Large-Scale Biogeographical Shifts of Abundance of Antibiotic Resistance Genes and Marine Bacterial Communities as Their Carriers along a Trophic Gradient.

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 (sul1, sul2), tetracyclines (tetA, tetB), ß-lactams (blaCTX-M, blaTEMblaVIM), macrolides (ermB, mphA), aminoglycosides (aac3-2), and integrase gene (intl1) 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, intl1, followed by blaTEM and blaVIM, were strongly associated with anthropogenic influence and Gammaproteobacteria 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 Gammaproteobacteria in winter to Marine group II in summer.

Marine resistome of a temperate zone: Distribution, diversity, and driving factors across the trophic gradient.

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.

Microbiome profiling and characterization of virulent and vancomycin-resistant Enterococcus faecium from treated and untreated wastewater, beach water and clinical sources.

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.

Impacts of Atmospheric and Anthropogenic Factors on Microbiological Pollution of the Recreational Coastal Beaches Neighboring Shipping Ports.

A comparative study of the two northeastern ports of the Adriatic Sea indicated that the port of Rijeka is microbiologically more loaded than the port of Pula and posing a greater threat to other ports through a potential transfer of pathogens by ballast water. Fecal indicator bacteria, Escherichia coli and intestinal enterococci, were investigated seasonally in 2014-2015 in the ports and during the bathing season monitoring in the two bays where ports are located in 2009-2020. In addition, the indicators and pathogens related to human health were determined in the ports' seawater and sediment. The determined factors contributing to microbiological pollution were higher number of tourists and locals, potential wastewater and ballast water discharge and enclosed port configuration, with high solar radiation and low precipitation reducing the negative effects. Our research points to the necessity of including Clostridium perfringens in monitoring beach sand during the bathing seasons and a wider list of pathogens in port monitoring due to a potential transfer by shipping ballast water.

Staphylococcus aureus-An Additional Parameter of Bathing Water Quality for Crowded Urban Beaches.

During the last years, the report of the occurrence of waterborne disease symptoms related to non-enteric pathogens has increased, without any record of higher levels of indicator bacteria (Escherichia coli and intestinal enterococci). Therefore, the use of current indicators is not always adequate when assessing the overall potential health risk and the inclusion of additional parameters needs to be examined. This paper reports on the incidence and levels of Staphylococcus aureus at 258 locations in Primorje-Gorski Kotar County (Croatia) recorded by official bathing water quality monitoring, as well as supplemental monitoring carried out at the two most frequented beaches in the City of Rijeka. The number of bathers was found to be the main factor affecting S. aureus levels (r = 0.321, p < 0.05). The share of S. aureus positive samples from the official monitoring was significantly lower, when compared to the share of samples from supplemental monitoring (2.2% and 36.3%, respectively; p < 0.01). Besides the number of bathers, one of the main reasons is likely the higher sampling frequency. No correlation was found between S. aureus levels and the indicator bacteria. The results indicate that the determination of S. aureus and increased sampling frequency is recommended for overcrowded beaches.

Changes in the Trophic Pathways within the Microbial Food Web in the Global Warming Scenario: An Experimental Study in the Adriatic Sea.

A recent analysis of the Mediterranean Sea surface temperature showed significant annual warming. Since small picoplankton microorganisms play an important role in all major biogeochemical cycles, fluxes and processes occurring in marine systems (the changes at the base of the food web) as a response to human-induced temperature increase, could be amplified through the trophic chains and could also significantly affect different aspects of the structure and functioning of marine ecosystems. In this study, manipulative laboratory growth/grazing experiments were performed under in situ simulated conditions to study the structural and functional changes within the microbial food web after a 3 °C increase in temperature. The results show that a rise in temperature affects the changes in: (1) the growth and grazing rates of picoplankton, (2) their growth efficiency, (3) carrying capacities, (4) sensitivity of their production and grazing mortality to temperature, (5) satisfying protistan grazer carbon demands, (6) their preference in the selection of prey, (7) predator niche breadth and their overlap, (8) apparent uptake rates of nutrients, and (9) carbon biomass flow through the microbial food web. Furthermore, temperature affects the autotrophic and heterotrophic components of picoplankton in different ways.

Is TBX agar a suitable medium for monitoring Escherichia coli in bathing water using the membrane filtration method?

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.

Temperature and phosphorus interacts in controlling the picoplankton carbon flux in the Adriatic Sea: an experimental versus field study.

Temperature and phosphorus positively interacted in controlling picoplankton biomass production and its transfer towards higher trophic levels. Two complementary approaches (experimental and field study) indicated several coherent patterns: (1) the impact of temperature on heterotrophic bacteria was high at temperatures lower than 16°C and levelled off at higher temperatures, whereas this impact on autotrophic picoplankton was linear along the entire range of the investigated temperatures; (2) the addition of phosphorus increased the values of picoplankton production and grazing, but did not change the nature of their relationships with temperature substantially; (3) the picoplankton carbon flux towards higher trophic levels was larger during the warmer months (grazing by HNF dominated during the warmer period and by ciliates during the colder period) and also strengthened in conditions without phosphorus limitation; (4) the hypothesis that the available phosphorus can be better utilized at higher temperatures was confirmed for both autotrophic and heterotrophic picoplankton; (5) the hypothesis that the rise in temperature stimulates growth only in conditions of sufficient phosphorus was confirmed only for heterotrophic bacteria. Therefore, in the global warming scenario, an increase of the picoplankton carbon flux towards higher trophic levels can be expected in the Adriatic Sea, particularly under unlimited phosphorus conditions.

Relations between mercury fractions and microbial community components in seawater under the presence and absence of probable phosphorus limitation conditions.

Microbial transformations of toxic monomethylmercury (MMHg) and dissolved gaseous mercury (DGM) at the lower levels of the marine food web are not well understood, especially in oligotrophic and phosphorus-limited seas. To examine the effects of probable phosphorus limitation (PP-limitation) on relations between mercury (Hg) fractions and microorganisms, we determined the total mercury (THg), total methylated mercury (MeHg), DGM, and microbiological and chemical parameters in the Central Adriatic Sea. Using statistical analysis, we assessed the potential microbial effects on Hg transformations and bioaccumulation. Only in the absence of PP-limitation conditions (NO-PP-limitation) is MeHg significantly related to most chemical and microbial parameters, indicating metabolism-dependent Hg transformations. The heterotrophic activity of low nucleic acid bacteria (abundant in oligotrophic regions) seems responsible for most of Hg methylation under NO-PP-limitation. Under these conditions, DGM is strongly related to microbial fractions and chlorophyll a, indicating biological DGM production, which is probably not metabolically induced, as most of these relations are also observed under PP-limitation. MMHg biomagnification was observed through an increased bioaccumulation factor from microseston to mesozooplankton. Our results indicate that Hg transformations and uptake might be enhanced under NO-PP-limitation conditions, emphasizing their impact on the transfer of Hg to higher trophic levels.

Status of faecal pollution in ports: A basin-wide investigation in the Adriatic Sea.

Ports are subject to a variety of anthropogenic impacts, and there is mounting evidence of faecal contamination through several routes. Yet, little is known about pollution in ports by faecal indicator bacteria (FIB). FIB spatio-temporal dynamics were assessed in 12 ports of the Adriatic Sea, a semi-enclosed basin under strong anthropogenic pressure, and their relationships with environmental variables were explored to gain insight into pollution sources. FIB were abundant in ports, often more so than in adjacent areas; their abundance patterns were related to salinity, oxygen, and nutrient levels. In addition, a molecular method, quantitative (q)PCR, was used to quantify FIB. qPCR enabled faster FIB determination and water quality monitoring that culture-based methods. These data provide robust baseline evidence of faecal contamination in ports and can be used to improve the management of routine port activities (dredging and ballast water exchange), having potential to spread pathogens in the sea.

Performance characteristics of the temperature-modified ISO 9308-1 method for the enumeration of Escherichia coli in marine and inland bathing waters.

This study defines performance characteristics of the temperature-modified ISO 9308-1 method for E. coli enumeration in bathing water. After a 4-hour resuscitation period at 36 ±â€¯2 °C, the incubation temperature was changed to 44 ±â€¯0.5 °C. Elevated incubation temperature significantly suppressed the growth of thermo-intolerant bacteria, and enhanced the selectivity of Chromogenic Coliform Agar (CCA) up to 49.5% for inland and up to 66.0% for coastal water. Consequently, most of the selectivity-related performance characteristics are improved. Relative recovery was determined by comparing an alternative method against the reference, ISO 9308-1:2014 method, following the criteria set out in ISO 17994:2014. Temperature modification did not significantly alter the results and the methods were evaluated as "not different" for both, coastal and inland waters. Chromogenic Coliform Agar was assessed as a suitable medium for reliable E. coli enumeration in bathing water when incubated for 17-19 h at 44 ±â€¯0.5 °C after the 4-5 h resuscitation period at 36 ±â€¯2 °C.

The effect of temperature increase on microbial carbon fluxes in the Adriatic Sea: an experimental approach.

An assessment of the temperature increase effect on processes within the microbial food web provides a better insight into the carbon transfer and energy flow processes in marine environments in the global warming perspective. Modified laboratory dilution experiments that allow simultaneous estimates of protozoan grazing and viral lysis on picoplankton groups (bacteria, Prochlorococcus, Synechococcus and pico-eukaryotic algae) under in situ and 3°C above in situ temperatures were performed at seasonal scale. Picoplankton mortality due to grazing was generally higher than that caused by viral lysis, especially in the cold months. The largest part of HNF carbon demand was satisfied by grazing on bacteria throughout the year. Although ciliates satisfied their carbon demand predominantly through grazing on HNF and bacteria, the role of autotrophic picoplankton (APP) as their prey increased significantly in the cold months. Bacteria constituted the most important host for viruses throughout the year. However, during the warm months, APP groups were also significant hosts for viral infection. Under the warming condition the amount of picoplankton biomass transferred to protozoan grazers exceeded the lysed biomass, suggesting that global warming could further increase picoplankton carbon flow toward higher trophic levels in the Adriatic Sea.

Temporal variations analyses and predictive modeling of microbiological seawater quality.

Bathing water quality is a major public health issue, especially for tourism-oriented regions. Currently used methods within EU allow at least a 2.2 day period for obtaining the analytical results, making outdated the information forwarded to the public. Obtained results and beach assessment are influenced by the temporal and spatial characteristics of sample collection, and numerous environmental parameters, as well as by differences of official water standards. This paper examines the temporal variation of microbiological parameters during the day, as well as the influence of the sampling hour, on decision processes in the management of the beach. Apart from the fecal indicators stipulated by the EU Bathing Water Directive (E. coli and enterococci), additional fecal (C. perfringens) and non-fecal (S. aureus and P. aeriginosa) parameters were analyzed. Moreover, the effects of applying different evaluation criteria (national, EU and U.S. EPA) to beach ranking were studied, and the most common reasons for exceeding water-quality standards were investigated. In order to upgrade routine monitoring, a predictive statistical model was developed. The highest concentrations of fecal indicators were recorded early in the morning (6 AM) due to the lack of solar radiation during the night period. When compared to enterococci, E. coli criteria appears to be more stringent for the detection of fecal pollution. In comparison to EU and U.S. EPA criteria, Croatian national evaluation criteria provide stricter public health standards. Solar radiation and precipitation were the predominant environmental parameters affecting beach water quality, and these parameters were included in the predictive model setup. Predictive models revealed great potential for the monitoring of recreational water bodies, and with further development can become a useful tool for the improvement of public health protection.

Escherichia coli in marine water: Comparison of methods for the assessment of recreational bathing water samples.

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.

Autochthonous Bacterial Isolates Successfully Stimulate In vitro Peripheral Blood Leukocytes of the European Sea Bass (Dicentrarchus labrax).

Commercially available probiotics are routinely administered as feed supplements in aquaculture important species. Among them, the European sea bass (Dicentrarchus labrax) is the most widely reared fish in the Mediterranean, whose rearing systems are highly variable between countries, affecting at some level the sustainability of production. After random isolation of autochthonous gut bacteria of the sea bass, their identification and pathogenicity testing, we have selected three potentially probiotic isolates; Pseudoalteromonas sp., Alteromonas sp., and Enterovibrio coralii. Selected isolates were tested and their immunostimulative efficiency was compared with a commercially available Lactobacillus casei isolate, inferring inflammatory, apoptotic and anti-pathogen response of sea bass' peripheral blood leukocytes. Phagocytic activity, respiratory burst, and expression of lysozyme, Mx protein, caspase 3, TNF-α, IL-10 genes was measured 1, 3, 5, and 12 h post-stimulation by four bacterial isolates to evaluate early kinetics of the responses. Best immunostimulative properties were observed in Pseudoalteromonas-stimulated leukocytes, followed by Alteromonas sp. and L. casei, while Enterovibrio coralii failed to induce significant stimulation. Based on such in vitro assay intestinal autochthonous bacterial isolates showed to have better immunostimulative effect in sea bass compared to aquaculture-widely used L. casei, and further steps need to engage tank and field feeding trials to evaluate long-term prophylactic suitability of the chosen isolates. A panel of biomarkers that represent pro-/anti-inflammatory, pro-/anti-apoptotic, and anti-bacteria/viral responses of the fish should be taken into consideration when evaluating the usefulness of the potential probiotic in aquaculture.