Vehicle transmission of antibiotic-resistant pathogens mediated by plastic debris in aquatic ecosystems.

Plastic materials are emerging environmental pollutants acting as potential vehicles for accumulation and spread of multidrug-resistant bacteria. The current study investigates the role of plastics in favoring the dispersal of specific pathogens and their associated antibiotic resistant genes (ARGs). Artificial plastic substrates (APSs) were submerged in seven sampling points of Lake Bracciano (Italy), and after one-month both APSs and raw water (RW) samples were collected. Through the combination of standard microbiological and biochemical techniques, 272 bacterial strains were identified and characterized for antibiotic resistant profiling. Our results revealed a notable difference in terms of diversity and abundance of pathogenic bacteria recovered from APSs, compared to RW. In addition, higher resistance patterns were detected in APSs isolates, with frequent appearance of relevant ARGs and class 1 integrons. These findings reinforce the idea that plastic materials in aquatic ecosystems serve as a reservoir for superbugs, significantly contributing to the dissemination of ARGs.

Emerging Issues on Antibiotic-Resistant Bacteria Colonizing Plastic Waste in Aquatic Ecosystems.

Antibiotic-resistant bacteria (ARB) adhesion onto plastic substrates is a potential threat to environmental and human health. This current research investigates the prevalence of two relevant human pathogens, Staphylococcus spp. and Klebsiella spp., and their sophisticated equipment of antibiotic-resistant genes (ARGs), retrieved from plastic substrates submerged into an inland water body. The results of microbiological analysis on selective and chromogenic media revealed the presence of colonies with distinctive phenotypes, which were identified using biochemical and molecular methods. 16S rDNA sequencing and BLAST analysis confirmed the presence of Klebsiella spp., while in the case of Staphylococcus spp., 63.6% of strains were found to be members of Lysinibacillus spp., and the remaining 36.3% were identified as Exiguobacterium acetylicum. The Kirby-Bauer disc diffusion assay was performed to test the susceptibility of the isolates to nine commercially available antibiotics, while the genotypic resistant profile was determined for two genes of class 1 integrons and eighteen ARGs belonging to different classes of antibiotics. All isolated bacteria displayed a high prevalence of resistance against all tested antibiotics. These findings provide insights into the emerging risks linked to colonization by potential human opportunistic pathogens on plastic waste commonly found in aquatic ecosystems.

Teratogenic effects of environmental concentration of plastic particles on freshwater organisms.

Given the widespread presence of plastics, especially in micro- and nanoscale sizes, in freshwater systems, it is crucial to identify a suitable model organism for assessing the potential toxic and teratogenic effects of exposure to plastic particles. Until now, the early life stage of freshwater organisms and the regeneration capacity in relation to plastic particles exposure is a still poorly investigated topic. In this study, we examine the teratogenic effect on diatom Cocconeis placentula and cnidarian Hydra vulgaris under controlled exposure conditions of poly(styrene-co-methyl methacrylate) (P(S-co-MMA)) particles. Significant effects were observed at the lowest concentrations (0.1 µg/L). A significant increase in the teratological frequency in C. placentula and a significant decrease in the regeneration rate in H. vulgaris were found at the lowest concentration. The delay in hydra regeneration impaired the feeding capacity and tentacles reactivity at 96 h of exposure. No effects on diatom growth were observed upon exposure to P(S-co-MMA) particles (0.1, 1, 100, 10,000 µg/L) for 28 days and these findings agree with other studies investigating algal growth. The application of the Teratogenic Risk Index, modified for diatoms, highlighted a moderate risk for the lowest concentration evaluating C. placentula and low risk at the lowest and the highest concentrations considering H. vulgaris. This study suggests the importance of testing organisms belonging to different trophic levels as diverse teratogenic effects can be found and the need to evaluate environmentally relevant concentrations of plastic particles.

Detection of Morganella morganii bound to a plastic substrate in surface water.

OBJECTIVES: Around the globe, escalation in rare opportunistic microbial infections is alarming as they are heading steadily towards 'superbug' status. In aquatic ecosystems, plastic fosters multidrug-resistant pathogenic bacteria and plays a significant role in trafficking antibiotic-resistant genes. In this study, we focused on a multidrug-resistant bacterial strain isolated from microbial communities found on plastic substrates of a volcanic lake in central Italy. METHODS: Extended-spectrum beta-lactamase-producing strains were isolated from both raw water and plastic substrates for a comparative investigation using microbiological and molecular methods, and antibiotic susceptibility profiling was performed against a panel of ten antibiotics. RESULTS: Molecular identification and Basic Local Alignment Search Tool analysis confirmed an almost identical sequencing pattern of two isolated strains and their homology with Morganella morganii. Antibiotic susceptibility tests revealed their resistance to almost all tested antibiotics. Class 1 integron-associated gene (intI1) and seven antibiotic resistance genes were detected in both strains, confirming their superbug status. CONCLUSION: To our knowledge, this is the first study on the characterization of extended-spectrum beta-lactamase-producing M. morganii isolated from the biofilm of plastic substrates, depicting the potential toxicity of plastic in harbouring and dispersing virulent, multidrug-resistant, opportunistic human pathogens.

COVID-19 Lockdown Pandemic Period Effects in Highly Impacted Aquatic Ecosystems.

Human activities severely affect aquatic ecosystems: the lockdown restrictions due to SARS-CoV-2 (COVID-19) have proved that reducing anthropogenic activities can positively influence the environment and mitigate atmospheric pollution. In addition, studies on aquatic ecosystems have highlighted the decrease in suspended matter and pollutant concentrations associated with reduced marine transport and industrial activities. We evaluated the effects of the reduction of anthropogenic activities in highly impacted water ecosystems. Three of the sampling sites were on the Tiber River's lower stretch close to the urban area of Rome (Italy), and the other two were transitional waters located in the southern Latium region coastal area. These sampling sites have been studied by our research group for at least 20 years. The present study involved analysis of chemical and physicochemical parameters, biological communities, and land use. Microbiological fecal indicators and Salmonella spp. were also analyzed. There were two sampling campaigns, the first one in May 2020 (ID II) at the end of lockdown measures, and the second one a year later, in June 2021 (ID III). The data were compared with our old data, collected from 2002 to 2015 (ID I), to evaluate the potential effects of the restrictive measures. Biotic communities were dominated by pollution-tolerant taxa, indicating eutrophication and organic pollution. Furthermore, the high concentrations of microbiological indicators was mainly due to sewage from the urban area. The results of the research indicated no significant changes from past years (ID I) to 2020 (ID II) and 2021 (ID III). In conclusion, the restrictions adopted in Italy during the pandemic period were not sufficient to mitigate the heavy pressure of urbanization and agriculture that have long affected the studied aquatic ecosystems. Environ Toxicol Chem 2023;42:966-977. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Can Medical Devices Help Mitigate Global Environmental Change Effects on Human and Animal Health? A Pilot Study.

Globalization and urbanization are new challenges for the ability to protect public health. Indeed, the anthropogenic impact is changing the environment on a global scale. These changes can have direct and indirect health effects on both human and animal populations, introducing new diseases. Heat waves and floods are an example of these changes. Global Environmental Change (GEC) consequences on human health and well-being are stronger in urban areas, which are inhabited by 70% of the European population. In this context, the use of appropriate medical devices can also help mitigate the effects of climate change. Studies into lifestyle, environment quality and potential fields of application can be useful tools to identify possible types of medical device that could help to support the therapeutic needs and the prevention of health both in everyday life, and in the case of environmental alerts. A study was carried out on the potential role of medical devices (MDs) in mitigating the effects of GEC on human and animal health, by issuing two different questionnaires to specific professional clusters: the first to doctors, pharmacists, and veterinarians, the second to MD manufacturers. The data obtained from this study confirm the strong connection between GEC and the increase in the use of some MDs. Results obtained from questionnaires circulated to MD manufacturers confirmed this trend. MD manufacturers also declared that there are no longer any seasonal trends in market demand for some medical devices. This is a pilot study to consider MDs as a mitigation tool for CEGs.

Interaction between bacterial enteric pathogens and aquatic macrophytes. Can Salmonella be internalized in the plants used in phytoremediation processes?

The environment is considered a reservoir of pathogens and a possible source of infection for animals and humans. The association between enteric pathogens and food plants has been demonstrated in several studies, while few studies have addressed possible interactions between human pathogens and aquatic plants. This study, performed by setting mesocosms, evaluates the interaction between an enteric pathogen (Salmonella enterica serovar Napoli, S. Napoli) and a macrophyte (Phragmites australis (Cav.) Trin. ex Steudel) and the possible ability of the bacterium to internalize into the plant. The results show that S. Napoli concentration decreased gradually in growth solution without plants (control) while it was able to persist adhering to submerged parts of plants in treated mesocosms. The adhesion of the bacterium remained stable for 20 days, then decreased gradually until the end of the experiment. In addition, S. Napoli was able to internalize and colonize stems and leaves. In conclusion, the study suggests that macrophytes can represent an alternative environmental reservoir of pathogens for humans and animals. The adhesion to roots and rhizomes and the internalization could contribute to the bacterial persistence in the aquatic ecosystems by playing an important role in ecology and transmission of pathogens.

Need for a sustainable use of medicinal products: environmental impacts of ivermectin.

It is worldwide recognized that the use of pharmaceuticals for human and veterinary purposes could lead to unsustainable effects on the environment. A strategy to reduce the impact of pharmaceuticals on the environment has been recently established at European level, where guidelines to evaluate the impacts of veterinary drugs used to treat animal diseases are in place. The aim of this article is to focus on the worldwide used antiparasitic drug ivermectin (IVM) and its potential impact on the environment. A specific section is related to the IVM resistance that the massive use of this drug could generate enhancing the risk scenarios also for human health. The application of stringent measures for the veterinary use of this substance, in line with the recommendations provided by International frameworks such as One Health and EcoHealth, is recommended.

Health and Climate Change: science calls for global action.

Climate changes affect social and environmental health determinants such as clean air, ecosystems health, safe drinking water and safe sufficient food. Globally, people at greatest risk of adverse health effects associated with climate change include children, the elderly and other vulnerable groups. Temperature-related death and illness, extreme events, polluted or stressed ecosystems represent relevant issues raising concern for both health and economic consequences. The aim of the Symposium "Health and Climate Change" (Istituto Superiore di Sanità, Rome 3-5 December 2018) was to promote an inter-sectoral and multidisciplinary approach to estimate and prevent climate change-related events as well as to call the authorities to put in place measures to reduce adverse health effects. At the end of the Symposium the Rome International Charter on Health and Climate Change was presented. It includes a series of actions and recommendations, discussed and shared by all the participants, intended to inform policy makers and all the stakeholders involved in the management of climate changes.

A Patented Rapid Method for Identification of Italian Diatom Species.

The study of diatoms-unicellular algae of the class Bacillariophyceae-has several applications, first and foremost the evaluation of freshwater ecosystem quality according to the Water Frame Directive 2000/60/EC (WFD). Identification at the species level is a crucial step in diatom studies, considering that species belonging to the same genus have different geographical distributions and different ecological requirements. The Rapid Method for Identification of Italian Diatom Species is aimed at guiding users in the classification of freshwater diatom species. It consists of a digitized flow chart that leads, step by step, to the identification, starting with an image capture by light or electron microscopy. This rapid and easy tool could be useful to workers of an environmental agency when performing the operational monitoring required by the WFD to classify surface waters. It will also expand the application of diatoms in numerous fields. This method has been patented in Italy.

Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface.

Contact lens (CL) wear is a risk factor for development of microbial keratitis, a vision threatening infection of the eye. Adverse events associated with colonization of lenses, especially by the multi-drug resistant and biofilm forming bacterium Pseudomonas aeruginosa remain a major safety issue. Therefore, novel strategies and compounds to reduce the onset of CL-associated ocular infections are needed. Recently, the activity of the frog skin-derived antimicrobial peptide Esc(1-21) and its diastereomer Esc(1-21)-1c was evaluated against both planktonic and sessile forms of this pathogen. Furthermore, Esc(1-21) was found to significantly reduce the severity of P. aeruginosa keratitis in a mouse model and preserve antipseudomonal activity in the presence of human basal tears. Here, we have analyzed the activity of the peptides on P. aeruginosa biofilm formed on soft CLs. Microbiological assays and scanning electron microscopy analysis indicated that the peptides were able to disrupt the bacterial biofilm, with the diastereomer having the greater efficacy (up to 85% killing vs no killing at 4 µM for some strains). Furthermore, upon covalent immobilization to the CL, the two peptides were found to cause more than four log reduction in the number of bacterial cells within 20 minutes and to reduce bacterial adhesion to the CL surface (77%-97% reduction) in 24 hours. Importantly, peptide immobilization was not toxic to mammalian cells and did not affect the lens characteristics. Overall, our data suggest that both peptides have great potential to be developed as novel pharmaceuticals for prevention and treatment of CL-associated P. aeruginosa keratitis.

Detection of Coxiella burnetii in Urban River Water.

Previous molecular-based studies have identified microorganisms of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. Water samples were taken from the River Tiber in and out of the city of Rome. Genetic analysis of the sequences obtained showed the presence of Coxiella burnetii in both the analyzed sites. Blast analysis showed that two sequences were identical to each other. Sequences from the polluted site showed high homology with different strains of C. burnetii. In this article, we report for the first time the presence of C. burnetii in environmental waters.

Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods.

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project µAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654. © 2016 SETAC.

Detection of Human Enteric Viruses in Freshwater from European Countries.

The transmission of water-borne pathogens typically occurs by a faecal-oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.

A validated UPLC-MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems.

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2-9.6% and 1.3-12.0% respectively. The method was applied to the analysis of aquatic samples (n=206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n=22), cylindrospermopsin (n=25), microcystin-RR (n=17), microcystin-LR (n=12), microcystin-LY (n=1), microcystin-LF (n=1) and nodularin (n=5). For microcystins, the levels detected ranged from 0.001 to 1.51µg/L, with two samples showing combined levels above the guideline set by the WHO of 1µg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.

A pilot study of bacterial regrowth in water pipelines.

Bacterial regrowth in water distribution systems results in deterioration of bacteriological quality of drinking water, as well as accelerated corrosion of the pipelines. The aim of the present study was to analyze the phenomena of colonization and bacterial regrowth in source water and in the water distribution systems of three distribution networks in the province of Catania (Sicily, Italy). The BART™ (Biological Activity Reaction Test) method was used, which is also capable of determining the potential aggressiveness of microbial species in water. We searched for sulfate reducing bacteria, iron-related bacteria, nitrifying and denitrifying bacteria, heterotrophic bacteria, slime-forming bacteria and fluorescent Pseudomonads. A high concentration of heterotrophic bacteria was found in almost every water sample analyzed. Sulfate reducing bacteria and iron-related bacteria were found in all three distribution networks, while non-fluorescing Pseudomonas were detected in source water of only one of the distribution networks. The BART™ method was found to be a practical and easy to use tool to detect the different bacteria groups involved in regrowth phenomena.

Evaluation of two methods for the use of diatoms in drowning cases.

In this study, we compare digestive methods used in a forensic context to extract diatoms (37 % hydrochloric acid) to a method recently described in Italian protocols for analysis of benthic diatoms for ecological assessment of surface water (hydrogen peroxide digestion). The two digestive methods were performed using 5 g of brain, lung, liver, kidney, and bone marrow taken from the bodies of 10 drowning victims recovered from three different aquatic environments (ocean, lakes, and rivers). Postmortem examination was performed on all bodies, but aquatic samples were only analyzed in two cases. Tissue digestion was equal by both methods. Hydrogen peroxide (H2O2) digestion resulted in better diatom preservation, enabling identification of nine genera in all samples examined versus three obtained using hydrochloride digestion. The ideal digestive method to provide evidence for corroboration of a diagnosis of drowning still needs to be established. However, the benthic diatoms protocol can be useful because it is less chemically hazardous to the laboratory operator and supports better diatom preservation for reliable taxonomic analysis.

Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on Mycobacterium spp.

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected-24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012-2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

Molecular detection of a potentially toxic diatom species.

A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.