SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfish.

The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.

Removal of emerging pathogenic bacteria using metal-exchanged natural zeolite bead filter.

Hospital wastewaters can become a route for dissemination of antibiotic-resistant bacteria to the environment if not properly treated. Some of these bacteria are able to survive conventional disinfection treatments (e.g. chlorination, UV irradiation), which evokes the need for novel disinfection methods. The metal-exchanged zeolites were tested as novel antibacterial agents for wastewater treatment. The natural zeolite clinoptilolite enriched with silver (AgNZ) showed far better antibacterial activity towards hospital pathogenic bacterium Acinetobacter baumannii when compared with copper-exchanged zeolite (CuNZ), with minimal bactericidal concentration of 0.25-2 (AgNZ) compared with 32-64 mg L-1 (CuNZ) in a batch system and respective log 5.6 reduction compared with log 0.5 reduction in a flow system with pure bacterial culture. In the flow system with real effluent wastewater from the treatment plant, the removal of carbapenem-resistant bacteria using AgNZ was 90-100% during the 4 days of the experimental run. These results indicate that the AgNZ efficiently removes pathogenic bacteria from the wastewater, including A. baumannii, and is promising as a disinfectant material in a bead filter system.

Carbapenem-resistant isolates of Acinetobacter baumannii in a municipal wastewater treatment plant, Croatia, 2014.

Acinetobacter baumannii is an emerging hospital pathogen. Whereas A. baumannii isolated from patients or hospitals has been reported, there are few data regarding propagation of viable A. baumannii in the natural environment. This study investigates the occurrence and antimicrobial susceptibility of viable A. baumannii in municipal wastewater and its persistence through the wastewater treatment process. A total of 21 A. baumannii isolates were recovered at a secondary type of municipal wastewater treatment plant in Zagreb, Croatia: 15 from raw influent wastewater and six from final effluent. All isolates were carbapenem- and multidrug-resistant. Among 14 isolates tested for blaOXA genes, all harboured the constitutive blaOXA-51-like gene, while the acquired blaOXA-23-like and blaOXA-40-like genes were found in 10 and three isolates respectively. Six A. baumannii isolates recovered from effluent wastewater multiplied and survived in sterilised effluent wastewater up to 50 days. These findings support the idea that multidrug-resistant A. baumannii can occur and have the ability to survive in the environment.

Emerging Trends in the Epidemiology of COVID-19: The Croatian 'One Health' Perspective.

During the four pandemic waves, a total of 560,504 cases and 10,178 deaths due to COVID-19 were reported in Croatia. The Alpha variant, dominant from March 2021 (>50% of positive samples), was rapidly replaced by Delta variants (>90%) by August 2021. Several seroprevalence studies were conducted in different populations (general population, children/adolescents, professional athletes, healthcare workers, veterinarians) and in immunocompromised patients (hemodialysis patients, liver/kidney transplant recipients). After the first pandemic wave, seroprevalence rates of neutralizing (NT) antibodies were reported to be 0.2-5.5%. Significantly higher seropositivity was detected during/after the second wave, 2.6-18.7%. Two studies conducted in pet animals (February-June 2020/July-December 2020) reported SARS-CoV-2 NT antibodies in 0.76% of cats and 0.31-14.69% of dogs, respectively. SARS-CoV-2 NT antibodies were not detected in wildlife. Environmental samples taken in the households of COVID-19 patients showed high-touch personal objects as most frequently contaminated (17.3%), followed by surfaces in patients' rooms (14.6%), kitchens (13.3%) and bathrooms (8.3%). SARS-CoV-2 RNA was also detected in 96.8% affluent water samples, while all effluent water samples tested negative. Detection of SARS-CoV-2 in humans, animals and the environment suggests that the 'One Health' approach is critical to controlling COVID-19 and future pandemics.

Industrial wastewater treatment plant enriches antibiotic resistance genes and alters the structure of microbial communities.

Antibiotic resistance is an emerging global health crisis, driven largely by overuse and misuse of antibiotics. However, there are examples in which the production of these antimicrobial agents has polluted the environment with active antibiotic residues, selecting for antibiotic resistant bacteria and the genes they carry. In this work, we have used shotgun metagenomics to investigate the taxonomic structure and resistance gene composition of sludge communities in a treatment plant in Croatia receiving wastewater from production of the macrolide antibiotic azithromycin. We found that the total abundance of antibiotic resistance genes was three times higher in sludge from the treatment plant receiving wastewater from pharmaceutical production than in municipal sludge from a sewage treatment plant in Zagreb. Surprisingly, macrolide resistance genes did not have higher abundances in the industrial sludge, but genes associated with mobile genetic elements such as integrons had. We conclude that at high concentrations of antibiotics, selection may favor taxonomic shifts towards intrinsically resistant species or strains harboring chromosomal resistance mutations rather than acquisition of mobile resistance determinants. Our results underscore the need for regulatory action also within Europe to avoid release of antibiotics into the environment.

The fate of carbapenem-resistant bacteria in a wastewater treatment plant.

Wastewater treatment plants have been considered potential sources of antibiotic resistance gene exchange and release into the environment. The aim of our study was to quantify environmental and human-associated carbapenem-resistant bacterial populations (CRBPs) across wastewater treatment stages and correlate bacterial counts to physicochemical and other bacteriological parameters in order to see their behaviour in wastewater and sludge and their potential dissemination in the environment. Samples were taken from five sites (treatment stages) of the largest Croatian wastewater treatment plant (20 per site) over 10 months of monitoring. CRBPs were found at all wastewater treatment stages save for the lime-treated, stabilised sludge, which underlines the importance of effluent and digested sludge disinfection. Secondary sludge settling removed 99% of CRBP from the effluent, but the relative proportion of CRBP in the total bacterial count significantly increased in the effluent (0.0020%) and digested sludge (0.0019%) compared to the influent (0.0006%), indicating selection for resistant bacteria in these settings. CRBP counts did not correlate with measured carbapenem concentrations in wastewater, which suggests that antibiotic concentrations were not the reason for CRBP selection. Negative correlation between activated sludge retention time and CRBP indicated that their number could be reduced by increasing the retention time during secondary treatment. Despite the indications that WWTPs select for antibiotic-resistant bacteria, wastewater treatment is very efficient in reducing their absolute numbers, and proper effluent and sludge disinfection can significantly reduce dissemination of antibiotic-resistant bacteria into the environment.