Antibiotic Resistances of Clostridioides difficile.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are a matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances, and most of the epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways or biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Clostridioides difficile in Pigs and Dairy Cattle in Northern Italy: Prevalence, Characterization and Comparison between Animal and Human Strains.

It has been observed that novel strains of Clostridioides difficile can rapidly emerge and move between animal and human hosts. The aim of this study was to investigate the prevalence of C. difficile in pigs and dairy cattle in northern Italy and to characterize and compare C. difficile animal strains with those from patients from the same geographical area. The C. difficile strains were isolated from animals from farms and slaughterhouses (cross-sectional studies) and from neonatal animals with enteric disorders in routine diagnostic investigations (passive surveillance). Samples positive for C. difficile were found in 87% of the pig farms and in 40% of the cattle farms involved in the cross-sectional studies, with a 20% prevalence among suckling piglets and 6.7% prevalence in neonatal calves, with no significant difference between animals with and without diarrheal symptoms. The prevalence of C. difficile in older animal categories was significantly lower. This result suggests that young age is an important risk factor for C. difficile colonization. In cross-sectional studies at slaughterhouses, in both the heavy pigs and dairy cows examined, only 2% of the intestinal content samples were positive for C. difficile and no contamination was found on the surface of the carcasses. Considering passive surveillance, the prevalence rates of positive samples were 29% in piglets and 1.4% in calves. Overall, 267 strains of animal origin and 97 from humans were collected. In total, 39 ribotypes (RTs) were identified, with RT 078 and RT 018 being predominant among animals and humans, respectively. Several RTs overlapped between animals and patients. In particular, RT 569 was identified as an emergent type in our country. Resistance to erythromycin and moxifloxacin was widely diffused among C. difficile strains, regardless of origin. This study supports C. difficile as a pathogen of one-health importance and highlights the need for a collaborative approach between physicians and veterinarians to control and prevent infections that are able to cross species and geographical barriers.

Comparison of microbiological profile of enterotoxigenic Bacteroides fragilis (ETBF) isolates from subjects with colorectal cancer (CRC) or intestinal pre-cancerous lesions versus healthy individuals and evaluation of environmental factors involved in intestinal dysbiosis.

OBJECTIVE: The aim of this study was to analyze enterotoxigenic Bacteroides fragilis (ETBF) isolates from colorectal biopsies of subjects with a histological analysis positive for colorectal cancer (CRC), pre-cancerous lesions (pre-CRC) or with a healthy intestinal tissue and to evaluate the environmental factors that may not only concur to CRC development but may also affect gut microbiota composition. METHODS: ETBF isolates were typed using the ERIC-PCR method, while PCR assays were performed to investigate the bft alleles, the B. fragilis pathogenicity island (BFPAI) region and the cepA, cfiA and cfxA genes. Susceptibility to antibiotics was tested using the agar dilution method. Environmental factors that could play a role in promoting intestinal dysbiosis were evaluated throughout a questionnaire administered to the subjects enrolled. RESULTS: Six different ERIC-PCR types were identified. The type denominated C in this study was the most prevalent, in particular among the biopsies of subjects with pre-CRC, while an isolate belonging to a different type, denominated F, was detected in a biopsy from a subject with CRC. All the ETBF isolates from pre-CRC or CRC subjects had a B. fragilis pathogenicity island (BFPAI) region pattern I, while those from healthy individuals showed also different patterns. Furthermore, 71% of isolates from subjects with pre-CRC or CRC were resistant to two or more classes of antibiotics vs 43% of isolates from healthy individuals. The B. fragilis toxin BFT1 was the most frequently detected in this study, confirming the constant circulation of this isoform strains in Italy. Interestingly, BFT1 was found in 86% of the ETBF isolates from patients with CRC or pre-CRC, while the BFT2 was prevalent among the ETBF isolates from healthy subjects. No substantial differences based on sex, age, tobacco and alcohol consumption were observed between healthy and non-healthy individuals included in this study, while most of the subjects with CRC or pre-CRC lesions were subjected to pharmacological therapy (71%) and showed a body mass index (BMI) that falls within the overweight range (86%). CONCLUSIONS: Our data suggest that some types of ETBF seem to better adapt and colonize the human gut and that the selective pressure exerted by factors related to lifestyle, such as pharmacological therapy and weight, could facilitate their persistence in the gut and their possible involvement in CRC development.

Clostridioides difficile infection (CDI) during the COVID-19 pandemic.

The ongoing coronavirus disease (COVID-19) pandemic has dramatically tested healthcare systems around the world, with serious repercussions on the measures of prevention and control of hospital-acquired infections (HAIs). Among HAIs, Clostridioides difficile infection (CDI) represents one of the most important global public health threats. Although the full impact of the COVID-19 pandemic on CDI remains undetermined, depending on the development of the pandemic in the coming months, in this review literature studies of the last three years have been considered in order to depict the current situation, and make some considerations about possible future developments. If on the one hand, a general reduction in CDI incidence has been reported in several settings, mainly due to the extraordinary reinforcement of infection prevention measures, on the other hand, the critical circumstances experienced in many hospitals have limited the effectiveness of these measures, particularly in the intensive care units (ICUs), increasing the possibility of the occurrence of hospital-acquired CDI (HA-CDI). New concerns have arisen from the decrease in C. difficile testing and the increased use of broad-spectrum antibiotics reported during the pandemic. In particular, overuse of antibiotics and disinfectants may lead to a selection of resistant C. difficile strains not only in hospitals but also in the community. Furthermore, patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and patients that have survived COVID-19 may represent a new group of frail patients potentially at a higher risk of CDI, a group that could potentially increase in size due to SARS-CoV-2 evolution. In the dramatic COVID-19 era, the multifactorial nature of CDI has emerged more clearly than before, highlighting the necessity of a strong refocus on efforts to improve prevention strategies and to integrate CDI surveillance in a One Health prospective in order to curtail the public health threat posed by this infection in the next future.

Association of Polygenic Risk Score and Bacterial Toxins at Screening Colonoscopy with Colorectal Cancer Progression: A Multicenter Case-Control Study.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci.

Mechanisms of antibiotic resistance of Clostridioides difficile.

Clostridioides difficile (CD) is one of the top five urgent antibiotic resistance threats in USA. There is a worldwide increase in MDR of CD, with emergence of novel strains which are often more virulent and MDR. Antibiotic resistance in CD is constantly evolving with acquisition of novel resistance mechanisms, which can be transferred between different species of bacteria and among different CD strains present in the clinical setting, community, and environment. Therefore, understanding the antibiotic resistance mechanisms of CD is important to guide optimal antibiotic stewardship policies and to identify novel therapeutic targets to combat CD as well as other bacteria. Epidemiology of CD is driven by the evolution of antibiotic resistance. Prevalence of different CD strains and their characteristic resistomes show distinct global geographical patterns. Understanding epidemiologically driven and strain-specific characteristics of antibiotic resistance is important for effective epidemiological surveillance of antibiotic resistance and to curb the inter-strain and -species spread of the CD resistome. CD has developed resistance to antibiotics with diverse mechanisms such as drug alteration, modification of the antibiotic target site and extrusion of drugs via efflux pumps. In this review, we summarized the most recent advancements in the understanding of mechanisms of antibiotic resistance in CD and analysed the antibiotic resistance factors present in genomes of a few representative well known, epidemic and MDR CD strains found predominantly in different regions of the world.

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte, Liguria e Valle d'Aosta, Italy.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016-2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

COVID-19 and Clostridioides difficile infection (CDI): Possible implications for elderly patients.

COVID-19 dramatically affects the elderly. Due to the large usage of antibiotics during the current pandemic and the gastrointestinal manifestations of COVID-19, the elderly population, hospitalized patients, residents in LTCFs and persons that survived the COVID-19 might be more prone to Clostridioides difficile infections (CDI). A renewed attention to CDI is necessary during the ongoing COVID-19 pandemic.

Characterization of Scardovia wiggsiae Biofilm by Original Scanning Electron Microscopy Protocol.

Early childhood caries (ECC) is a severe manifestation of carious pathology with rapid and disruptive progression. The ECC microbiota includes a wide variety of bacterial species, among which is an anaerobic newly named species, Scardovia wiggsiae, a previously unidentified Bifidobacterium. Our aim was to provide the first ultrastructural characterization of S. wiggsiae and its biofilm by scanning electron microscopy (SEM) using a protocol that faithfully preserved the biofilm architecture and allowed an investigation at very high magnifications (order of nanometers) and with the appropriate resolution. To accomplish this task, we analyzed Streptococcus mutans' biofilm by conventional SEM and VP-SEM protocols, in addition, we developed an original procedure, named OsO4-RR-TA-IL, which avoids dehydration, drying and sputter coating. This innovative protocol allowed high-resolution and high-magnification imaging (from 10000× to 35000×) in high-vacuum and high-voltage conditions. After comparing three methods, we chose OsO4-RR-TA-IL to investigate S. wiggsiae. It appeared as a fusiform elongated bacterium, without surface specialization, arranged in clusters and submerged in a rich biofilm matrix, which showed a well-developed micro-canalicular system. Our results provide the basis for the development of innovative strategies to quantify the effects of different treatments, in order to establish the best option to counteract ECC in pediatric patients.

Microbiological characteristics of human and animal isolates of Clostridioides difficile in Italy: Results of the Istituto Superiore di Sanità in the years 2006-2016.

The increased incidence of Clostridioides difficile infection (CDI) and the emergence of highly virulent types highlight the need of microbiological characterization to gain insight CDI epidemiological changes. This paper, reporting data obtained by the Istituto Superiore di Sanità Central Laboratory Service for C. difficile (ISS-CLSCD) in 2006-2016, provides a first long-term microbiological analysis of human and animal C. difficile strains circulating in Italy. The number of human isolates analyzed by ISS-CLSCD significantly increased over the time (170 in 2006-2011 vs 661 in 2012-2016). Independently from the year of isolation, 42% of the clinical isolates belonged to the PCR-ribotype (RT) 018-lineage (RT 018, RT 607, RT 541, PR07661 and PR14328), with RT 018 and RT 607 grouping the majority of isolates. This lineage was significantly associated to CDIs occurred in the General Medicine Units, Clinic Units or Long-Term Care Facilities, while it was rarely found in pediatric patients. Although the percentage of isolates positive for the binary toxin (CDT) was stable during the study (20%), several CDT-positive RTs emerged in 2012-2016, including RT 027. In total, 32 RTs overlapped between animals and humans and six of these RTs were non-toxigenic. The two lineages prevalent in animals, the RT 078-lineage and the RT 569-lineage (RT 569, RT 049, RT 056 and RT 727), were also found in humans, while the RT 018-lineage was rarely detected in animals, suggesting that it is prevalently associated to human infections. Sixty-two percent of clinical isolates showed a multidrug-resistance (MDR) phenotype, with resistance to rifampicin characterizing successful RTs. A MDR phenotype was also observed in 18% of animal isolates, in particular from dogs, supporting animals as potential reservoirs of resistant C. difficile strains. Interestingly, multiple resistances were observed in both human and animal non-toxigenic isolates suggesting their contribution to antibiotic resistance spread among C. difficile population. All these data indicate that CDI is an issue of growing concern in Italy, highlighting the need for a standardized surveillance in our Country and an interdisciplinary approach to deal successfully with this infection.

Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance.

Clostridioides difficile (Clostridium difficile) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn6194 [ermB]), tetracycline resistance (Tn6190 [tetM] and Tn6164 [tet44]), and fluoroquinolone resistance (gyrA/B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large "open" pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance.IMPORTANCE Historically, Clostridioides difficile (Clostridium difficile) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance.

Survey, characterization and antimicrobial susceptibility of Clostridium difficile from marine bivalve shellfish of North Adriatic Sea.

Clostridium difficile is a major cause of infectious diarrhea associated to healthcare settings. Community-acquired infections are increasingly reported in the last decade and exposure other than to symptomatic patients rather to contaminated foods or animals is feasible. Occurrence of C. difficile in shellfish raises concern because spores can survive the cooking temperatures given that shellfish is often consumed poorly cooked or raw. Aim of our study was to investigate whether shellfish represents a reservoir of C. difficile human PCR-ribotypes (RTs). 702 shellfish samples of farmed and wild bivalve mollusc species were collected over the 2015-2017 period in North Adriatic Italian Sea to investigate contamination with C. difficile and characterize the isolates in terms of genotypic variability and antimicrobial resistance profile. C. difficile was detected in 16.9% (CI: 14.1%-19.8%) samples: 11.6% mussels and 23.2% clams. Compared to mussels, clams were significantly associated with detection of C. difficile (OR = 2.4, P < 0.01). Overall 113 C. difficile isolates were genotyped and 75 (66.4%) were toxigenic. Fifty-three different RTs were identified. 40.7% C. difficile isolates were among the RTs most commonly involved in human infection in Europe. The profile of antimicrobial susceptibility was determined by E-test; microbiological resistance was frequent against clindamycin (17%), erythromycin (23%), rifampicin (8.8%) and moxifloxacin (10.6%). All isolates were susceptible to metronidazole and one showed MIC > ECOFF for vancomycin. C. difficile strains showed high variety in RTs, most of them already detected in other animals or known as highly virulent and epidemic in humans. These results prompt towards investigating on specific risk mitigation measures against C. difficile and are preliminary for any source attribution and risk assessment study.

Direct detection and characterization of Clostridium difficile from a novel collection device to improve laboratory workflow.

Emergence of Clostridium difficile strains with increased virulence emphasizes the importance of early diagnosis and surveillance of C. difficile infection (CDI). In this study, the new FecalSwab™ collection and transport system was evaluated to improve C. difficile diagnosis. The FecalSwab™ was used for direct C. difficile molecular detection, C. difficile culture/toxigenic culture (TC) and bacterial genomic DNA (bgDNA) extraction. Our results demonstrated that the FecalSwab™ medium could be successfully used as template for Xpert C. difficile binary toxin (BT), regardless of the bacterial load of samples, and for C. difficile culture also after a long storage (30 days) of FecalSwab™ tubes at 4 °C. Furthermore, good-quality bgDNA was extracted from the FecalSwab™ medium for the majority (75%) of the samples analyzed. Typing was performed to fully characterize C. difficile strains isolated during this study and 17 different PCR-ribotypes (RTs) were identified. The results obtained indicate that the FecalSwab™ can be successfully used not only in daily diagnostic routine of C. difficile but also in surveillance and retrospective studies.

Multidrug-resistant infections in long-term care facilities: extended-spectrum ß-lactamase-producing Enterobacteriaceae and hypervirulent antibiotic resistant Clostridium difficile.

Infections due to multidrug-resistant (MDR) organisms in long-term care facilities (LTCFs) residents constitute a public health concern. This multicenter study investigated the frequency of ESBL-producing pathogens and MDR Clostridium difficile in clinical specimens from LTCF residents in Italy. During October 2014-March 2015, all urine and diarrheic fecal samples from LTCF residents (≥65 years) with suspected urinary tract infection or C. difficile infection, respectively, received for diagnosis by 4 hospital laboratories located in different cities were analyzed. Antibiotic susceptibility testing, characterization of resistance genes, and molecular typing of pathogens were performed. Of 806 urine cultures collected from 626 residents at 44 different LTCFs, 492 were positive for microbial infection. Of these, 158 were positive for at least an ESBL-producing Enterobacteriaceae species (32.1%), with Escherichia coli as the most frequent ESBL pathogen (23.4%) followed by Klebsiella pneumoniae (4.5%). Furthermore, 4 carbapenemase producers (0.8%) (1 E. coli with VIM-1and 3 K. pneumoniae with KPC-3) were detected. The CTX-M-15 type ESBL predominated in both E. coli (71.3%) and K. pneumoniae (77.3%). Most E. coli isolates (82.6%) belonged to the ST131/H30 clone/subclone. For K. pneumoniae, ST307 and ST15 were frequent (31.8% and 22.7%, respectively), but isolates harboring blaKPC-3 belonged to CC258. Of 136 diarrheic fecal samples collected from 111 residents at 26 different LTCFs, 21 (15.4%) were positive for toxigenic C. difficile; of these, 13 (62%) were MDR (resistant to 3 or more antimicrobial agents of different classes). The predominant C. difficile polymerase chain reaction ribotype was 356/607 (42.9%), followed by 018, 449, and 078 (14% each). Public health efforts are needed to contain the diffusion of CTX-M-producing Enterobacteriaceae and MDR C. difficile in LTCF settings.

Antibiotic Resistances of Clostridium difficile.

The rapid evolution of antibiotic resistance in Clostridium difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances and most of epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways and biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, recent data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Clostridium difficile causing pediatric infections: New findings from a hospital-based study in Italy.

Recent studies support a change of Clostridium difficile infections (CDIs) epidemiology in pediatric patients. Since limited information is available about C. difficile in this population, we investigated the epidemiology of CDI in a large pediatric hospital that acts as reference centre in Italy and analyzed C. difficile isolates to identify the prevalent PCR-ribotypes (RTs), the binary toxin (CDT)-positive strains and the antibiotic susceptibility patterns. The CDI incidence was 6.6 cases/1000 admissions and the majority (92%) of CDI were healthcare-associated (47% occurred in the Hematology-Oncology and in the Gastroenterology units). Most of symptomatic children <3 years with a positive culture for C. difficile were negative for other gastrointestinal pathogens, supporting C. difficile as cause of disease in these patients, including those showing recurrences. Strains RT020 (16%) and RT014 (14%) were identified as the main cause of infection, while RT356/607 and RT018, predominant in Italian adult patients, were absent (RT356/607) or rarely found (RT018) among children. CDT-positive strains represented the 20% of the total number of isolates analyzed. In particular, two emerging types, RT033 and RT442, were recognized as Toxin A-/Toxin B-/CDT+. Resistance to antibiotics characterized almost 50% of the toxigenic isolates analyzed in this study and, in particular, 20% of them were multidrug resistant (MDR). The emergence and circulation of strains with peculiar toxins profiles and/or MDR strongly highlight the necessity of a rapid CDI diagnosis, a careful monitoring of C. difficile in pediatric patients and a more strict control of antibiotics usage in the Italian pediatric hospitals.

Epidemiology and outcome of Clostridium difficile infections in patients hospitalized in Internal Medicine: findings from the nationwide FADOI-PRACTICE study.

BACKGROUND: Clostridium difficile (CD) is a leading cause of diarrhoea among hospitalized patients. The objective of this study was to evaluate the rate, the optimal diagnostic work-up, and outcome of CD infections (CDI) in Internal Medicine (IM) wards in Italy. METHODS: PRACTICE is an observational prospective study, involving 40 IM Units and evaluating all consecutive patients hospitalized during a 4-month period. CDI were defined in case of diarrhoea when both enzyme immunoassay for GDH, and test for A/B toxin were positive. Patients with CDI were followed-up for recurrences for 4 weeks after the end of therapy. RESULTS: Among the 10,780 patients observed, 103 (0.96 %) showed CDI, at admission or during hospitalization. A positive history for CD, antibiotics in the previous 4 weeks, recent hospitalization, female gender and age were significantly associated with CDI (multivariable analysis). In-hospital mortality was 16.5 % in CD group vs 6.7 % in No-CD group (p < 0.001), whereas median length of hospital stay was 16 (IQR = 13) vs 8 (IQR = 8) days (p < 0.001) among patients with or without CDI, respectively. Rate of CD recurrences was 14.6 %. As a post-hoc evaluation, 23 out of 34 GDH+/Tox- samples were toxin positive, when analysed by molecular method (a real-time PCR assay). The overall CD incidence rate was 5.3/10,000 patient-days. CONCLUSIONS: Our results confirm the severity of CDI in medical wards, showing high in-hospital mortality, prolonged hospitalization and frequent short-term recurrences. Further, our survey supports a 2-3 step algorithm for CD diagnosis: EIA for detecting GDH, A and B toxin, followed by a molecular method in case of toxin-negative samples.

Characterization of Clostridium difficile PCR-ribotype 018: A problematic emerging type.

Recent surveys indicate that the majority of toxigenic Clostridium difficile strains isolated in European hospitals belonged to PCR-ribotypes (RTs) different from RT 027 or RT 078. Among these types, RT 018 has been reported in Italy and, more recently, in Korea and Japan. In Italy, strains RT 018 have become predominant in the early 2000s, whereas the majority of strains isolated before were RT 126, a type belonging to the same lineage as the RT 078. In this study, we have found that Italian strains RT 018 are resistant to erythromycin, clindamycin, moxifloxacin and rifampicin. Rifampicin resistance is rarely observed in strains RT 018 from other countries and in Italian strains RT 078 and RT 126, therefore the decennial use of rifamycin antibiotics in Italy may be one of the driving factors for the spread of RT 018 in our country. The strains RT 018 examined showed a significant higher adhesion to Caco-2 cells compared to strains RT 078 and RT 126. Furthermore, strains RT 018 became predominant in in vitro competition assays with strains RT 078 or RT 126. If maintained in vivo, these characteristics could lead to a rapid colonization of the intestine by strains RT 018. Under the conditions used, isolates RT 018 produced significantly higher toxins levels compared to strains RT 078 and RT 126, while heat-resistant CFUs production seems to be strain-dependent. Robust toxin production and enhanced sporulation could in part explain the high diffusion and interpatient transmissibility observed for strains RT 018 in the hospital environment. In conclusion, the characteristics observed in the Italian isolates RT 018 seem to contribute in conferring an adaptive advantage to these strains, allowing their successful spread in our country.

Transfer of Clostridium difficile Genetic Elements Conferring Resistance to Macrolide-Lincosamide-Streptogramin B (MLSB) Antibiotics.

Molecular analysis is an important tool to investigate Clostridium difficile resistance to macrolide-lincosamide-streptogramin B (MLSB). In particular, the protocols described in this chapter have been designed to investigate the genetic organization of erm(B)-containing elements and to evaluate the capability of these elements to transfer in C. difficile recipient strains using filter mating assay.