Whole Sequencing and Detailed Analysis of SARS-CoV-2 Genomes in Southeast Spain: Identification of Recurrent Mutations in the 20E (EU1) Variant with Some Clinical Implications.

During the COVID-19 pandemic caused by SARS-CoV-2, new waves have been associated with new variants and have the potential to escape vaccinations. Therefore, it is useful to conduct retrospective genomic surveillance research. Herein, we present a detailed analysis of 88 SARS-CoV-2 genomes belonging to samples taken from COVID-19 patients from October 2020 to April 2021 at the "Reina Sofía" Hospital (Murcia, Spain) focused to variant appeared later. The results at the mentioned stage show the turning point since the 20E (EU1) variant was still prevalent (71.6%), but Alpha was bursting to 14.8%. Concern mutations have been found in 5 genomes classified as 20E (EU1), which were not characteristic of this still little evolved variant. Most of those mutations are found in the spike protein, namely Δ69-70, E484K, Q675H and P681H. However, a relevant deletion in ORF1a at positions 3675-3677 was also identified. These mutations have been reported in many later SARS-CoV-2 lineages, including Omicron. Taken together, our data suggest that preferential emergence mutations could already be present in the early converging evolution. Aside from this, the molecular information has been contrasted with clinical data. Statistical analyses suggest that the correlation between age and severity criteria is significantly higher in the viral samples with more accumulated changes.

Occurrence of Clostridioides (Clostridium) difficile in Poultry Giblets at Slaughter and in Retail Pork and Poultry Meat in Southeastern Spain.

ABSTRACT: Raw meat and meat products contaminated with Clostridioides difficile could be a vehicle for spreading community-associated C. difficile infection. This study was conducted to determine the occurrence of C. difficile in pork and poultry meat samples (n = 325) from retail establishments and in edible giblet samples (n = 36) from a poultry processing plant in Murcia (southeastern Spain). C. difficile was isolated after selective enrichment from 2% (6 of 361) of the samples, all of which were from the poultry processing plant. These isolates were recovered from 17% (6 of 36) of the edible chicken giblets, i.e., 28% (5 of 18) of the gizzard samples and 6% (1 of 18) of the liver samples. All six C. difficile isolates were negative for toxin A and B genes by PCR assay. These findings indicate that C. difficile can survive in the gastric acid of the chicken gizzard and could be transmitted to other meat products. However, the very low prevalence of C. difficile in the tested samples indicates that retail meat may not be an important source for transmission of C. difficile to humans.

Presence of toxigenic Clostridioides (Clostridium) difficile in edible bivalve mollusks in Spain.

Clostridioides difficile reservoirs other than humans are becoming increasingly recognized, and the occurrence of the pathogen in shellfish raises concern because spores can survive cooking temperature and edible bivalve mollusks are often consumed raw or poorly cooked. This study was conducted to determine the occurrence of pathogenic C. difficile in retail bivalve mollusks. The microbiological quality of samples was also checked through the isolation of Salmonella spp. and Escherichia coli. We analyzed 129 mollusk samples from different fishmongers and grocery stores in Murcia. C. difficile was isolated from 8.53% (11/129) of the mollusks investigated. Four C. difficile isolates harbored genes for the production of toxin A and B. Salmonella spp. were not isolated from any sample and E. coli was isolated from 1.55% (2/129) of the samples, in both cases in accordance with the current legal requirements for consumption. Our findings indicate that the intake of raw or poorly cooked contaminated bivalve mollusks could be a potential source of C. difficile, leading to a risk for human health.

Effect of Specimen Type and Processing on the Detection of Clostridioides [Clostridium] difficile in Piglet Fecal Samples.

Subclinical Clostridioides difficile colonization in piglets could be a potential source of this bacterium for community-acquired C. difficile infection. The purposes of this study were to assess the effect of specimen type and processing on C. difficile isolation, culture, and detection by polymerase chain reaction (PCR), and to determine the occurrence of C. difficile in piglets of different ages. We compared different culture procedures-direct plating, ethanol shock, and an enrichment step-to isolate C. difficile from swine feces and rectal swabs. DNA was isolated directly from feces, processed feces, and bacterial isolates to detect the triose phosphate isomerase (tpi) gene and identify the toxins A and B genes. The results show that ethanol shock increased the C. difficile isolation from feces, while it decreased it for rectal swabs, in comparison with direct plating. The use of the enrichment broth gave the highest C. difficile recovery from both types of specimen. Our findings show low sensitivity for tpi gene detection after the DNA extraction directly from feces and an increase in PCR-positive samples when feces were processed before the DNA extraction. The overall prevalence of C. difficile was 16.9% (22/130), of which 100% were found to be toxigenic as assessed by the enrichment culture of fecal samples. The rate of isolation of positive samples decreased with the animal age, regardless of the presence or absence of diarrhea. Our results demonstrate the persistent reservoir of toxigenic C. difficile in fecal samples of piglets and support the impact of specimen processing on its isolation.

A review of Clostridioides [Clostridium] difficile occurrence through the food chain.

The epidemiology and transmission of Clostridioides difficile, particularly for community-associated infections, are not completely understood. Although there have been no confirmed cases of any foodborne disease caused by C. difficile, its occurrence in livestock and foods suggests that contaminated food products with spores could be a vehicle to spread C. difficile infection. This review proposes potential sources of C. difficile infection in the community and contamination routes of food products. Based on European research, it also summarizes the occurrence and organism characterization of C. difficile in animals at slaughterhouses and in human foods. Most of the analyzed literature reported prevalence in retail foods of less than 8%, including microorganism belonging to the ribotype 078, an important hypervirulent strain involved in disease in humans. This prevalence in Europe is underestimated, being lower that reported in North America (rates up to 42%), probably due of the lack of an ISO procedure for the detection of C. difficile in food products that preclude the comparison of prevalence data from different studies. The survival and growth of vegetative C. difficile cells and the resistance of its spores in foods are discussed as well as the risk factors of acquisition CDI from food products.