Minimum inhibitory concentrations increase in Clostridioides difficile isolates from patients with recurrence: results from a retrospective single-centre cohort study

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The aim of this study was to assess the rate of antimicrobial resistance to four antimicrobials (vancomycin, metronidazole, tigecycline and ciprofloxacin), to assess risk factors for antimicrobial resistance and to evaluate MIC variation in patients with recurrence. Methods Data from consecutive patients with CDI admitted to our Istitution between the 1st of January 2022 and the 30th of April 2023 were collected. We performed AST with gradient diffusion and NAAT to evaluate presumptive presence of R027/NAP1 and toxin production genes. Results Antimicrobial susceptibility testing was performed on 108 available isolates. We didn’t found any isolate resistant to vancomycin, metronidazole and tigecycline, while all the isolates were resistant to ciprofloxacin. For 8 patients isolates from both first episode and recurrence were available: 3 (37.5%) displayed 2 fold MIC increase for vancomycin, 6 (75%) for metronidazole and 3 (37.5%) for tigecycline. Conclusions Our results were concordant with European surveillance data. MIC increase to all tested antibiotics in patients with CDI may be due to biofilm formation and its possible role warrant further research, especially considering reports of clinical failure due to vancomycin resistance. Clostridioides difficile antimicrobial resistance vancomycin metronidazole Figures Figure 1 Figure 2 Figure 3 Introduction Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium. Disruptions to gut microbiota, such as antibiotic use or proton pump inhibitors, can promote C. difficile colonization and the proliferation of its vegetative cells. It is the primary cause of Clostridioides difficile infection (CDI), often linked to toxigenic strains.( 1 ) A 2016–2017 report by the European Centre for Disease Control and Prevention showed a CDI incidence of 3.48 cases per 10,000 patient-days, higher in tertiary hospitals. The 1-year attributable mortality is around 7.9%, with a high risk of recurrence, especially in older patients, those with comorbidities, healthcare-associated CDI, or proton pump inhibitor use. ( 2 ), ( 3 ) First-line therapy for CDI, per guidelines from the European Society on Microbiology and Infectious Disease (ESCMID) and the Infectious Disease Society of America, recommends fidaxomicin as the preferred treatment, with oral vancomycin as an alternative. New treatment options available to prevent recurrence after previous episodes of CDI are monoclonal antibodies directed toward toxin B (bezlotoxumab) and faecal microbiota transplantation. ( 4 ), ( 5 ) The diagnosis of CDI is based on glutamate dehydrogenase (GDH) antigen detection, Nucleic Acid Amplification Test (NAAT) and research of toxin A and B via enzyme immunoassay (EIA) rather than microbiological culture. Therefore, antimicrobial susceptibility testing (AST) is not routinely performed. ( 6 ) Nevertheless, AST performed for surveillance purpose has shown an increase in resistance rates in recent years. A metanalysis revealed pooled resistance rates of 3% for vancomycin and 5% for metronidazole. ( 7 ), ( 8 ) Metronidazole resistance may be related to metabolic pathway alterations, while vancomycin resistance could stem from mutations in peptidoglycan biosynthesis, binding site alterations, or acquisition of resistance plasmids. Fidaxomicin resistance is rare and linked to mutations in the RNA polymerase β subunit. However, these mutations impair bacterial fitness.( 7 )–( 10 ) Although fluoroquinolones, like ciprofloxacin, are not recommended for CDI, its resistance seems to be a hallmark of hypervirulent strains and has been associated with higher mortality. It is mediated by amino acidic substitutions harboured in two DNA gyrase subunits, acquired when the environmental concentration of fluoroquinolones is not able to inhibit C. difficile .( 8 ), ( 10 ) The clinical impact of antimicrobial resistance in CDI remains uncertain, as the high concentrations of vancomycin and fidaxomicin in the stool may offset the effects of resistance. However, metronidazole resistance has been associated with treatment failure, likely due to lower stool concentrations. ( 11 ) With this in mind, the study's primary aim was to assess the prevalence of C. difficile resistance to vancomycin, metronidazole, ciprofloxacin, and tigecycline in a cohort of adult patients admitted with CDI. Secondary objectives include evaluating risk factors for resistance and to evaluate MIC variation in patients with recurrence. Materials and Methods Study procedures This is a retrospective single centre, cohort observational study. The study population included patients aged 18 years or more admitted to IRCCS Fondazione Policlinico San Matteo with a microbiologically confirmed diagnosis of CDI between the 1st of January 2022 and the 30th of April 2023. The study period was chosen considering the incidence of CDI in the centre and to ensure an adequate sample size for the primary objective of the study. Stool samples of patient admitted to Fondazione IRCCS Policlinico San Matteo with clinical suspicion of CDI were processed by the Microbiology laboratory. According to the ESCMID guidelines for the management of CDI, an episode of CDI was defined as clinical findings compatible with CDI and microbiological evidence of C. difficile free toxins by EIA without reasonable evidence of another cause of diarrhoea or a clinical picture compatible with CDI and a positive NAAT, or positive toxigenic C. difficile culture or pseudomembranous colitis as diagnosed during endoscopy, after colectomy or on autopsy, in combination with a positive test for the presence of toxigenic C. difficile . Diarrhoea was defined as ≥ 3 loose stools in 24 hours. Patients with positive microbiological findings for C. difficile without clinical findings compatible with CDI were excluded Recurrence was defined as symptoms recurrence within 8 weeks after a previous CDI episode, provided the symptoms from the previous episode resolved after completion of initial treatment ( 4 ). For each included patients, clinical data were retrieved from medical charts and electronic records for each patient. C. difficile positive stool samples were subjected to alcohol shock by preparing a 1:1 suspension of fecal sample and 95% ethanol, followed by vortexing and incubation at room temperature for 1 hour. Subsequently, 50–100 microliters of the suspension were inoculated onto Schaedler Agar + 5% sheep blood (Biomérieux, Marcy L’Etoile, France) and incubated at 37°C under anaerobic conditions for 3 days. AST was performed using gradient diffusion (MIC Test Strip, Liofilchem srl, Roseto degli Abruzzi (TE), Italy) and results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCSAT) reference breakpoints ( 12 ). According to previous reports, gradient diffusion may result in underestimation of MICs ( 13 ), ( 14 ), which is why the agar dilution test is the reference for C. difficile antimicrobial susceptibility testing. Nevertheless, gradient diffusion has shown good concordance with agar dilution, it is more practical and allows testing of multiple antimicrobials ( 14 ). NAAT (GeneXpert, Cepheid, Sunnyvale, CA, USA) was used to identified presumptive NAP01/R027 (due to tcdCΔ117 deletion) and toxin production genes. Statistics The sample size was calculated on the primary endpoint of vancomycin and/or metronidazole resistance prevalence. The statistical unit was the positive stool sample corresponding to an infectious episode. We hypothesized a prevalence of 5% according to the results of a recent meta-analysis. Therefore, the study would require a sample size of 130 for estimating the expected proportion with 5% absolute precision and 99% confidence ( 7 ), ( 15 ). All analysis were performed using RStudio version 4.4.0 ( 17 ). We described continuous variables with the mean and standard deviation or the median and 25th-75th percentiles for skewed distributions; we described categorical data as counts and percent. We compared characteristics between groups with the Student t test or Kruskal wallis test and the Fisher exact test, respectively. A 2-sided p-value of 0.05 was considered statistically significant. Prevalence of antimicrobial resistance was calculated by dividing the number of resistant isolates for the total samples tested. Results Patients We enrolled 108 patients, 46% of them males, 54% females. Median age was 76 years (interquartile range (IQR) 62–84). Patients are described in Table 1 : the median Charlson Comorbidity Index (CCI) was 5 (IQR: 4–7) and the most prevalent comorbidities were cardiovascular disease, COPD and solid tumors. Most patients had severe, hospital-acquired CDI. For 78% of the patients it was the first episode, and for 16% it was the first recurrence, while 6% had two or more recurrences. Nearly 90% of the patients had undergone an antibiotic therapy in last three months, and nearly 20% of patients had already been treated with vancomycin. For the current episode, the preferred treatment was vancomycin, followed by combination therapy of vancomycin and metronidazole. Thirty percent of patients had presumptive NAP1/027 strain, and seven percent of patients died. Table 1 Patients description with counts and frequency for absolute numbers, and median and first-third interquartile ranges for continuous numbers.CDI: Clostridioides difficile infection. Patients (total) 108 Epidemiology Age [years] 76 (62–84) Males/females 50/58 (46.0/54.0) Charlson comorbidity index 5 ( 4 – 7 ) Cardiovascular disease 32 (37.0) Peripheral venous disease 12 (13.8) Stroke 12 (13.8) Chronic Obstructive Pulmonary Disease 21 (24.1) Liver disease 13 (14.9) Solid malignancy 16 (18.4) Hematological malignancy 2 (2.4) Previous hospitalization 38 (44.2) Current episode of CDI Hospital/community acquired CDI 74/12 (86.0/14.0) Severe CDI 39 (47.0) Episode: first/second/third or more 67/14/5 (77.9/16.3/5.8) Duration of therapy [days] 10 (10-13.5) NAP1/R027 30 (30.0) Binary Toxin 42 (42.0) TcdB+ 83 (83.0) ICU admission 2 (2.4) Recurrence 14 (16.9) Lenght of stay [days] 26 (15–42) 28 days mortality 6 (7.3) Therapies Use of Proton Pump Inhibitors before CDI 58 (69.1) Proton Pump Inhibitors started during CDI 7 (8.4) Recent use of antibiotic 76 (89.4) Previous use of vancomycin 17 (19.8) Previous use of metronidazole 7 (8.4) Previous use of fidaxomicin 3 (3.5) Treatment with vancomycin 59 (74.7) Treatment with fidaxomicin 5 (6.3) Treatment with metronidazole 1 (1.3) Treatment with vancomycin and metronidazole 14 (17.7) MICs of vancomycin, metronidazole, tigecycline and ciprofloxacin All the 108 isolates were available for antimicrobial susceptibility testing. The MICs’ distribution is represented in Fig. 1 . The median MICs for vancomycin, metronidazole, fidaxomicin and ciprofloxacin were respectively 0.5, 1, 0.016 and 32. All the isolates were resistant to ciprofloxacin but sensitive to the other three antibiotics, although MIC distribution varied. MIC variation in subsequent episodes For 8 patients isolates from both first episode and recurrence were available: 3 (37.5%) displayed a twofold MIC increase for vancomycin, 6 (75%) a twofold increase for metronidazole and 3 (37.5%) a twofold increase for tigecycline. The variation in MIC distributions is represented in Fig. 2 . Variables associated with MIC distributions We did not find any significant correlation between the antibiotic’s MICs and various possible risk factors; episode number, previous vancomycin administration, mode of acquisition (healthcare versus community). Similarly, we didn’t found any statistically significant MIC according to the presence of presumptive NAP1/027 or toxin production genes (Supplementary Table 1). MICs values according to NAP1/027 and toxin production genes are displayed in Fig. 3 . Discussion The aim of the present study was to evaluate the rate of antimicrobial resistance of C. difficile isolates recovered from patients admitted with CDI in our Institution and to evaluate risk factors associated with decreased antimicrobial susceptibility. We didn’t find any isolate resistant to vancomycin, metronidazole and tigecycline, while all the isolates were resistant to ciprofloxacin. While worldwide weighted pool resistance to vancomycin and metronidazole were found to be 3% and 5% ( 7 ), their frequency is actually variable according to different world regions, reaching up to 34% in a cohort of patients with CDI in the US ( 18 ). Our results are consistent with those reported in Southern and Western Europe, while increased metronidazole resistance has been described in Eastern Europe ( 19 ), ( 20 ). It is worth noting that metronidazole MICs in isolates from our cohort were close to the breakpoint and frequently higher than the geometric mean metronidazole MIC from a recent collection of European isolates (0.29 mg/L). This can be explained by the high proportion of presumptive NAP01/R027 in our cohort, as this ribotype was associated with higher geometric mean metronidazole MIC (1.87 mg/L) ( 20 ). Interestingly, all the isolates demonstrated low MICs for tigecycline, that has been recently introduced in the ESCMID guidelines as a possible therapeutic option in severe CDI due to favourable outcomes in a retrospective cohort study. ( 5 ),( 21 ) Resistance to ciprofloxacin was found in all the isolates, confirming a trend of decreased susceptibility described in Italy and Europe during the last decades. ( 22 ),( 23 ) Fluoroquinolones resistance is mediated by mutation in the gyr genes, encoding the DNA gyrase subunits GyrA and GyrB due to exposure to subinhibitory concentrations of this antimicrobial class and to their widespread use both in the healthcare setting and in the community. Furthermore, fluoroquinolone exposure is a well-recognized risk factor for CDI and ciprofloxacin upregulates the expression of both TcdA and TcdB in hypervirulent strains of C. difficile. ( 24 ),( 25 ) Interestingly, when comparing isolates from first episode to those of the subsequent recurrence from the same patients, we noticed a frequent increase in the MIC to all the tested antibiotics, even those not used for the treatment of the index CDI episode. Similar MICs increases have been previously described in retrospective studies and were associated with hypervirulent strains and binary toxin production. ( 26 ),( 27 ) Vancomycin MIC increase in recurrent CDIs was similarly shown in a phase 2 clinical trial evaluating the efficacy of rinidazole compared to vancomycin. ( 28 ) Biofilm formation production is frequent in C. difficile isolates, it is associated with reduced susceptibility to vancomycin and metronidazole and may explain this result.( 8 ),( 10 ),( 29 ) Biofilm may help selecting more tolerant strains after treatment of the first episode that will cause subsequent recurrence. Unfortunately, we didn’t evaluate biofilm production in our isolates and we can’t prove this hypothesis. Several bacterial factors, such as ribotype 027, binary toxin and Toxin B production, have reduced antimicrobial susceptibility to both metronidazole and vancomycin. ( 30 ) R027 strains tend to be associated with increased vancomycin resistance due to frequent association with a Thr115Ala mutation in the regulatory gene VanR that result in constitutive expression of vanG leading to decrease vancomycin affinity (MIC 50 and MIC 90 values of 2 and 4 µg/ml). ( 30 ),( 31 ) When stratifying MIC values according to virulence genes, we didn’t found any significant differences associated with their expression. It should be considered that all the isolates in our cohort were susceptible to vancomycin and therefore our R027 strains were probably harboring wild type Thr115, which is characterized by MIC 50 and MIC 90 of 0,5 and 2 ug/ml, respectively. ( 31 ) Therefore, the impact of virulence genes on antimicrobial susceptibility is likely less relevant when assessing values below the breakpoint. The principal strength of this study is that show MICs increase in isolates from recurrence in a subset (albeit limited) of real life patients with CDI. Several limitations of this study must be acknowledged. Firstly, the retrospective and single centre design make these findings poorly generalizable to other settings and implied the presence of missing data. Secondly, we didn’t perform susceptibility testing for fidaxomicin, that represents the recommended treatment for CDI. The reports of fidaxomicin resistance have been anecdotal and associated with fitness costs for C. difficile , an therefore its impact seems to be limited at the moment. ( 29 ) Another limitation was the use of a molecular test that detects only some genes compared to other techniques such as whole genome sequencing that enables more precise genotyping and ribotyping of C. difficile strains.( 6 ) Antimicrobial resistance has become more frequent in CDI, but its influence on clinical outcomes has been considered limited due to the high stool concentration of vancomycin that overcomes the MIC of resistant strains.( 11 ) This assumption has been recently questioned by a retrospective cohort study performed in a setting with high prevalence of vancomycin resistant strains, where decreased susceptibility was associated with lower sustained clinical cure. Interestingly, the higher rates of failure seemed to be driven by antibiotic failure rather than increased recurrence. ( 18 ) Conclusions The results of this study are important not only in terms of resistance surveillance, but also on a clinical perspective. In patients with recurrence, hypervirulent strains, and binary or TcdB production genes, antimicrobial resistance should be taken into account, especially in settings where its frequence is high. Abbreviations CDI Clostridioides difficile infection ESCMID European Society on Microbiology and Infectious Disease GDH glutamate dehydrogenase NAAT Nucleic Acid Amplification Test EIA enzyme immunoassay AST antimicrobial susceptibility testing Declarations Ethics approval and consent to participate The study was approved by the Fondazione IRCCS Policlinico S.Matteo Internal Review Board (protocol number 39948/2023). Informed Consent was withdrawn due to the retrospective nature of the study. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding Not applicable Authors' contributions Conceptualization, P.V. and E.A.; methodology, P.V. and P.C.; software, P.V.; validation, P.C. and A.D.S. and F.B. and R.B.; formal analysis, P.V.; investigation, F.Co. and F. Ca. and P.S. and M.C.; resources, R.B.; data curation, M.M. and N.A. and G.B and R.D. and F.V..; writing—original draft preparation, P.V. and M.M. and E.A.; writing—review and editing, E.A. and P.S. and P.C.; visualization, P.V..; supervision, P.S. and P.C. and A.D.S and F.B.; project administration, R.B.;. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable References Lawson PA, Citron DM, Tyrrell KL, Finegold SM. Reclassification of Clostridium difficile as Clostridioides dif-ficile (Hall and O’Toole 1935) Prévot 1938. Anaerobe. Aug. 2016;40:95–9. van Rossen TM et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5997394","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":414220030,"identity":"526e52a5-cccf-4148-aa9e-032b10365e3a","order_by":0,"name":"Pietro Valsecchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYLCCByBCgrHxAbIgD14tCRAtzQYgCqaUB68eiBYGNglkLTitMWfgMfyQ8MdGnn92c1vlzxy7Onv25gPMhW0MMvY4tFg28BhLJLalGc64c7DtNu+2ZAkenmMJzDPbcDvM4ABbgkRiw+EEA6DG24zbmCV4JHIMmHnO4NWS/CPhz3+wlsKf2+oleOTfENLCfEwige0AWAsD77bDQFt4gFoqcGuxbGY+ZpHYlmw440ZiszTvtuOSPWfSEg7PqAD66QCOEGNvbL7x4Y+dPP+M9Icff26r5mdvP3zwcYGBjT17Aw6HMWMTPQyKJlzAAKsoVnNGwSgYBaNgxAIAajJOhMtGnFMAAAAASUVORK5CYII=","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":true,"prefix":"","firstName":"Pietro","middleName":"","lastName":"Valsecchi","suffix":""},{"id":414220031,"identity":"0110e095-12bd-4941-a32c-f8bb12b7fc45","order_by":1,"name":"Erika Asperges","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"","lastName":"Asperges","suffix":""},{"id":414220032,"identity":"8264ed3a-ba95-4d22-b62f-efacc6375593","order_by":2,"name":"Marta Corbella","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Corbella","suffix":""},{"id":414220033,"identity":"20ecca66-4993-474d-ab50-4db6d688e42c","order_by":3,"name":"Greta Banfi","email":"","orcid":"","institution":"University of Pavia","correspondingAuthor":false,"prefix":"","firstName":"Greta","middleName":"","lastName":"Banfi","suffix":""},{"id":414220034,"identity":"e0313dcd-7db7-4b2c-8e2b-7f6dc31fc33f","order_by":4,"name":"Marcello Maffezzoni","email":"","orcid":"","institution":"ASST-Sette Laghi","correspondingAuthor":false,"prefix":"","firstName":"Marcello","middleName":"","lastName":"Maffezzoni","suffix":""},{"id":414220035,"identity":"87bc1890-f11b-46bb-8b13-6150d9c348d5","order_by":5,"name":"Nicolò Amarasinghe","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Nicolò","middleName":"","lastName":"Amarasinghe","suffix":""},{"id":414220036,"identity":"e9ee9119-0def-4d24-8936-f769f24da55d","order_by":6,"name":"Roberta Drago","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Drago","suffix":""},{"id":414220037,"identity":"bd580788-4418-4c36-ba99-83ad3baf5ff7","order_by":7,"name":"Flavia Virga","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Flavia","middleName":"","lastName":"Virga","suffix":""},{"id":414220038,"identity":"87465b2f-cc5b-41c3-8945-090075f21b5c","order_by":8,"name":"Filippo Costanzo","email":"","orcid":"","institution":"Ospedale Civile di Voghera - ASST Pavia","correspondingAuthor":false,"prefix":"","firstName":"Filippo","middleName":"","lastName":"Costanzo","suffix":""},{"id":414220039,"identity":"12a92141-9772-407f-b3c8-b79759bc29cc","order_by":9,"name":"Francesca Calabretta","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Calabretta","suffix":""},{"id":414220040,"identity":"5c69aeea-2cae-4c8c-951d-9ed6eaddc6e9","order_by":10,"name":"Paolo Sacchi","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Sacchi","suffix":""},{"id":414220042,"identity":"c0a6370f-23b4-4a47-b891-7f77b58cba27","order_by":11,"name":"Patrizia Cambieri","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Patrizia","middleName":"","lastName":"Cambieri","suffix":""},{"id":414220044,"identity":"6dbdada9-b4a4-4658-8f19-62bf899a37a0","order_by":12,"name":"Antonio Di Sabatino","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"Di","lastName":"Sabatino","suffix":""},{"id":414220045,"identity":"ed471a9d-405a-4fad-897a-831788a16ee6","order_by":13,"name":"Fausto Baldanti","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Fausto","middleName":"","lastName":"Baldanti","suffix":""},{"id":414220046,"identity":"c17e6b60-0ff5-4703-a5e7-76eb1454ffdc","order_by":14,"name":"Raffaele Bruno","email":"","orcid":"","institution":"IRCCS Fondazione Policlinico S. Matteo - Pavia","correspondingAuthor":false,"prefix":"","firstName":"Raffaele","middleName":"","lastName":"Bruno","suffix":""}],"badges":[],"createdAt":"2025-02-10 08:53:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5997394/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5997394/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76193496,"identity":"ef0fbc57-697b-4740-bdcb-a57fcba7359b","added_by":"auto","created_at":"2025-02-13 09:59:12","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64179,"visible":true,"origin":"","legend":"\u003cp\u003eMICs distribution of the isolates. The red line represents the breakpoint.\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5997394/v1/39659e6e64ad0c3bcbafb041.jpeg"},{"id":76193507,"identity":"4f935475-3d29-47ca-b2d5-015f2e2a091b","added_by":"auto","created_at":"2025-02-13 09:59:13","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41840,"visible":true,"origin":"","legend":"\u003cp\u003eMICs distribution of the isolates in the first episode (blue) and the recurrence (orange), showing a trend toward MIC increase.\u003c/p\u003e","description":"","filename":"groupimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5997394/v1/70cdc419a57b475571cdd31e.jpeg"},{"id":76193499,"identity":"355aef3a-8d72-4187-a48c-9dc69ee4abf6","added_by":"auto","created_at":"2025-02-13 09:59:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71629,"visible":true,"origin":"","legend":"\u003cp\u003eViolin plots of the MICs distribution for vancomycin, metronidazole and tigecycline in relation to the presence of strain NAP1/027 strain, binary toxin and toxin b. The black square is set at the median value.\u003c/p\u003e","description":"","filename":"groupimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5997394/v1/c27860d0d4d262b7c7132efb.jpeg"},{"id":79968562,"identity":"f98404a3-440b-48f9-8ec4-b6213daaaa02","added_by":"auto","created_at":"2025-04-05 15:01:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":892194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5997394/v1/24061690-6e7f-4c08-a6f2-1e9ff22cb915.pdf"},{"id":76194105,"identity":"0d93e758-1cf8-4d1c-a06f-9c35beab4384","added_by":"auto","created_at":"2025-02-13 10:07:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24153,"visible":true,"origin":"","legend":"","description":"","filename":"CDDramaSupplementv2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5997394/v1/f01a4e68b3f892b8b6ee1151.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Minimum inhibitory concentrations increase in Clostridioides difficile isolates from patients with recurrence: results from a retrospective single-centre cohort study","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eClostridioides difficile\u003c/em\u003e is a Gram-positive, anaerobic, spore-forming bacterium. Disruptions to gut microbiota, such as antibiotic use or proton pump inhibitors, can promote \u003cem\u003eC. difficile\u003c/em\u003e colonization and the proliferation of its vegetative cells. It is the primary cause of \u003cem\u003eClostridioides difficile\u003c/em\u003e infection (CDI), often linked to toxigenic strains.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) A 2016\u0026ndash;2017 report by the European Centre for Disease Control and Prevention showed a CDI incidence of 3.48 cases per 10,000 patient-days, higher in tertiary hospitals. The 1-year attributable mortality is around 7.9%, with a high risk of recurrence, especially in older patients, those with comorbidities, healthcare-associated CDI, or proton pump inhibitor use. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e First-line therapy for CDI, per guidelines from the European Society on Microbiology and Infectious Disease (ESCMID) and the Infectious Disease Society of America, recommends fidaxomicin as the preferred treatment, with oral vancomycin as an alternative. New treatment options available to prevent recurrence after previous episodes of CDI are monoclonal antibodies directed toward toxin B (bezlotoxumab) and faecal microbiota transplantation. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe diagnosis of CDI is based on glutamate dehydrogenase (GDH) antigen detection, Nucleic Acid Amplification Test (NAAT) and research of toxin A and B via enzyme immunoassay (EIA) rather than microbiological culture. Therefore, antimicrobial susceptibility testing (AST) is not routinely performed. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eNevertheless, AST performed for surveillance purpose has shown an increase in resistance rates in recent years. A metanalysis revealed pooled resistance rates of 3% for vancomycin and 5% for metronidazole. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) Metronidazole resistance may be related to metabolic pathway alterations, while vancomycin resistance could stem from mutations in peptidoglycan biosynthesis, binding site alterations, or acquisition of resistance plasmids. Fidaxomicin resistance is rare and linked to mutations in the RNA polymerase β subunit. However, these mutations impair bacterial fitness.(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u0026ndash;(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Although fluoroquinolones, like ciprofloxacin, are not recommended for CDI, its resistance seems to be a hallmark of hypervirulent strains and has been associated with higher mortality. It is mediated by amino acidic substitutions harboured in two DNA gyrase subunits, acquired when the environmental concentration of fluoroquinolones is not able to inhibit \u003cem\u003eC. difficile\u003c/em\u003e.(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe clinical impact of antimicrobial resistance in CDI remains uncertain, as the high concentrations of vancomycin and fidaxomicin in the stool may offset the effects of resistance. However, metronidazole resistance has been associated with treatment failure, likely due to lower stool concentrations. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWith this in mind, the study's primary aim was to assess the prevalence of \u003cem\u003eC. difficile\u003c/em\u003e resistance to vancomycin, metronidazole, ciprofloxacin, and tigecycline in a cohort of adult patients admitted with CDI. Secondary objectives include evaluating risk factors for resistance and to evaluate MIC variation in patients with recurrence.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy procedures\u003c/h2\u003e \u003cp\u003eThis is a retrospective single centre, cohort observational study.\u003c/p\u003e \u003cp\u003eThe study population included patients aged 18 years or more admitted to IRCCS Fondazione Policlinico San Matteo with a microbiologically confirmed diagnosis of CDI between the 1st of January 2022 and the 30th of April 2023.\u003c/p\u003e \u003cp\u003eThe study period was chosen considering the incidence of CDI in the centre and to ensure an adequate sample size for the primary objective of the study.\u003c/p\u003e \u003cp\u003eStool samples of patient admitted to Fondazione IRCCS Policlinico San Matteo with clinical suspicion of CDI were processed by the Microbiology laboratory.\u003c/p\u003e \u003cp\u003eAccording to the ESCMID guidelines for the management of CDI, an episode of CDI was defined as clinical findings compatible with CDI and microbiological evidence of \u003cem\u003eC. difficile\u003c/em\u003e free toxins by EIA without reasonable evidence of another cause of diarrhoea or a clinical picture compatible with CDI and a positive NAAT, or positive toxigenic \u003cem\u003eC. difficile\u003c/em\u003e culture or pseudomembranous colitis as diagnosed during endoscopy, after colectomy or on autopsy, in combination with a positive test for the presence of toxigenic \u003cem\u003eC. difficile\u003c/em\u003e. Diarrhoea was defined as \u0026ge;\u0026thinsp;3 loose stools in 24 hours. Patients with positive microbiological findings for \u003cem\u003eC. difficile\u003c/em\u003e without clinical findings compatible with CDI were excluded Recurrence was defined as symptoms recurrence within 8 weeks after a previous CDI episode, provided the symptoms from the previous episode resolved after completion of initial treatment (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor each included patients, clinical data were retrieved from medical charts and electronic records for each patient.\u003c/p\u003e \u003cp\u003e\u003cem\u003eC. difficile\u003c/em\u003e positive stool samples were subjected to alcohol shock by preparing a 1:1 suspension of fecal sample and 95% ethanol, followed by vortexing and incubation at room temperature for 1 hour. Subsequently, 50\u0026ndash;100 microliters of the suspension were inoculated onto Schaedler Agar\u0026thinsp;+\u0026thinsp;5% sheep blood (Biom\u0026eacute;rieux, Marcy L\u0026rsquo;Etoile, France) and incubated at 37\u0026deg;C under anaerobic conditions for 3 days. AST was performed using gradient diffusion (MIC Test Strip, Liofilchem srl, Roseto degli Abruzzi (TE), Italy) and results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCSAT) reference breakpoints (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). According to previous reports, gradient diffusion may result in underestimation of MICs (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), which is why the agar dilution test is the reference for \u003cem\u003eC. difficile\u003c/em\u003e antimicrobial susceptibility testing. Nevertheless, gradient diffusion has shown good concordance with agar dilution, it is more practical and allows testing of multiple antimicrobials (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNAAT (GeneXpert, Cepheid, Sunnyvale, CA, USA) was used to identified presumptive NAP01/R027 (due to tcdCΔ117 deletion) and toxin production genes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eThe sample size was calculated on the primary endpoint of vancomycin and/or metronidazole resistance prevalence. The statistical unit was the positive stool sample corresponding to an\u003c/p\u003e \u003cp\u003einfectious episode. We hypothesized a prevalence of 5% according to the results of a recent meta-analysis. Therefore, the study would require a sample size of 130 for estimating the expected proportion with 5% absolute precision and 99% confidence (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll analysis were performed using RStudio version 4.4.0 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). We described continuous variables with the mean and standard deviation or the median and 25th-75th percentiles for skewed distributions; we described categorical data as counts and percent. We compared characteristics between groups with the Student t test or Kruskal wallis test and the Fisher exact test, respectively. A 2-sided p-value of 0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003ePrevalence of antimicrobial resistance was calculated by dividing the number of resistant isolates for the total samples tested.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eWe enrolled 108 patients, 46% of them males, 54% females. Median age was 76 years (interquartile range (IQR) 62\u0026ndash;84). Patients are described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: the median Charlson Comorbidity Index (CCI) was 5 (IQR: 4\u0026ndash;7) and the most prevalent comorbidities were cardiovascular disease, COPD and solid tumors.\u003c/p\u003e \u003cp\u003eMost patients had severe, hospital-acquired CDI. For 78% of the patients it was the first episode, and for 16% it was the first recurrence, while 6% had two or more recurrences. Nearly 90% of the patients had undergone an antibiotic therapy in last three months, and nearly 20% of patients had already been treated with vancomycin. For the current episode, the preferred treatment was vancomycin, followed by combination therapy of vancomycin and metronidazole. Thirty percent of patients had presumptive NAP1/027 strain, and seven percent of patients died.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatients description with counts and frequency for absolute numbers, and median and first-third interquartile ranges for continuous numbers.CDI: Clostridioides difficile infection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients (total)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpidemiology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge [years]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76 (62\u0026ndash;84)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMales/females\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50/58 (46.0/54.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharlson comorbidity index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCardiovascular disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (37.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeripheral venous disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (13.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStroke\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (13.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronic Obstructive Pulmonary Disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (24.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (14.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolid malignancy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (18.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematological malignancy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (2.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious hospitalization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38 (44.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCurrent episode of CDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHospital/community acquired CDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74/12 (86.0/14.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere CDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 (47.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpisode: first/second/third or more\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67/14/5 (77.9/16.3/5.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of therapy [days]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (10-13.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAP1/R027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (30.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBinary Toxin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42 (42.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTcdB+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83 (83.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICU admission\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (2.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecurrence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (16.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLenght of stay [days]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26 (15\u0026ndash;42)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28 days mortality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (7.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTherapies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUse of Proton Pump Inhibitors before CDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58 (69.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProton Pump Inhibitors started during CDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (8.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecent use of antibiotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76 (89.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious use of vancomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (19.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious use of metronidazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (8.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious use of fidaxomicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (3.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment with vancomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59 (74.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment with fidaxomicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (6.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment with metronidazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment with vancomycin and metronidazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (17.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMICs of vancomycin, metronidazole, tigecycline and ciprofloxacin\u003c/h3\u003e\n\u003cp\u003eAll the 108 isolates were available for antimicrobial susceptibility testing. The MICs\u0026rsquo; distribution is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median MICs for vancomycin, metronidazole, fidaxomicin and ciprofloxacin were respectively 0.5, 1, 0.016 and 32. All the isolates were resistant to ciprofloxacin but sensitive to the other three antibiotics, although MIC distribution varied.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMIC variation in subsequent episodes\u003c/h2\u003e \u003cp\u003eFor 8 patients isolates from both first episode and recurrence were available: 3 (37.5%) displayed a twofold MIC increase for vancomycin, 6 (75%) a twofold increase for metronidazole and 3 (37.5%) a twofold increase for tigecycline. The variation in MIC distributions is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVariables associated with MIC distributions\u003c/h3\u003e\n\u003cp\u003eWe did not find any significant correlation between the antibiotic\u0026rsquo;s MICs and various possible risk factors; episode number, previous vancomycin administration, mode of acquisition (healthcare \u003cem\u003eversus\u003c/em\u003e community). Similarly, we didn\u0026rsquo;t found any statistically significant MIC according to the presence of presumptive NAP1/027 or toxin production genes (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eMICs values according to NAP1/027 and toxin production genes are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of the present study was to evaluate the rate of antimicrobial resistance of \u003cem\u003eC. difficile\u003c/em\u003e isolates recovered from patients admitted with CDI in our Institution and to evaluate risk factors associated with decreased antimicrobial susceptibility.\u003c/p\u003e \u003cp\u003eWe didn\u0026rsquo;t find any isolate resistant to vancomycin, metronidazole and tigecycline, while all the isolates were resistant to ciprofloxacin.\u003c/p\u003e \u003cp\u003eWhile worldwide weighted pool resistance to vancomycin and metronidazole were found to be 3% and 5% (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), their frequency is actually variable according to different world regions, reaching up to 34% in a cohort of patients with CDI in the US (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Our results are consistent with those reported in Southern and Western Europe, while increased metronidazole resistance has been described in Eastern Europe (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). It is worth noting that metronidazole MICs in isolates from our cohort were close to the breakpoint and frequently higher than the geometric mean metronidazole MIC from a recent collection of European isolates (0.29 mg/L). This can be explained by the high proportion of presumptive NAP01/R027 in our cohort, as this ribotype was associated with higher geometric mean metronidazole MIC (1.87 mg/L) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e Interestingly, all the isolates demonstrated low MICs for tigecycline, that has been recently introduced in the ESCMID guidelines as a possible therapeutic option in severe CDI due to favourable outcomes in a retrospective cohort study. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e),(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eResistance to ciprofloxacin was found in all the isolates, confirming a trend of decreased susceptibility described in Italy and Europe during the last decades. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e),(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) Fluoroquinolones resistance is mediated by mutation in the \u003cem\u003egyr\u003c/em\u003e genes, encoding the DNA gyrase subunits GyrA and GyrB due to exposure to subinhibitory concentrations of this antimicrobial class and to their widespread use both in the healthcare setting and in the community. Furthermore, fluoroquinolone exposure is a well-recognized risk factor for CDI and ciprofloxacin upregulates the expression of both TcdA and TcdB in hypervirulent strains of \u003cem\u003eC. difficile.\u003c/em\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e),(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eInterestingly, when comparing isolates from first episode to those of the subsequent recurrence from the same patients, we noticed a frequent increase in the MIC to all the tested antibiotics, even those not used for the treatment of the index CDI episode.\u003c/p\u003e \u003cp\u003eSimilar MICs increases have been previously described in retrospective studies and were associated with hypervirulent strains and binary toxin production. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e),(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) Vancomycin MIC increase in recurrent CDIs was similarly shown in a phase 2 clinical trial evaluating the efficacy of rinidazole compared to vancomycin. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eBiofilm formation production is frequent in \u003cem\u003eC. difficile\u003c/em\u003e isolates, it is associated with reduced susceptibility to vancomycin and metronidazole and may explain this result.(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e),(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e),(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) Biofilm may help selecting more tolerant strains after treatment of the first episode that will cause subsequent recurrence. Unfortunately, we didn\u0026rsquo;t evaluate biofilm production in our isolates and we can\u0026rsquo;t prove this hypothesis.\u003c/p\u003e \u003cp\u003eSeveral bacterial factors, such as ribotype 027, binary toxin and Toxin B production, have reduced antimicrobial susceptibility to both metronidazole and vancomycin. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eR027 strains tend to be associated with increased vancomycin resistance due to frequent association with a Thr115Ala mutation in the regulatory gene VanR that result in constitutive expression of vanG leading to decrease vancomycin affinity (MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values of 2 and 4 \u0026micro;g/ml). (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e),(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e When stratifying MIC values according to virulence genes, we didn\u0026rsquo;t found any significant differences associated with their expression. It should be considered that all the isolates in our cohort were susceptible to vancomycin and therefore our R027 strains were probably harboring wild type Thr115, which is characterized by MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e of 0,5 and 2 ug/ml, respectively. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTherefore, the impact of virulence genes on antimicrobial susceptibility is likely less relevant when assessing values below the breakpoint.\u003c/p\u003e \u003cp\u003eThe principal strength of this study is that show MICs increase in isolates from recurrence in a subset (albeit limited) of real life patients with CDI.\u003c/p\u003e \u003cp\u003eSeveral limitations of this study must be acknowledged. Firstly, the retrospective and single centre design make these findings poorly generalizable to other settings and implied the presence of missing data.\u003c/p\u003e \u003cp\u003eSecondly, we didn\u0026rsquo;t perform susceptibility testing for fidaxomicin, that represents the recommended treatment for CDI. The reports of fidaxomicin resistance have been anecdotal and associated with fitness costs for \u003cem\u003eC. difficile\u003c/em\u003e, an therefore its impact seems to be limited at the moment. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eAnother limitation was the use of a molecular test that detects only some genes compared to other techniques such as whole genome sequencing that enables more precise genotyping and ribotyping of \u003cem\u003eC. difficile\u003c/em\u003e strains.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eAntimicrobial resistance has become more frequent in CDI, but its influence on clinical outcomes has been considered limited due to the high stool concentration of vancomycin that overcomes the MIC of resistant strains.(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThis assumption has been recently questioned by a retrospective cohort study performed in a setting with high prevalence of vancomycin resistant strains, where decreased susceptibility was associated with lower sustained clinical cure. Interestingly, the higher rates of failure seemed to be driven by antibiotic failure rather than increased recurrence. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of this study are important not only in terms of resistance surveillance, but also on a clinical perspective. In patients with recurrence, hypervirulent strains, and binary or TcdB production genes, antimicrobial resistance should be taken into account, especially in settings where its frequence is high.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCDI\u003cem\u003e\u0026nbsp;Clostridioides difficile\u003c/em\u003e infection\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eESCMID European Society on Microbiology and Infectious Disease\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGDH glutamate dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNAAT Nucleic Acid Amplification Test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEIA enzyme immunoassay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAST antimicrobial susceptibility testing \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Fondazione IRCCS Policlinico S.Matteo Internal Review Board (protocol number 39948/2023). Informed Consent was withdrawn due to the retrospective nature of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, P.V. and E.A.; methodology, P.V. and P.C.; software, P.V.; validation, P.C. and A.D.S. and F.B. and R.B.; formal analysis, P.V.; investigation, F.Co. and F. Ca. and P.S. and M.C.; resources, R.B.; data curation, M.M. and N.A. and G.B and R.D. and F.V..; writing\u0026mdash;original draft preparation, P.V. and M.M. and E.A.; writing\u0026mdash;review and editing, E.A. and P.S. and P.C.; visualization, P.V..; supervision, P.S. and P.C. and A.D.S and F.B.; project administration, R.B.;. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLawson PA, Citron DM, Tyrrell KL, Finegold SM. Reclassification of Clostridium difficile as Clostridioides dif-ficile (Hall and O\u0026rsquo;Toole 1935) Pr\u0026eacute;vot 1938. Anaerobe. Aug. 2016;40:95\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Rossen TM et al. 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Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates. J Antimicrob Chemother. 2020;75(4):859\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jac/dkz513\u003c/span\u003e\u003cspan address=\"10.1093/jac/dkz513\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 31873741; PMCID: PMC7069472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchandin H, Anjou C, Poulen G, et al. In vivo emergence of a still uncommon resistance to fidaxomicin in the urgent anti-microbial resistance threat Clostridioides difficile. J Antimicrob Chemother Published online August. 2023;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jac/dkad194\u003c/span\u003e\u003cspan address=\"10.1093/jac/dkad194\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Clostridioides difficile, antimicrobial resistance, vancomycin, metronidazole","lastPublishedDoi":"10.21203/rs.3.rs-5997394/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5997394/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWhile antimicrobial susceptibility testing (AST) isn\u0026rsquo;t routinely performed for \u003cem\u003eC. difficile\u003c/em\u003e infection (CDI), reports of antimicrobial resistance have increased in surveillance studies. The aim of this study was to assess the rate of antimicrobial resistance to four antimicrobials (vancomycin, metronidazole, tigecycline and ciprofloxacin), to assess risk factors for antimicrobial resistance and to evaluate MIC variation in patients with recurrence.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eData from consecutive patients with CDI admitted to our Istitution between the 1st of January 2022 and the 30th of April 2023 were collected. We performed AST with gradient diffusion and NAAT to evaluate presumptive presence of R027/NAP1 and toxin production genes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAntimicrobial susceptibility testing was performed on 108 available isolates. We didn\u0026rsquo;t found any isolate resistant to vancomycin, metronidazole and tigecycline, while all the isolates were resistant to ciprofloxacin. For 8 patients isolates from both first episode and recurrence were available: 3 (37.5%) displayed 2 fold MIC increase for vancomycin, 6 (75%) for metronidazole and 3 (37.5%) for tigecycline.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results were concordant with European surveillance data. MIC increase to all tested antibiotics in patients with CDI may be due to biofilm formation and its possible role warrant further research, especially considering reports of clinical failure due to vancomycin resistance.\u003c/p\u003e","manuscriptTitle":"Minimum inhibitory concentrations increase in Clostridioides difficile isolates from patients with recurrence: results from a retrospective single-centre cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-13 09:59:07","doi":"10.21203/rs.3.rs-5997394/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a6a0997-3ba2-411b-91c5-2ba5437c68fa","owner":[],"postedDate":"February 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-05T14:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-13 09:59:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5997394","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5997394","identity":"rs-5997394","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}