Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system. To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

The Addition of Co into CuO-ZnO Oxides Triggers High Antibacterial Activity and Low Cytotoxicity.

In the present work, a simple two-step method is proposed for mixed oxide synthesis aimed at the achievement of antibacterial nanomaterials. In particular, Cu, Zn and Co have been selected to achieve single-, double- and triple-cation oxides. The synthesized samples are characterized by XRD, IR, SEM and EDX, indicating the formation of either crystalline or amorphous hydrocarbonate precursors. The oxides present one or two crystalline phases, depending on their composition; the triple-cation oxides form a solid solution of tenorite. Also, the morphology of the samples varies with the composition, yielding nanoparticles, filaments and hydrangea-like microaggregates. The antibacterial assays are conducted against E. coli and indicate an enhanced efficacy, especially displayed by the oxide containing 3% Co and 9% Zn incorporated into the CuO lattice. The oxides with the highest antibacterial properties are tested for their cytotoxicity, indicating a low toxicity impact, in line with literature data.

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 outbreak of cryptosporidiosis associated with drinking water in north-eastern Italy, August 2019: microbiological and environmental investigations.

Cryptosporidium is a leading global cause of waterborne disease, with many reported outbreaks related to main water supplies. In August 2019, an outbreak of cryptosporidiosis involving 80 cases occurred among 114 vacationers in a small municipality located in the Tuscan-Emilian Apennines, north-eastern Italy. After excluding a potential food-borne outbreak, the epidemiological investigation focussed on the hypothesis of a waterborne outbreak. This was confirmed by the finding of Cryptosporidium oocysts in stools of the cases and in water samples from the municipal water network. Molecular characterisation revealed the zoonotic species Cryptosporidium parvum as the causative agent. A single subtype (IIdA25G1) was found among all cases, and in one of two positive water samples. The municipality's water supply used spring water that only received a disinfection treatment insufficient to inactivate the parasite. Possible entry means into the water mains were found through further environmental investigations. As these types of water supplies are particularly vulnerable to various environmental factors, a control system based on the risk assessment of each phase of the water supply chain is required to guarantee water safety. Effective methods for detection of protozoan pathogens, which are generally excluded from routine water supply analysis, should be applied.

Microbial Air Quality in Healthcare Facilities.

There is increasing evidence that indoor air quality and contaminated surfaces provide an important potential source for transmission of pathogens in hospitals. Airborne hospital microorganisms are apparently harmless to healthy people. Nevertheless, healthcare settings are characterized by different environmental critical conditions and high infective risk, mainly due to the compromised immunologic conditions of the patients that make them more vulnerable to infections. Thus, spread, survival and persistence of microbial communities are important factors in hospital environments affecting health of inpatients as well as of medical and nursing staff. In this paper, airborne and aerosolized microorganisms and their presence in hospital environments are taken into consideration, and the factors that collectively contribute to defining the infection risk in these facilities are illustrated.

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.

Quantitative Microbial Risk Assessment as support for bathing waters profiling.

Profiling bathing waters supported by Quantitative Microbial Risk Assessment (QMRA) is key to the WHO's recommendations for the 2020/2021 revision of the European Bathing Water Directive. We developed an area-specific QMRA model on four pathogens, using fecal indicator concentrations (E. coli, enterococci) for calculating pathogen loads. The predominance of illness was found to be attributable to Human Adenovirus, followed by Salmonella, Vibrio, and Norovirus. Overall, the cumulative illness risk showed a median of around 1 case/10000 exposures. The risk estimates were strongly influenced by the indicators that were used, suggesting the need for a more detailed investigation of the different sources of fecal contamination. Area-specific threshold values for fecal indicators were estimated on a risk-basis by modelling the cumulative risk against E. coli and enterococci concentrations. To improve bathing waters assessment, we suggest considering source apportionment, locally estimating of pathogen/indicator ratios, and calculating site-specific indicators thresholds based on risk assessment.

[Some considerations on revision of legislation Hygiene-sanitary for the management of swimming pools for swimming-pool use and the National Consultation of the Ministry of Health]. / Alcune considerazioni sulla revisione della normativa igienico-sanitaria per la gestione di piscine ad uso natatorio e la Consultazione Nazionale del Ministero della Salute.

After over a decade the Italian regulation on hygiene and surveillance in swimming pools is under revision based on WHO guidelines and current laws from other European countries. The Ministry of Health is proposing an update of the Annex 1 that contains physical, chemical and microbiological indicators for monitoring swimming pool hygiene. The authors review current regulations and the new proposal for updating surveillance. A comparison of Annex 1 as approved in 2003 and the new version as proposed in 2016 allowed to enlighten innovative improvements. The suggested updates underwent a public consultation through an online questionnaire involving citizens and stakeholders, following the required procedure for accessing the final approval of a new regulation. The updates include new requirements and changes involving several controls and microbiological parameters. Safety of recreational waters is presently under revision in different European countries. This updating process is a valuable opportunity to implement critical control points and improve monitoring. The final aim is an optimization of surveillance in terms of efficacy and costs, with an advantage for both the National Health Service as the swimming pool management.

[World Health Organization (WHO) Guidelines for coastal and fresh waters: Italian translation by the Society of Hygiene, Preventive Medicine and Public Health (SItI) Working Group "Movement Sciences for Health"]. / Linee Guida dell'Organizzazione Mondiale della Sanità per acque costiere ed acque dolci: traduzione a cura del Gruppo di Lavoro SItI - "Scienze Motorie per la Salute"

The Working Group "Movement Sciences for Health" of the Italian Society of Hygiene, Preventive Medicine and Public Health has promoted the Italian translation of the WHO Guidelines for Safe Recreational Water Environments, relating to coastal and freshwater environments. In this article the authors briefly summarize the main areas covered in the guidelines, namely drowning and injury, exposure to cold, heat and sunlight, water quality, contamination of beach sand, exposure to algae and their products, aesthetic issues, exposure to chemical and physical agents, dangerous aquatic organisms, monitoring and assessment hazards and risks.

Zootechnical wastewater reuse: constructed wetland as a challenge for protozoan parasite removal.

Samples of soil, well water, and wastewater from a zootechnical farm, water after phytodepuration and maize plants (Zea mays) grown on soils irrigated with these different kinds of water were analysed for indicator bacteria and the protozoa Giardia and Cryptosporidium. Protozoa and bacterial indicators, except coliforms, were not recovered from well water samples. In the effluent from the zootechnical farm, high parasitological concentrations were observed, whilst water after the phytodepuration process showed a concentration reduction of two orders of magnitude. The high numbers of Giardia and Cryptosporidium in the zootechnical effluent could represent a potential risk for the spread of the pathogens. Nevertheless the environmental spread is minimized when data on soils and plants are observed. From the study, it emerges that this water treatment system could represent an alternative option to other conventional wastewater treatments and an economic and environmental advantage.

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.

An Italian study on Cryptosporidium and Giardia in wastewater, fresh water and treated water.

Cryptosporidium oocysts and Giardia cysts has been isolated from waters worldwide. In Italy, studies on these parasites in the environment are still limited due to absence of epidemiological evidence and difficulty of adequate methodologies of sampling and analysis. The new Drinking Water Directive 98/83/CE states that Cryptosporidium has to be determined in water intended for human consumption if Clostridium perfringens is detected. This investigation contributes to the knowledge of both Cryptosporidium and Giardia occurrences in Italian aquatic environments through a two-year monitoring program. In addition, indicator bacteria and Clostridium perfringens were monitored in sewage, surface waters, drinking water, and swimming pools and possible correlations were calculated among all the selected parameters. Cysts and oocysts were detected in sewage and surface water and Giardia numbers always prevailed over Cryptosporidium. The parasites were not detected in drinking water, while a positive sample was obtained from the analysis of swimming pools. Pearson's correlation coefficients evidenced a reciprocal correlation between both the protozoa and the Enterococci counts.

Detection and genotypic characterization of Giardia cysts in water samples.

Water samples were collected from a wastewater treatment plant and from two lakes in the vicinity of Rome (Italy) for detection and genotypic characterization of Giardia. All wastewater samples and all but two of the lake samples were scored by immunofluorescence as positive for Giardia. In addition, Giardia cysts were detected, by nested PCR, in all of the examined samples. Analysis of the SSU-rRNA gene sequences showed that all Giardia isolates belonged to Assemblage A. The presence of Assemblage A genotype in wastewater and surface bathing water represents an important public health concern. In fact, isolates showing this genotype are considered to be potential human pathogens and also have zoonotic implications.

[Microbial indicators and fresh water quality assessment]. / Indicatori microbiologici e valutazione della qualità delle acque superficiali.

Traditionally, the microbiological quality of waters has been measured by the analysis of indicator microorganisms. The article reviews the sanitary significance of traditional indicators of faecal contamination (total coliforms, faecal coliforms and faecal streptococci) and points out their limits. For some characteristics Escherichia coli may be considered a more useful indicator then faecal coliforms and recently it has been included in all recent laws regarding fresh, marine and drinking water. A clearer taxonomic definition of faecal streptococci evidenced the difficulty into defining a specific standard methodology of enumeration and suggested the more suitable role of enterococci as indicator microorganisms. Several current laws require the detection of enterococci. The resistance of Clostridium perfringens spores may mean that they would serve as a useful indicator of the sanitary quality of sea sediments.

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.

Occurrence of Cryptosporidium oocysts in sewage effluents and correlation with microbial, chemical and physical water variables.

Contaminated water are recognized as a potential source of Cryptosporidium oocysts. In Italy studies on the environmental spread of the parasite are still scarce even if a high frequency of serological responses could suggest high rates of endemic Cryptosporidium infections. In this investigation the occurrence of the parasite in water samples from a municipal wastewater treatment plant was monitored with the aim to evaluate possible correlation between oocysts and bacterial indicators, pathogens and physico-chemical parameters using a multivariate analysis. Data collected showed high numbers of oocysts, with a high percentage of positive samples and a significant correlation with pH, redox potential and total organic carbon. No correlation was found between Cryptosporidium concentration and that of the other microrganisms. The results, through a probing statistical analysis, show that none of the selected microrganisms was a reliable predictor of the presence of the parasite.

[Microbiological quality of seaside sands: a beach in Latium]. / Qualità microbiologica delle sabbie marine: una spiaggia del Lazio.

This study is focused on the microbiological quality of a sandy beach in the coastal area around Rome, Italy. The microbiological surveys were carried out on the sands collected both on the beach and on the waterline. A low-concentration of faecal bacteria (streptococci outnumbered Escherichia coli) and a constant rate of staphylococci were detected over the sampling period. Significant statistical correlations were calculated between yeasts and moulds, Escherichia coli and streptococci, streptococci and sulfite-reducing clostridium spores. This survey's data could be a baseline for future studies.