Hidden threats in the plastisphere: Carbapenemase-producing Enterobacterales colonizing microplastics in river water.

Carbapenem resistance poses a significant burden on healthcare systems worldwide. Microplastics (MPs) have emerged as potential contributors to antibiotic resistance spread in the environment. However, the link between MPs and carbapenem resistance remains unexplored. We investigated the prevalence of carbapenem-resistant bacteria colonizing MPs placed in a river. Three replicates of a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET) and of PET alone were placed both upstream and downstream a wastewater treatment plant (WWTP) discharge. Carbapenem-resistant Enterobacterales (CRE) were further characterized by phenotypic tests and whole-genome sequencing. The abundance of carbapenem-resistant bacteria on MPs increased significantly downstream the WWTP. Their prevalence was higher in the MPs mixture compared to PET alone. CRE strains colonizing MPs included Klebsiella pneumoniae (n = 3), Klebsiella quasipneumoniae (n = 3), Raoultella ornithinolytica (n = 2), Enterobacter kobei (n = 1) and Citrobacter freundii (n = 1), most (n = 8) recovered after the WWTP discharge. All strains exhibited at least one of the tested virulence traits (biofilm formation at 37 °C, haemolytic activity and siderophore production), were multi-drug resistant and carried carbapenemase-encoding genes [blaKPC-3 (n = 5), blaGES-5 (n = 2) or blaKPC-3 + blaGES-5 (n = 3)]. Uncommon phenotypes of resistance to imipenem/relebactam (n = 3) and ceftazidime/avibactam (n = 2) were observed. Two blaKPC-3-positive K. pneumoniae successfully transfer this gene trough conjugation. Genome analysis predicted all strains as human pathogens. The blaKPC-3 was associated with the Tn4401d transposon on a pBK30683-like plasmid in most of the isolates (n = 7). The blaGES-5 was mostly linked to class 3 integrons. A K. pneumoniae strain belonging to the outbreak-causing high-risk clone ST15 carried both blaKPC-3 and blaCTX-M-15. Two K. quasipneumoniae isolates carried the plasmid-mediated colistin resistance gene mcr-9. Our results underscore the role of MPs as vectors for CRE dissemination, particularly following WWTPs discharges. MPs may act as carriers, facilitating the dissemination of carbapenemase-encoding genes and potentially contributing to increased CRE incidence in the environment.

Microplastics accumulate priority antibiotic-resistant pathogens: Evidence from the riverine plastisphere.

Microplastics (MPs) might accumulate and transport antibiotic-resistant bacteria (ARB) in aquatic systems. We determined the abundance and diversity of culturable ciprofloxacin- and cefotaxime-resistant bacteria in biofilms covering MPs placed in river water, and characterized priority pathogens from these biofilms. Our results showed that the abundance of ARB colonizing MPs tends to be higher compared to sand particles. Also, higher numbers were cultivated from a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET), compared to PP and PET alone. Aeromonas and Pseudomonas isolates were the most frequently retrieved from MPs placed before a WWTP discharge while Enterobacteriaceae dominated the culturable plastisphere 200 m after the WWTP discharge. Ciprofloxacin- and/or cefotaxime-resistant Enterobacteriaceae (n = 54 unique isolates) were identified as Escherichia coli (n = 37), Klebsiella pneumoniae (n = 3), Citrobacter spp. (n = 9), Enterobacter spp. (n = 4) and Shigella sp. (n = 1). All isolates presented at least one of the virulence features tested (i.e. biofilm formation, haemolytic activity and production of siderophores), 70% carried the intI1 gene and 85% exhibited a multi-drug resistance phenotype. Plasmid-mediated quinolone resistance genes were detected in ciprofloxacin-resistant Enterobacteriaceae [aacA4-cr (40% of the isolates), qnrS (30%), qnrB (25%), and qnrVC (8%)], along with mutations in gyrA (70%) and parC (72%). Cefotaxime-resistant strains (n = 23) harbored blaCTX-M (70%), blaTEM (61%) and blaSHV (39%). Among CTX-M producers, high-risk clones of E. coli (e.g. ST10 or ST131) and K. pneumoniae (ST17) were identified, most of which carrying blaCTX-M-15. Ten out of 16 CTX-M producers were able to transfer blaCTX-M to a recipient strain. Our results demonstrated the occurrence of multidrug resistant Enterobacteriaceae in the riverine plastisphere, harboring ARGs of clinical concern and exhibiting virulence traits, suggesting a contribution of MPs to the dissemination of antibiotic-resistant priority pathogens. The type of MPs and especially water contamination (e.g. by WWTPs discharges) seem to determine the resistome of the riverine plastisphere.

Bioremediation of coastal aquaculture effluents spiked with florfenicol using microalgae-based granular sludge - a promising solution for recirculating aquaculture systems.

Aquaculture is a crucial industry in the agri-food sector, but it is linked to serious environmental problems. There is a need for efficient treatment systems that allow water recirculation to mitigate pollution and water scarcity. This work aimed to evaluate the self-granulation process of a microalgae-based consortium and its capacity to bioremediate coastal aquaculture streams that sporadically contain the antibiotic florfenicol (FF). A photo-sequencing batch reactor was inoculated with an autochthonous phototrophic microbial consortium and was fed with wastewater mimicking coastal aquaculture streams. A rapid granulation process occurred within ca. 21 days, accompanied by a substantially increase of extracellular polymeric substances in the biomass. The developed microalgae-based granules exhibited high and stable organic carbon removal (83-100%). Sporadically wastewater contained FF which was partially removed (ca. 5.5-11.4%) from the effluent. In periods of FF load, the ammonium removal slightly decreased (from 100 to ca. 70%), recovering 2 days after FF feeding ceased. A high-chemical quality effluent was obtained, complying with ammonium, nitrite, and nitrate concentrations for water recirculation within a coastal aquaculture farm, even during FF feeding periods. Members belonging to the Chloroidium genus were predominant in the reactor inoculum (ca. 99%) but were replaced from day-22 onwards by an unidentified microalga from the phylum Chlorophyta (>61%). A bacterial community proliferated in the granules after reactor inoculation, whose composition varied in response to feeding conditions. Bacteria from the Muricauda and Filomicrobium genera, Rhizobiaceae, Balneolaceae, and Parvularculaceae families, thrived upon FF feeding. This study demonstrates the robustness of microalgae-based granular systems for aquaculture effluent bioremediation, even during periods of FF loading, highlighting their potential as a feasible and compact solution in recirculation aquaculture systems.

Report and Comparative Genomics of an NDM-5-Producing Escherichia coli in a Portuguese Hospital: Complex Class 1 Integrons as Important Players in blaNDM Spread.

BACKGROUND: New Delhi metallo-beta-lactamase (NDM) has been spreading across the globe, but the causes of its success are poorly understood. We characterized a blaNDM-5-positive Escherichia coli strain from a Portuguese hospital and conducted comparative genomic analyses to understand the role of clonal background and horizontal gene transfer in blaNDM-5 dissemination. METHODS: After blaNDM PCR screening and genome sequencing, Ec355340 was subjected to mating, transformation, and plasmid curing assays and MICs determination for several antibiotics. Comparison with data compiled from public databases was performed. RESULTS: blaNDM-5 was in a complex integron co-located in a FIB-FII plasmid (pEc355340_NDM-5). The mating assays were unsuccessful, but plasmid transformation into a susceptible host led to resistance to all beta-lactams and to sulfamethoxazole-trimethoprim. The profile of virulence genes (n = 73) was compatible with extraintestinal pathogenesis. An analysis of genomes from public databases suggested that blaNDM-5 has rarely been associated with ST156 strains (such as Ec355340), while is has frequently been found on strains of the ST10 clonal complex. However, ST156 may play a role in the co-spreading of blaNDM and mcr genes. Regardless, comparative genomics confirmed the presence of blaNDM in similar complex integrons in plasmids (48/100 plasmids most similar to pEc355340_NDM-5) and ST156 genomes (20/41 blaNDM-positive genomes). CONCLUSIONS: blaNDM-5 and other blaNDM variants were more frequently associated to complex integrons than previously reported and, therefore, these platforms may be important drivers in their dissemination. The identification of blaNDM-5 for the first time in Portugal could be a game-changer in the current Portuguese antibiotic resistance scenario, as this gene encodes a higher-level resistance phenotype, and its spread may be facilitated due to the association with complex integrons.

CTX-M-Producing Bacteria Isolated from a Highly Polluted River System in Portugal.

Enterobacteriaceae resistant to third-generation cephalosporins are a great concern for public health, as these are first-line drugs to treat infections. The production of carbapenemases and extended spectrum beta-lactamases (ESBLs) and/or the overexpression of AmpC ß-lactamases are the main mechanisms of resistance to these antibiotics. Among the ESBLs, CTX-M ß-lactamases are the most prevalent worldwide. Our aims were to determine the prevalence of cefotaxime-resistant Enterobacteriaceae along a heavily polluted river and characterize blaCTX-M carriers. River water was collected in 11 sites along the main course and tributaries, in two sampling moments. Water quality was evaluated and a collection of cefotaxime-resistant isolates was obtained. blaCTX-M carriers were characterized regarding phylogenetic affiliation, clonality, antibiotic susceptibility, gene diversity, and context. Water presented very low quality in all sites. From 147 cefotaxime-resistant isolates, 46% carried blaCTX-M and were affiliated with Escherichia, Klebsiella, Enterobacter, and Citrobacter. Molecular typing revealed clonal isolates in different sites and over the two years, suggesting survival of the strains in the river or continuous pollution inputs from the same sources. Eight variants of blaCTX-M were found, with blaCTX-M-15 being the most prevalent (52.5%). Sites with a lower water quality showed the highest resistance rates and prevalence of blaCTX-M, suggesting that river water may embody human health risks.

Genome Analyses of Two Blueberry Pathogens: Diaportheamygdali CAA958 and Diaporthe eres CBS 160.32.

The genus Diaporthe includes pathogenic species distributed worldwide and affecting a wide variety of hosts. Diaporthe amygdali and Diaporthe eres have been found to cause cankers, dieback, or twig blights on economically important crops such as soybean, almond, grapevine, and blueberry. Despite their importance as plant pathogens, the strategies of species of Diaporthe to infect host plants are poorly explored. To provide a genomic basis of pathogenicity, the genomes of D. amygdali CAA958 and D. eres CBS 160.32 were sequenced and analyzed. Cellular transporters involved in the transport of toxins, ions, sugars, effectors, and genes implicated in pathogenicity were detected in both genomes. Hydrolases and oxidoreductases were the most prevalent carbohydrate-active enzymes (CAZymes). However, analyses of the secreted proteins revealed that the secretome of D. eres CBS 160.32 is represented by 5.4% of CAZymes, whereas the secreted CAZymes repertoire of D. amygdali CAA958 represents 29.1% of all secretomes. Biosynthetic gene clusters (BGCs) encoding compounds related to phytotoxins and mycotoxins were detected in D. eres and D. amygdali genomes. The core gene clusters of the phytotoxin Fusicoccin A in D. amygdali are reported here through a genome-scale assembly. Comparative analyses of the genomes from 11 Diaporthe species revealed an average of 874 CAZymes, 101 secondary metabolite BGCs, 1640 secreted proteins per species, and genome sizes ranging from 51.5 to 63.6 Mbp. This study offers insights into the overall features and characteristics of Diaporthe genomes. Our findings enrich the knowledge about D. eres and D. amygdali, which will facilitate further research into the pathogenicity mechanisms of these species.

KPC-3-, GES-5-, and VIM-1-Producing Enterobacterales Isolated from Urban Ponds.

Carbapenems are antibiotics of pivotal importance in human medicine, the efficacy of which is threatened by the increasing prevalence of carbapenem-resistant Enterobacterales (CRE). Urban ponds may be reservoirs of CRE, although this hypothesis has been poorly explored. We assessed the proportion of CRE in urban ponds over a one-year period and retrieved 23 isolates. These were submitted to BOX-PCR, PFGE, 16S rDNA sequencing, antibiotic susceptibility tests, detection of carbapenemase-encoding genes, and conjugation assays. Isolates were affiliated with Klebsiella (n = 1), Raoultella (n = 11), Citrobacter (n = 8), and Enterobacter (n = 3). Carbapenemase-encoding genes were detected in 21 isolates: blaKPC (n = 20), blaGES-5 (n = 6), and blaVIM (n = 1), with 7 isolates carrying two carbapenemase genes. Clonal isolates were collected from different ponds and in different campaigns. Citrobacter F6, Raoultella N9, and Enterobacter N10 were predicted as pathogens from whole-genome sequence analysis, which also revealed the presence of several resistance genes and mobile genetic elements. We found that blaKPC-3 was located on Tn4401b (Citrobacter F6 and Enterobacter N10) or Tn4401d (Raoultella N9). The former was part of an IncFIA-FII pBK30683-like plasmid. In addition, blaGES-5 was in a class 3 integron, either chromosomal (Raoultella N9) or plasmidic (Enterobacter N10). Our findings confirmed the role of urban ponds as reservoirs and dispersal sites for CRE.

Occurrence and distribution of Carbapenem-resistant Enterobacterales and carbapenemase genes along a highly polluted hydrographic basin.

We determined the distribution and temporal variation of Carbapenem Resistant Enterobacterales (CRE), carbapenemase-encoding genes and other antibiotic resistance genes (ARGs) in a highly polluted river (Lis River; Portugal), also assessing the potential influence of water quality to this distribution. Water samples were collected in two sampling campaigns performed one year apart (2018/2019) from fifteen sites and water quality was analyzed. CRE were isolated and characterized. The abundance of four ARGs (blaNDM, blaKPC, tetA, blaCTX-M), two Microbial Source Tracking (MST) indicators (HF183 and Pig-2-Bac) and the class 1 integrase gene (IntI1) was measured by qPCR. RESULTS: confirmed the poor quality of the Lis River water, particularly in sites near pig farms. A collection of 23 CRE was obtained: Klebsiella (n = 19), Enterobacter (n = 2) and Raoultella (n = 2). PFGE analysis revealed a clonal relationship between isolates obtained in different sampling years and sites. All CRE isolates exhibited multidrug resistance profiles. Klebsiella and Raoultella isolates carried blaKPC while Enterobacter harbored blaNDM. Conjugation experiments were successful for only four Klebsiella isolates. All ARGs were detected by qPCR on both sampling campaigns. An increase in ARGs and IntI1 abundances was detected in sites located downstream of wastewater treatment plants. Strong correlations were observed between blaCTX-M, IntI1 and the human-pollution marker HF183, and also between tetA and the pig-pollution marker Pig-2-bac, suggesting that both human- and animal-derived pollution in the Lis River are a potential source of ARGs. Plus, water quality parameters related to eutrophication and land use were significantly correlated with ARGs abundances. Our findings demonstrated that the Lis River encloses high levels of antibiotic resistant bacteria and ARGs, including CRE and carbapenemase-encoding genes. Overall, this study provides a better understanding on the impacts of water pollution resulting from human and animal activities on the resistome of natural aquatic systems.

Genome and Metabolome MS-Based Mining of a Marine Strain of Aspergillus affinis.

Aspergillus section Circumdati encompasses several species that express both beneficial (e.g., biochemical transformation of steroids and alkaloids, enzymes and metabolites) and harmful compounds (e.g., production of ochratoxin A (OTA)). Given their relevance, it is important to analyze the genetic and metabolic diversity of the species of this section. We sequenced the genome of Aspergillus affinis CMG 70, isolated from sea water, and compared it with the genomes of species from section Circumdati, including A. affinis's strain type. The A. affinis genome was characterized considering secondary metabolites biosynthetic gene clusters (BGCs), carbohydrate-active enzymes (CAZymes), and transporters. To uncover the biosynthetic potential of A. affinis CMG 70, an untargeted metabolomics (LC-MS/MS) approach was used. Cultivating the fungus in the presence and absence of sea salt showed that A. affinis CMG 70 metabolite profiles are salt dependent. Analyses of the methanolic crude extract revealed the presence of both unknown and well-known Aspergillus compounds, such as ochratoxin A, anti-viral (e.g., 3,5-Di-tert-butyl-4-hydroxybenzoic acid and epigallocatechin), anti-bacterial (e.g., 3-Hydroxybenzyl alcohol, l-pyroglutamic acid, lecanoric acid), antifungal (e.g., lpyroglutamic acid, 9,12,13-Trihydroxyoctadec-10-enoic acid, hydroxyferulic acid), and chemotherapeutic (e.g., daunomycinone, mitoxantrone) related metabolites. Comparative analysis of 17 genomes from 16 Aspergillus species revealed abundant CAZymes (568 per species), secondary metabolite BGCs (73 per species), and transporters (1359 per species). Some BGCs are highly conserved in this section (e.g., pyranonigrin E and UNII-YC2Q1O94PT (ACR toxin I)), while others are incomplete or completely lost among species (e.g., bikaverin and chaetoglobosins were found exclusively in series Sclerotiorum, while asperlactone seemed completely lost). The results of this study, including genome analysis and metabolome characterization, emphasize the molecular diversity of A. affinis CMG 70, as well as of other species in the section Circumdati.

Tetracycline-Resistant Bacteria Selected from Water and Zebrafish after Antibiotic Exposure.

The emergence of antibiotic-resistant pathogens due to worldwide antibiotic use is raising concern in several settings, including aquaculture. In this work, the selection of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) was evaluated after exposure of zebrafish to oxytetracycline (OTC) for two months, followed by a recovery period. The selection of ARB in water and fish was determined using selective media. The abundance of tetA genes was estimated through qPCR. Higher prevalence of ARB was measured in all samples exposed to the antibiotic when compared to control samples, although statistical significance was only achieved five days after exposure. Isolates recovered from samples exposed to the antibiotic were affiliated with Pseudomonas and Stenotrophomonas. Various antibiotic susceptibility profiles were detected and 37% of the isolates displayed multidrug resistance (MDR). The selection of the tetA gene was confirmed by qPCR at the highest OTC concentration tested. Two MDR isolates, tested using zebrafish embryos, caused significant mortality, indicating a potential impact on fish health and survival. Overall, our work highlights the potential impact of antibiotic contamination in the selection of potential pathogenic ARB and ARGS.

Genome analysis of two multidrug-resistant Escherichia coli O8:H9-ST48 strains isolated from lettuce.

Vegetables may become contaminated with antibiotic-resistant bacteria from farm-to-fork. Here we report draft genome sequences of two multidrug-resistant Escherichia coli isolated from lettuce. Whole genomes of strains Y15 V.22 and Y15 V.54 were sequenced. Available tools were used to inspect for virulence factors (VF), metals tolerance, resistome and mobilome features. The predicted genome sizes were 5,4 Mb and 6,2 Mb for Y15 V.22 and Y15 V.54, respectively, both with 50.7% GC content, ST48 and serotype O8:H9. Resistome analysis showed genes encoding resistance to ß-lactams, sulphonamides, trimethoprim, tetracyclines and macrolides. Cobalt, cadmium, zinc and copper tolerance determinants were identified in both. VF detected included genetic determinants related to toxin production, adherence and invasion. SNPs and VF content analysis showed a close relatedness to ETEC. Putative genomic islands, prophage and CRISPR sequences were predicted. The genome sequences here reported will aid in understanding antibiotic resistance transfer between vegetables consumed raw and humans.

Draft Genome Resources Sequences of Six Pseudomonas syringae pv. actinidiae Strains Isolated from Actinidia chinensis var. deliciosa Leaves in Portugal.

Pseudomonas syringae pv. actinidiae is a quarantine bacterium affecting all the Portuguese main areas of kiwifruit production. We report the draft genome of six P. syringae pv. actinidiae strains isolated from symptomatic leaves of Actinidia chinensis var. deliciosa in a study that determined the genetic population structure of the endophytic and epiphytic populations in two consecutive seasons. Average nucleotide identity values were above 99% similarity with reference strains from P. syringae pv. actinidiae biovar 3. The genomic differences found between these strains confirm the genetic diversity described for P. syringae pv. actinidiae population in Portugal. Furthermore, data provide evidence that the initial clonal expansion of P. syringae pv. actinidiae in Europe was followed by a genomic diversification constituting a valuable resource for epidemiological and evolutionary studies, namely when adopting strategies for epidemics management.

Selection of antibiotic resistance by metals in a riverine bacterial community.

Antibiotic resistance is a health challenge across human, animal and environmental settings. In the environment, metals may contribute to antibiotic resistance selection. This study aimed to investigate the role of copper and zinc in the selection of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in a riverine bacterial community. Using a microcosm approach, bacteria in water samples were exposed to 50 µg L-1 and 100 µg L-1 of copper and zinc, for 20 days. The prevalence of ARB was determined from colony forming units counts in media with and without antibiotics. A significant increase in the prevalence of cefotaxime-resistant (from 2.3% in control to 9.5% in Cu50 and 16.8% in Cu100) and tetracycline-resistant bacteria (from 0.03% to 0.23% in Cu100) was observed in communities exposed to copper. Zinc exposure resulted in an increase in the prevalence of cefotaxime-resistant bacteria (from 24.6% to 91.3% in Zn50 and 72.4% in Zn100) and of kanamycin-resistant bacteria (from 6.1% to 24.1% in Zn50 and 43% in Zn100). Cefotaxime- and kanamycin-resistant bacteria belonged to genera intrinsically resistant to these compounds. DGGE profiling confirmed that metal exposure altered the structure and diversity of bacterial communities. Changes in the abundance of genes usually associated with mobile genetic elements (blaCTX-M, blaTEM, tet(A) and intI1) were not detected after exposure. Results demonstrated the selection of bacteria intrinsically resistant to antibiotics imposed by copper and zinc exposure, suggesting an important role played by cross-resistance mechanisms.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Occurrence, antibiotic-resistance and virulence of E. coli strains isolated from mangrove oysters (Crassostrea gasar) farmed in estuaries of Amazonia.

Concentration of bacterial species indicative of fecal contamination in the gut of mangrove oysters (Crassostrea gasar) is a major concern for public health and food surveillance. Our work aimed to determine the occurrence, antibiotic-resistance, phylogenetic profile and virulence of Escherichia coli strains isolated from C. gasar farmed in four estuaries of Amazonia. Santo Antônio de Urindeua was the sampling point with the highest number of E. coli cells in oyster samples (104 per 100 g of sample). Twenty-four isolates (52.2%) showed resistance to cephalotin and 18 to amoxicillin (39.1%). Eighteen clonal populations were determined by rep-PCR and were mainly affiliated to the pathogenic and commensal phylo-groups B1 and D. The presence of elt genes suggests that 10 of these clones belong to the Enterotoxigenic Escherichia coli pathotype. Plasmids, mostly of the F incompatibility group, were detected in the majority of the strains. All isolates were susceptible to last-resort antibiotics.

Genotypic and phenotypic traits of blaCTX-M-carrying Escherichia coli strains from an UV-C-treated wastewater effluent.

Wastewater treatment plants (WWTPs) are relevant sources of antibiotic resistance into aquatic environments. Disinfection of WWTPs' effluents (e.g. by UV-C irradiation) may attenuate this problem, though some clinically relevant bacteria have been shown to survive disinfection. In this study we characterized 25 CTX-M-producing Escherichia coli strains isolated from a WWTP's UV-C-irradiated effluent, aiming to identify putative human health hazards associated with such effluents. Molecular typing indicated that the strains belong to the phylogroups A, B2 and C and clustered into 9 multilocus sequence types (STs), namely B2:ST131 (n = 7), A:ST58 (n = 1), A:ST155 (n = 4), C:ST410 (n = 2), A:ST453 (n = 2), A:ST617 (n = 2), A:ST744 (n = 1), A:ST1284 (n = 3) and a putative novel ST (n = 3). PCR-screening identified 9 of the 20 antibiotic resistance genes investigated [i.e. sul1, sul2, sul3, tet(A), tet(B), blaOXA-1-like, aacA4, aacA4-cr and qnrS1]. The more prevalent were sul1, sul2 (n = 15 isolates) and tet(A) (n = 14 isolates). Plasmid restriction analysis indicated diverse plasmid content among strains (14 distinct profiles) and mating assays yielded cefotaxime-resistant transconjugants for 8 strains. Two of the transconjugants displayed a multi-drug resistance (MDR) phenotype. All strains were classified as cytotoxic to Vero cells (9 significantly more cytotoxic than the positive control) and 10 of 21 strains were invasive towards this cell line (including all B2:ST131 strains). The 10 strains tested against G. mellonella larvae exhibited a virulent behaviour. Twenty-four and 7 of the 25 strains produced siderophores and haemolysins, respectively. Approximately 66% of the strains formed biofilms. Genome analysis of 6 selected strains identified several virulence genes encoding toxins, siderophores, and colonizing, adhesion and invasion factors. Freshwater microcosms assays showed that after 28 days of incubation 3 out of 6 strains were still detected by cultivation and 4 strains by qPCR. Resistance phenotypes of these strains remained unaltered. Overall, we confirmed WWTP's UV-C-treated outflow as a source of MDR and/or virulent E. coli strains, some probably capable of persisting in freshwater, and that carry conjugative antibiotic resistance plasmids. Hence, disinfected wastewater may still represent a risk for human health. More detailed evaluation of strains isolated from wastewater effluents is urgent, to design treatments that can mitigate the release of such bacteria.

Genomic analysis of Chromobacterium haemolyticum: insights into the species resistome, virulence determinants and genome plasticity.

The increasing number of Chromobacterium haemolyticum human infection reports, especially in tropical regions and connected with environmental sources, resulted in an urge to better describe this species. This study aimed to characterize the C. haemolyticum resistome, virulence determinants and genetic platforms related with genome plasticity. A comparative genomic analysis was conducted between clinical C. haemolyticum genomes publicly available and the genome of an environmental isolate obtained in this study. The pangenome of C. haemolyticum was calculated and a total of 3378 core genes were predicted in its core genome, corresponding to 51.7% of the pangenome. Genetic determinants putatively encoding resistance to beta-lactams, fosfomycin, aminoglycosides and trimethoprim were predicted in all genomes, possibly constituting the intrinsic resistome of this species. In terms of resistance to beta-lactams, 4 genes were predicted encoding beta-lactamases of classes A, C and D. Moreover, the analysis of Chromobacterium genomes and C. haemolyticum environmental isolates reinforced the role of this genus as progenitor of the blaKPC gene. Putative virulence factors (VFs) were predicted in all genomes, related to adherence, toxins production, colonization and cell invasion. Secretion systems, including type III, were detected. A significant number of transposases and genomic islands were predicted in C. haemolyticum, in some cases above the average reported for Gram-negative bacterial genomes. We conclude that C. haemolyticum strains, including those of environmental origin, present a noteworthy collection of antibiotic resistance genes and VFs. Furthermore, sequences related to gene mobility and genome plasticity suggest high adaptability potential and a possible role as disseminator of antibiotic resistance.

Occurrence of carbapenemase-producing Enterobacteriaceae in a Portuguese river: blaNDM, blaKPC and blaGES among the detected genes.

Carbapenems are used as last-resort drugs to treat infections caused by multidrug-resistant bacteria. Despite the increasing number of reports of carbapenem-resistant Enterobacteriaceae (CRE), there is still limited information on their distribution or prevalence in the environment. Our aim was to assess the occurrence of CRE in the Lis river (Portugal) and to characterize the genetic platforms linked to carbapenemase genes. We collected six water samples from sites near a wastewater treatment plant (n = 4 samples) and livestock farms (n = 2). Twenty-four CRE were characterized by BOX element-polymerase chain reaction (BOX-PCR), and thirteen representative isolates were analysed by Pulsed-Field Gel Electrophoresis (PFGE) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility testing, PCR screening for carbapenemase-encoding genes, conjugation experiments and plasmid analysis were performed. Four isolates were chosen for whole-genome sequencing. All water samples contained CRE (4.0 CFU/mL on average). Representative isolates were multidrug-resistant (resistant to ciprofloxacin, trimethoprim-sulfamethoxazole and to all ß-lactams tested) and were identified as K. pneumoniae, Enterobacter and Citrobacter. Isolates carried plasmids and harboured carbapenemase-encoding genes: blaKPC-3 in K. pneumoniae (n = 9), blaNDM-1 in Enterobacter (n = 3) and blaGES-5 in Citrobacter (n = 1). Conjugation experiments were successful in two Klebsiella isolates. Enterobacter PFGE profiles grouped in one cluster while Klebsiella were divided in three clusters and a singleton. Whole-genome sequencing analysis revealed blaGES-5 within a novel class 3 integron (In3-16) located on an IncQ/pQ7-like plasmid in Citrobacter freundii CR16. blaKPC-3 was present on IncFIA-FII pBK30683-like plasmids, which were subsequently confirmed in all K. pneumoniae (n = 9). Furthermore, blaKPC-3 was part of a genomic island in K. pneumoniae CR12. In E. roggenkampii CR11, blaNDM-1 was on an IncA/C2 plasmid. The carbapenemase-encoding plasmids harboured other resistance determinants and mobile genetic elements. Our results demonstrate that Lis river is contaminated with CRE, highlighting the need for monitoring antibiotic resistance in aquatic environments, especially to last-resort drugs.

Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil.

Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the bla CTX-M was the most prevalent (n = 12 positive isolates), followed by bla TEM (n = 5) and bla SHV (n = 4). bla CTX-M-15 (n = 5), bla CTX-M-14 (n = 1) and bla CTX-M-2 (n = 1) variants were detected in conserved genomic contexts: bla CTX-M-15 flanked by ISEcp1 and Orf477; bla CTX-M-14 flanked by ISEcp1 and IS903; and bla CTX-M-2 associated to an ISCR element. For 4 strains the transfer of bla CTX-M was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. bla CTX-M-15 as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., bla OXA-208 and bla ADC-like). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains.

Long-term effects of oxytetracycline exposure in zebrafish: A multi-level perspective.

Oxytetracycline (OTC) is a broad-spectrum antibiotic widely used in livestock production. Like many other pharmaceuticals, OTC is not completely metabolized by the organism and thus, increasing amounts of the compound are being detected in the aquatic environment. The assessment of the environmental risk of pharmaceuticals is hindered by their very low concentrations and specific modes of action and thus relevant exposure scenarios and sensitive endpoints are needed. Thus, this work aimed to study the long-term effect of OTC exposure in zebrafish (at behavior and biochemical levels) and associated bacterial communities (fish gut and water bacterial communities). Results revealed that at behavioral level, boldness increase (manifested by increased exploratory behavior of a new environment) was observed in fish exposed to low OTC concentrations. Moreover, changes in fish swimming pattern were observed in light periods (increased stress response: hyperactivity and freezing) probably due to photo-sensibility conferred by OTC exposure. Effects at biochemical level suggest that long-term exposure to OTC interfere with cellular energy allocation mainly by reducing lipids levels and increasing energy consumption. Moreover, evidences of oxidative damage were also observed (reduced levels of TG, GST and CAT). The analysis of water and gut microbiome revealed changes in the structure and diversity of bacterial communities potentially leading to changes in communities' biological function. Some of the effects were observed at the lowest concentration tested, 0.1 µg/L which is a concentration already detected in the environment and thus clearly demonstrating the need of a serious ecotoxicological assessment of OTC effects on non-target organisms.