], as well in pets, farm animals and exotic species such as domestic reptiles (Table 1) underscores its capacity for rapid dissemination and its potential to act as a zoonotic agent [13].It is strongly advised that general antimicrobial resistance monitoring and surveillance in clinical settings be conducted from a "One Health" perspective [13].However, it is crucial to exercise caution when interpreting AST results, considering the epidemiological and methodological context.Consequently, animals have been demonstrated to serve as potential reservoirs or amplifiers of AMR [96], yet the AMR profiles of C. difficile isolates from animals and humans may diverge from each other [87].Consequently, the roles of the community [97], and the environment [73] in C. difficile epidemiology should be carefully considered.
C. difficile is also regarded as an AMR vector and amplifier due to its dynamic genomic asset of mobile elements [12,69,98].It is of the utmost importance to comprehend these dynamics and to evaluate the genomic relationships between veterinary, human, and environmental strains, as well as the evolutionary processes that have shaped its contemporary epidemiology.It is possible that human and animal outbreak isolates with highly related AMR characteristics may have a common source of contamination, namely the acquisition of AMR genes.Indeed, independent genomic transfer of antimicrobial resistance genes from other Gram-positive bacteria, such as Enterococcus spp. or Erysipelothrix rhusiopathiae to C. difficile has been observed [99], in addition to transmission between humans and animals [51,69].
The occurrence of resistance to vancomycin and moxifloxacin, in addition to resistance and heteroresistance to metronidazole, has been documented in C. difficile strains over time, irrespective of host species and geographical region (Fig. 4).Furthermore, resistance to ciprofloxacin or enrofloxacin has been identified in animal isolates, as evidenced by data presented in Table 1.This suggests that selective pressure may be exerted in animal healthcare environments [76], or alternatively, that therapeutic administration may contribute to genetic mutations and quinolone resistance Consequently, it is imperative to conduct further investigation into this genetic evolution.
With regard to metronidazole, the results of the MIC assessments from our evaluated collection must be interpreted in light of the methodological variations resulting from the media dependency, heterogeneity, and instability of metronidazole resistance in C. difficile strains [100].The various methodologies could potentially influence the results of antimicrobial resistance.It is evident that the implementation of standardized or harmonized methodological approaches for AST and MIC reporting assessment will significantly contribute to a more comprehensive understanding of the epidemiological dynamics underlying the obtained results.

Strengths and limitations
To the best of our knowledge, this review presents the first comprehensive collection of a large and complex dataset on antimicrobial resistance in Clostridioides difficile from animal or animal-related sources.It describes the minimum inhibitory concentrations instead of the clinical interpretation categories.This approach offers a more straightforward yet comprehensive overview of antimicrobial resistance.When available, laboratory techniques and genomic profiling were also included for assessment.The identification of potential avenues for further research endeavors, in addition to areas for Fig. 6.Clostridioides difficile antimicrobial susceptibility testing methodologies assessment.Inoculum concentrations along with variations of media supplements were described across the analyzed literature.E-test was the most frequent AST method implemented.improvement, was also outlined.Consequently, recommendations for a more comprehensive and harmonized reporting style were also presented.
In our view, a limiting factor that could contribute to the underestimation of high MICs and thus to the assessment of antimicrobial resistance is the inherent nature of the E-test (E-TEST™ or M.I.C.E.®) method.This method does not provide the final MIC of any commercially available antimicrobial agent when compared to the agar dilution method.Furthermore, the data in the analyzed collections may be incomplete or ambiguous.Moreover, although some studies present and analyze data exclusively from animal sources, some also include data from other related sources that undoubtedly contribute to the assessment of antimicrobial resistance in different settings.Nevertheless, the inclusion of all analyzed literature in this study is essential to highlight the research efforts made on this topic and to provide valuable insights for a more comprehensive understanding of C. difficile AMR in veterinary medicine.

Conclusions
In recent years, there has been a significant increase in the publication of detailed data on C. difficile antimicrobial resistance and its genetic determinants, particularly within the field of human medicine [12,98].However and despite the existence of standardized techniques for AMR testing proposed by CLSI and EUCAST [94,95] or a simplified disk-diffusion technique proposed by the CA-SFM (Committee of the Antibiogram of the French Society of Microbiology) [101], the lack of a commonly accepted and used approach for both, phenotypic and genotypic AMR characterization is still noted.A standardized approach would facilitate the development of a comprehensive and globally comparable knowledge base on the ecology of C. difficile resistome.
The genetic diversity of C. difficile and its ability to infect a wide range of hosts observed in our analysis indicates a multifactorial epidemiology.The high degree of similarity observed between strains from animal and human hosts [51,69], provides a compelling rationale for research into a holistic epidemiological context.This is a perspective that has already been proposed in the current literature [13].In light of the perplexing data regarding AMR and MIC patterns in veterinary medicine, which exhibit striking parallels to those observed in isolates from humans, it becomes evident that a "One Health" approach is imperative.This necessitates the formation of multidisciplinary teams to evaluate and validate practical approaches to combat C. difficile AMR infections in humans and animals.Such approaches should include optimal surveillance, testing, and control strategies, as well as the search for evidence-based therapeutic strategies that have a favorable impact on the health of patients and are in balance with animal health and a safe environment.
Consequently, it is now evident that there is an urgent need to harmonize AMR testing and to intensify the search for genomic pathways determining phenotypic behavior.In this context, it is imperative to develop a comprehensive protocol for future research that encompasses the following elements: a) The number and source of tested strains (a comprehensive metadata report) b) Standard operating procedures for antimicrobial susceptibility testing c) Minimum inhibitory concentrations, which could be reported individually or expressed in ranges along with concentration, and that should be grouped by metadata d) Citation of interpretation criteria with a proper rationale e) Proportion of resistant or susceptible strains for each tested antimicrobial agent f) A cohesive report in respect to phenotype for genetic AMR elements There is an urgent need for greater collaborative efforts to establish a robust interdisciplinary research network that integrates insights from environmental, veterinary, and human medicine to accelerate the dissemination of research findings from the academic and research communities.This will ensure a long-term and sustainable environment for C. difficile research and control.

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Maps used in our infographics, available thru repository access, were created for information and academic purposes and do not represent a geopolitical statement, from the authors and the correspondent affiliate institutions, regarding the aggrupation or representation of countries and territories.QGIS 3.

Fig. 3 .
Fig. 3. Global characterization and distribution of Clostridioides difficile sources.From analyzed literature that assesses antimicrobial resistance data in veterinary medicine, it was determined that most of samples from production animals came from pigs.Further information is available in Table1and S1.

Fig. 4 .
Fig. 4. Clostridioides difficile worldwide publications and MIC ranges.Antimicrobial susceptibility testing was performed in 4694 isolates from 22 countries against 57 antimicrobial agents or combinations of those.With some exceptions, MIC ranges correspond to the lowest and highest values reported across the analyzed regions.Detailed information regarding final MICs is available in Supplemental Materials (TableS1 and S2).

Fig. 5 .
Fig. 5. Metronidazole, Vancomycin and Moxifloxacin High-MICs timeline.As expected from a highly diverse collection of AST data, the MIC values for the selected agents are consistently variable.The sources of isolation for antimicrobial resistant strains include different animal species and the environment and show the wide-range of C. difficile AMR hosts and reservoirs.MICs are expressed in μg/ml.A complete description of MIC sources is available in TableS1.

Funding
MAM holds a research grant by DAAD (Research Grants -Doctoral Programmes in Germany, Code 57135739).ID is funded by European Union's Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme.