Predominance of Clostridioides difficile PCR ribotype 181 in northern Greece, 2016-2019.

OBJECTIVES: The epidemiology of Clostridioides difficile infection (CDI) has undergone many changes since the beginning of this century and continues to evolve based on recent studies. Here, we performed a molecular analysis of C. difficile isolates in northern Greece across 10 health-care facilities, spanning from 2016 to 2019. METHODS: 221 C. difficile isolates were cultured from stool samples of hospitalized patients with diarrhea and screened by PCR for the presence of the toxin A (tcdA), toxin B (tcdB), the binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC. PCR ribotyping of the cultured isolates was performed by a standardized protocol for capillary gel-based PCR ribotyping and an international database with well-documented reference strains. RESULTS: Thirty-five different PCR ribotypes were identified. The most common RTs identified were: 181 (36%, 80/221), 017 (10%, 21/221), 126 (9%, 19/221), 078 (4%, 9/221) and 012 (4%, 8/221). Notably, the predominant RT181, with toxin profile tcdA+tcdB+cdtA+cdtB+, was identified in seven out of ten participating hospitals. CONCLUSIONS: Multiple C. difficile ribotypes have been circulating in the northern Greece region with RTs 181 (closely related to 027), 017, 126 and 078 being predominant.

Short communication: Bovine mastitis caused by a multidrug-resistant, mcr-1-positive (colistin-resistant), extended-spectrum ß-lactamase-producing Escherichia coli clone on a Greek dairy farm.

We performed a survey aimed at analyzing milk samples collected from cows with mastitis for the presence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Single-quarter mastitic milk samples obtained from 400 cows in 23 Greek dairy herds with a history of E. coli mastitis were processed for the selective isolation of ESBL-producing E. coli. The antimicrobial susceptibility of the ESBL-producing isolates was analyzed using agar disk diffusion, and minimum inhibitory concentrations of colistin were determined by broth microdilution. We used PCR followed by DNA sequencing to characterize the ß-lactamases and mcr-1 (colistin resistance) genes, and for phylotyping and multilocus sequence typing. We found a total of 89/400 (22.25%) E. coli isolates from 12/23 (52%) farms. Six isolates originating from 6 cows on a single farm were ESBL producers and were resistant to cefquinome, amoxicillin-clavulanic acid, aztreonam, ampicillin, and colistin. Five of these isolates were resistant to trimethoprim-sulfamethoxazole, and 5 to streptomycin. The 6 ESBL producers were mcr-1-positive and carried blaTEM-1 genes; 3 also carried blaCTX-M genes, and 3 carried blaSHV genes. All of the ESBL producers belonged to phylogroup A, multilocus sequence type ST666 (n = 5), or a single locus variant of ST666 (n = 1). To our knowledge, this is the first report of endemic bovine mastitis caused by mcr-1-positive, ESBL-producing E. coli. These results highlight the value of active surveillance of antimicrobial resistance not commonly tested by diagnostic laboratories for the early detection of novel resistant strains.

Epidemiology and antimicrobial susceptibility of Staphylococcus lugdunensis in a Greek tertiary-care hospital.

Staphylococcus lugdunensis is considered more pathogenic than other coagulase-negative Staphylococci (CoNS), with its virulence resembling that of Staphylococcus aureus. We report a retrospective study of all S. lugdunensis infection cases during a 3.5-year period in a large tertiary university hospital in Greece. S.lugdunensis was susceptible to most tested antibiotics, although a high resistance percentage was found to clindamycin (27%) and erythromycin (25%). The susceptibility rate to penicillin was 49%, much lower than previously reported elsewhere, indicating that penicillin may not be an optimal treatment choice for S. lugdunensis infections in our region.

Bacteremia due to a toxin A-negative, B-positive Clostridioides difficile ribotype 017 strain.

Clostridioides difficile is the leading cause of healthcare-associated diarrhea. Although the incidence of C. difficile-associated diarrhea is increasing worldwide, bacteremia due to C. difficile is relatively rare and represents the least frequent extra-colonic manifestation of C. difficile infection. To date, only 60 C. difficile bacteremia cases with detailed clinical patient characteristics have been reported in the literature, and another 77 cases have been identified in epidemiological reports. We report a rare and fatal case of bacteremia due to C. difficile from a Greek hospital.

An outbreak of Clostridioides difficile infections due to a 027-like PCR ribotype 181 in a rehabilitation centre: Epidemiological and microbiological characteristics.

Clostridioides difficile is one of the most important healthcare-associated pathogens. Recently, several new 027-like types have been found that all belong to the multilocus sequence typing (MLST) Clade 2. We report a rapidly spreading outbreak of C. difficile infections (CDI) due to a newly identified PCR ribotype (RT) 181 in a Rehabilitation Centre (RC). Genomic analysis revealed the outbreak strain, not previously identified in Greece, belonged to clade 2, sequence type (ST) 1 and had a 18bp deletion in tcdC at position 311 together with a single nucleotide deletion at position 117, similarly to RT 027. The presence of a clonal outbreak was confirmed by whole genome sequencing, yet the source of this ribotype remained unclear. The emergence and rapid spread of new C. difficile ribotypes highlights the need for ongoing C. difficile surveillance and better understanding of overall Clade 2 phylogeny.

Clostridium difficile infection: New insights into therapeutic options.

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings and represents a major social and economic burden. The major virulence determinants are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), encoded within the pathogenicity locus. Traditional therapies, such as metronidazole and vancomycin, frequently lead to a vicious circle of recurrences due to their action against normal human microbiome. New disease management strategies together with the development of novel therapeutic and containment approaches are needed in order to better control outbreaks and treat patients. This article provides an overview of currently available CDI treatment options and discusses the most promising therapies under development.

Wide dissemination of linezolid-resistant Staphylococcus epidermidis in Greece is associated with a linezolid-dependent ST22 clone.

OBJECTIVES: Dependence on linezolid was recently described as significant growth acceleration of linezolid-resistant Staphylococcus epidermidis (LRSE) isolates upon linezolid exposure. We investigated the possible contribution of linezolid dependence to LRSE dissemination in Greece. METHODS: Linezolid resistance rates were estimated in six tertiary hospitals located throughout Greece between 2011 and 2013. Sixty-three randomly selected LRSE recovered in these hospitals during this period were studied. Growth curve analysis was conducted with and without linezolid. Clonality of the isolates was investigated by PFGE and MLST. RESULTS: During the study period, the LRSE rate in the participating hospitals rose significantly from 6.9% to 9% (P = 0.006); the increase was more prominent in ICUs (from 15.1% to 20.9%; P = 0.005). Forty-seven (74.6%) of the 63 LRSE, derived from all study hospitals, clearly exhibited linezolid dependence, growing significantly faster in the presence of 16 and 32 mg/L linezolid. Of note, 61 (96.8%) LRSE exhibited a single macrorestriction pattern and belonged to ST22, which included all linezolid-dependent LRSE. The remaining two LRSE belonged to unique STs. Five of six linezolid-dependent isolates tested also exhibited linezolid dependence upon exposure to 8 mg/L linezolid. Interestingly, five of six ST22 linezolid-non-dependent isolates tested developed linezolid dependence when linezolid exposure preceded growth analysis. CONCLUSIONS: The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.

Molecular epidemiology of noroviruses in Northern Greece, 2005-2006.

Noroviruses (NoVs) are important human pathogens associated with acute viral gastroenteritis worldwide, displaying significant genetic heterogeneity. Genotype GII.4 is responsible for the majority of outbreaks reported to date. A total of 460 faecal samples from sporadic gastroenteritis cases were screened for the presence of NoV RNA. Four additional human samples collected during a waterborne NoV gastroenteritis outbreak observed in 2005 in northern Greece, were also included in the study. All PCR-positive samples were tested further using a multiplex RT-PCR, which targets the viral capsid VP1 region D. PCR products from all outbreak samples and from 20 randomly selected samples were sequenced. Phylogenetic analysis revealed that GII.4 genotype predominated (70%), while genotypes GII.2 (10%), GII.7 (15%), and GI.1 (5%) were also detected. All the outbreak NoV strains belonged to the GI.1 genotype. The present study provides a first insight into the epidemiology and genetic diversity of NoVs in Greece and shows that various strains are circulating in the country and cause sporadic cases or outbreaks.

Clostridioides difficile infection epidemiology during the COVID-19 pandemic in Greece.

Aim: The aim was to highlight the incidence and epidemiology of C. difficile infections (CDI) in a tertiary Greek hospital during the COVID-19 pandemic. Methods: A single-center prospective observational cohort study was conducted (October 2021 until April 2022). 125 C. difficile isolates were cultured from hospitalized patients stool samples and screened by PCR for toxin A (tcdA), toxin B (tcdB), binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC. Results: The incidence of CDI increased to 13.1 infections per 10,000 bed days. The most common PCR ribotypes identified included hypervirulent RT027-related RT181 (73.6%), presumably hypervirulent RT126 (8.0%) and toxin A negative RT017 (7.2%). Conclusion: Although the incidence of CDI increased significantly, the CDI epidemiology remained stable.

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Lemierre's syndrome presenting to the ED: rapidly fatal sepsis caused by methicillin-susceptible Staphylococcus aureus Staphylococcus protein A type t044.

We describe the case of a fatal septic illness in a previously healthy young man caused by community-acquired methicillin-susceptible Staphylococcus aureus of Staphylococcus protein A (spa) type t044. The patient developed a devastating Lemierre-like syndrome with extensive thrombosis of inferior vena cava and iliac veins with multiple metastatic septic emboli of the lungs. He presented to the emergency department with rapidly progressing sepsis followed by multiple organ dysfunction syndrome. Recognition of such virulent community-acquired strains is of great importance because they could prove to be emerging pathogens for life-threatening diseases.

Implication of COVID-19 pandemic on the incidence of Clostridioides difficile infection in a Greek tertiary hospital.

Introduction. C. difficile infection (CDI) represents an important global threat. In the COVID-19 era, the multifactorial nature of CDI has emerged.Hypothesis - Aim. The aim was to assess the impact of COVID-19 pandemic on the incidence of CDI in a Greek hospital.Methodology. A retrospective study was performed throughout a 51 month period (January 2018 to March 2022), divided into two periods: pre-pandemic (January 2018 to February 2020) and COVID-19 pandemic (March 2020 to March 2022). The effects of the pandemic compared to the pre-pandemic period on the incidence of CDI [expressed as infections per 10 000 bed days (IBD)] were studied using interrupted time-series analysis.Results. Throughout the study, there was an increase in the monthly CDI incidence from 0.00 to 11.77 IBD (P<0.001). Interrupted time-series disclosed an increase in CDI incidence during the pre-pandemic period from 0.00 to 3.36 IBD (P<0.001). During the COVID-19 pandemic period the linear trend for monthly CDI rose from 2.65 to 13.93 IBD (P<0.001). The increase rate was higher during the COVID-19 pandemic period (r2 = +0.47) compared to the pre-pandemic period (r1 = +0.16).Conclusion. A significant increase of CDI incidence was observed, with the rate of the rise being more intense during the COVID-19 pandemic.

Diabetic Patient Adherence to Yearly Influenza Vaccination in Northern Greece.

Background Influenza virus infection is associated with increased morbidity and mortality in patients with diabetes mellitus. Public health authorities recommend yearly vaccination of diabetic patients against seasonal influenza. Methods We surveyed to define the adherence to influenza vaccination and associated factors among diabetic patients in Thessaloniki, Greece. Predictors of adherence to yearly influenza vaccination were assessed with logistic regression models. Results A total of 206 patients were enrolled, with 47.1% reporting yearly vaccination against influenza (95% confidence interval, CI:40.3% to 53.9%). In univariate models, the absence of additional indications for vaccination was associated with a decreased likelihood of vaccination uptake (OR:0.29, 95% CI:0.11 to 0.68, p=0.007); older diabetic patients were more likely to receive influenza vaccination (34% increase per 10 years of age). These associations were attenuated in multivariable analysis. Conclusion Our study demonstrates a significant gap in influenza vaccination coverage rate in diabetic patients. Our data could be extrapolated to enhance the uptake of vaccines against SARS-CoV-2: emphasis should be placed on patient education.

Metabolic Phenotyping Study of Mouse Brain Following Microbiome Disruption by C.difficile Colonization.

Clostridioides difficile infection (CDI) is responsible for an increasing number of cases of post-antibiotic diarrhea worldwide, which has high severity and mortality among hospitalized elderly patients. The disruption of gut microbiota due to antibacterial medication facilitates the intestinal colonization of C. difficile. In the present study, a murine model was used to investigate the potential effects of antibiotic administration and subsequent colonization by C. difficile, as well as the effects of three different 10-day treatments (metronidazole, probiotics, and fecal microbiota transplantation), on the brain metabolome for the first time. Four different metabolomic-based methods (targeted HILIC-MS/MS, untargeted RP-LC-HRMS/MS, targeted GC-MS/MS, and untargeted GC-MS) were applied, resulting in the identification of 217 unique metabolites in the brain extracts, mainly glycerophospholipids, glycerolipids, amino acids, carbohydrates, and fatty acids. Univariate and multivariate statistical analysis revealed that CDI, as well as the subsequent treatments, altered significantly several brain metabolites, probably due to gut dysbiosis, and affected the brain through the gut-brain axis. Notably, none of the therapeutic approaches completely restored the brain metabolic profile to the original, healthy, and non-infected phenotype, even after 10 days of treatment.

Molecular epidemiology of Clostridium difficile strains in children compared with that of strains circulating in adults with Clostridium difficile-associated infection.

Molecular analysis of Clostridium difficile (28 isolates) from children (n = 128) in Oxfordshire, United Kingdom, identified eight toxigenic genotypes. Six of these were isolated from 27% of concurrent adult C. difficile-associated infections studied (n = 83). No children carried hypervirulent PCR ribotype 027. Children could participate in the transmission of some adult disease-causing genotypes.

Clostridium difficile infection: a comprehensive review.

Clostridium difficile is one of the most important causes of healthcare acquired diarrhea. The disease spectrum caused by C. difficile infection ranges from mild, self-limited, illness to a severe, life-threatening colitis. The incidence of C. difficile associated disease has risen dramatically over the last decade, leading to increased research interest aiming at the discovery of new virulence factors and the development of new treatment and prevention regimens. This review summarizes the pathogenesis and changing epidemiology of C. difficile associated disease, the clinical spectrum and laboratory methods to diagnose C. difficile infection, and current treatment strategies.

Multilocus sequence typing of Clostridium difficile.

A robust high-throughput multilocus sequence typing (MLST) scheme for Clostridium difficile was developed and validated using a diverse collection of 50 reference isolates representing 45 different PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (http://pubmlst.org/cdifficile/) to facilitate the dissemination and comparison of C. difficile MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of C. difficile in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.

Insights into the Role of Human Gut Microbiota in Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) has emerged as a major health problem worldwide. A major risk factor for disease development is prior antibiotic use, which disrupts the normal gut microbiota by altering its composition and the gut's metabolic functions, leading to the loss of colonization resistance and subsequent CDI. Data from human studies have shown that the presence of C. difficile, either as a colonizer or as a pathogen, is associated with a decreased level of gut microbiota diversity. The investigation of the gut's microbial communities, in both healthy subjects and patients with CDI, elucidate the role of microbiota and improve the current biotherapeutics for patients with CDI. Fecal microbiota transplantation has a major role in managing CDI, aiming at re-establishing colonization resistance in the host gastrointestinal tract by replenishing the gut microbiota. New techniques, such as post-genomics, proteomics and metabolomics analyses, can possibly determine in the future the way in which C. difficile eradicates colonization resistance, paving the way for the development of new, more successful treatments and prevention. The aim of the present review is to present recent data concerning the human gut microbiota with a focus on its important role in health and disease.

Clostridioides (Clostridium) Difficile in Food-Producing Animals, Horses and Household Pets: A Comprehensive Review.

Clostridioides (Clostridium) difficile is ubiquitous in the environment and is also considered as a bacterium of great importance in diarrhea-associated disease for humans and different animal species. Food animals and household pets are frequently found positive for toxigenic C. difficile without exposing clinical signs of infection. Humans and animals share common C. difficile ribotypes (RTs) suggesting potential zoonotic transmission. However, the role of animals for the development of human infection due to C. difficile remains unclear. One major public health issue is the existence of asymptomatic animals that carry and shed the bacterium to the environment, and infect individuals or populations, directly or through the food chain. C. difficile ribotype 078 is frequently isolated from food animals and household pets as well as from their environment. Nevertheless, direct evidence for the transmission of this particular ribotype from animals to humans has never been established. This review will summarize the current available data on epidemiology, clinical presentations, risk factors and laboratory diagnosis of C. difficile infection in food animals and household pets, outline potential prevention and control strategies, and also describe the current evidence towards a zoonotic potential of C. difficile infection.

A two-step approach improves the diagnosis of Clostridium difficile infection.

Clostridium difficile infection (CDI) is a major cause of health care-associated diarrhea. The aim of the present study was to evaluate a two-step approach for the diagnosis of CDI. The two-step procedure consisted of GDH-toxin A/B EIA (Enzyme immunoassay targeting enterotoxin A and Cytotoxin B), followed by PCR detecting toxigenic C. difficile. Results indicate that EIAs provide a rapid screening assay for the laboratory diagnosis of CDI but, in GDH-positive and toxins-negative samples, EIA should be always followed by PCR to distinguish toxigenic vs nontoxigenic strains. GDH-toxin A/B EIA-rapid test has high specificity but low sensitivity to detect CDI. The implementation of a two-step procedure significantly increases the diagnostic accuracy to detect CDI and provides a toxigenic type characterization of C. difficile isolates.

Clostridium difficile infections in a university hospital in Greece are mainly associated with PCR ribotypes 017 and 126.

PURPOSE: Data regarding the incidence and molecular epidemiology of Clostridium difficile infections (CDIs) in Greece are limited. METHODOLOGY: A retrospective study of all laboratory-confirmed CDI cases in a university hospital during a 9-month period. Stool samples from inpatients with diarrhoea were tested with a combined glutamate dehydrogenase (GDH) and toxin enzyme immunoassay (EIA) test, as part of a two-step algorithm for CDI testing. All GDH-positive samples were cultured and isolates were further tested for the presence of toxin genes and characterized by PCR ribotyping. RESULTS: The incidence of CDI in our hospital was 25 per 10 000 hospital admissions. Of 33 CDI cases, 72.7 % were hospital-acquired. Fourteen different PCR ribotypes were identified, of which 017 (21.2 %), 078/126 (15.1 %) and RT202 and RT106 (9 %) were the most prevalent. Most patients had a risk profile of recent antibiotic use, older age and comorbidities. Despite mild CDI clinical characteristics, six cases showed complications and led to 18.2 % mortality. CONCLUSION: The CDI incidence was comparable to that in other European countries. The hypervirulent PCR ribotype 027 was not found, whereas ribotypes 017 and 126 predominated. Most CDI cases were in patients who used antibiotics, emphasizing that antimicrobial stewardship should be considered as a cornerstone for the prevention of CDI.