Occurrence and risk assessment of pharmaceuticals in hospital wastewater in Costa Rica.

This work aims to determine the occurrence, hazard and prioritization of pharmaceuticals from hospital wastewater in Costa Rica through the monitoring of 70 compounds and assessing their environmental risk through a hazard quotient approach (HQ). Moreover, the quantification of selected antibiotic resistance genes (ARGs) was conducted for the first time in this matrix in this geographical location. Thirty-four pharmaceuticals were detected, being caffeine, 1,7-dimethylxanthine, acetaminophen, ibuprofen, naproxen, ciprofloxacin and ketoprofen the most frequent (>50% of the samples). Eighteen pharmaceuticals exhibited high hazard (HQ ≥ 1), while five more showed medium hazard (1 > HQ ≥ 0.1). Prioritization, which also included frequency parameters, revealed caffeine, lovastatin, diphenhydramine, acetaminophen, ibuprofen, ciprofloxacin, and sildenafil as the compounds of major concern. Similarly, cumulative hazard per sample (ΣHQ) estimated high hazard towards aquatic organisms in every sample. All selected ARGs, except mcr-1 (polymyxin resistance), were detected. Among genes conferring resistance to beta-lactams, blaCTX-M and blaKPC were the most abundant, related to resistance to cephalosporins and carbapenems. Ecotoxicological evaluation showed mostly low toxicity towards Daphnia magna and Vibrio fischeri, contrary to the marked effect observed towards Lactuca sativa. These findings provide relevant and novel information on the risk posed by hospital wastewater and their pharmaceutical content in the Latin American environmental context.

Occurrence of Aeromonas Species in the Cutaneous Mucus of Barbour's Seahorses (Hippocampus barbouri) as Revealed by High-Throughput Sequencing.

Although several studies have described the bacterial community composition associated with marine fish, there is limited information related to seahorses. Moreover, previous studies have demonstrated that the skin microbiota is useful for determining health status and common disorders in the host. This study, therefore, aimed to explore the skin bacterial community composition in Barbour's seahorse (Hippocampus barbouri) using high-throughput sequencing of 16S ribosomal RNA genes. Water and sediment samples from the surrounding environment were also analyzed for comparative purposes. The results revealed that sequences affiliated with the Shewanellaceae family were dominant in the skin of female Barbour's seahorses and sediment samples, whereas sequences affiliated with the Bacillaceae family were dominant in the skin of male Barbour's seahorses. Interestingly, sequences affiliated with the Aeromonas genus were found in the skin of Barbour's seahorses, whose abundance was slightly similar between the female and male specimens. Further comparative analysis showed that the presence of Aeromonas species in the skin of Barbour's seahorses was strongly influenced by the surrounding sediment. Given that some Aeromonas species are known to be important pathogens in humans and fish, these results may be used for further research on the dependency of the skin microbial composition on the environment as well as determine whether the presence of Aeromonas and other detected species has implications on seahorse health.

A Broad-Host-Range Phage Cocktail Selectively and Effectively Eliminates Vibrio Species from Shrimp Aquaculture Environment.

The protective effects of a phage cocktail composed of vB_Vc_SrVc2 and vB_Vc_SrVc9 were tested in Pacific white shrimp (Litopenaeus vannamei) postlarvae, which were originally isolated from diseased shrimps and selected due to their broad-host-range properties against several pathogenic Vibsrio species. We used culture-dependent and culture-independent approaches to explore its effect on bacterial communities associated with shrimp postlarvae. Both methods revealed that the levels of Vibrio species were significantly reduced after phage cocktail administration. Phage-treated shrimp also exhibisuppted lesser damage and higher lipid accumulation in B cells of the hepatopancreas, as revealed by histopathological examination. Taken together, this study provides clear evidence that phage therapy can selectively and effectively reduce Vibrio species, thereby providing an environmentally safe alternative to the prophylactic use of antibiotics in shrimp aquaculture.

Effects of two host-associated probiotics Bacillus mojavensis B191 and Bacillus subtilis MRS11 on growth performance, intestinal morphology, expression of immune-related genes and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcusiniae.

The intensification and diversification of production systems have increased the incidence of diseases, which are usually treated with antibiotics. However, its use should be restricted due to the increasing prevalence of antibiotic-resistant bacteria. Probiotics represent therefore an alternative environmentally friendly strategy for improving growth and disease resistance in aquaculture. Considering that host-derived probiotics may offer greater advantages than those from other environments in terms of safety and efficacy, two potential host-associated probiotic strains (Bacillus mojavensis B191 and Bacillus subtilis MRS11) were used in the present study, which were previously isolated from intestinal mucus of Nile tilapia (Oreochromis niloticus). This study was conducted to assess the effects of dietary administration of two Bacillus strains on growth performance, intestinal morphology, immunity, and disease resistance of Nile tilapia. A total of 375 fish were randomly divided into five groups in triplicate. Nile tilapia were fed a basal diet (control group) or a basal diet supplemented with Bacillus mojavensis B191 (BM) or Bacillus subtilis MRS11 (BS) spores at different concentrations of 1 × 106 (BM6 and BS6, respectively) and 1 × 108 (BM8 and BS8, respectively) CFU/g of feed for 60 days. Moreover, the survival rate of tilapia upon challenge with Streptococcus iniae was determined following the feeding trial. After the feeding trial, the growth performances were significantly improved in all probiotic-fed groups, with the BS8 group being the highest. Light and electron microscopy observations revealed elevated goblet cells, intestinal villus length (except BM8), microvilli length, microvilli density, and perimeter ratio increase in the intestine of all probiotic-fed groups compared with the control group. Regarding the expression analysis, HSP70 gene was only up-regulated in the BM8 group and a general trend of up-regulation of some immune-related cytokines (TGF-ß, IL-10, TNF-α and IL-1ß) was observed in all probiotic-fed groups. Likewise, the best protection against Streptococcus iniae was observed in the BS8 group, followed by BS6, BM6 and BM8 groups. Altogether, dietary probiotic supplementation with BS8 and BM6 may improve growth performance, intestinal morphology, immunity, and disease resistance in Nile tilapia.

Bacteriophage cocktail as a promising bio-enhancer for methanogenic activities in anaerobic membrane bioreactors.

This study aimed to explore the effect of a bacteriophage cocktail, pyophage, on the treatment of wastewater containing antibiotics in an anaerobic membrane bioreactor (AnMBR). During the operational period, performance of the AnMBR was monitored through the changes in chemical oxygen demand (COD), antibiotic removal, transmembrane pressure, and biogas production. Microbial community structure and composition, as well as the occurrence of antibiotic resistance genes were analyzed through shotgun metagenomics analysis. When exposed to pyophage, COD removal efficiency was enhanced up to 96%, whereas membrane fouling was delayed by 25%. Average biogas production was doubled from 224.2 mL/d in control with antibiotics to 447.3 mL/d when exposed to pyophage cocktail with considerable alterations to the archaeal and bacterial community structures. Most notably, the methanogenic community shifted from dominance of Methanothermobacter to Methanoculleus, along with syntrophic bacteria. The results provide insight into the synergistic effects of phage-bacteria and methanogenic communities and illustrate the potential of bacteriophages as bio-enhancers.

Isolation of Salmonella spp. from black spiny-tailed iguana (Ctenosaura similis) meat commercialised in markets of León city, Nicaragua.

BACKGROUND: The black spiny-tailed iguana (Ctenosaura similis) is an endemic animal in Mesoamerica, whose meat is consumed by the local population. OBJECTIVES: Because the black spiny-tailed iguana may be potential reservoirs of pathogens, this study aimed to isolate and characterise Salmonella spp. in their meat commercialised in markets of the city of León, Nicaragua. METHODS: Thirteen specimens were analysed for the isolation of Salmonella spp., as well as their antimicrobial resistance patterns, including the presence of genes encoding extended-spectrum ß-lactamases. RESULTS: Salmonella spp. isolates were found in eight out of 13 samples, with S. enterica serovar Enteritidis being found in six out of eight samples. Moreover, eight Salmonella spp. isolates were resistant to amoxicillin plus clavulanic acid and cephalexin, but sensitive to other tested antibiotics. The blaSHV gene was detected in seven out of eight Salmonella spp. isolates, followed by the blaTEM (two out of eight) and blaCXT-M (one out of eight) genes. CONCLUSIONS: These findings represent an important contribution to the implementation of appropriate strategies to prevent foodborne diseases.

Characterization of the genetic structure of mcr-1 gene among Escherichia coli isolates recovered from surface waters and sediments from Ecuador.

Although anthropogenic activities contribute to the selection and spread of antibiotic resistance in aquatic environments, limited information is available from countries with absent or incomplete sewage treatment systems and the impact of their discharges onto water bodies. This study therefore aimed to characterize the genetic structure of colistin resistance (mcr) genes among Escherichia coli isolates recovered from surface waters and sediments in Ecuador. Out of 459 isolates, four Escherichia coli showed multidrug-resistant phenotypes, which harbored the mcr-1 gene and ß-lactamases, such as blaTEM, blaCTX-M-15, blaCTX-M-55, or blaCTX-M-65 genes. Three E. coli isolates (U20, U30 and U144) shared a similar genetic environment surrounding the mcr-1 gene, which was located on plasmids. Only one E. coli isolate (U175) showed that the mcr-1 gene was chromosomally located. Moreover, the core genome multilocus sequence typing (cgMLST) analysis revealed that these isolates belong to different lineages. This study represents the first detection of the mcr-1 gene in multidrug-resistant E. coli isolates from environmental samples in Ecuador.

Making waves: How does the emergence of antimicrobial resistance affect policymaking?

This article considers current trends in antimicrobial resistance (AMR) research and knowledge gaps relevant to policymaking in the water sector. Specifically, biological indicators of AMR (antibiotic-resistant bacteria and their resistance genes) and detection methods that have been used so far are identified and discussed, as well as the problems with and solutions to the collection of AMR data, sewage surveillance lessons from the COVID-19 pandemic, and the financial burden caused by AMR, which could be synergically used to improve advocacy on AMR issues in the water sector. Finally, this article proposes solutions to overcoming existing hurdles and shortening the time it will take to have an impact on policymaking and regulation in the sector.

Assessment of bacteriophage vB_Pd_PDCC-1 on bacterial dynamics during ontogenetic development of the longfin yellowtail (Seriola rivoliana).

The Seriola genus includes species of worldwide commercial importance due to its rapid growth and easy adaptability to confinement conditions. However, like other fish species, large mortalities occur during their early life stages, where the main problems are caused by opportunistic bacteria. Disease control strategies are thus urgently needed. The present study aimed to evaluate the efficacy of phage vB_Pd_PDCC-1 during the early development of longfin yellowtail (Seriola rivoliana), as well as its effect on microbial communities. This broad-host-range phage was added to the culture every 3 days starting from the egg-stage until 12 days after hatching (DAH) at a concentration of 1.41×1010 plaque-forming units (PFU) per mL and at a multiplicity of infection (MOI) of 1. The results showed positive effects (p<0.05) on egg hatching, survival, growth, and pigmentation area in treated larvae. Moreover, high-throughput sequencing analysis of 16S rRNA genes showed that phage administration did not produce significant changes (p>0.05) in the composition and structure of the associated microbiota. However, sequences affiliated to the Gammaproteobacteria class were displaced by those belonging to the Alphaproteobacteria class over time regardless of the treatment received. At the family level, there was a decrease in Rhodobacteraceae, Pseudoalteromonadaceae, and Flavobacteriaceae in both groups over time. To our best knowledge, this study represents the first attempt to evaluate the effect of a phage as a biological control agent during ontogenetic development of longfin yellowtail larvae. KEY POINTS: • Phages can be used against proliferation of Vibrio in fish cultures. • Seriola includes several important commercial fish species due to its rapid growth. • Phages do not cause significant changes in the associated microbiota.

Metagenomic analysis of urban wastewater resistome and mobilome: A support for antimicrobial resistance surveillance in an endemic country.

In developing countries, where high levels of antimicrobial resistance are observed in hospitals, the surveillance of this phenomenon in wastewater treatment plants (WWTPs) and the environment is very limited, especially using cutting-edge culture-independent methods. In this study, the composition of bacterial communities, the resistome and mobilome (the pool of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), respectively) at a WWTP were determined using shotgun metagenomics and culture-based approaches. Wastewater samples were collected at four sampling points of a WWTP in Antioquia, Colombia. A total of 24 metagenomes were analyzed. Specifically, there were marked differences in bacterial community composition, resistome, and mobilome, according to the WWTP sampling points. Bacterial families of clinical importance such as Moraxellaceae, Aeromonadaceae, and Enterobacteriaceae were mainly detected in the WWTP influent and effluent samples. Genes encoding resistance to macrolide-lincosamide-streptogramin, ß-lactams, and those conferring multidrug resistance (e.g., acrB, adeG, and mexD) were the most abundant. Moreover, some clinically important ARGs such as blaKPC-2 and blaCTX-M, and others not reported locally, such as blaTEM-196, blaGES-23, blaOXA-10, mcr-3, and mcr-5 were frequently detected. Co-occurrence network analyses indicated a significant association of ARGs such as blaOXA-58 and blaKPC genes with Aeromonadaceae and Enterobacteriaceae. Among the markers of MGEs, intI1 and ISCR8 were the most frequently detected. Altogether, this work reveals the importance of shotgun metagenomics and culture-based approaches in antimicrobial resistance studies. The findings also support that WWTPs are hotspots for antimicrobial resistance, whose analysis constitutes a powerful tool to predict the impact of antimicrobial resistance in a population.

Administration of Probiotics Improves the Brine Shrimp Production and Prevents Detrimental Effects of Pathogenic Vibrio Species.

In this study, we evaluated a consortium of probiotic bacteria as an environmentally-friendly strategy for controlling pathogenic Vibrio species during the brine shrimp incubation period. Probiotic strains were initially selected on basis of (i) their ability to colonize the cyst surfaces, (ii) their absence of cross-inhibitory effects, and (iii) no detrimental effect on cyst hatching. The cysts and nauplius surfaces were immediately colonized after the application of selected probiotic strains, without detrimental effects on survival. Ten probiotic strains were mixed at similar proportions (probiotic consortium) and evaluated at different concentrations into brine shrimp cultures during incubation and early stages of development. Subsequently, these cultures were challenged with Vibrio parahaemolyticus and Vibrio harveyi. The probiotic consortium was effective to reduce the abundance of pathogenic Vibrio species and to prevent the mortality during Vibrio challenges; however, its effect was concentration-dependent and was successful at a starting concentration of 1.8 × 106 CFU/ml. Our results suggest that this probiotic consortium offers an alternative to antimicrobial agents routinely used to reduce the incidence and prevalence of pathogenic Vibrio species in brine shrimp production.

Isolation, characterization and evaluation of probiotic lactic acid bacteria for potential use in animal production.

In livestock production, lactic acid bacteria (LAB) are the most common microorganisms used as probiotics. For such use, these bacteria must be correctly identified and characterized to ensure their safety and efficiency. In the present study, LAB were isolated from broiler excreta, where a fermentation process was used. Nine among sixteen isolates were identified by biochemical and molecular (sequencing of the 16S rRNA gene) methods as Lactobacillus crispatus (n=1), Lactobacillus pentosus (n=1), Weissella cibaria (n=1), Pediococcus pentosaceus (n=2) and Enterococcus hirae (n=4). Subsequently, these bacteria were characterized for their growth capabilities, lactic acid production, acidic pH and bile salts tolerance, cell surface hydrophobicity, antimicrobial susceptibility and antagonistic activity. Lactobacillus pentosus strain LB-31, which showed the best characteristics, was selected for further analysis. This strain was administered to broilers and showed the ability of modulating the immune response and producing beneficial effects on morpho-physiological, productive and health indicators of the animals.

The role of biofilms as environmental reservoirs of antibiotic resistance.

Antibiotic resistance has become a significant and growing threat to public and environmental health. To face this problem both at local and global scales, a better understanding of the sources and mechanisms that contribute to the emergence and spread of antibiotic resistance is required. Recent studies demonstrate that aquatic ecosystems are reservoirs of resistant bacteria and antibiotic resistance genes as well as potential conduits for their transmission to human pathogens. Despite the wealth of information about antibiotic pollution and its effect on the aquatic microbial resistome, the contribution of environmental biofilms to the acquisition and spread of antibiotic resistance has not been fully explored in aquatic systems. Biofilms are structured multicellular communities embedded in a self-produced extracellular matrix that acts as a barrier to antibiotic diffusion. High population densities and proximity of cells in biofilms also increases the chances for genetic exchange among bacterial species converting biofilms in hot spots of antibiotic resistance. This review focuses on the potential effect of antibiotic pollution on biofilm microbial communities, with special emphasis on ecological and evolutionary processes underlying acquired resistance to these compounds.

Phylogenetic characterization and in situ detection of bacterial communities associated with seahorses (Hippocampus guttulatus) in captivity.

Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that Vibrionaceae was the dominant population in Artemia sp. (live prey) and intestinal content of the seahorses, while Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described.

Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms.

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.