Freshwater salmon aquaculture in Chile and transferable antimicrobial resistance.

Large amounts of antimicrobials are used in salmonid aquaculture in Chile. Most are used in marine aquaculture, but appreciable amounts are also employed in freshwater aquaculture. Much research and many publications have examined transferable antimicrobial resistance in bacteria isolated from marine salmon farms, but much less attention has been paid to this area in freshwater salmon farming. A recent paper by Domínguez et al. (2019) has as least in part remedied this situation. We now comment on some of its interpretations and have attempted to point out its areas of strength and weakness in light of the published scientific literature. Seen in this setting, the important results presented by Domínguez et al. (2019) underline the need for increased awareness of the challenge to animal and human health posed by excessive use of antimicrobials in aquaculture.

Antimicrobial resistance in Chile and The One Health paradigm: Dealing with threats to human and veterinary health resulting from antimicrobial use in salmon aquaculture and the clinic. / Resistencia a los antimicrobianos en Chile y el paradigma de Una Salud: manejando los riesgos para la salud pública humana y animal resultante del uso de antimicrobianos en la acuicultura del salmón y en medicina.

The emergence and dissemination of antimicrobial-resistant bacteria (ARB) is currently seen as one of the major threats to human and animal public health. Veterinary use of antimicrobials in both developing and developed countries is many-fold greater than their use in human medicine and is an important determinant in selection of ARB. In light of the recently outlined National Plan Against Antimicrobial Resistance in Chile, our findings on antimicrobial use in salmon aquaculture and their impact on the environment and human health are highly relevant. Ninety-five percent of tetracyclines, phenicols and quinolones imported into Chile between 1998 and 2015 were for veterinary use, mostly in salmon aquaculture. Excessive use of antimicrobials at aquaculture sites was associated with antimicrobial residues in marine sediments 8 km distant and the presence of resistant marine bacteria harboring easily transmissible resistance genes, in mobile genetic elements, to these same antimicrobials. Moreover, quinolone and integron resistance genes in human pathogens isolated from patients in coastal regions adjacent to aquaculture sites were identical to genes isolated from regional marine bacteria, consistent with genetic communication between bacteria in these different environments. Passage of antimicrobials into the marine environment can potentially diminish environmental diversity, contaminate wild fish for human consumption, and facilitate the appearance of harmful algal blooms and resistant zoonotic and human pathogens. Our findings suggest that changes in aquaculture in Chile that prevent fish infections and decrease antimicrobial usage will prove a determining factor in preventing human and animal infections with multiply-resistant ARB in accord with the modern paradigm of One Health.

Plasmid-Mediated Quinolone Resistance (PMQR) Genes and Class 1 Integrons in Quinolone-Resistant Marine Bacteria and Clinical Isolates of Escherichia coli from an Aquacultural Area.

Antimicrobial usage in aquaculture selects for antimicrobial-resistant microorganisms in the marine environment. The relevance of this selection to terrestrial animal and human health is unclear. Quinolone-resistance genes qnrA, qnrB, and qnrS were chromosomally located in four randomly chosen quinolone-resistant marine bacteria isolated from an aquacultural area with heavy quinolone usage. In quinolone-resistant uropathogenic clinical isolates of Escherichia coli from a coastal area bordering the same aquacultural region, qnrA was chromosomally located in two E. coli isolates, while qnrB and qnrS were located in small molecular weight plasmids in two other E. coli isolates. Three quinolone-resistant marine bacteria and three quinolone-resistant E. coli contained class 1 integrons but without physical association with PMQR genes. In both marine bacteria and uropathogenic E. coli, class 1 integrons had similar co-linear structures, identical gene cassettes, and similarities in their flanking regions. In a Marinobacter sp. marine isolate and in one E. coli clinical isolate, sequences immediately upstream of the qnrS gene were homologous to comparable sequences of numerous plasmid-located qnrS genes while downstream sequences were different. The observed commonality of quinolone resistance genes and integrons suggests that aquacultural use of antimicrobials might facilitate horizontal gene transfer between bacteria in diverse ecological locations.

Resistencia a los antimicrobianos en Chile y el paradigma de Una Salud: manejando los riesgos para la salud pública humana y animal resultante del uso de antimicrobianos en la acuicultura del salmón y en medicina / Antimicrobial resistance in Chile and The One Health paradigm: dealing with threats to human and veterinary health resulting from antimicrobial use in salmon aquaculture and the clinic

El aumento de la resistencia bacteriana múltiple a antimicrobianos es considerado una gran amenaza para la salud pública mundial y como generador de una importante crisis en el funcionamiento de los sistemas de salud. Esta crisis es discutida diariamente por los gobiernos y los parlamentos, las instituciones globales de salud, fundaciones benéficas y de científicos y de profesionales de la salud y también de consumidores de productos animales. En todos los países del orbe se ha identificado al uso de antimicrobianos en la crianza industrial de animales como un importante determinante en la selección de esta resistencia. Aprovechando la oportunidad que se ha planteado en Chile con el diseño del Plan Nacional Contra la Resistencia a los Antimicrobianos, hemos creído importante revisitar y actualizar sumariamente nuestros estudios sobre el uso de antimicrobianos en la acuicultura del salmón y de su potencial impacto en el ambiente y la salud humana y animal. Estos estudios indican que 95% de tres grupos de antimicrobianos importados al país, que incluyen tetraciclinas, fenicoles y quinolonas, son usados en medicina veterinaria y mayormente en la acuicultura del salmón. Nuestros estudios indican que el excesivo uso de estos antimicrobianos genera la presencia de residuos de antimicrobianos en el ambiente marino hasta 8 km de los sitios de acuicultura, los que seleccionan a bacterias con resistencia múltiple en dicho ambiente, ya que ellas contienen variados genes de resistencia a estos antimicrobianos. Estos genes de resistencia están contenidos en elementos genéticos móviles incluyendo plásmidos e integrones, los que son trasmitidos a otras bacterias permitiendo su potencial diseminación epidémica entre poblaciones bacterianas. Bacterias del ambiente marino contienen genes idénticos a los genes de resistencia a quinolonas e integrones similares a los de patógenos humanos, sugiriendo comunicación genética entre estas bacterias de diversos ambientes. Alrededor de los recintos de acuicultura, este uso exagerado de antimicrobianos contamina con ellos también a peces silvestres para consumo humano y potencialmente selecciona BRA en su carne y en los productos de acuicultura. El consumo de estos productos selecciona bacterias resistentes en el microbioma humano y facilita también el intercambio genético entre bacterias del ambiente acuático y la microbiota comensal y patógena humana. El pasaje de antimicrobianos al ambiente marino disminuye la diversidad en él, y potencialmente podría facilitar la aparición de florecimientos de algas nocivas, la infección de peces por patógenos piscícolas resistentes los antimicrobianos y la aparición de patógenos zoonóticos resistentes, incluyendo a Vibrio parahaemolyticus. Estos hallazgos sugieren que la prevención de infecciones en peces y la disminución del uso de antimicrobianos en su crianza, será en Chile un factor determinante en la prevención de infecciones humanas y animales con resistencia múltiple a los antimicrobianos, de acuerdo con el paradigma moderno e integral de Una Salud.

The emergence and dissemination of antimicrobial-resistant bacteria (ARB) is currently seen as one of the major threats to human and animal public health. Veterinary use of antimicrobials in both developing and developed countries is many-fold greater than their use in human medicine and is an important determinant in selection of ARB. In light of the recently outlined National Plan Against Antimicrobial Resistance in Chile, our findings on antimicrobial use in salmon aquaculture and their impact on the environment and human health are highly relevant. Ninety-five percent of tetracyclines, phenicols and quinolones imported into Chile between 1998 and 2015 were for veterinary use, mostly in salmon aquaculture. Excessive use of antimicrobials at aquaculture sites was associated with antimicrobial residues in marine sediments 8 km distant and the presence of resistant marine bacteria harboring easily transmissible resistance genes, in mobile genetic elements, to these same antimicrobials. Moreover, quinolone and integron resistance genes in human pathogens isolated from patients in coastal regions adjacent to aquaculture sites were identical to genes isolated from regional marine bacteria, consistent with genetic communication between bacteria in these different environments. Passage of antimicrobials into the marine environment can potentially diminish environmental diversity, contaminate wild fish for human consumption, and facilitate the appearance of harmful algal blooms and resistant zoonotic and human pathogens. Our findings suggest that changes in aquaculture in Chile that prevent fish infections and decrease antimicrobial usage will prove a determining factor in preventing human and animal infections with multiply-resistant ARB in accord with the modern paradigm of One Health.

Antimicrobial resistance genes in marine bacteria and human uropathogenic Escherichia coli from a region of intensive aquaculture.

Antimicrobials are heavily used in Chilean salmon aquaculture. We previously found significant differences in antimicrobial-resistant bacteria between sediments from an aquaculture and a non-aquaculture site. We now show that levels of antimicrobial resistance genes (ARG) are significantly higher in antimicrobial-selected marine bacteria than in unselected bacteria from these sites. While ARG in tetracycline- and florfenicol-selected bacteria from aquaculture and non-aquaculture sites were equally frequent, there were significantly more plasmid-mediated quinolone resistance genes per bacterium and significantly higher numbers of qnrB genes in quinolone-selected bacteria from the aquaculture site. Quinolone-resistant urinary Escherichia coli from patients in the Chilean aquacultural region were significantly enriched for qnrB (including a novel qnrB gene), qnrS, qnrA and aac(6')-1b, compared with isolates from New York City. Sequences of qnrA1, qnrB1 and qnrS1 in quinolone-resistant Chilean E. coli and Chilean marine bacteria were identical, suggesting horizontal gene transfer between antimicrobial-resistant marine bacteria and human pathogens.

Genome Sequence of Borrelia chilensis VA1, a South American Member of the Lyme Borreliosis Group.

Borrelia chilensis strain VA1 is a recently described South American member of the Borrelia burgdorferi sensu lato complex from Chile. Whole-genome sequencing analysis determined its linear chromosome and plasmids lp54 and cp26, confirmed its membership in the Lyme borreliosis group, and will open new research avenues regarding its pathogenic potential.

Plasmid-related quinolone resistance determinants in epidemic Vibrio parahaemolyticus, uropathogenic Escherichia coli, and marine bacteria from an aquaculture area in Chile.

Marine bacteria from aquaculture areas with industrial use of quinolones have the potential to pass quinolone resistance genes to animal and human pathogens. The VPA0095 gene, related to the quinolone resistance determinant qnrA, from clinical isolates of epidemic Vibrio parahaemolyticus conferred reduced susceptibility to quinolone after cloning into Escherichia coli K-12 either when acting alone or synergistically with DNA gyrase mutations. In addition, a plasmid-mediated quinolone resistance gene from marine bacteria, aac(6')-Ib-cr, was identical to aac(6')-Ib-cr from urinary tract isolates of E. coli, suggesting a recent flow of this gene between these bacteria isolated from different environments. aac(6')-Ib-cr from E. coli also conferred reduced susceptibility to quinolone and kanamycin when cloned into E. coli K-12.

Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non-aquaculture sites.

Antimicrobial resistance (AR) detected by disc diffusion and antimicrobial resistance genes detected by DNA hybridization and polymerase chain reaction with amplicon sequencing were studied in 124 marine bacterial isolates from a Chilean salmon aquaculture site and 76 from a site without aquaculture 8 km distant. Resistance to one or more antimicrobials was present in 81% of the isolates regardless of site. Resistance to tetracycline was most commonly encoded by tetA and tetG; to trimethoprim, by dfrA1, dfrA5 and dfrA12; to sulfamethizole, by sul1 and sul2; to amoxicillin, by blaTEM ; and to streptomycin, by strA-strB. Integron integrase intl1 was detected in 14 sul1-positive isolates, associated with aad9 gene cassettes in two from the aquaculture site. intl2 Integrase was only detected in three dfrA1-positive isolates from the aquaculture site and was not associated with gene cassettes in any. Of nine isolates tested for conjugation, two from the aquaculture site transferred AR determinants to Escherichia coli. High levels of AR in marine sediments from aquaculture and non-aquaculture sites suggest that dispersion of the large amounts of antimicrobials used in Chilean salmon aquaculture has created selective pressure in areas of the marine environment far removed from the initial site of use of these agents.

Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the Southern Hemisphere.

Borrelia burgdorferi sensu lato (s.l.), transmitted by Ixodes spp. ticks, is the causative agent of Lyme disease. Although Ixodes spp. ticks are distributed in both Northern and Southern Hemispheres, evidence for the presence of B. burgdorferi s.l. in South America apart from Uruguay is lacking. We now report the presence of culturable spirochetes with flat-wave morphology and borrelial DNA in endemic Ixodes stilesi ticks collected in Chile from environmental vegetation and long-tailed rice rats (Oligoryzomys longicaudatus). Cultured spirochetes and borrelial DNA in ticks were characterized by multilocus sequence typing and by sequencing five other loci (16S and 23S ribosomal genes, 5S-23S intergenic spacer, flaB, ospC). Phylogenetic analysis placed this spirochete as a new genospecies within the Lyme borreliosis group. Its plasmid profile determined by polymerase chain reaction and pulsed-field gel electrophoresis differed from that of B. burgdorferi B31A3. We propose naming this new South American member of the Lyme borreliosis group B. chilensis VA1 in honor of its country of origin.

Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health.

The worldwide growth of aquaculture has been accompanied by a rapid increase in therapeutic and prophylactic usage of antimicrobials including those important in human therapeutics. Approximately 80% of antimicrobials used in aquaculture enter the environment with their activity intact where they select for bacteria whose resistance arises from mutations or more importantly, from mobile genetic elements containing multiple resistance determinants transmissible to other bacteria. Such selection alters biodiversity in aquatic environments and the normal flora of fish and shellfish. The commonality of the mobilome (the total of all mobile genetic elements in a genome) between aquatic and terrestrial bacteria together with the presence of residual antimicrobials, biofilms, and high concentrations of bacteriophages where the aquatic environment may also be contaminated with pathogens of human and animal origin can stimulate exchange of genetic information between aquatic and terrestrial bacteria. Several recently found genetic elements and resistance determinants for quinolones, tetracyclines, and ß-lactamases are shared between aquatic bacteria, fish pathogens, and human pathogens, and appear to have originated in aquatic bacteria. Excessive use of antimicrobials in aquaculture can thus potentially negatively impact animal and human health as well as the aquatic environment and should be better assessed and regulated.

Salmon aquaculture and antimicrobial resistance in the marine environment.

Antimicrobials used in salmon aquaculture pass into the marine environment. This could have negative impacts on marine environmental biodiversity, and on terrestrial animal and human health as a result of selection for bacteria containing antimicrobial resistance genes. We therefore measured the numbers of culturable bacteria and antimicrobial-resistant bacteria in marine sediments in the Calbuco Archipelago, Chile, over 12-month period at a salmon aquaculture site approximately 20 m from a salmon farm and at a control site 8 km distant without observable aquaculture activities. Three antimicrobials extensively used in Chilean salmon aquaculture (oxytetracycline, oxolinic acid, and florfenicol) were studied. Although none of these antimicrobials was detected in sediments from either site, traces of flumequine, a fluoroquinolone antimicrobial also widely used in Chile, were present in sediments from both sites during this period. There were significant increases in bacterial numbers and antimicrobial-resistant fractions to oxytetracycline, oxolinic acid, and florfenicol in sediments from the aquaculture site compared to those from the control site. Interestingly, there were similar numbers of presumably plasmid-mediated resistance genes for oxytetracycline, oxolinic acid and florfenicol in unselected marine bacteria isolated from both aquaculture and control sites. These preliminary findings in one location may suggest that the current use of large amounts of antimicrobials in Chilean aquaculture has the potential to select for antimicrobial-resistant bacteria in marine sediments.

[Injudicious and excessive use of antibiotics: public health and salmon aquaculture in Chile]. / Uso inadecuado y excesivo de antibióticos: Salud pública y salmonicultura en Chile.

Salmon aquaculture was one of the major growing and exporting industries in Chile. Its development was accompanied by an increasing and excessive use of large amounts of antimicrobials, such as quinolones, tetracyclines and florfenicol. The examination of the sanitary conditions in the industry as part of a more general investigation into the uncontrolled and extensive dissemination of the ISA virus epizootic in 2008, found numerous and wide-ranging shortcomings and limitations in management of preventive fish health. There was a growing industrial use of large amounts of antimicrobials as an attempt at prophylaxis of bacterial infections resulting from widespread unsanitary and unhealthy fish rearing conditions. As might be expected, these attempts were unsuccessful and this heavy antimicrobial use failed to prevent viral and parasitic epizootics. Comparative analysis of the amounts of antimicrobials, especially quinolones, consumed in salmon aquaculture and in human medicine in Chile robustly suggests that the most important selective pressure for antibiotic resistant bacteria in the country will be excessive antibiotic use in this industry. This excessive use will facilitate selection of resistant bacteria and resistance genes in water environments. The commonality of antibiotic resistance genes and the mobilome between environmental aquatic bacteria, fish pathogens and pathogens of terrestrial animals and humans suggests that horizontal gene transfer occurs between the resistome of these apparently independent and isolated bacterial populations. Thus, excessive antibiotic use in the marine environment in aquaculture is not innocuous and can potentially negatively affect therapy of bacterial infections of humans and terrestrial animals.

Uso inadecuado y excesivo de antibióticos: Salud pública y salmonicultura en Chile / Injudicious and excessive use of antibiotics: Public health and salmon aquaculture in Chile

Salmón aquaculture was one ofthe major growing and exporting industries in Chile. Its development was accompanied by an increasing and excessive use oflarge amounts of antimicrobials, such as quinolones, tetracyclines and florfenicol. The examination of the sanitary conditions in the industry as part of a more general investigation into the uncontrolled and extensive dissemination of the ISA virus epizootic in 2008, found numerous and wide-ranging shortcomings and limitations in management of preventive fish health. There was a growing industrial use of large amounts of antimicrobials as an attempt at prophylaxis of bacterial infections resulting from widespread unsanitary and unhealthy fish rearing conditions. As might be expected, these attempts were unsuccessful and this heavy antimicrobial use failed to prevent viral and parasitic epizootics. Comparative analysis of the amounts of antimicrobials, especially quinolones, consumed in salmón aquaculture and in human medicine in Chile robustly suggests that the most important selective pressurefor antibiotic resistant bacteria in the country will be excessive antibiotic use in this industry. This excessive use will facilitate selection of resistant bacteria and resistance genes in water environments. The commonality of antibiotic resistance genes and the mobilome between environmental aquatic bacteria, fishpathogens and pathogens of terrestrial animáis and humans suggests that horizontal gene transfer occurs between the resistome of these apparently independent and isolated bacterial populations. Thus, excessive antibiotic use in the marine environment in aquaculture is not innocuous and can potentially negatively affect therapy of bacterial infections of humans and terrestrial animáis.

BmpA is a surface-exposed outer-membrane protein of Borrelia burgdorferi.

BmpA is an immunodominant protein of Borrelia burgdorferi as well as an arthritogenic factor. Rabbit antirecombinant BmpA (rBmpA) antibodies were raised, characterized by assaying their cross reactivity with rBmpB, rBmpC and rBmpD, and then rendered monospecific by absorption with rBmpB. This monospecific reagent reacted only with rBmpA in dot immunobinding and detected a single 39 kDa, pI 5.0, spot on two-dimensional immunoblots. It was used to assess the BmpA cellular location. BmpA was present in both detergent-soluble and -insoluble fractions of Triton X-114 phase-partitioned borrelial cells, suggesting that it was a membrane lipoprotein. Immunoblots of proteinase K-treated intact and Triton X-100 permeabilized cells showed digestion of BmpA in intact cells, consistent with surface exposure. This exposure was confirmed by dual-label immunofluorescence microscopy of intact and permeabilized borrelial cells. Conservation and surface localization of BmpA in all B. burgdorferi sensu lato genospecies could point to its playing a key role in this organism's biology and pathobiology.

TNF-alpha and gamma-irradiation induced activation of the Salmonella typhimurium reproduction in the organs of infected animals.

The goal of the current work is to determine the role of the TNF-alpha on the activation of the bacterial growth in an in vivo system. In order to reach this goal we studied the dynamics of the reproduction of vegetative forms of Salmonella and the recultivation of non-cultivating forms of Salmonella in infected animals. Experiments have been conducted both on animals that had been injected with exogenous TNF together with bacterial suspension and on animals that had been exposed to gamma-radiation before infection, since it is known that irradiation increases the secretion of TNF. We demonstrate that in all cases the increase in the level of TNF-alpha in animals leads to the activation of the Salmonella growth. Moreover, in this paper we present the data obtained by the method of molecular display on the identification of genes that are highly expressed in the Salmonella cells cultivated in vitro in the presence of TNF.