In accordance with the global action plan on antimicrobial resistance adopted by the World Health Assembly in 2015, there is a need to develop surveillance programs for antimicrobial resistant bacteria. In this context, we have analyzed the clonal diversity of Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) isolated from aquatic environments and human and food samples in Spain, with the aim of determining possible clonal complexes (CCs) that act as markers of the potential risk of transmission of these resistant bacteria. The phylogenetic groups, sequence types (STs) and CCs were determined by different Polymerase Chain Reaction (PCR) and Multilocus Sequence Typing (MLST) techniques. Phylogroup A was prevalent and was mainly present in food and water strains, while human strains were mostly associated with phylogroup B2. According to the observed prevalence in the different niches, CC23 and CC10 are proposed as markers of phylogroups A and C, related with the spread of bla(CTX-M1) and bla(CTX-M15) genes. Similarly, CC131 and CC38 could be associated to the dissemination of pathogenic strains (phylogroups B2 and D) carrying mainly bla(CTX-M14) and bla(CTX-M15) genes. Some strains isolated from wastewater treatment plants (WWTPs) showed identical profiles to those isolated from other environments, highlighting the importance that water acquires in the dissemination of bacterial resistance. In conclusion, the detection of these genetic markers in different environments could be considered as an alert in the spread of ESBL.

According to the search for alternatives to replace antibiotics in animal production suggested in the antimicrobial resistance action plans around the world, the objective of this work was to evaluate the bactericidal effect of kaolin-silver nanomaterial for its possible inclusion as an additive in animal feed. The antibacterial activity of the C3 (kaolin-silver nanomaterial) product was tested against a wide spectrum of Gram-negative and Gram-positive bacteria (including multidrug resistant strains) by performing antibiograms, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), as well as growth inhibition curves against seven strains causing infections in animals. The C3 product generated inhibition halos in all the tested strains, and a higher activity against Gram-negative bacteria was found, with MBC values ranged from 7.8 µg/mL (P. aeruginosa) to 15.6 µg/mL (E. coli and Salmonella). In contrast, it was necessary to increase the concentration to 31.3 µg/mL or 250 µg/mL to eliminate 99.9% of the initial population of S. aureus ATCC 6538 and E. faecium ATCC 19434, respectively. Conversely, the inhibition growth curves showed a faster bactericidal activity against Gram-negative bacteria (between 2 and 4 h), while it took at least 24 h to observe a reduction in cell viability of S. aureus ATCC 6538. In short, this study shows that the kaolin-silver nanomaterials developed in the framework of the INTERREG POCTEFA EFA183/16/OUTBIOTICS project exhibit antibacterial activity against a wide spectrum of bacteria. However, additional studies on animal safety and environmental impact are necessary to evaluate the effectiveness of the proposed alternative in the context of One Health.

Brucella ovisis a non-zoonotic roughBrucellathat causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors ofB. ovisis necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smoothB. melitensisRev1 vaccine is banned. Although severalB. ovisvirulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate -cc; phosphoenolpyruvate) inB. ovisPA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, thisppdKmutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in aB. ovisPA CO2-independent construct previously proposed for developingB. ovisvaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independentppdKmutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis ofB. ovisovine brucellosis.

Due to the global progress of antimicrobial resistance, the World Health Organization (WHO) published the list of the antibiotic-resistant "priority pathogens" in order to promote research and development of new antibiotics to the families of bacteria that cause severe and often deadly infections. In the framework of the One Health approach, the surveillance of these pathogens in different environments should be implemented in order to analyze their spread and the potential risk of transmission of antibiotic resistances by food and water. Therefore, the objective of this work was to determine the presence of high and critical priority pathogens included in the aforementioned list in different aquatic environments in the POCTEFA area (North Spain-South France). In addition to these pathogens, detection of colistin-resistant Enterobacteriaceae was included due its relevance as being the antibiotic of choice to treat infections caused by multidrug resistant bacteria (MDR). From the total of 80 analyzed samples, 100% of the wastewater treatment plants (WWTPs) and collectors (from hospitals and slaughterhouses) and 96.4% of the rivers, carried antibiotic resistant bacteria (ARB) against the tested antibiotics. Fifty-five (17.7%) of the isolates were identified as target microorganisms (high and critical priority pathogens of WHO list) and 58.2% (n= 32) of them came from WWTPs and collectors. Phenotypic and genotypic characterization showed that 96.4% were MDR and resistance to penicillins/cephalosporins was the most widespread. The presence ofblagenes, KPC-type carbapenemases,mcr-1andvanBgenes has been confirmed. In summary, the presence of clinically relevant MDR bacteria in the studied aquatic environments demonstrates the need to improve surveillance and treatments of wastewaters from slaughterhouses, hospitals and WWTPs, in order to minimize the dispersion of resistance through the effluents of these areas.

One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum beta-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 x 10(-1) +/- 5 x 10(-1), being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 x 10(-4) +/- 2.85 x 10(-4) and 9.61 x 10(-4) +/- 1.96 x 10(-3), respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs.

The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum beta-plactamase (ESBL) producing Enterobacteriaceae in food products purchased in Navarra, northern Spain. A total of 174 samples of fish and chicken were analyzed from September 2015 to September 2016, including raw and ready-to-eat products: trout (n = 25), salmon (n = 28), panga (n = 13), chicken nuggets and chicken scalopes (n = 32), sushi (n = 31) and sliced cooked poultry (n = 45). Cefpodoxime-resistant strains were isolated on ChromlD ESBL agar and further phenotypic (antimicrobial study on MicroScan(C) NM37 panel) and genotypic characterization (multiplex PCR, sequencing and multi-locus sequence typing, MLST) was performed to confirm and characterize ESBL producers. Raw chicken and sushi have been determined as the most risky products regarding transmission of ESBL-producing Enterobacteriaceae (occurrence 53.1% and 19.4%, respectively), while sliced cooked poultry products appear to be a safe product in this aspect. With regard to raw fish, prevalence in salmon was lower (3.6%) than in trout and panga (16.0%). Ninety-eight per cent of ESBL isolates (n = 50) show multidrug-resistant profiles, highlighting the high resistances against quinolones and tetracyclines observed in chicken isolates, as well as against ertapenem and chloramphenicol in sushi strains. Predominant 13-lactamase type was SHV-12 (50.1%), followed by TEM-type (24.5%) and CTX-M (20.8%). In addition, CTX-M type was only detected in chicken products. The phylogenetic study showed the prevalence of groups A (35%), F (25%) and B1 (15%), usually related to nonvirulent strains. MIST E. coil isolates (n = 20) were grouped into 5 clonal complexes (CC) and 15 sequence types (ST), showing high clonal diversity. ST117 was the prevalent sequence type, while the human pathogen ST131 was not detected in this study. The high prevalence of ESBL-producing multidrug-resistant Enterobacteriaceae detected in products of widespread consumption such as chicken and sushi, increases the concern regarding human exposure to superbugs and encourages the need to improve surveillance of this public health issue.

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum -lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26-ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1-IS26-IS903-ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and beta-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.

Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5-10% CO2 necessity for growth (CO2-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1-4 strains. Although molecular tests are replacing phenotypic methods, CO2-dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO2-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO2 to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO2-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO2-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO2-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO2-independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media.