Clostridioides difficile from Fecally Contaminated Environmental Sources: Resistance and Genetic Relatedness from a Molecular Epidemiological Perspective.

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3')-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven "hypervirulent" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.

Discrimination between hypervirulent and non-hypervirulent ribotypes of Clostridioides difficile by MALDI-TOF mass spectrometry and machine learning.

Hypervirulent ribotypes (HVRTs) of Clostridioides difficile such as ribotype (RT) 027 are epidemiologically important. This study evaluated whether MALDI-TOF can distinguish between strains of HVRTs and non-HVRTs commonly found in Europe. Obtained spectra of clinical C. difficile isolates (training set, 157 isolates) covering epidemiologically relevant HVRTs and non-HVRTs found in Europe were used as an input for different machine learning (ML) models. Another 83 isolates were used as a validation set. Direct comparison of MALDI-TOF spectra obtained from HVRTs and non-HVRTs did not allow to discriminate between these two groups, while using these spectra with certain ML models could differentiate HVRTs from non-HVRTs with an accuracy >95% and allowed for a sub-clustering of three HVRT subgroups (RT027/RT176, RT023, RT045/078/126/127). MALDI-TOF combined with ML represents a reliable tool for rapid identification of major European HVRTs.

Occurrence, resistance patterns, and management of carbapenemase-producing bacteria in war-wounded refugees from Ukraine.

We analyzed consecutive clinical cases of infections due to carbapenemase-producing gram-negative bacteria detected in war-wounded patients from Ukraine who were treated at one university medical center in southwest Germany between June and December 2022. The isolates of multiresistant gram-negative bacteria were subjected to a thorough microbiological characterization and whole genome sequencing (WGS). We identified five war-wounded Ukrainian patients who developed infections with New Delhi metallo-ß-lactamase 1-positive Klebsiella pneumoniae. Two isolates also carried OXA-48 carbapenemases. The bacteria were resistant to novel antibiotics, such as ceftazidime/avibactam and cefiderocol. The used treatment strategies included combinations of ceftazidime/avibactam + aztreonam, colistin, or tigecycline. WGS suggested transmission during primary care in Ukraine. We conclude that there is an urgent need for thorough surveillance of multiresistant pathogens in patients from war zones.

Epidemiology of Clostridioides difficile Infections in Germany, 2010-2019: A Review from Four Public Databases.

INTRODUCTION: Clostridioides difficile infection (CDI) is a recognized global threat especially for vulnerable populations. It is of particular concern to healthcare providers as it is found in both hospital and community settings, with severe courses, frequent recurrence, high mortality and substantial financial impact on the healthcare system. The CDI burden in Germany has been described and compared by analysing data from four different public databases. METHODS: Data on hospital burden of CDI have been extracted, compared, and discussed from four public databases for the years 2010-2019. Hospital days due to CDI were compared to established vaccine preventable diseases, such as influenza and herpes zoster, and also to CDI hospitalisations in the United States (US). RESULTS: All four databases reported comparable incidences and trends. Beginning in 2010, population-based hospitalised CDI incidence increased to a peak of > 137/100,000 in 2013. Then, incidence declined to 81/100,000 in 2019. Hospitalised patients with CDI were predominantly > 50 years of age. The population-based incidence of severe CDI was between 1.4 and 8.4/100,000 per year. Recurrence rates were between 5.9 to 6.5%. More than 1,000 CDI deaths occurred each year, with a peak of 2,666 deaths in 2015. Cumulative CDI patient days (PD) were between 204,596 and 355,466 each year, which exceeded cumulated PD for influenza and herpes zoster in most years, though year-to-year differences were observed. Finally, hospitalized CDI incidence was higher in Germany than in the US, where the disease is well recognized as a public health threat. CONCLUSIONS: All four public sources documented a decline in CDI cases since 2013, but the disease burden remains substantial and warrants continued attention as a severe public health challenge.

RNA-based stable isotope probing provides no indication for rapid α-synuclein assimilation by murine gut bacteria.

In Parkinson's disease (PD), α-synuclein is a key protein in the process of neurodegeneration. Besides motor symptoms, most PD patients additionally suffer from gastrointestinal tract (GIT) dysfunctions, even several years before the onset of motor disabilities. Studies have reported a dysbiosis of gut bacteria in PD patients compared to healthy controls and have suggested that the enteric nervous system (ENS) can be involved in the development of the disease. As α-synuclein was found to be secreted by neurons of the ENS, we used RNA-based stable isotope probing (RNA-SIP) to identify gut bacteria that might be able to assimilate this protein. The gut contents of 24 mice were pooled and incubated with isotopically labelled (13C) and unlabelled (12C) α-synuclein. After incubation for 0, 4 and 24 h, RNA was extracted from the incubations and separated by density gradient centrifugation. However, RNA quantification of density-resolved fractions revealed no incorporation of the 13C isotope into the extracted RNA, suggesting that α-synuclein was not assimilated by the murine gut bacteria. Potential reasons and consequences for follow-up-studies are discussed.

Antigen-Specific vs. Neutralizing Antibodies Against Conditioned Media of Patients With Clostridioides difficile Infection: A Prospective Exploratory Study.

The immunological response against Clostridioides difficile (C. difficile) is crucial for an improved understanding of disease mechanisms and the development of novel therapeutic strategies. From April 2014 to February 2015, adult patients with C. difficile infection (CDI) were recruited, and the clinical course and treatment response were carefully monitored. On day 1, 3, and 6 after diagnosis, patient plasma samples were screened for anti-GDH (glutamate dehydrogenase), anti-TcdA, anti-TcdB, and anti-CWP84 (cell-wall protein 84) antibodies by ELISA. Additionally, neutralization assays of toxins from conditioned media of clinical isolates (RT010, RT014, and RT027) were performed. Most patients with CDI (n = 46) had antibodies against GDH (85%) and CWP84 (61%), but only few had antibodies against TcdA (11%) and TcdB (28%). We found patients with neutralizing antibodies against C. difficile toxins (conditioned media) produced by RT027 (26%). A subgroup of these samples could neutralize both toxins from RT027 and RT014 [11%, (5/46)]; however, no single sample neutralized only RT014. Overall, neutralizing antibody titers were low (≤1:16). In a one week follow-up of acute infection, we never observed an early booster effect with seroconversion or antibody increases, irrespective of disease severity. No correlation was found between the presence of antigen-specific (ELISA) or neutralizing antibodies and the clinical course of disease. Anti-TcdB but not anti-TcdA antibodies correlated with the occurrence of neutralizing antibodies. In conclusion, natural antibody titers against C. difficile toxins were absent or low and were not associated with disease severity. The correlation between the anti-TcdB with toxin neutralization confirms the importance of TcdB for virulence of CDI. Alternative sensitization strategies, e.g., through vaccine development, are required to overcome the regular low-titer antibody production following natural intestinal C. difficile exposure.

Implementation of a Clostridioides difficile sentinel surveillance system in Germany: First insights for 2019-2021.

OBJECTIVES: Clostridioides difficile is a major cause of nosocomial diarrhea. Several "hypervirulent" lineages such as ribotype 027 (RT027) and RT078 are of high epidemiological importance, leading to outbreaks and more severe courses of disease. An active surveillance system targeting molecular epidemiology and corresponding antimicrobial resistance has not been established in Germany. METHODS: Since October 2019, University Hospitals throughout Germany collected by two dates every year (1st April and October, respectively) their first ten unselected samples being tested positive for C. difficile. RESULTS: Out of 1026 samples received from 29 sites, 876 toxigenic C. difficile strains could be cultivated. PCR ribotyping of these strains revealed a large strain diversity with RT014 (17.5%) dominating, followed by isolates of the major nosocomial lineage RT001 (7.1%) and the "hypervirulent" lineage RT078 (5.9%). Notably, prevalence of RT027 was low with ∼3.5% at all time points analyzed, while the abundance of RT001 isolates significantly declined from 12.3% to 3.7% during the sampling period (P < 0.001). Antimicrobial resistance against clarithromycin, moxifloxacin, and rifampicin was detected in 18%, 15%, and 4% of the tested isolates, respectively. Highest resistance rates were found among RT027 isolates (83%, 87% and 63% for clarithromycin, moxifloxacin, and rifampicin, respectively). Vancomycin resistance was not detected, and metronidazole resistance was observed only for a single RT027 isolate. CONCLUSIONS: This Germany-wide continuing surveillance effort with a standardized mode of isolate acquisition indicates that isolates of RT027 were only sporadically detected under these strain acquisition conditions, and RT001 seems to become less important in the hospital setting, being replaced by other RTs.

Combined antibiotic stewardship and infection control measures to contain the spread of linezolid-resistant Staphylococcus epidermidis in an intensive care unit.

BACKGROUND: The unrestricted use of linezolid has been linked to the emergence of linezolid-resistant Staphylococcus epidermidis (LRSE). We report the effects of combined antibiotic stewardship and infection control measures on the spread of LRSE in an intensive care unit (ICU). METHODS: Microbiological data were reviewed to identify all LRSE detected in clinical samples at an ICU in southwest Germany. Quantitative data on the use of antibiotics with Gram-positive coverage were obtained in defined daily doses (DDD) per 100 patient-days (PD). In addition to infection control measures, an antibiotic stewardship intervention was started in May 2019, focusing on linezolid restriction and promoting vancomycin, wherever needed. We compared data from the pre-intervention period (May 2018-April 2019) to the post-intervention period (May 2019-April 2020). Whole-genome sequencing (WGS) was performed to determine the genetic relatedness of LRSE isolates. RESULTS: In the pre-intervention period, LRSE were isolated from 31 patients (17 in blood cultures). The average consumption of linezolid and daptomycin decreased from 7.5 DDD/100 PD and 12.3 DDD/100 PD per month in the pre-intervention period to 2.5 DDD/100 PD and 5.7 DDD/100 PD per month in the post-intervention period (p = 0.0022 and 0.0205), respectively. Conversely, vancomycin consumption increased from 0.2 DDD/100 PD per month to 4.7 DDD/100 PD per month (p < 0.0001). In the post-intervention period, LRSE were detected in 6 patients (4 in blood cultures) (p = 0.0065). WGS revealed the predominance of one single clone. CONCLUSIONS: Complementing infection control measures by targeted antibiotic stewardship interventions was beneficial in containing the spread of LRSE in an ICU.

Molecular epidemiology and antimicrobial resistance of Clostridioides difficile in Germany, 2014-2019.

Clostridioides difficile is a Gram positive spore-forming rod and mainly responsible for nosocomial diarrhea in developed nations. Molecular and antimicrobial surveillance is important for monitoring the strain composition including genotypes of high epidemiological importance such as ribotype 027 (RT027) and corresponding resistance patterns. 1535 isolates obtained from samples sent between 2014 and 2019 to the German National Reference Center (NRC) for diagnostic reasons (NRC strain set), and 1143 isolates from a Tertiary Care University Center in Saarland, Germany (non-NRC strain set), were evaluated using antibiotic susceptibility testing and ribotyping. In the NRC strain set, RT027 overtook RT001, the main RT found in the preceding studies, and dominated with 36.2%, followed by RT001 (13.3%), and RT014 (8.5%). Of note, since 2016 a constant decrease of RT027 could be noticed. In the non-NRC strain set a large strain diversity was present with RT014 (18%) and RT001 (8.9%) being most prevalent. In NRC samples, resistance towards metronidazole, vancomycin, moxifloxacin, clarithromycin and rifampicin was 2.7%, 0%, 57.1%, 53.2% and 19.2%, respectively. Metronidazole resistance was almost exclusively found in RT027 isolates. Rifampicin resistance was also observed predominantly in isolates of RT027, constituting an almost four-fold increase, when compared to preceeding studies in this region. In conclusion these data demonstrate that RT027 is a driver for rifampicin and metronidazole resistance, underlining the importance of continuous surveillance efforts.

Application and clinical impact of the RESIST-4 O.K.N.V. rapid diagnostic test for carbapenemase detection in blood cultures and clinical samples.

Invasive infections caused by carbapenemase-producing bacteria are associated with excess mortality. We applied a rapid diagnostic test (RDT) on clinical samples with an elevated likelihood of carbapenemase-producing bacteria and documented its impact on antibiotic treatment decisions. Among 38 patients, twelve tested positive for infections caused by carbapenemase-producing bacteria (31.6%), mainly in blood cultures. KPC (n = 10) was more frequent than OXA-48 (n = 2). RDT-based carbapenemase detection led to a treatment modification to ceftazidime/avibactam-containing regimens in all patients before detailed antibiotic testing results became available. Eleven patients (92%) survived the acute infection, whereas one patient with a ceftazidime/avibactam- and colistin-resistant OXA-48-positive isolate died.

Clostridioides (Clostridium) difficile Pacemaker Infection.

Clostridioides difficile is the leading cause of antibiotic-associated nosocomial diarrhea, but extra-intestinal manifestations are rare. We describe the first documented case of bacteraemia with pacemaker pocket and lead infection with the toxigenic C. difficile ribotype 014 with a lack of abdominal symptoms. The patient underwent pacemaker extraction and treatment with intravenous and oral vancomycin. Genotyping and molecular subtyping revealed clonality between pacemaker and intestinal isolates. This case illustrates the risk of intravascular device infections due to C. difficile. Even asymptomatic C. difficile colonization might pose a risk for prosthetic material infection.

Simple Questionnaires to Improve Pooling Strategies for SARS-CoV-2 Laboratory Testing.

Background: Liberal PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to contain the coronavirus disease 2019 (COVID-19) pandemic. Combined multi-sample testing in pools instead of single tests might enhance laboratory capacity and reduce costs, especially in low- and middle-income countries. Objective: The purpose of our study was to assess the value of a simple questionnaire to guide and further improve pooling strategies for SARS-CoV-2 laboratory testing. Methods: Pharyngeal swabs for SARS-CoV-2 testing were obtained from healthcare and police staff, hospital inpatients, and nursing home residents in the southwestern part of Germany. We designed a simple questionnaire, which included questions pertaining to a suggestive clinical symptomatology, recent travel history, and contact with confirmed cases to stratify an individual's pre-test probability of having contracted COVID-19. The questionnaire was adapted repeatedly in face of the unfolding pandemic in response to the evolving epidemiology and observed clinical symptomatology. Based on the response patterns, samples were either tested individually or in multi-sample pools. We compared the pool positivity rate and the number of total PCR tests required to obtain individual results between this questionnaire-based pooling strategy and randomly assembled pools. Findings: Between March 11 and July 5, 2020, we processed 25,978 samples using random pooling (n = 6,012; 23.1%) or questionnaire-based pooling (n = 19,966; 76.9%). The overall prevalence of SARS-CoV-2 was 0.9% (n = 238). Pool positivity (14.6% vs. 1.2%) and individual SARS-CoV-2 prevalence (3.4% vs. 0.1%) were higher in the random pooling group than in the questionnaire group. The average number of PCR tests needed to obtain the individual result for one participant was 0.27 tests in the random pooling group, as compared to 0.09 in the questionnaire-based pooling group, leading to a laboratory capacity increase of 73% and 91%, respectively, as compared to single PCR testing. Conclusions: Strategies that combine pool testing with a questionnaire-based risk stratification can increase laboratory testing capacities for COVID-19 and might be important tools, particularly in resource-constrained settings.

Do written diagnosis-treatment recommendations on microbiological test reports improve the management of Staphylococcus aureus bacteremia? A single-center, retrospective, observational study.

The objective of this study was to assess the impact of microbiological test reports that provide specific written recommendations on the appropriate management of Staphylococcus aureus bacteremia (SAB). We performed a retrospective analysis of laboratory and clinical data of all SAB patients treated at one German University hospital, 2012-2015. Among 467 included patients, methicillin-resistant S. aureus (MRSA) accounted for 15.2% of all SAB cases. All-cause in-hospital mortality was 25.2%, and was significantly elevated in individuals aged >55 years, in MRSA bacteremia and if the source of infection remained unidentified. Focus identification was achieved in 71.1%, with the most prevalent foci being catheter-associated bloodstream infection (23.1%), soft tissue infection (15.4%), osteomyelitis (5.1%) and endocarditis (4.9%). Standardized written recommendations on microbiological test reports led to a significant increase of transesophageal echocardiography, additional imaging studies for focus identification and more frequent follow-up blood cultures, but no significant effect on mortality was observed.

Risk Factors for Primary Clostridium difficile Infection; Results From the Observational Study of Risk Factors for Clostridium difficile Infection in Hospitalized Patients With Infective Diarrhea (ORCHID).

Background: There are inconsistent data on the risk factors for Clostridium difficile infection (CDI) in the literature. Aims: To use two C. difficile infection (CDI) case-control study groups to compare risk factors in hospitalized patients with diarrhea across different countries. Methods: A multi-center group of CDI cases/controls were identified by standardized testing from seven countries from the prior EUropean, multi-center, prospective bi-annual point prevalence study of CLostridium difficile Infection in hospitalized patients with Diarrhea (EUCLID). A second group of CDI cases/controls was identified from a single center in Germany [parallel study site (PSS)]. Data were extracted from the medical notes to assess CDI risk factors. Univariate analyses and multivariate logistic regression models were used to identify and compare risk factors between the two groups. Results: There were 253 and 158 cases and 921 and 584 controls in the PSS and EUCLID groups, respectively. Significant variables from univariate analyses in both groups were age ≥65, number of antibiotics (OR 1.2 for each additional antibiotic) and prior hospital admission (all p < 0.001). Congestive heart failure, diabetes, admission from assisted living or Emergency Department, proton pump inhibitors, and chronic renal disease were significant in PSS (all p < 0.05) but not EUCLID. Dementia and admitted with other bacterial diseases were significant in EUCLID (p < 0.05) but not PSS. Following multivariate analyses, age ≥ 65, number of antibiotics and prior hospital admission were consistently identified as CDI risk factors in each individual group and combined datasets. Conclusion: Our results show that the same CDI risk factors were identified across datasets. These were age ≥ 65 years, antibiotic use and prior hospital admission. Importantly, the odds of developing CDI increases with each extra antibiotic prescribed.

N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases.

In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant reduction of collagen degradation in an ex vivo pig-skin model.

Molecular epidemiology and antimicrobial resistance of Clostridioides difficile detected in chicken, soil and human samples from Zimbabwe.

BACKGROUND: Clostridioides difficile is the major cause of infectious nosocomial diarrhoea in industrialized nations. Data on the occurrence of C. difficile in Africa, ribotype (RT) distribution, antimicrobial susceptibility patterns and potential zoonotic transmission are scarce. METHODS: 80 Zimbabwean C. difficile isolates from different sources (chicken [n=30], soil [n=21] and humans [n=29]) were investigated using ribotyping, toxin gene detection, resistance testing, multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing (WGS). RESULTS: Among chicken isolates, the most common RTs were RT103 (6/30), RT025 (5/30) and RT070 (4/30). Within soil samples, RT025 and RT056 were most common (3/21 each). In contrast, the non-toxigenic RT084 was most frequently found in human isolates (4/29). Toxin genes were detected in only 19/29 human isolates. Susceptibility testing showed no resistance against metronidazole and vancomycin, and resistance against macrolides and rifampicin was scarce (3/80 and 2/80, respectively); however, 26/80 isolates showed moxifloxacin resistance. MLVA and WGS of strains with identical RTs stemming from different sources revealed clustering of RT025 and RT084 isolates from human und non-human samples. CONCLUSION: No "hypervirulent" strains were found. The detected clusters between human, chicken and soil isolates indicate ongoing transmission between humans and environmental sources and might point towards a zoonotic potential.

Factors affecting reported Clostridioides difficile infection rates; the more you look the more you find, but should you believe what you see?

Reported rates of C. difficile infection (CDI) have increased in many settings; however, these can be affected by factors including testing density (test-density) and diagnostic methods. We aimed to describe the impact of multiple factors on CDI rates. Hospitals (n = 182) across five countries (France, Germany, Italy, Spain, and UK) provided data on; size and type of institution, CDI testing methodology, number of tests/month and patient-bed-days (pbds)/month over one year. Incidence rates were compared between countries, different sized institutions, types of institutions and testing method. After univariate analyses, the highest CDI rates were observed in Italy (average 11.8/10,000pbds/hospital/month), acute/primary hospitals (12.3/10,000pbds/hospital/month), small hospitals (16.7/10,000pbds/hospital/month), and hospitals using methods that do not detect toxin (NO-TOXIN) (e.g. GDH/NAAT or standalone NAAT) (10.7/10,000pbds/hospital/month). After adjusting for test-density, highest incidence rates were still in Italy, acute/primary hospitals and those using NO-TOXIN. The relative rate in long-term healthcare facilities (LTHCFs) increased, but size of institution no longer influenced the CDI rate. Test-density appears to have the largest effect on reported CDI rates. NO-TOXIN testing still influences CDI rates, even after adjusting for test-density, which is consistent with tests that 'overcall' true CDI. Low test-density can mask the true burden of CDI, e.g. in LTHCFs, highlighting the importance of good quality surveillance.

Quality assurance for genotyping and resistance testing of Clostridium (Clostridioides) difficile isolates - Experiences from the first inter-laboratory ring trial in four German speaking countries.

Clostridium (Clostridioides) difficile is a major cause of nosocomial diarrhoea. A first inter-laboratory ring trial was performed in four European countries to evaluate the genotyping and antibiotic susceptibility testing (AST) accuracy. Six C. difficile isolates representing the epidemiologic important ribotypes (RT), RT001, RT002, RT010, RT014, RT027, and RT078 were blinded and send to 21 participating laboratories. Participants tested the samples with their genotyping and AST methods in use for concordance with reference. A total of 21 genotyping- and 14 antimicrobial susceptibility data sets were obtained. Ribotyping (11 participants) correctly identified most RTs (median 91% concordance rate) except for RT002, which was misidentified in 4/11 reports. However, this isolate was correctly asserted to RT002 after an update of a publicly available ribotyping database. Multilocus sequence typing, surface layer sequence typing, DNA microarray based genotyping, and whole genome sequencing, which were used by 1-3 participants, identified all six isolates correctly. AST was done by epsilometry by the participants and compared to agar dilution data determined by the coordinating reference centre. Susceptibilities against metronidazole, moxifloxacin, and vancomycin were correctly identified in 235 of 237 cases and in accordance to agar dilution as the gold standard. Genotyping of the C. difficile test strains revealed a remarkable high concordance on the level of ribotypes with a wide variety of methods. Epsilometry appears to be a reliable method for AST of C. difficile isolates in routine clinical microbiology laboratories.

Incidence, outcome, and risk factors for recurrence of nosocomial Clostridioides difficile infection in adults: A prospective cohort study.

BACKGROUND: Nosocomial Clostridioides difficile infection (CDI) complicates up to 1% of all hospital admissions and is associated with considerable health burden. AIMS: To determine the incidence and outcomes of nosocomial CDI at a major University Medical Center. METHODS: Consecutive adult nosocomial CDI cases were prospectively identified. Stool samples were collected for ribotyping and antibiotic resistance testing. Patients were followed for eight weeks after discharge for relapse. RESULTS: Over a 2-year period, 215 patients developed nosocomial CDI (incidence 2:1000) and 200 (mean age 62.2±19.6 years) gave informed consent. Mean hospital stay was 23.3±28.9 days (range 0-278). Infection was diagnosed within 7 days of admission (range 0-95) in 129 patients (64.5%). More than two-thirds (69.0%) were previously hospitalized within 12 weeks of the index hospitalization. Twenty five percent received prior antibiotics within eight weeks. Fifty-two patients (26.0%) did not receive antibiotics prior to diagnosis. Considerable comorbidities (Charlson Comorbidity Index ≥8) were noted in 33.5% of patients. Recurrence occurred in 43 patients (21.5%). On multivariate logistic regression, fluoroquinolone exposure was the only predictor of recurrence (OR=2.9, 95%CI 1.1-7.7). Overall mortality was 14.0% and CCI ≥8 was the only predictor on multivariate analysis (p=0.004). Genotyping did not identify any known hypervirulent strains and all isolates were susceptible to metronidazole and vancomycin. CONCLUSION: Antibiotic exposure, comorbidities, and prior hospitalization constitute the major risk factors for nosocomial CDI. Recurrence is common and is associated with fluoroquinolones exposure. High baseline comorbidity score was the only predictor of increased mortality in this prospective cohort.