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This paper presents a validated protocol, using a novel, specifically formulated medium, to perform broth microdilution antimicrobial susceptibility assays of the salmonid bacterial pathogen Piscirickettsia salmonis. The minimum inhibitory concentrations (MIC) for florfenicol and oxytetracycline against 58 P. salmonis isolates recovered from various outbreaks occurred in Chilean salmonid farms were determined using this protocol. Normalized resistance interpretation (NRI) analysis was applied to these data to calculate appropriate protocol-specific epidemiological cut-off values. These cut-off values allow the isolates to be categorized as either fully susceptible wild type (WT) members of this species, or as manifesting reduced susceptibility non-wild type (NWT). The distribution of MIC values of florfenicol was bimodal and the distribution of the normalized values for the putative WT observation had a standard deviation of 0.896 log2 mg mL-1. This analysis calculated a cut-off value of ≤0.25 mg mL-1 and categorized 33 (56%) of the isolates as manifesting reduced susceptibility to florfenicol. For the oxytetracycline MIC data the NRI analysis also treated the distribution as bimodal. The distribution of the normalized values for the putative WT observation had a standard deviation of 0.951 log2 mg mL-1. This analysis gave a cut-off value of ≤0.5 mg mL-1 and categorized five isolates (9%) as manifesting reduced susceptibility to oxytetracycline. The susceptibility testing protocol developed in this study was capable of generating MIC data from all the isolates tested. On the basis of the precision of the data it generated, and the degree of separation of values for WT and NWT it achieved, it is argued that this protocol has the performance characteristics necessary for it to be considered as a standard protocol. [ABSTRACT FROM AUTHOR]

The administration of antimicrobials to control bacterial pathologies in Chilean scallop hatcheries is a frequent practice, but their effects on these cultures remained unknown. This study was undertaken to obtain information on the effect of the administration of florfenicol and oxytetracycline on the growth, survival and bacterial content of scallop larvae under farming conditions. Florfenicol-treated cultures exhibited high survival rates (44% after 17 days of culture), whereas cultures not treated or treated with oxytetracycline collapsed after 11 days of culture. Surprisingly, no significant differences in the heterotrophic (Tukey test; P = 0.226) and Vibrio (Tukey test; P = 0.666) concentrations between the oxytetracycline-treated and untreated larval cultures were observed. Otherwise, florfenicol administered directly into rearing tanks produced significantly higher larval growth (Tukey test; P = 0.0001) and survival (Tukey test; P = 0.011) than bath treatment. When 2 and 4 mg L−1 of florfenicol were compared, no significant differences in growth ( t-test; P = 0.4596) and survival (Tukey test; P = 0.057) were observed, suggesting that a concentration of 2 mg L−1 is sufficient to ensure larval production. The present results demonstrate the efficacy of florfenicol-based therapy to increase larval survival and growth at commercial scale and prompt the necessity to standardize its use in Chilean scallop hatcheries. [ABSTRACT FROM AUTHOR]

The use of oxytetracycline for preventing and controlling bacterial pathogens in salmon farming is frequent in Chile, yet no studies have been performed to evaluate the ecological impact of its intensive and prolonged use. In this work, the frequency of oxytetracycline-resistant bacteria from water, pelletized feed and fingerlings from four Chilean freshwater Atlantic salmon farms, as well as the level of resistance of selected strains was investigated. Viable bacterial counts were performed by a spread plate method and antibiotic-resistant bacteria were counted in culture media supplemented with the selected antibiotic. Resistance levels of selected strains isolated in media containing antibiotic were determined using an agar plate dilution method. High proportions of low- and high-level oxytetracycline-resistant bacteria (selected in agar plates containing 30 and 100 μg/ml, respectively), mainly from pelletized feed and effluent samples of the fish farms were found. The highest proportions of resistant bacteria were found in the effluent samples, and were significantly higher (P<0.05, Tukey''s test) than those from the other samples studied. On the contrary, influent samples exhibited the lowest proportions of resistant bacteria. One hundred and three resistant Gram-negative isolates, which represented the oxytetracycline-resistant bacterial population, were randomly selected on TSA containing 30 or 100 μg/ml of oxytetracycline, from salmon farms and pellet samples, and streaked for purification on TSA plates without oxytetracycline. A large number of non-fermenting bacteria (77.7%) were identified. Among these, Pseudomonas fluorescens (28.2%), Stenotrophomonas maltophilia (5.8%), Sphingomonas paucimobilis (5.8%), Acinetobacter lwoffii (4.8%), and Pseudomonas putida (4.8%) were the most frequent. Also, an important number of strains of Aeromonas hydrophila (9.7%), Burkholderia cepacia (3.9%), Brevundimonas vesicularis (3.9%), Acinetobacter johnsonii (2.9%), Pantoea sp. (2.9%) and Moraxella sp. (2.9%) were found. P. fluorescens and A. hydrophila predominated in salmon fingerlings, whereas A. lwoffii and S. maltophilia were predominant in pellet samples. Selected strains exhibited high levels of oxytetracycline resistance, with minimum inhibitory concentrations (MICs) ranging from 64 to 2048 μg/ml, whereas MIC90 of oxytetracycline varied between 1024 and 2048 μg/ml. This study shows the presence of an important population of oxytetracycline-resistant bacteria in the microflora of Chilean salmon farms. Therefore, the environment of these farms might play important roles as reservoirs of bacteria carrying genetic determinants for high-level tetracycline resistance, prompting an important risk to public health for workers involved in fish culturing and processing. [Copyright &y& Elsevier]

The intensive use of antimicrobial agents, mainly oxytetracycline, to prevent and control bacterial pathologies in Chilean salmon culture is a frequent practice. A total of 103 Gram-negative oxytetracycline-resistant bacteria recovered from various sources of 4 Chilean freshwater salmon farms were identified and investigated for their susceptibility patterns to various antibacterial agents, by using an agar disk diffusion method. Antibacterial resistance patterns of isolates were not correlated with bacterial species or strain source. A high number of bacteria resistant to amoxicillin, ampicillin, erythromycin, and furazolidone, as well as an important frequency of bacterial resistance to florfenicol, chloramphenicol, cefotaxime and trimethoprim-sulfamethoxazole was found. On the contrary, the proportion of bacteria resistant to gentamicin, kanamycin, flumequine and enrofloxacin was rather low. Resistant microflora showed a high taxonomic variability and mainly consisted of non-fermenting bacteria (77.7%). These strains mainly belonged to the species Pseudomonas fluorescens (29), Aeromonas hydrophila (10), Stenotrophomonas maltophilia (6), isolated from salmon fingerlings, and Acinetobacter lwoffii (5) isolated from pelletized feed. The occurrence of simultaneous resistance to various antibacterials was frequent. We observe a high frequency of bacteria resistant to 6–10 antibacterials (74 strains), and antibiotic resistance index (ARI) values ranging from 0.38 to 0.48 for the four salmon farms studied. These results suggest that Chilean salmon farms might play a role as reservoirs of antibacterial multiresistant bacteria, thus prompting the necessity for a more restrictive attitude towards the intensive use of antibacterials in salmon farming. [Copyright &y& Elsevier]

Simple Summary: The culture of the marine fish 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 antibiotics, mainly florfenicol. In this study, the role of live prey (rotifers and the brine shrimp Artemia franciscana) used to feed fish larvae as a source of antibacterial-resistant bacteria in a commercial culture of G. chilensis was investigated. Samples of live feeds were collected during the larval growth period and their bacterial contents were determined. High levels of potentially opportunistic pathogens, such as Vibrio spp., as well as florfenicol-resistant bacteria, were detected. Sixty-five florfenicol-resistant isolates were recovered from these cultures and identified as Vibrio (81.5%) and Pseudoalteromonas (15.4%), which exhibited a high incidence of co-resistance to the antibiotics streptomycin, oxytetracycline, co-trimoxazole, and kanamycin. The majority of them carried the florfenicol-resistance encoding genes floR and fexA. The high prevalence of antibiotic-resistant bacteria and the associated genetic elements in live feed administered to reared fish larvae requires the prompt implementation of efficient management strategies to prevent future therapy failures in fish larval cultures and the spread of antibiotic-resistant bacteria to associated aquatic environments. 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. [ABSTRACT FROM AUTHOR]