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.

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.

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.

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.

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.

Interobserver reproducibility of cytologic p16INK4a /Ki-67 dual immunostaining in human papillomavirus-positive women.

BACKGROUND: The accumulation of cyclin-dependent kinase inhibitor 2A (p16ink4a ) protein in a cell is associated with neoplastic progression in precancerous cervical lesions. Dual staining for p16ink4a and Ki-67 has been proposed as a triage test in cervical cancer screening for women who test positive for human papillomavirus DNA. In this study, interobserver reproducibility of the interpretation of this test was assessed. METHODS: Forty-two immunostained, liquid-based cytology slides were divided into 2 sets and were interpreted by 17 to 21 readers from 9 different laboratories, yielding a total of 816 reports. Immunostaining results were classified as positive, negative, inconclusive, or inadequate. After evaluation of the first set of slides and before circulation of the second set, the results were discussed in a plenary meeting. The 10 slides with the most discordant results were evaluated again by selected expert cytopathologists. RESULTS: The overall κ value was 0.612 (95% confidence interval [CI], 0.523-0.701), it was higher for the positive and negative categories (κ = 0.692 and κ = 0.641, respectively), and it was almost null for the inconclusive category (κ = 0.058). Considering only readers from laboratories with documented experience, the κ value was higher (κ = 0.747; 95% CI, 0.643-0.839) compared with nonexperienced centers (κ = 0.498; 95% CI, 0.388-0.616). The results were similar in both sets of slides (κ = 0.505 [95% CI, 0.358-0.642] and κ = 0.521 [95% CI, 0.240-0.698] for the first and second sets, respectively). Reinterpretation of the slides with the most discordant results did not provide any improvement (first evaluation, κ = 0.616 [95% CI, 0.384-0.866]; second evaluation, κ = 0.403 [95% CI, 0.182-0.643]). CONCLUSIONS: Dual staining for p16 ink4a and Ki-67 demonstrated good reproducibility, confirming its robustness, which is a necessary prerequisite for its adoption as a triage test in cervical cancer screening programs that use human papillomavirus DNA as a primary test. Cancer Cytopathol 2017;125:212-220. © 2016 American Cancer Society.

Interpretation of p16(INK4a) /Ki-67 dual immunostaining for the triage of human papillomavirus-positive women by experts and nonexperts in cervical cytology.

BACKGROUND: The triage of human papillomavirus (HPV)-positive women is needed to avoid overreferral to colposcopy. p16(INK4a) immunostaining is an efficient triage method. p16(INK4a) /Ki-67 dual staining was introduced mainly to increase reproducibility and specificity compared with stand-alone p16(INK4a) staining. METHODS: Within a pilot project, HPV-positive women were referred to colposcopy if cytology was abnormal or unsatisfactory or HPV testing was still positive after 1 year. For 500 consecutive women, a slide obtained during colposcopy was immunostained for p16(INK4a) /Ki-67 and independently interpreted by 7 readers without previous experience with dual staining. Four of these readers were experts in cervical pathology and 3 were not. Kappa values for multiple raters, sensitivity, and specificity for cervical intraepithelial neoplasia type 2-positive histology were computed. Because women with normal cytology were underrepresented, estimates for all HPV-positive women were obtained as weighted means of cytology-specific estimates. RESULTS: The overall kappa for HPV-positive women was 0.70 (95% confidence interval [95% CI], 0.60-0.77). Kappa values were not found to be significantly different between expert and nonexpert readers with regard to cervical cytology but were significantly increased (P =. 0066) after consensus discussion. The overall specificity estimate for HPV-positive women was 64.0% (95% CI, 57.4%-70.2%): 66.7% (95% CI, 59.8%-73.0%) for experts and 60.5% (95% CI, 59.8%-73.0%) for nonexperts. Among women with abnormal cytology, the sensitivity was 85.5% (95% CI, 77.9%-90.8%): 85.8% (95% CI, 77.9%-91.2%) for experts and 85.1% (95% CI, 76.6%-90.9%) for nonexperts. CONCLUSIONS: p16(INK4a) /Ki-67 immunostaining demonstrated good reproducibility and specificity when triaging HPV-positive women. Dual-staining interpretation can be performed, after short training, even by staff who are not experts in cervical cytology. This allows HPV-based screening with triage to be performed in settings in which such expert staff is not available.

Airborne microorganisms associated with waste management and recovery: biomonitoring methodologies.

This paper presents preliminary results from a year-long indoor bioaerosol monitoring performed in three working environments of a municipal composting facility treating green and organic waste. Composting, whereby organic matter is stabilized through aerobic decomposition, requires aeration, causing the dispersion of microbial particles (microorganisms and associated toxins). Waste can, therefore, become a potential source of biological hazard. Bioaerosol samples were collected on a monthly basis. Through a comparison of results obtained using two samplers - the Surface Air System DUO SAS 360 and the BioSampler - the study aimed at assessing the presence of biological pollutants, and at contributing to the definition of standard sampling methods for bioaerosols leading, eventually, to the establishment of exposure limits for these occupational pollutants.

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.

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.