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 β-lactams and glycopeptides in the Bacillaceae family, to quinolones and mupirocin in the Staphylococcaceae family, and mostly to β-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.

The frequent carriage of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), by wild animals along with its zoonotic potential poses a public health problem. Furthermore, the repeated detection of the mecA gene homologue, mecC, in wildlife raises the question whether these animals may be a reservoir for mecC-MRSA. Thus, we aimed to isolate S. aureus and MRSA from wild rodents living in port areas and to characterize their antimicrobial resistance and genetic lineages. Mouth and rectal swab samples were recovered from 204 wild rodents. The samples were incubated in BHI broth with 6.5% of NaCl and after 24 h at 37 °C the inoculum was seeded onto Baird-Parker agar, Mannitol Salt agar and ORSAB (supplemented with 2 mg/L of oxacillin) plates. Species identification was confirmed by MALDI-TOF MS. The antimicrobial susceptibility testing was performed by the Kirby–Bauer disc diffusion method against 14 antibiotics. The presence of virulence and resistance genes was performed by PCR. The immune evasion cluster (IEC) system was investigated in all S. aureus. All isolates were characterized by MLST, spa- and agr typing. From 204 samples, 38 S. aureus were isolated of which six MRSA were detected. Among the six MRSA isolates, three harbored the mecC gene and the other three, the mecA gene. All mecC-MRSA isolates were ascribed to sequence type (ST) 1945 (which belongs to CC130) and spa-type t1535 whereas the mecA isolates belonged to ST22 and ST36 and spa-types t747 and t018. Twenty-five S. aureus were susceptible to all antibiotics tested. S. aureus isolates were ascribed to 11 MLST and 12 spa-types. S. aureus presents a great diversity of genetic lineages in wild rodents. This is the first report of mecC-MRSA in Portugal.

Livestock-associated MRSA (LA-MRSA) is a zoonotic pathogen that has been reported in several animals, and it is often associated with clonal complex (CC) 398. We aimed to isolate MRSA from quails and to characterize their antimicrobial resistance and genetic lineages. One hundred swab samples were recovered from quails at the slaughterhouse. The swabs were inoculated onto CHROMagar™ MRSA agar plates for MRSA isolation. The presence of antimicrobial-resistant genes and virulence factors was investigated by PCR. All strains were typed by MLST, SCCmec-, spa- and agr-typing. From the 100 samples, 29 MRSA were isolated. All strains were resistant to penicillin, cefoxitin, ciprofloxacin, erythromycin and clindamycin and carried the blaZ, mecA, ermB and ermC genes. All strains, except one, showed resistance to tetracycline and harbored the tetM, tetK and tetL genes in different combinations. Twenty strains belonged to ST398 and SCCmec type V, and nine strains belonged to the new ST6831. Twenty-eight out of twenty-nine strains were ascribed to t011 and one to t108. As far as we know, this is the first report of MRSA from quails slaughtered for human consumption. Most strains belonged to ST398-t011, which is the most common LA-MRSA clone found in livestock in Europe.

Natural aquatic environments represent one of the most important vehicles of bacterial dissemination. Therefore, we aimed to isolate staphylococci from surface waters and to investigate the presence of antimicrobial resistance genes and virulence factors as well as the genetic lineages of all Staphylococcus aureus isolates. Staphylococci were recovered from water samples collected from 78 surface waters, including rivers, streams, irrigation ditches, dams, lakes, and fountains. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Multilocus sequence typing and spa-typing were performed in all S. aureus isolates. From the 78 water samples, 33 S. aureus, one S. pseudintermedius, and 51 coagulase-negative staphylococci (CoNS) were identified. Among the S. aureus isolates, four MRSA were identified, and all harbored the mecC gene. Fourteen S. aureus were susceptible to all antimicrobials tested and the remaining showed resistance to penicillin, erythromycin and/or tetracycline encoded by the blaZ, ermT, msr(A/B), tetL, and vgaA genes. Regarding the clonal lineages, one mecC-MRSA isolate belonged to spa-type t843 and sequence type (ST) 130 and the other three to t742 and ST425. The remaining S. aureus were ascribed 14 spa-types and 17 sequence types. Eleven species of CoNS were isolated: S. sciuri, S. lentus, S. xylosus, S. epidermidis, S. cohnii spp. urealyticus, S. vitulinus, S. caprae, S. carnosus spp. Carnosus, S. equorum, S. simulans, and S. succinus. Thirteen CoNS isolates had a multidrug resistance profile and carried the following genes: mecA, msr(A/B), mph(C), aph(3′)-IIIa, aac(6′)-Ie–aph(2′’)-Ia, dfrA, fusB, catpC221, and tetK. A high diversity of staphylococci was isolated from surface waters including mecCMRSA strains and isolates presenting multidrug-resistance profiles. Studies on the prevalence of antibiotic-resistant staphylococci in surface waters are still very scarce but extremely important to estimate the contribution of the aquatic environment in the spread of these bacteria.

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.