PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES.

The WRN protein mutated in the hereditary premature aging disorder Werner syndrome plays a vital role in handling, processing, and restoring perturbed replication forks. One of its most abundant partners, Replication Protein A (RPA), has been shown to robustly enhance WRN helicase activity in specific cases when tested in vitro. However, the significance of RPA-binding to WRN at replication forks in vivo has remained largely unexplored. In this study, we have identified several conserved phosphorylation sites in the acidic domain of WRN that are targeted by Casein Kinase 2 (CK2). Surprisingly, these phosphorylation sites are essential for the interaction between WRN and RPA, both in vitro and in human cells. By characterizing a CK2-unphosphorylatable WRN mutant that lacks the ability to bind RPA, we have determined that the WRN-RPA complex plays a critical role in fork recovery after replication stress whereas the WRN-RPA interaction is not necessary for the processing of replication forks or preventing DNA damage when forks stall or collapse. When WRN fails to bind RPA, fork recovery is impaired, leading to the accumulation of single-stranded DNA gaps in the parental strands, which are further enlarged by the structure-specific nuclease MRE11. Notably, RPA-binding by WRN and its helicase activity are crucial for countering the persistence of G4 structures after fork stalling. Therefore, our findings reveal for the first time a novel role for the WRN-RPA interaction to facilitate fork restart, thereby minimizing G4 accumulation at single-stranded DNA gaps and suppressing accumulation of unreplicated regions that may lead to MUS81-dependent double-strand breaks requiring efficient repair by RAD51 to prevent excessive DNA damage.

Survival of different microbial strains in pure and diluted tattoo inks.

Several microorganisms can be found in tattoo inks injected into the skin, despite the ink matrix being considered inhospitable to microbial growth. Studies on the microbial quality of tattoo inks have reported the presence of microorganisms in most of the samples. This study aimed to assess the survival of environmental and human microbial species, selected on the specific criteria, in tattoo inks. Undiluted sterile black ink and serial dilutions (10-fold/100-fold) were each separately seeded with four bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Mycobacterium fortuitum), one yeast (Candida albicans), and one mould (Fusarium solani). Their survival was periodically tested using cultural methods. No tested microorganisms were able to survive in undiluted ink, except for B. pumilus that survived up to 3 weeks. All the tested species, except for S. aureus, showed survivability for up to 10 weeks in 100-fold diluted inks, and P. aeruginosa, M. fortuitum, and C. albicans were even able to grow. B. pumilus and F. solani had good rates of survival even at the smallest dilution. The ability of microorganisms to survive and grow in tattoo inks could have health implications if contaminated ink dilutions are used during tattooing practices and stored for a long time.

An Italian survey on the microbiological safety of toys containing aqueous media.

AIMS: The purpose of the present investigation is to fill the current gap in information regarding the microbiological quality of toys containing aqueous media and the related risks for users. METHODS: Over 18 years, a total of 491 sealed toys containing aqueous media were analysed using conventional microbial culture methods. In addition, molecular methods (PCR/nested RT-PCR, followed by Sanger sequencing) were employed to test for enteric viruses (enteroviruses and adenoviruses) in a subset of toys; subsequently, the infectivity of the positive samples was tested on cell cultures. RESULTS: Of the examined toys, 23.8% were noncompliant with the limits of the European guideline. The most frequently exceeded limits were those for Aerobic bacteria (84.6%), and Pseudomonas aeruginosa (29.9%). Other opportunistic bacterial species that were frequently detected were Stenotrophomonas maltophilia, Pseudomonas fluorescens, Burkholderia cepacia, Sphingomonas paucimobilis and Comamonas acidovorans. In a subset of 28 samples, adenovirus (25%) and enterovirus (11%) genome was also found to be present, although the samples with viral positivity did not show infectivity after inoculation on appropriate cell monolayers. CONCLUSIONS: The results indicate a condition of microbial exposure related to the use of toys containing aqueous media. SIGNIFICANCE AND IMPACT OF STUDY: The investigation highlights the need for more stringent monitoring during the production, packaging and storage of toys containing aqueous matrices in order to safeguard children's health.

Behavior of Pseudomonas aeruginosa and Enterobacter aerogenes in Water from Filter Jugs.

Careless use conditions of filter jugs were applied to simulate and evaluate the behavior of two ubiquitous aquatic bacterial species, Pseudomonas aeruginosa and Enterobacter aerogenes. According to a reference protocol, nine different jugs of popular brands sold in the Italian market were used for the test. Separately, a suspension of the two bacteria was spiked in water used for filling the jugs. The concentration of the test organisms and total aerobic microbial count (TAMC) was measured daily in the filtered water along a period corresponding to the cartridge lifetime. Results showed a different trend of bacterial behavior. E. aerogenes was detectable exclusively on the first day after jug filling, while P. aeruginosa confirmed its persistence over time in all the jugs and its ability to potentially colonize surfaces and cartridges. The TAMC was detected at a concentration range from 102 to 107 CFU/100 mL in all the tests, high values that were not far from those raised in bottled flat natural mineral water weeks after bottling.

Evaluation of an MPN test for the rapid enumeration of Pseudomonas aeruginosa in hospital waters.

In this study, the performance of a new most probable number (MPN) test (Pseudalert(®)/Quanti-Tray(®)) for the enumeration of Pseudomonas aeruginosa from hospital waters was compared with both international and national membrane filtration-based culture methods for P. aeruginosa: ISO 16266:2006 and UK The Microbiology of Drinking Water - Part 8 (MoDW Part 8), which both use Pseudomonas CN agar. The comparison based on the calculation of mean relative differences between the two methods was conducted according to ISO 17994:2014. Using both routine hospital water samples (80 from six laboratories) and artificially contaminated samples (192 from five laboratories), paired counts from each sample and the enumeration method were analysed. For routine samples, there were insufficient data for a conclusive assessment, but the data do indicate at least equivalent performance of Pseudalert(®)/Quanti-Tray(®). For the artificially contaminated samples, the data revealed higher counts of P. aeruginosa being recorded by Pseudalert(®)/Quanti-Tray(®). The Pseudalert(®)/Quanti-Tray(®) method does not require confirmation testing for atypical strains of P. aeruginosa, saving up to 6 days of additional analysis, and has the added advantage of providing confirmed counts within 24-28 hours incubation compared to 40-48 hours or longer for the ISO 16266 and MoDW Part 8 methods.

A case study of sanitary survey on community drinking water supplies after a severe (post-Tsunami) flooding event.

This report presents a case study of a comprehensive sanitary survey on ca. 160 community drinking water supplies after a severe (post-Tsunami) flooding event in Sri Lanka. Sanitary inspection and microbiological and chemical water quality analyses were performed according to specifically-designed procedures established on the World Health Organization (WHO) guidelines. Significant hazards and critical points were identified in almost all the investigated water supplies. The overall results showed a significant level of microbiological and chemical risk associated with drinking water consumption within the investigated areas. The criteria and methods practised in this study are proposed as a model to assure an effective and reliable monitoring in post-emergencies involving possible deterioration of water quality and to identify health priorities related to water consumption.

Non-tuberculous mycobacteria and microbial populations in drinking water distribution systems.

Data on the occurrence of non-tuberculous mycobacteria (NTM), in parallel with those obtained for bacterial indicators and amoebae, are presented with the aim to collect information on the spread of NTM in drinking water distribution systems in Italy. Samples were collected from taps of hospitals and households in Central and Southern Italy. The concentration values obtained for the more traditional microbial parameters complied with the mandatory requirements for drinking water. Conversely, moderate-to-high microbial loads (till 300 CFU/L) were observed for the NTM. Positive samples were obtained from 62% of the investigated water samples. Analogous results were observed for amoebae showing a higher percentage of positive samples (76%). In terms of public health, the presence of mycobacteria in water distribution systems may represent a potential risk especially for vulnerable people such as children, the elderly or immunocompromised individuals.

Microbial characterization of water and biofilms in drinking water distribution systems at sport facilities.

The present study investigated the in situ microbial indicators' occurrence in water and biofilm in drinking water distribution systems of sport facilities such as gyms. The presence of Legionella, such as a potential pathogen, was also verified. Water and biofilm were contemporaneously collected and microbiologically analyzed. Few colonies of coliforms were detected in biofilm but not in the corresponding water samples. Conversely, some colonies of heterotrophs were counted at 22 degrees C in both the 80% biofilm samples and the 53% water samples. Legionella was detected in 29% of the hot water systems, and, in particular, L. pneumophila in 21% and L. non pneumophila in 9% of the samples. Higher counts were found in biofilm samples. As it was recommended for swimming pools, it is to be hoped that hygiene risk assessment criteria and safety plans could be also developed for gyms to reduce potential hazards to health for all attendants.

Assessment of microbiological and parasitological quality of composted wastes: health implications and hygienic measures.

Feedstock and compost samples were collected from twenty composting plants and analysed from the microbiological point of view. Faecal indicator organisms were determined in order to evaluate the efficacy of processes for the removal of pathogenic micro-organisms with similar survival characteristics and to verify their suitability as appropriate markers of microbial quality of composted products. In addition to the classical bacterial indicators, selected organisms, such as Salmonella, Giardia, Cryptosporidium, Clostridium spores and helminth ova, were investigated. Statistically significant differences in the removal of the different micro-organisms were observed with regard to both the different composting plants (P < 0.05) and feedstock composition (P < 0.05). In fact, compost obtained by feedstock containing sewage sludge was shown to have a better hygienic quality in comparison with compost containing green discards and municipal solid waste as raw matter. Giardia cysts, Cryptosporidium oocysts and helminth ova were not effective indicators of hygienic quality of compost, whereas Clostridium perfringens spores, because of their high resistance to treatments, could be considered as an additional model for assessing the composting process, especially with regard to more resistant pathogen reduction.

[A new method for the detection of coliforms and Escherichia coli in water intended for human consumption.]. / Il nuovo metodo rapido per la determinazione di coliformi ed Escherichia coli nelle acque destinate al consumo umano.

The ISO reference method, defined by the European Drinking Water Directive for the enumeration of total coliforms and Escherichia coli in water intended for human consumption, has various limitations, especially related to discrepancies observed with the new taxonomic classification of the coliform group. A study was therefore performed to compare the above reference method with another rapid method, the DST/Colilert, and to evaluate the phenotypical characteristics of isolated microrganisms. The ISO reference method failed to detect a significant proportion of coliforms and E. coli in water and furthermore, it enumerated microrganisms belonging to other groups. The DST/Colilert method was found instead to be a suitable alternative method for the detection of bacterial indicators.

Occurrence of potential bacterial pathogens in coastal areas of the Adriatic Sea.

A monitoring program was carried out in bathing waters along the sea-coast of the Adriatic Sea. The investigation was aimed at obtaining a quali-quantitative picture of microorganisms of anthropic origin and of a more strictly autochthonous origin and at verifying the existence of possible reciprocal correlations and combinations of relationship between them. Results showed that the concentrations of indicators of faecal contamination, whether within or over the values established by legislation, are not always related to the presence and density of microorganisms of environmental origin but the regression models used can help to describe how these latter microorganisms can be in relation to levels of the other parameters. The difficulty is evident of the predictability of health effects swimming-associated; for this reason more complex models that are currently used should be taken into account for minimising the risk for bathers.