Intense rainfall can affect bathing water quality, especially in areas with poorly developed sewage systems or combined sewer overflows (CSOs). The aim of this study was to assess the impact of ...precipitation on coastal bathing water quality in the area of Split and Kaštela (Adriatic Sea), the urban areas where CSOs were applied. The study was conducted during two bathing seasons, 2020 and 2021. The sampling of coastal waters and measurement of physical/chemical parameters was performed every two weeks and after a precipitation event of more than 2 mm. The impact of precipitation on the quality of coastal bathing waters was not noted in the Split area nor in Kaštela, probably due to the low amount of precipitation. The quality of bathing waters in the Kaštela area was significantly worse than in the Split area, which is due to the condition of the sewage system in these areas and not the precipitation effect. It was also revealed that bathing water quality depends on the timing of sampling and the indicator against which it is assessed. Escherichia coli (E. coli) proved to be a better indicator for early morning sampling, while intestinal enterococci were better for late morning sampling.
Bathing Water Directive (2006/7/EC) specifies two reference methods for Escherichia coli detection: ISO 9308-1 and 9308-3. The revised ISO 9308-1 is recommended only for waters with a low bacterial ...background flora. Considering the extended time needed for analysis and, generally, the lack of experience in using ISO 9308-3 in the Mediterranean, the suitability of ISO 9308-1 for the examination of E. coli in bathing water was evaluated. The present study was aimed at a comparison of data obtained by the reference method in seawater samples (110 beaches, N=477) with data received from six alternative methods. Results show that recently used TSA/TBA method may overestimate E. coli numbers in marine waters. The temperature modified ISO 9308-1 (44°C) did not significantly alter the results, but outperformed the antibiotic supplemented agar at reducing non-E. coli bacteria on the plates, allowing the use of the respective method for monitoring coastal water.
•477 seawater samples were examined for E. coli presence using reference method and six alternative methods•The temperature modified ISO 9308-1 (44 °C) seems to be a suitable method for marine bathing water quality assessment•Previously used TSA/TBA media possibly overestimate the E. coli count•Colilert-18 in marine water results inconclusive, hence need exists to expand research to diverse geographical locations•In order to improve health protection, application of rapid method is required
To understand the activity of marine viruses, experiments on viral production, viral decay and the percentage of lytic and lysogenic bacterial cells among the total number of bacterial cells were ...carried out seasonally at two stations in the Adriatic Sea with different trophic conditions. Additionally, we are providing an insight on the enrichment with dissolved and particulate organic matter by viral lysis in the studied area. Viral production was higher at the coastal station than at the open-sea station. Viral decay rates were also higher at the coastal sea station than at the open-sea station, and accounted for approximately 40% of viral production at both investigated stations. The percentage of lysogenic infection was lower than that of lytical infection, which indicates the prevalence of the lytic cycle at both stations. Viruses had a significant influence on bacterial mortality through high daily removal of the bacterial standing stock at the coastal and open-sea station. The viruses contributed to the restoration of dissolved organic carbon, nitrogen and phosphorus in the microbial loop by lysing the bacterial cells at the studied stations. All the above suggest that viruses are important in the microbial food web and an important factor in the control of bacterial populations within the study area.
The role of marine environments in the global spread of antibiotic resistance still remains poorly understood, leaving gaps in the One Health-based research framework. Antibiotic resistance genes ...(ARGs) encoding resistance to five major antibiotic classes, including sulfonamides (
,
), tetracyclines (
,
), β-lactams (
,
), macrolides (
,
), aminoglycosides (
), and integrase gene (
) were quantified by RT-qPCR, and their distribution was investigated in relation to environmental parameters and the total bacterial community in bottom layer and surface waters of the central Adriatic (Mediterranean), over a 68 km line from the wastewater-impacted estuary to coastal and pristine open sea. Seasonal changes (higher in winter) were observed for antibiotic resistance frequency and the relative abundances of ARGs, which were generally higher in eutrophic coastal areas. In particular,
, followed by
and
, were strongly associated with anthropogenic influence and
as their predominant carriers. Water column stratification and geographic location had a significant influence on ARGs distribution in the oligotrophic zone, where the bacterial community exhibited a seasonal shift from
in winter to Marine group II in summer.
Introduction: The Republic of Croatia (HR) has significantly invested in monitoring the quality of inland bathing areas during the past 10 years. The aim of this paper was to analyze the results ...obtained during the 2014-2019 period and to compare them with the Region (non-European Union EU countries neighboring Croatia) and EU. Moreover, bathing water quality data at two bathing areas of Zagreb (lakes Jarun and Bundek) were processed in more detail.Methods: The quality of inland bathing areas in the HR is monitored in five rivers and five lakes in nine Croatian counties; 19 of the 35 monitored sites are located in Zagreb County. Escherichia coli (EC) was determined by EN ISO 9308-3: 1998, while intestinal enterococci (ENT) were defined by EN ISO 7899-2: 2000. Statistical processing of the results was performed using the Microsoft Excel Statistical Package (Redmond, USA) and Statistica 13.5. (Stat.Sof.Inc., Tulsa, USA); the significance level was set to p < 0.05.Results: During the study period, the worst Croatian inland water quality was recorded in 2019 (excellent quality for only 25.9% of the bathing areas), while the best quality was recorded in 2015 (excellent quality in 57.1%). On average (2014-2019), the share of bathing areas with excellent quality in Croatia was 46.3% (i.e., under the EU average of 85.3%); with no poor locations (EU average was nearly 2%). A significant positive correlation was found between fecal indicators (EC&ENT) and the amount of precipitation, while a negative correlation with water and air temperature in lakes.Conclusions: The quality of Croatian inland bathing areas is significantly lower than the European average, while EU water quality is improving. Regarding the countries bordering Croatia, Italy, Slovenia, and Hungary have achieved better results, while Montenegro, Serbia, and Bosnia and Herzegovina are still in the process of developing the monitoring of the bathing water quality.
•Marine microbial diversity, resistome and environmental factors were investigated.•Proteobacteria were major contributors of marine resistome in a temperate zone.•Seasonality was a main driving ...factor of microbial and resistome diversity.•Impact of seasonality enhances by the distance from the coast.•Water column stratification highly modified the bacterial community and resistome.
Marine and ocean environments are the most widespread habitats in the world but are still the least studied from the aspect of antibiotic resistance. The indigenous and tetracycline (TET)- and sulfamethoxazole (SXT)-resistant planktonic bacterial communities were simultaneously investigated for the first time along a trophic gradient of a temperate zone, regarding their taxonomic and functional structures as well as biotic and abiotic factors affecting their dynamics as vehicles of antibiotic resistance genes (ARGs), thus impacting the ARGs distribution at seasonal and spatial scales.
A total of 80 microbiomes, recovered seasonally from bottom layer and surface waters along a 68-km transect from wastewater-impacted estuary to coastal and pristine open sea in the central Adriatic (Mediterranean Sea), were analysed using 16S rRNA amplicon sequencing, PICRUSt2 bioinformatics and extensive biostatistics. Eighty-one bacterial phyla were identified, with majority (n = 49) in summer when communities were found to be more species enriched across the gradient. Microbial diversity was more site-specific and pronounced in surface microbiomes in winter. Nevertheless, both richness and community diversity decreased with distance from the coast. Although the microbiomes from human-influenced sites significantly differed from those in oligotrophic offshore area, Proteobacteria were still the most abundant phylum during both seasons at the surface and seabed along the gradient, and the major contributors to the marine resistome regarding native and TET- and SXT-resistant microbial communities. Resistome structure was more diverse in winter, whereas peptide, vancomycin and multidrug resistance modules predominated regardless of season, trophic status, or antibiotic. However, multidrug, beta-lactam resistance modules as well as macrolide, phenicol, aminoglycoside, and particularly imipenem resistance genes were much more frequent in winter, suggesting that the diversity of indigenous resistomes is highly dependent on seasonal variations of the water column, driven by thermohaline stratification and nutrients. Moreover, several pathogenic genera stood out as important carriers of multiple resistance traits in TET- and SXT-related resistomes in both seasons, particularly Acinetobacter, Vibrio, Bacillus and Pseudomonas, beside which Proteus, Serratia and Bacteroides prevailed in native resistomes.
This study evidenced seasonal and spatial variations of the marine microbiome and resistome and their driving forces along the trophic gradient, providing a comprehensive insight into the diversity and distribution of antibiotic resistance in the marine ecosystem of a temperate zone.
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Predictive models of bathing water quality are a useful support to traditional monitoring and provide timely and adequate information for the protection of public health. When developing models, it ...is critical to select an appropriate model type and appropriate metrics to reduce errors so that the predicted outcome is reliable. It is usually necessary to conduct intensive sampling to collect a sufficient amount of data. This paper presents the process of developing a predictive model in Kaštela Bay (Adriatic Sea) using only data from regular (official) bathing water quality monitoring collected during five bathing seasons. The predictive modelling process, which included data preprocessing, model training, and model tuning, showed no silver bullet model and selected two model types that met the specified requirements: a neural network (ANN) for Escherichia coli and a random forest (RF) for intestinal enterococci. The different model types are probably the result of the different persistence of two indicator bacteria to the effects of marine environmental factors and consequently the different die-off rates. By combining these two models, the bathing water samples were classified with acceptable performances, an informedness of 71.7%, an F-score of 47.1%, and an overall accuracy of 80.6%.
•Responses of MFW structure to temperature change are reproducible in time.•Similar changes in MFW structure occurred regardless of the trophic status.•Importance of microbial heterotrophic ...activities increases with temperature.•Importance of autotrophic picoplankton increases with temperature.
Global and atmospheric climate change is altering the thermal conditions in the Adriatic Sea and, consequently, the marine ecosystem. Along the eastern Adriatic coast sea surface temperature (SST) increased by an average of 1.03 °C during the period from 1979 to 2015, while in the recent period, starting from 2008, a strong upward almost linear trend of 0.013 °C/month was noted. Being mainly oligotrophic, the middle Adriatic Sea is characterized by the important role played by the microbial food web in the production and transfer of biomass and energy towards higher trophic levels. It is very important to understand the effect of warming on microbial communities, since small temperature increases in surface seawater can greatly modify the microbial role in the global carbon cycle. In this study, the Self-Organizing Map (SOM) procedure was used to analyse the time series of a number of microbial parameters at two stations with different trophic status in the central Adriatic Sea. The results show that responses of the microbial food web (MFW) structure to temperature changes are reproducible in time. Furthermore, qualitatively similar changes in the structure of the MFW occurred regardless of the trophic status. The rise in temperature was associated with: (1) the increasing importance of microbial heterotrophic activities (increase bacterial growth and bacterial predator abundance, particularly heterotrophic nanoflagellates) and (2) the increasing importance of autotrophic picoplankton (APP) in the MFW.
The use of a suitable method for the enumeration of indicator microorganisms is of crucial importance for reliable monitoring and assessment of the quality of bathing waters. Among other ...characteristics, the method should be selective enough and ensure acceptable relative recovery of target microorganisms. This study presents the basic parameters, relative recovery and categorical performance characteristics of Tryptone Bile X-glucuronide (TBX) agar for
Escherichia coli
(
E. coli
) enumeration in bathing water samples using the membrane filtration method.
The results of the relative recovery study, in which TBX agar was compared against temperature-modified ISO 9308-1:
2014
, showed that in order to achieve a satisfactory relative recovery of
E. coli
with TBX agar at 44 ± 0.5 °C, the resuscitation period on a non-selective medium (Minerals Modified Glutamate Agar, MMGA) at 36 ± 2 °C is crucial. Incubation on a double-layer MMGA/TBX medium with a 6-h resuscitation period and alternating incubation on single-layer MMGA and TBX agar with a 4-h resuscitation period resulted in acceptable and very similar relative recovery. The achieved performance characteristics of the tested medium, double-layer MMGA/TBX agar, are acceptable. The selectivity was matrix-dependent and was 60.6% for inland and 69.9% for coastal waters. No significant effect of the resuscitation period on selectivity was recorded. Finally, the results showed that when the resuscitation period on a non-selective medium is included, TBX agar is a suitable medium for
E. coli
enumeration in bathing water samples using the membrane filtration method and that its use, theoretically, would not have negative effects on the assessment of bathing water quality.
Vancomycin-resistant Enterococcus faecium (VREfm) is an opportunistic pathogen among the highest global priorities regarding public and environmental health. Following One Health approach, we ...determined for the first time the antibiotic resistance and virulence genes, and sequence types (STs) affiliation of VREfm recovered simultaneously from marine beach waters, submarine outfall of a wastewater treatment plant and an offshore discharge of untreated sewage, and compared them with the surveillance VREfm from regional university hospital in Croatia to assess the hazard of their transmission and routes of introduction into the natural environment. Importantly, VREfm recovered from wastewater, coastal bathing waters and hospital shared similar virulence, multidrug resistance, and ST profiles, posing a major public health threat. All isolates carried the vanA gene, while one clinical isolate also possessed the vanC2/C3 gene. The hospital strains largely carried the aminoglycoside-resistance genes aac(6′)-Ie-aph(2″)-Ia, and aph(2″)-Ib and aph(2″)-Id, which were also predominant in the environmental isolates. The hyl gene was the most prevalent virulence gene. The isolates belonged to 10 STs of the clonal complex CC17, a major epidemic lineage associated with hospital infections and outbreaks, with ST117 and ST889 common to waterborne and hospital isolates, pointing to their sewage-driven dissemination.
To gain better insight into the diversity of accompanying taxons in the surveyed water matrices, microbiome taxonomic profiling was carried out using Illumina-based 16S rDNA sequencing and their resistome features predicted using the PICRUSt2 bioinformatics tool. An additional 60 pathogenic bacterial genera were identified, among which Arcobacter, Acinetobacter, Escherichia-Shigella, Bacteroides and Pseudomonas were the most abundant and associated with a plethora of antibiotic resistance genes and modules, providing further evidence of the hazardous effects of wastewater discharges, including the treated ones, on the natural aquatic environment that should be adequately addressed from a sanitary and technological perspective.
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•Hospital and environmental vancomycin-resistant Enterococcus faecium were studied.•VREfm enter marine environment by outfalls of treated and untreated wastewater.•VanA genotype predominates among hospital and waterborne isolates in Croatia.•Hospital and environmental VREfm carry similar virulence and resistance genes.•Microbiome analysis revealed accompanying pathogenic taxa and resistance genes.
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