Aquatic Environments as Hotspots of Transferable Low-Level Quinolone Resistance and Their Potential Contribution to High-Level Quinolone Resistance.

The disposal of antibiotics in the aquatic environment favors the selection of bacteria exhibiting antibiotic resistance mechanisms. Quinolones are bactericidal antimicrobials extensively used in both human and animal medicine. Some of the quinolone-resistance mechanisms are encoded by different bacterial genes, whereas others are the result of mutations in the enzymes on which those antibiotics act. The worldwide occurrence of quinolone resistance genes in aquatic environments has been widely reported, particularly in areas impacted by urban discharges. The most commonly reported quinolone resistance gene, qnr, encodes for the Qnr proteins that protect DNA gyrase and topoisomerase IV from quinolone activity. It is important to note that low-level resistance usually constitutes the first step in the development of high-level resistance, because bacteria carrying these genes have an adaptive advantage compared to the highly susceptible bacterial population in environments with low concentrations of this antimicrobial group. In addition, these genes can act additively with chromosomal mutations in the sequences of the target proteins of quinolones leading to high-level quinolone resistance. The occurrence of qnr genes in aquatic environments is most probably caused by the release of bacteria carrying these genes through anthropogenic pollution and maintained by the selective activity of antimicrobial residues discharged into these environments. This increase in the levels of quinolone resistance has consequences both in clinical settings and the wider aquatic environment, where there is an increased exposure risk to the general population, representing a significant threat to the efficacy of quinolone-based human and animal therapies. In this review the potential role of aquatic environments as reservoirs of the qnr genes, their activity in reducing the susceptibility to various quinolones, and the possible ways these genes contribute to the acquisition and spread of high-level resistance to quinolones will be discussed.

Characterization of a Novel Variant of the Quinolone-Resistance Gene qnrB (qnrB89) Carried by a Multi-Drug Resistant Citrobacter gillenii Strain Isolated from Farmed Salmon in Chile.

The main objective of this study was to characterize using whole-genome sequencing analysis, a new variant of the qnrB gene (qnrB89) carried by a fluoroquinolone-susceptible bacterium isolated from mucus of farmed Salmo salar fingerling in Chile. Citrobacter gillenii FP75 was identified by using biochemical tests and 16S ribosomal gene analysis. Nucleotide and amino acid sequences of the qnrB89 gene exhibited an identity to qnrB of 81.24% and 91.59%, respectively. The genetic environment of qnrB89 was characterized by the upstream location of a sequence encoding for a protein containing a heavy metal-binding domain and a gene encoding for a N-acetylmuramoyl-L-alanine amidase protein, whereas downstream to qnrB89 gene were detected the csp and cspG genes, encoding cold-shock proteins. The qnrB89 gene was located on a large chromosomal contig of the FP75 genome and was not associated with the 10-kb plasmid and class 1 integron harbored by the FP75 strain. This study reports for the first time the carriage of a qnrB gene by the C. gillenii species, and its detection in a bacterial strain isolated from farmed salmon in Chile.

Live Feeds Used in the Larval Culture of Red Cusk Eel, Genypterus chilensis, Carry High Levels of Antimicrobial-Resistant Bacteria and Antibiotic-Resistance Genes (ARGs).

The culture of red cusk eel Genypterus chilensis is currently considered a priority for Chilean aquaculture but low larval survival rates have prompted the need for the continuous use of antibacterials. The main aim of this study was to evaluate the role of live feed as a source of antibacterial-resistant bacteria in a commercial culture of G. chilensis. Samples of rotifer and Artemia cultures used as live feed were collected during the larval growth period and culturable bacterial counts were performed using a spread plate method. Rotifer and Artemia cultures exhibited high levels of resistant bacteria (8.03 × 104 to 1.79 × 107 CFU/g and 1.47 × 106 to 3.50 × 108 CFU/g, respectively). Sixty-five florfenicol-resistant isolates were identified as Vibrio (81.5%) and Pseudoalteromonas (15.4%) using 16S rRNA gene sequence analysis. A high incidence of resistance to streptomycin (93.8%), oxytetracycline (89.2%), co-trimoxazole (84.6%), and kanamycin (73.8%) was exhibited by resistant isolates. A high proportion of isolates (76.9%) carried the florfenicol-resistance encoding genes floR and fexA, as well as plasmid DNA (75.0%). The high prevalence of multiresistant bacteria in live feed increases the incidence of the resistant microbiota in reared fish larvae, thus proper monitoring and management strategies for live feed cultures appear to be a priority for preventing future therapy failures in fish larval cultures.

Occurrence of Transferable Integrons and sul and dfr Genes Among Sulfonamide-and/or Trimethoprim-Resistant Bacteria Isolated From Chilean Salmonid Farms.

Salmon farming industry in Chile currently uses a significant quantity of antimicrobials to control bacterial pathologies. The main aims of this study were to investigate the presence of transferable sulfonamide- and trimethoprim-resistance genes, sul and dfr, and their association with integrons among bacteria associated to Chilean salmon farming. For this purpose, 91 Gram-negative strains resistant to sulfisoxazole and/or trimethoprim recovered from various sources of seven Chilean salmonid farms and mainly identified as belonging to the Pseudomonas genus (81.0%) were studied. Patterns of antimicrobial resistance of strains showed a high incidence of resistance to florfenicol (98.9%), erythromycin (95.6%), furazolidone (90.1%) and amoxicillin (98.0%), whereas strains exhibited minimum inhibitory concentrations (MIC90) values of sulfisoxazole and trimethoprim of >4,096 and >2,048 µg mL-1, respectively. Strains were studied for their carriage of these genes by polymerase chain reaction, using specific primers, and 28 strains (30.8%) were found to carry at least one type of sul gene, mainly associated to a class 1 integron (17 strains), and identified by 16S rRNA gene sequencing as mainly belonging to the Pseudomonas genus (21 strains). Of these, 22 strains carried the sul1 gene, 3 strains carried the sul2 gene, and 3 strains carried both the sul1 and sul2 genes. Among these, 19 strains also carried the class 1 integron-integrase gene intI1, whereas the dfrA1, dfrA12 and dfrA14 genes were detected, mostly not inserted in the class 1 integron. Otherwise, the sul3 and intI2 genes were not found. In addition, the capability to transfer by conjugation these resistance determinants was evaluated in 22 selected strains, and sul and dfr genes were successfully transferred by 10 assayed strains, mainly mediated by a 10 kb plasmid, with a frequency of transfer of 1.4 × 10-5 to 8.4 × 10-3 transconjugant per recipient cell, and exhibiting a co-transference of resistance to florfenicol and oxytetracycline, currently the most used in Chilean salmon industry, suggesting an antibacterial co-selection phenomenon. This is the first report of the characterization and transferability of integrons as well as sul and dfr genes among bacteria associated to Chilean salmon farms, evidencing a relevant role of this environment as a reservoir of these genes.

Purification and characterization of indochrome type blue pigment produced by Pseudarthrobacter sp. 34LCH1 isolated from Atacama desert.

The interest in and demand for natural dyes has increased significantly in recent years; however, very few natural blue dyes are commercially available, because blue colored compounds in nature are relatively rare. In this study, a blue pigment-producing bacteria from Lake Chungará (Atacama Desert, Chile) was isolated, and its blue pigment was purified and chemically characterized. The pigment-producing strain was identified as Pseudarthrobacter sp. by 16S rRNA gene sequencing. The pigment was separated from the filtered culture medium by column chromatography/solid-phase extraction using different resins (ionic exchange, C-18, size exclusion). The strain produced up to 2.5 g L-1 of blue pigment, which was very soluble in water, partially soluble in methanol and insoluble in other organic solvents. The pigment was analyzed and characterized by analytical HPLC, UV-Vis, FT-IR, and H-NMR, and purified by semi-preparative HPLC. The pigment was non-toxic to brine shrimp (LD50 > 2.3 g L-1) and was stable at pH 6-10 at temperatures below 60 °C. HPLC analysis shows that the pigment is composed of four major blue fractions. The physicochemical properties and structural analysis demonstrate that this pigment belongs to the indochrome isomers, whose properties have yet to have been characterized. The high solubility in water, good stability in neutral and basic pH, and negligible toxicity of the blue pigment make it a good candidate suitable for several industrial and possibly some food applications.

Current Status of the Use of Antibiotics and the Antimicrobial Resistance in the Chilean Salmon Farms.

The Chilean salmon industry has undergone a rapid development making the country the world's second largest producer of farmed salmon, but this growth has been accompanied by an intensive use of antibiotics. This overuse has become so significant that Chilean salmon aquaculture currently has one of the highest rates of antibiotic consumption per ton of harvested fish in the world. This review has focused on discussing use of antibiotics and current status of scientific knowledge regarding to incidence of antimicrobial resistance and associated genes in the Chilean salmonid farms. Over recent years there has been a consistent increase in the amount of antimicrobials used by Chilean salmonid farms, from 143.2 tons in 2010 to 382.5 tons in 2016. During 2016, Chilean companies utilized approximately 0.53 kg of antibiotics per ton of harvested salmon, 363.4 tons (95%) were used in marine farms, and 19.1 tons (5%) in freshwater farms dedicated to smolt production. Florfenicol and oxytetracycline were by far the most frequently used antibiotics during 2016 (82.5 and 16.8%, respectively), mainly being used to treat Piscirickettsia salmonis, currently considered the main bacterial threat to this industry. However, the increasing development of this industry in Chile, as well as the intensive use of antimicrobials, has not been accompanied by the necessary scientific research needed to understand the impact of the intensive use of antibiotics in this industry. Over the last two decades several studies assessing antimicrobial resistance and the resistome in the freshwater and marine environment impacted by salmon farming have been conducted, but information on the ecological and environmental consequences of antibiotic use in fish farming is still scarce. In addition, studies reporting the antimicrobial susceptibility of bacterial pathogens, mainly P. salmonis, have been developed, but a high number of these studies were aimed at setting their epidemiological cut-off values. In conclusion, further studies are urgently required, mainly focused on understanding the evolution and epidemiology of resistance genes in Chilean salmonid farming, and to investigate the feasibility of a link between these genes among bacteria from salmonid farms and human and fish pathogens.

Sulphur-cycling bacteria and ciliated protozoans in a Beggiatoaceae mat covering organically enriched sediments beneath a salmon farm in a southern Chilean fjord.

The colourless mat covering organically enriched sediments underlying an intensive salmon farm in Estero Pichicolo, southern Chile, was surveyed by combined 454 PyroTag and conventional Sanger sequencing of 16S/18S ribosomal RNA genes for Bacteria and Eukarya. The mat was dominated by the sulphide-oxidizing bacteria (SOB) Candidatus Isobeggiatoa, Candidatus Parabeggiatoa and Arcobacter. By order of their abundances, sulphate-reducing bacteria (SRB) were represented by diverse deltaproteobacterial Desulfobacteraceae, but also within Desulfobulbaceae, Desulfuromonadaceae and Desulfovibrionaceae. The eukaryotic PyroTags were dominated by polychaetes, copepods and nematodes, however, ciliated protozoans were highly abundant in microscopy observations, and were represented by the genera Condylostoma, Loxophyllum and Peritromus. Finally, the abundant Sulfurimonas/Sulfurovum also suggest the occurrence of zero-valence sulphur oxidation, probably derived from Beggiatoaceae as a result of bacteriovorus infaunal activity or generated as free S(0) by the Arcobacter bacteria. The survey suggests an intense and complex sulphur cycle within the surface of salmon-farm impacted sediments.

Distribution and growth of Vibrio parahaemolyticus in southern Chilean clams (Venus antiqua) and blue mussels (Mytilus chilensis).

We evaluated the distribution and growth of Vibrio parahaemolyticus in the inland sea of southern Chile, where the world's largest foodborne gastroenteritis outbreak by the pandemic strain O3:K6 occurred in 2005. Intertidal samples of Mytilus chilensis and Venus antiqua were collected around port towns between 41°28'S and 43°07'S, during April to May 2011 and January to March 2012. We used most probable number real-time polymerase chain reaction (MPN-PCR) for enumeration of the tlh, tdh, and trh genes in freshly harvested bivalves and after a controlled postharvest temperature abuse. Pathogenic markers (tdh+ or trh+) were not detected. Total V. parahaemolyticus (tlh+) in freshly harvested samples reached up to 0.38 and 3.66 log MPN/g in 2011 and 2012, respectively, with values close to or above 3 log MPN/g only near Puerto Montt (41°28'S, 72°55'W). Enrichments by temperature abuse (>2 log MPN/g) occurred mainly in the same zone, regardless of the year, suggesting that both natural or anthropogenic exposure to high temperatures were more critical. Lower salinity and higher sea surface temperature in Reloncaví Sound and Reloncaví Estuary were consistent with our observations and allowed confirmation of the existence of a high-risk zone near Puerto Montt. Based on the results, a strategy focused on risk management inside this defined hazard zone is recommended.

Bacteriostatic anti-Vibrio parahaemolyticus activity of Pseudoalteromonas sp. strains DIT09, DIT44 and DIT46 isolated from Southern Chilean intertidal Perumytilus purpuratus.

We characterised the anti-Vibrio parahaemolyticus (anti-V. parahaemolyticus) marine bacteria DIT09, DIT44 and DIT46 isolated from the intertidal mussel Perumytilus purpuratus. The 16S rRNA gene sequences identify a Pseudoalteromonas sp. that form a clade with P. prydzensis and P. mariniglutinosa. The strains produced bacteriostatic anti-V. parahaemolyticus agents during the exponential growth phase, which were also active against V. cholerae and V. anguillarum, but not on other Gram positive and Gram negative bacteria. Bacteriostatic agents could be permeated by analytic ultra-filtration with 3.5 kDa cut-off, partially precipitated with 70 and 90 % ammonium sulphate, but not extracted with ethyl acetate. Reverse-phase HPLC revealed the production of a set of 5-6 active compounds by each strain (elution from 20 to 40 % acetonitrile), with similar but non identical HPLC patterns. Additionally, V. parahaemolyticus was able to progressively overcome the inhibition of antibiotics in trypticase soy agar with Fe(III) 0.5 up to 2 mM, suggesting the involvement of a set of novel siderophore or active molecules targeted at different Fe-siderophore uptake systems. The overall findings suggest that Pseudoalteromonas sp. DIT strains produce a putatively novel class of bacteriostatic and probably amphiphilic anti-Vibrio agents, indicating the need for further studies with chemical purification followed by their structural and functional characterization. Finally, the crude cell-free extracts, as well as the strains incubated at 10(3) and 10(5) c.f.u./mL, did not cause mortality in Artemia franciscana nauplii, suggesting that these bacteria are serious candidates for further probiotic evaluations with shellfish and fish cultures.