Misunderstandings and misinterpretations: Antimicrobial use and resistance in salmon aquaculture.

The exponential growth of aquaculture over the past 30 years has been accompanied by a parallel increase in the use of antimicrobials. This widespread use has had negative effects on animal, human and environmental health and affected the biodiversity of the environments where aquaculture takes place. Results showing these harmful effects have been resisted and made light of by the aquaculture industry and their scientific supporters through introduction of misunderstandings and misinterpretations of concepts developed in the evolution, genetics, and molecular epidemiology of antimicrobial resistance. We focus on a few of the most obvious scientific shortcomings and biases of two recent attempts to minimise the negative impacts of excessive antimicrobial use in Chilean salmon aquaculture on human and piscine health and on the environment. Such open debate is critical to timely implementation of effective regulation of antimicrobial usage in salmon aquaculture in Chile, if the negative local and worldwide impacts of this usage are to be avoided.

Biological Effects of Quinolones: A Family of Broad-Spectrum Antimicrobial Agents.

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi.

Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants of S. Typhi were screened. S. Typhi zxx::EZ-Tn5 mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn5 transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene glnA. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the glnA mutant. Expression of ompF increased four times in the glnA null mutant compared to WT strain. To understand the relationship between the expression of glnA and ompF, a strain with the glnA gene under control of the tetracycline-inducible Ptet promoter was created, to modulate glnA expression. Induction of glnA decreased expression of ompF, at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the glnA mutant, compared to WT strain. In addition, expression of glnL and glnG genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the glnA mutant. Further studies indicate that deletion of glnG decreases susceptibility to CIP, while deletion of micF gene increases susceptibility CIP. Our findings indicate that glnA inactivation promotes ompF expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.

Cysteine auxotrophy drives reduced susceptibility to quinolones and paraquat by inducing the expression of efflux-pump systems and detoxifying enzymes in S. Typhimurium.

Currently, Salmonella enterica serovar Typhimurium (S. Typhimurium), is a major global public health problem, which has caused food-borne illnesses in many countries. Today, with the extensive use of antimicrobials, antimicrobial resistance is increasing at a serious rate in S. Typhimurium isolates. The present study sought the role of cysteine (Cys) auxotrophy on the resistance to quinolones and paraquat in S. Typhimurium. Cys auxotrophy was achieved by deleting either the cysDNC, cysJIH or cysQ loci. Deletion of these loci resulted in loss of susceptibility against nalidixic acid, levofloxacin, ciprofloxacin (CIP) and paraquat. Further studies with cysJIH mutant indicated increased expression of multi-antibiotic resistance genes marA and ramA, and consequently increased expression of efflux-pump systems. The cysJIH mutant presented a smaller increase of reactive oxygen species (ROS) in presence of paraquat or CIP. Expression of katG and sodA (expressing for a catalase and a superoxide dismutase, respectively) genes was increased in presence of paraquat in the cysJIH mutant; while expression of the superoxide dismutase gene sodB was decreased. These results indicate that deletion of cysDNC, cysJIH or cysQ genes of S. Typhimurium renders Cys auxotrophy along with decreased susceptibility in response to quinolone and paraquat. Overexpression of efflux-pump systems AcrB-TolC and SmvA-OmpD and antioxidant enzymes KatG and SodA could explain the mechanisms of antimicrobial resistance in the Cys auxotrophic mutants.

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.

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.