Norovirus, Hepatitis A and SARS-CoV-2 surveillance within Chilean rural wastewater treatment plants based on different biological treatment typologies.

During the COVID-19 pandemic, wastewater from WWTPs became an interesting source of epidemiological surveillance. However, there is uncertainty about the influence of treatment type on virus removal. The aim of this study was to assess viral surveillance within wastewater treatment plants (WWTPs) based on different biological treatments. Seasonal monitoring (autumn-winter and spring-summer) was conducted in 10 Chilean rural WWTPs, which were based on activated sludge, aerated lagoons, bio-discs, constructed wetlands, vermifilters and mixed systems. Viruses were measured (influent/effluent) by the RT-qPCR technique, using a commercial kit for SARS-CoV-2, NoV GI, NoV GII, and HAV. The detection of SARS-CoV-2 viral variants by genotyping was performed using SARS-CoV-2 Mutation Assays (ThermoFisher Scientific, USA). JC polyomavirus detection (control), as well as a qPCR technique. Results showed that SARS-CoV-2, NoV GI and GII were detected in influents at values between <5 and 462, 0 to 28, and 0 to 75 GC/mL, respectively. HAV was not detected among the studied WWTPs. The monitored WWTPs removed these viruses at percentages between 0 and 100 %. WWTPs based on activated sludge with bio-discs demonstrated to be the most efficient at removing SARS-CoV-2 (up to 98 %) and NoV GI and GII (100 %). Meanwhile, bio-discs technologies were the least efficient for viral removal, due to biofilm detachment, which could also adsorb viral aggregates. A correlation analysis established that solids, pH, and temperature are the most influential parameters in viral removal. Wastewater-based surveillance at WWTP allowed for the detection of Omicron before the Chilean health authorities notified its presence in the population. In addition, surveillance of viruses and other microorganisms could help assess the potential public health risk of wastewater recycling.

Comprehensive genome analyses of Sellimonas intestinalis, a potential biomarker of homeostasis gut recovery.

Sellimonas intestinalis is a Gram-positive and anaerobic bacterial species previously considered as uncultivable. Although little is known about this Lachnospiraceae family member, its increased abundance has been reported in patients who have recovered from intestinal homeostasis after dysbiosis events. In this context, the aim of the present study was to take advantage of a massive in vitro culture protocol that allowed the recovery of extremely oxygen-sensitive species from faecal samples, which led to isolation of S. intestinalis. Whole genome analyses of 11 S. intestinalis genomes revealed that this species has a highly conserved genome with 99.7 % 16S rRNA gene sequence similarity, average nucleotide polymorphism results >95, and 50.1 % of its coding potential being part of the core genome. Despite this, the variable portion of its genome was informative enough to reveal the existence of three lineages (lineage-I including isolates from Chile and France, lineage-II from South Korea and Finland, and lineage-III from China and one isolate from the USA) and evidence of some recombination signals. The identification of a cluster of orthologous groups revealed a high number of genes involved in metabolism, including amino acid and carbohydrate transport as well as energy production and conversion, which matches with the metabolic profile previously reported for microbiota from healthy individuals. Additionally, virulence factors and antimicrobial resistance genes were found (mainly in lineage-III), which could favour their survival during antibiotic-induced dysbiosis. These findings provide the basis of knowledge about the potential of S. intestinalis as a bioindicator of intestinal homeostasis recovery and contribute to advancing the characterization of gut microbiota members with beneficial potential.

Genome Sequence of Clostridium paraputrificum 373-A1 Isolated in Chile from a Patient Infected with Clostridium difficile.

Clostridium paraputrificum is a gut microbiota member reported in several cases of bacteremia and coinfections. So far, only one genome sequence of a C. paraputrificum (AGR2156) isolate is available. Here, we present the draft genome of C. paraputrificum strain 373-A1, isolated from stools from a patient with C. difficile infection.

Outcome of relapsing Clostridium difficile infections do not correlate with virulence-, spore- and vegetative cell-associated phenotypes.

One of the main clinical challenges of Clostridium difficile infections (CDI) is the high rate of relapse episodes. The main determinants involved in relapse of CDI include the presence of antibiotic-resistant C. difficile spores in the colonic environment and a permanent state of dysbiosis of the microbiota caused by antibiotic therapy. A possible scenario is that phenotypes related to the persistence of C. difficile spores might contribute to relapsing infections. In this study, 8 C. difficile isolates recovered from 4 cases with relapsing infection, and 9 isolates recovered from single infection cases were analyzed for PCR ribotyping and the presence of tcdA, tcdB and cdtAB genes. Factors associated to spore persistence, sporulation, spore adherence and biofilm formation and sporulation during biofilm formation were characterized. We also evaluated motility and cytotoxicity. However, we observed no significant difference in the analyzed phenotypes among the different clinical outcomes, most likely due to the high variability observed among strains within clinical backgrounds in each phenotype and the small sample size. It is noteworthy that C. difficile spores adhered to similar extents to undifferentiated and differentiated Caco-2 cells. By contrast, spores of all clinical isolates tested had increased germination efficiency in presence of taurocholate, while decreased sporulation rate during biofilm development in the presence of glucose. In conclusion, these results show that, at least in this cohort of patients, the described phenotypes are not detrimental in the clinical outcome of the disease.

Survival of Clostridium difficile spores at low temperatures.

Clostridium difficile's presence has been reported in meat products stored typically at low temperatures. This study evaluated the viability in phosphate buffer saline (PBS) of spores from epidemic C. difficile strain R20291 (4.6 log CFU/ml) and M120 (7.8 log CFU/ml). Viability was assessed during 4 months at -80 °C, -20 °C, 4 °C (refrigeration), and 23 °C (room temperature), and after 10 freeze (-20 °C)/thaw (+23 °C) cycles. Although spore viability decreased, significant viability was still observed after 4 months at -20 °C, i.e., 3.5 and 3.9 log CFU/ml and -80 °C, i.e., 6.0 and 6.1 log CFU/ml for strains R20291 and M120, respectively. The same trend was observed for M120 at 4 °C and 23 °C, while for R20291 the viability change was non-significant at 4 °C but increased significantly at 23 °C (p > 0.05). After 10 freeze-thaw cycles, viability of both strains decreased but a significant fraction remained viable (4.3 and 6.3 log CFU/ml for strain R20291 and M120, respectively). Strikingly, both strains showed higher viability in a meat model than in PBS. A small but significant decrease (p < 0.05) from 6.7 to 6.3 log CFU/ml in M120 viability was observed after 2-month storage in the meat model while the decrease from an initial 3.4 log CFU/ml observed for R20291 was non-significant (p = 0.12). In summary, C. difficile spores can survive low-temperature conditions for up to 4 months.

Rendimiento de índices pronósticos en infección asociada a Clostridium difficile grave: Análisis retrospectivo en un hospital universitario / Performance of prognostic index in severe Clostridium difficile-associated infection: Retrospective analysis in a university hospital

Introduction: By consensus severe, Clostridium difficile-associated infection (CDAI) is one that results in hospitalization in ICU, colectomy or death within 30 days. Multiple prognostic indices (IP) attempt to predict these adverse events. Objective: To evaluate the performance of 4 PI in predicting severe CDI. Methods: Hospitalized patients ≥ 18 years old with ICD were retrospectively evaluated. Patients with recurrent infection or hematological cancer were excluded. Four PI were evaluated: UPMC version 1, Calgary version 1, Hines VA and Calgary version 2. Results: Seven of 81 patients (8.1%) met the definition of severe CDI. Positive predicted value (PPV) and negative predicted value (NPV) of PI ranged from 20-75% and 91.3-95.7%, respectively. Only Hines VA index had a satisfactory Kappa index (0.74; 95% CI 0.41-1) with a PPV of 75% and NPV of 95,7%. However, because of the variables included, this PI could be calculated only in 32.6% of patients. Conclusion: Hines VA index has the best predicted value and agreement to rule out a severe CDI. Like others PI it has the limitation of including difficult variables to assess in all patients and tends to overestimate an unfavorable course.

Introducción: Por consenso, la infección asociada a Clostridium difficile (IACD) grave es aquella que resulta en hospitalización en unidad de cuidados intensivos, colectomía o muerte dentro de 30 días. Múltiples índices pronósticos (IP) intentan predecir estos eventos adversos. Objetivo: evaluar el rendimiento de cuatro IP en la predicción de IACD grave. Metodología: pacientes hospitalizados ≥ 18 años con IACD fueron evaluados retrospectivamente. Se excluyeron pacientes con infección recurrente o cáncer hematológico. Se evaluaron cuatro IP: UPMC versión 1, Calgary versión 1, Hines VA y Calgary versión 2. Resultados: Siete de 81 pacientes (8,1%) presentaron una IACD grave. El valor predictor positivo (VPP) y valor predictor negativo (VPN) de los IP varió entre 20-75% y 91,3-95,7%, respectivamente. Sólo el índice de Hines VA tuvo un índice Kappa satisfactorio (0,74;IC 95% 0,46-1) con un VPP de 75% y un VPN de 95,7%. Sin embargo, por las variables incluidas en este IP, sólo pudo ser calculado en 32,6% de los pacientes. Conclusión: El índice de Hines VA presenta el mejor valor predictor y concordancia para descartar una IACD grave. Como otros IP, tiene la limitación de incluir variables difícilmente evaluables en todos los pacientes y tiende a sobreestimar un curso desfavorable.

[Performance of prognostic index in severe Clostridium difficile-associated infection: retrospective analysis in a university hospital]. / Rendimiento de índices pronósticos en infección asociada a Clostridium difficile grave: Análisis retrospectivo en un hospital universitario.

INTRODUCTION: By consensus severe, Clostridium difficile-associated infection (CDAI) is one that results in hospitalization in ICU, colectomy or death within 30 days. Multiple prognostic indices (IP) attempt to predict these adverse events. OBJECTIVE: To evaluate the performance of 4 PI in predicting severe CDI. METHODS: Hospitalized patients ≥ 18 years old with ICD were retrospectively evaluated. Patients with recurrent infection or hematological cancer were excluded. Four PI were evaluated: UPMC version 1, Calgary version 1, Hines VA and Calgary version 2. RESULTS: Seven of 81 patients (8.1%) met the definition of severe CDI. Positive predicted value (PPV) and negative predicted value (NPV) of PI ranged from 20-75% and 91.3-95.7%, respectively. Only Hines VA index had a satisfactory Kappa index (0.74; 95% CI 0.41-1) with a PPV of 75% and NPV of 95,7%. However, because of the variables included, this PI could be calculated only in 32.6% of patients. CONCLUSION: Hines VA index has the best predicted value and agreement to rule out a severe CDI. Like others PI it has the limitation of including difficult variables to assess in all patients and tends to overestimate an unfavorable course.

The Clostridium difficile exosporium cysteine (CdeC)-rich protein is required for exosporium morphogenesis and coat assembly.

Clostridium difficile is an important nosocomial pathogen that has become a major cause of antibiotic-associated diarrhea. There is a general consensus that C. difficile spores play an important role in C. difficile pathogenesis, contributing to infection, persistence, and transmission. Evidence has demonstrated that C. difficile spores have an outermost layer, termed the exosporium, that plays some role in adherence to intestinal epithelial cells. Recently, the protein encoded by CD1067 was shown to be present in trypsin-exosporium extracts of C. difficile 630 spores. In this study, we renamed the CD1067 protein Clostridium difficile exosporium cysteine-rich protein (CdeC) and characterized its role in the structure and properties of C. difficile spores. CdeC is expressed under sporulation conditions and localizes to the C. difficile spore. Through the construction of an ΔcdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ΔcdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) ΔcdeC spores were more sensitive to lysozyme, ethanol, and heat treatment than wild-type spores; and (iv) despite the almost complete absence of the exosporium layer, ΔcdeC spores adhered at higher levels than wild-type spores to intestinal epithelium cell lines (i.e., HT-29 and Caco-2 cells). Collectively, these results indicate that CdeC is essential for exosporium morphogenesis and the correct assembly of the spore coat of C. difficile.