Snapshot of resistome, virulome and mobilome in aquaculture.

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with ß-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.

First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirs.

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.

Genomic Analysis of a mcr-9.1-Harbouring IncHI2-ST1 Plasmid from Enterobacter ludwigii Isolated in Fish Farming.

This study analyzed the resistome, virulome and mobilome of an MCR-9-producing Enterobacter sp. identified in a muscle sample of seabream (Sparus aurata), collected in a land tank from multitrophic fish farming production. Average Nucleotide Identity analysis identified INSAq77 at the species level as an Enterobacter ludwigii INSAq77 strain that was resistant to chloramphenicol, florfenicol and fosfomycin and was susceptible to all other antibiotics tested. In silico antimicrobial resistance analyses revealed genes conferring in silico resistance to ß-lactams (blaACT-88), chloramphenicol (catA4-type), fosfomycin (fosA2-type) and colistin (mcr-9.1), as well as several efflux pumps (e.g., oqxAB-type and mar operon). Further bioinformatics analysis revealed five plasmid replicon types, including the IncHI2/HI2A, which are linked to the worldwide dissemination of the mcr-9 gene in different antibiotic resistance reservoirs. The conserved nickel/copper operon rcnR-rcnA-pcoE-ISSgsp1-pcoS-IS903-mcr-9-wbuC was present, which may play a key role in copper tolerance under anaerobic growth and nickel homeostasis. These results highlight that antibiotic resistance in aquaculture are spreading through food, the environment and humans, which places this research in a One Health context. In fact, colistin is used as a last resort for the treatment of serious infections in clinical settings, thus mcr genes may represent a serious threat to human health.

Assessing the Bacterial Community Composition of Bivalve Mollusks Collected in Aquaculture Farms and Respective Susceptibility to Antibiotics.

Aquaculture is a growing sector, providing several products for human consumption, and it is therefore important to guarantee its quality and safety. This study aimed to contribute to the knowledge of bacterial composition of Crassostrea gigas, Mytilus spp. and Ruditapes decussatus, and the antibiotic resistances/resistance genes present in aquaculture environments. Two hundred and twenty-two bacterial strains were recovered from all bivalve mollusks samples belonging to the Aeromonadaceae, Bacillaceae, Comamonadaceae, Enterobacteriaceae, Enterococcaceae, Micrococcaceae, Moraxellaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Staphylococcaceae, Streptococcaceae, Vibrionaceae, and Yersiniaceae families. Decreased susceptibility to oxytetracycline prevails in all bivalve species, aquaculture farms and seasons. Decreased susceptibilities to amoxicillin, amoxicillin/clavulanic acid, cefotaxime, cefoxitin, ceftazidime, chloramphenicol, florfenicol, colistin, ciprofloxacin, flumequine, nalidixic acid and trimethoprim/sulfamethoxazole were also found. This study detected six qnrA genes among Shewanella algae, ten qnrB genes among Citrobacter spp. and Escherichia coli, three oqxAB genes from Raoultella ornithinolytica and blaTEM-1 in eight E. coli strains harboring a qnrB19 gene. Our results suggest that the bacteria and antibiotic resistances/resistance genes present in bivalve mollusks depend on several factors, such as host species and respective life stage, bacterial family, farm's location and season, and that is important to study each aquaculture farm individually to implement the most suitable measures to prevent outbreaks.

Genetic Relatedness and Diversity of Staphylococcus aureus from Different Reservoirs: Humans and Animals of Livestock, Poultry, Zoo, and Aquaculture.

The main aim of this study was the characterization of antibiotic resistance mechanisms in 82 Staphylococcus aureus strains isolated from humans and animals. Antibiotic susceptibility testing was performed on all S. aureus isolates accordingly, and antibiotic-resistant genes were investigated by genotypic methods. The genetic diversity of S. aureus was studied through spa, multilocus sequence typing (MLST), and agr typing methods. The majority of S. aureus from human sources were resistant to cefoxitin (and harbor the mecA gene) and fluoroquinolones, whereas only four strains of S. aureus from animal sources revealed resistance to ciprofloxacin. In the set of S. aureus isolated from humans, the most frequent spa, MLST, and agr group were t032, ST22, and I, respectively. In strains from animal origin the most common spa, MLST, and agr group found were t2383, ST398, and III/not typable, respectively. S. aureus from humans and animals were identified either in clonal complexes CC5, CC30, and CC398, suggesting that they have the same putative founder in their evolution. Considering the three CCs encompassing strains from human and animal reservoirs with different spa-types, we can hypothesize that this might reflect an adaptation to different phylogenetic lineages in those reservoirs (host species) probably associated to genetic diversification of pre-existing strains.

Bacterial Diversity and Antibiotic Susceptibility of Sparus aurata from Aquaculture.

In a world where the population continues to increase and the volume of fishing catches stagnates or even falls, the aquaculture sector has great growth potential. This study aimed to contribute to the depth of knowledge of the diversity of bacterial species found in Sparus aurata collected from a fish farm and to understand which profiles of diminished susceptibility to antibiotics would be found in these bacteria that might be disseminated in the environment. One hundred thirty-six bacterial strains were recovered from the S. aurata samples. These strains belonged to Bacillaceae, Bacillales Family XII. Incertae Sedis, Comamonadaceae, Enterobacteriaceae, Enterococcaceae, Erwiniaceae, Micrococcaceae, Pseudomonadaceae and Staphylococcaceae families. Enterobacter sp. was more frequently found in gills, intestine and skin groups than in muscle groups (p ≤ 0.01). Antibiotic susceptibility tests found that non-susceptibility to phenicols was significantly higher in gills, intestine and skin samples (45%) than in muscle samples (24%) (p ≤ 0.01) and was the most frequently found non-susceptibility in both groups of samples. The group of Enterobacteriaceae from muscles presented less decreased susceptibility to florfenicol (44%) than in the group of gills, intestine and skin samples (76%). We found decreased susceptibilities to ß-lactams and glycopeptides in the Bacillaceae family, to quinolones and mupirocin in the Staphylococcaceae family, and mostly to ß-lactams, phenicols and quinolones in the Enterobacteriaceae and Pseudomonadaceae families. Seven Enterobacter spp. and five Pseudomonas spp. strains showed non-susceptibility to ertapenem and meropenem, respectively, which is of concern because they are antibiotics used as a last resort in serious clinical infections. To our knowledge, this is the first description of species Exiguobacterium acetylicum, Klebsiella michiganensis, Lelliottia sp. and Pantoea vagans associated with S. aurata (excluding cases where these bacteria are used as probiotics) and of plasmid-mediated quinolone resistance qnrB19-producing Leclercia adecarboxylata strain. The non-synonymous G385T and C402A mutations at parC gene (within quinolone resistance-determining regions) were also identified in a Klebsiella pneumoniae, revealing decreased susceptibility to ciprofloxacin. In this study, we found not only bacteria from the natural microbiota of fish but also pathogenic bacteria associated with fish and humans. Several antibiotics for which decreased susceptibility was found here are integrated into the World Health Organization list of "critically important antimicrobials" and "highly important antimicrobials" for human medicine.