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The present study evaluated the capacity of three Bacillus species to improve health status and growth performance of Nile Tilapia fed with high levels of soybean meal and challenged with Aeromonas hydrophila. In vitro experiments showed that ß-hemolysin and metalloprotease enzymes were produced by A. hydrophila throughout the exponential growth phase. In vivo experiments showed that 107 colony-forming units (CFUs)/ml of this pathogen killed 50% of control group fishes in 13 days. To evaluate the influence of Bacillus strains on health status and growth performance in Nile Tilapia, 180 fishes (33.44 + 0.05 g) were distributed in 12 tanks of 200 L each, and animals were fed twice per day until satiety. 1) Control group without Bacillus, 2) Bacillus sp1, 3) Bacillus sp2, and 4) Bacillus sp3 groups were formulated containing 106 CFU/g. After 40 days of feeding, the fishes were intraperitoneally injected with 1 ml of A. hydrophila at 2 × 107 CFU/ml, and mortality was recorded. The results showed that cumulative mortality rate was significantly (p< 0.05) lower in the Bacillus sp1 (25%), sp2 (5%), and sp3 (15%) groups, than the control group (50%). Weight gain was also significantly better (p< 0.05) in the Bacillus sp1 (36%), sp2 (67%), and sp3 (55%) groups with respect to the control group (30%). In conclusion, functional diet formulated with high levels of soybean meal and supplemented with Bacillus sp2 could be an alternative to protect Nile tilapia cultures from A. hydrophila infections and improve fish growth performance.
Assuntos
Aeromonas hydrophila , Bacillus , Ciclídeos , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Animais , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Ciclídeos/crescimento & desenvolvimento , Ciclídeos/microbiologia , Aeromonas hydrophila/patogenicidade , Aeromonas hydrophila/crescimento & desenvolvimento , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Bactérias Gram-Negativas/prevenção & controle , Ração Animal , Probióticos/administração & dosagem , Glycine max/microbiologia , AquiculturaRESUMO
IncQ-type plasmids have become important vectors in the dissemination of blaGES among different bacterial genera and species from different environments around the world, and studies estimating the occurrence of Guiana extended-spectrum (GES)-type ß-lactamases are gaining prominence. We analyzed the genetic aspects of two IncQ1 plasmids harboring different blaGES variants from human and environmental sources. The blaGES variants were identified using polymerase chain reaction (PCR) in Aeromonas veronii isolated from hospital effluent and Klebsiella variicola isolated from a rectal swab of a patient admitted to the cardiovascular intensive care unit in a different hospital. Antimicrobial-susceptibility testing and transformation experiments were performed for phenotypic analysis. Whole-genome sequencing was performed using Illumina and Oxford Nanopore platforms. The comparative analysis of plasmids was performed using BLASTn, and the IncQ1 plasmids showed a high identity and similar size. A. veronii harbored blaGES-7 in a class 1 integron (In2061), recently described by our group, and K. variicola carried blaGES-5 in the known class 1 integron. Both integrons showed a fused gene cassette that encodes resistance to aminoglycosides and fluoroquinolones, with an IS6100 truncating the 3'-conserved segment. The fused genes are transcribed together, although the attC site is disrupted. These gene cassettes can no longer be mobilized. This study revealed a mobilome that may contribute to the dissemination of GES-type ß-lactamases in Brazil. Class 1 integrons are hot spots for bacterial evolution, and their insertion into small IncQ-like plasmids displayed successful recombination, allowing the spread of blaGES variants in various environments. Therefore, they can become prevalent across clinically relevant pathogens.
Assuntos
Plasmídeos , beta-Lactamases , Plasmídeos/genética , Brasil , beta-Lactamases/genética , Humanos , Genômica/métodos , Antibacterianos/farmacologia , Klebsiella/genética , Klebsiella/efeitos dos fármacos , Aeromonas/genética , Aeromonas/efeitos dos fármacos , Aeromonas/isolamento & purificação , Testes de Sensibilidade Microbiana , Sequenciamento Completo do Genoma , Genoma Bacteriano , Integrons/genéticaRESUMO
Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch's postulates, challenge experiments were conducted and median lethal dose (LD50) of L. garvieae and A. hydrophila was calculated as 1 × 105.91 CFU per mL and 1 × 105.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia.
Assuntos
Aeromonas hydrophila , Antibacterianos , Ciclídeos , Coinfecção , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Lactococcus , Filogenia , Animais , Aeromonas hydrophila/genética , Aeromonas hydrophila/isolamento & purificação , Aeromonas hydrophila/patogenicidade , Aeromonas hydrophila/classificação , Aeromonas hydrophila/efeitos dos fármacos , Ciclídeos/microbiologia , Índia , Doenças dos Peixes/microbiologia , Lactococcus/genética , Lactococcus/isolamento & purificação , Lactococcus/classificação , Lactococcus/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Coinfecção/microbiologia , Coinfecção/veterinária , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , RNA Ribossômico 16S/genética , Aquicultura , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismoRESUMO
Aeromonas trota is sensitive to most antibiotics and the sole species of this genus susceptible to ampicillin. This susceptibility profile could be related to its inability to acquire exogenous DNA. In this study, A. trota isolates were analyzed to establish their capacity to incorporate foreign DNA. Fourteen strains were identified as A. trota by multilocus phylogenetic analysis (MLPA). Minimal inhibitory concentrations of antibiotics (MIC) were assessed, confirming the susceptibility to most antibiotics tested. To explore their capacity to be transformed, A. trota strains were used as recipients in different horizontal transfer assays. Results showed that around fifty percent of A. trota strains were able to incorporate pBAMD1-2 and pBBR1MCS-3 plasmids after conjugal transfer. In all instances, conjugation frequencies were very low. Interestingly, several isoforms of plasmid pBBR1MCS-3 were observed in transconjugants. Strains could not receive pAr-32, a native plasmid from A. salmonicida. A. trota strains were unable to receive DNA by means of electroporation, natural transformation or vesiduction. These results confirm that A. trota species are extremely refractory to horizontal gene transfer, which could be associated to plasmid instability resulting from oligomerization or to the presence of defense systems against exogenous genetic material in their genomes. To explain the poor results of horizontal gene transfer (HGT), selected genomes were sequenced and analyzed, revealing the presence of defense systems, which could prevent the stable incorporation of exogenous DNA in A. trota.
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The study evaluated dietary supplementation with a feed additive composed of multi-strain Bacillus for Nile tilapia Oreochromis niloticus. In vitro and in vivo assays employing culture-based microbiological methods and metagenomics were performed. Additionally, the study assessed the haemato-immunology, intestinal microbiome, and growth performance of the animals. For this, 30 juvenile Nile tilapia were used in the in vitro assay and 180 (60 + 120) in the in vivo assays. In the in vitro assay, we found evidence of adhesion of the probiotic bacteria to the intestinal mucus of fish, corroborated in the 15-day in vivo assay, in which the count of B. licheniformis was significantly higher in fish fed with probiotic when compared to fish of the control group. Furthermore, in the 50-day in vivo trial, a metagenomic analysis provided evidence for the modulation of the intestine microbiome of Nile tilapia by dietary supplementation of the probiotic. In addition, there was an increase in species richness, higher abundance of potentially probiotic autochthonous species and a lower abundance of Aeromonas sp. when the animals were fed the supplemented diet. Finally, no significant differences were observed in growth performance and haemato-immunological analyses, suggesting no harm to fish health when the product was supplemented for 15 and 50 days. The in vitro results indicate that the multi-strain probiotics were able to adhere to the intestinal mucus of Nile tilapia. Additionally, a modulation of the intestinal microbiome was evidenced in the in vivo assay.
Assuntos
Ração Animal , Bacillus , Ciclídeos , Dieta , Microbioma Gastrointestinal , Probióticos , Animais , Probióticos/farmacologia , Probióticos/administração & dosagem , Ciclídeos/imunologia , Ciclídeos/crescimento & desenvolvimento , Ciclídeos/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Ração Animal/análise , Dieta/veterinária , Suplementos Nutricionais/análiseRESUMO
Aeromonas spp. are frequently encountered in aquatic environments, with Aeromonas veronii emerging as an opportunistic pathogen causing a range of diseases in both humans and animals. Recent reports have raised public health concerns due to the emergence of multidrug-resistant Aeromonas spp. This is particularly noteworthy as these species have demonstrated the ability to acquire and transmit antimicrobial resistance genes (ARGs). In this study, we report the genomic and phenotypic characteristics of the A. veronii TR112 strain, which harbors a novel variant of the Vietnamese Extended-spectrum ß-lactamase-encoding gene, blaVEB-28, and two mcr variants recovered from an urban river located in the Metropolitan Region of São Paulo, Brazil. A. veronii TR112 strain exhibited high minimum inhibitory concentrations (MICs) for ceftazidime (64 µg/mL), polymyxin (8 µg/mL), and ciprofloxacin (64 µg/mL). Furthermore, the TR112 strain demonstrated adherence to HeLa and Caco-2 cells within 3 h, cytotoxicity to HeLa cells after 24 h of interaction, and high mortality rates to the Galleria mellonella model. Genomic analysis showed that the TR112 strain belongs to ST257 and presented a range of ARGs conferring resistance to ß-lactams (blaVEB-28, blaCphA3, blaOXA-912) and polymyxins (mcr-3 and mcr-3.6). Additionally, we identified a diversity of virulence factor-encoding genes, including those encoding mannose-sensitive hemagglutinin (Msh) pilus, polar flagella, type IV pili, type II secretion system (T2SS), aerolysin (AerA), cytotoxic enterotoxin (Act), hemolysin (HlyA), hemolysin III (HlyIII), thermostable hemolysin (TH), and capsular polysaccharide (CPS). In conclusion, our findings suggest that A. veronii may serve as an environmental reservoir for ARGs and virulence factors, highlighting its importance as a potential pathogen in public health.
Assuntos
Aeromonas veronii , Antibacterianos , Testes de Sensibilidade Microbiana , Rios , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Humanos , Antibacterianos/farmacologia , Rios/microbiologia , Aeromonas veronii/genética , Aeromonas veronii/isolamento & purificação , Aeromonas veronii/efeitos dos fármacos , Brasil , Células HeLa , Células CACO-2 , Animais , Farmacorresistência Bacteriana Múltipla/genéticaRESUMO
AIMS: Although elasmobranchs are consumed worldwide, bacteriological assessments for this group are still sorely lacking. In this context, this study assessed bacteria of sharks and rays from one of the most important landing ports along the Rio de Janeiro coast. METHODS AND RESULTS: Bacteria were isolated from the cloacal swabs of the sampled elasmobranchs. They were cultured, and Vibrio, Aeromonas, and Enterobacterales were isolated and identified. The isolated bacteria were then biochemically identified and antimicrobial susceptibility assays were performed. Antigenic characterizations were performed for Salmonella spp. and Polymerase Chain Reaction (PCR) assays were performed to identify Escherichia coli pathotypes. Several bacteria of interest in the One Health context were detected. The most prevalent Enterobacterales were Morganella morganii and Citrobacter freundii, while Vibrio harveyi and Vibrio fluvialis were the most prevalent among Vibrio spp. and Aeromonas allosacharophila and Aeromonas veronii bv. veronii were the most frequent among Aeromonas spp. Several bacteria also displayed antimicrobial resistance, indicative of Public Health concerns. A total of 10% of Vibrio strains were resistant to trimethoprim-sulfamethoxazole and 40% displayed intermediate resistance to cefoxitin. Salmonella enterica strains displayed intermediate resistance to ciprofloxacin, nalidixic acid and streptomycin. All V. cholerae strains were identified as non-O1/non-O139. The detected E. coli strains did not exhibit pathogenicity genes. This is the first study to perform serology assessments for S. enterica subsp. enterica isolated from elasmobranchs, identifying the zoonotic Typhimurium serovar. Salmonella serology evaluations are, therefore, paramount to identify the importance of elasmobranchs in the epidemiological salmonellosis chain. CONCLUSIONS: The detection of several pathogenic and antibiotic-resistant bacteria may pose significant Public Health risks in Brazil, due to high elasmobranch consumption rates, indicating the urgent need for further bacteriological assessments in this group.
Assuntos
Aeromonas , Tubarões , Vibrio cholerae , Animais , Escherichia coli , Brasil , Salmonella/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Aeromonas/genéticaRESUMO
This study aimed to characterize 300 Aeromonas spp. strains isolated from 123 ornamental fish of 32 different species presenting with septicemia, skin lesions, and/or eye lesions. Within the 300 strains, 53.0% were identified as A. veronii, 41.3% as A. hydrophila, and 5.7% as A. caviae. Among the six virulence genes investigated, the most frequent were act (90.3%) and aer (79.3%). More than 50% of A. hydrophila strains were positive for all the studied genes. A total of 30 virulence profiles were identified, with the five main profiles identified comprising 75% of strains. Only five strains were negative for all genes and were identified as A. caviae and A. veronii. The antimicrobial susceptibility profile was performed for 234 strains, with sulfonamides presenting more than 50% of the resistance rates. Susceptibility was observed mainly for cephalosporins, aminoglycosides, chloramphenicol and piperacillin-tazobactam. Multidrug resistance was detected in 82.5% of the studied strains, including A. caviae with 100% multidrug resistance, and A. hydrophila with 90.9% multidrug resistance. The SE-AFLP analysis resulted in 66 genotypes of A. hydrophila, 118 genotypes of A. veronii, and 14 genotypes of A. caviae, demonstrating the greater heterogeneity of A. veronii and A. caviae. However, no direct correlation was observed between the genotypes and the strains' origins or virulence and resistance profiles.
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Sichuan is a significant aquaculture province in China, with a total aquaculture output of 1.72 × 106 tons in 2022. One of the most significant microorganisms hurting the Sichuan aquaculture is Aeromonas hydrophila, whose genotype and antibiotic resistance are yet unknown. This study isolated a total of 64 strains of A. hydrophila from various regions during September 2019 to June 2021 within Sichuan province, China. The technique of Multi-Locus Sequence Typing (MLST) was used for the purpose of molecular typing. Meanwhile, identification of antibiotic resistance phenotype and antibiotic resistance gene was performed. The findings of the study revealed that 64 isolates exhibited 29 sequence types (ST) throughout different regions in Sichuan, with 25 of these ST types being newly identified. Notably, the ST251 emerged as the predominant sequence type responsible for the pandemic. The resistance rate of isolated strains to roxithromycin was as high as 98.3%, followed by co-trimoxazole (87.5%), sulfafurazole (87.5%), imipenem (80%), amoxicillin (60%), and clindamycin (57.8%). Fifteen strains of A. hydrophila exhibited resistance to medicines across a minimum of three categories, suggesting the development of multidrug resistance in these isolates. A total of 63 ARGs were detected from the isolates, which mediated a range of antibiotic resistance mechanisms, with deactivation and efflux potentially serving as the primary mechanisms of antibiotic resistance. This study revealed the diversity of A. hydrophila genotypes and the risk of antibiotic resistance in Sichuan, providing reference for scientific and effective control of A. hydrophila infection.
Assuntos
Aeromonas hydrophila , Aeromonas , Aeromonas hydrophila/genética , Tipagem de Sequências Multilocus , Resistência Microbiana a Medicamentos , Genótipo , Fenótipo , Antibacterianos/farmacologia , China , Aeromonas/genéticaRESUMO
Abstract Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.
Resumo O Paquistão é um país agrícola, onde a pesca desempenha um papel muito importante para o desenvolvimento econômico. Diferentes doenças são prevalentes em peixes do Paquistão, mas as informações relacionadas aos agentes causadores não são bem conhecidas. Tendo em vista a importância dos patógenos bacterianos como agentes causadores de múltiplas doenças em peixes, o presente estudo foi conduzido para identificação, caracterização e análise de genes de virulência de isolados de Aeromonas spp. de peixes doentes. Foram coletadas 50 amostras de peixes com múltiplas indicações clínicas em diferentes fazendas do distrito de Kasur, Punjab, Paquistão. Para isolar Aeromonas spp., as amostras foram enriquecidas e inoculadas em meio de isolamento. Os isolados foram identificados e caracterizados por diferentes testes bioquímicos, kit Analytical Profile Index (API) 20E, e ensaios de reação em cadeia da polimerase (PCR). Todos os isolados foram selecionados para três genes de virulência putativos, incluindo aerolisina (aer), hemolisina (hyl) e enterotoxina citotônica termolábil (alt). Sete isolados de Aeromonas hydrophila foram recuperados e identificados com base no API 20E. Esses isolados foram posteriormente confirmados como A. hydrophila de acordo com ensaios de PCR. Três isolados indicaram a presença de genes de virulência (alt e hyl), enquanto o gene aerolisina (aer) não esteve presente em nenhum dos isolados de A. hydrophila. O presente estudo confirmou A. hydrophila como o agente causador da síndrome ulcerativa epizoótica e septicemia móvel por Aeromonas em fazendas de peixes, no distrito de Kasur, Punjab, Paquistão. Além disso, a detecção de dois genes de virulência (alt e hyl) em isolados de A. hydrophila é uma ameaça para os consumidores de peixes da área de estudo.
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Animais , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Bactérias Gram-Negativas/epidemiologia , Aeromonas/genética , Paquistão , Aeromonas hydrophila/genética , Enterotoxinas/genética , PeixesRESUMO
Abstract Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.
Resumo O Paquistão é um país agrícola, onde a pesca desempenha um papel muito importante para o desenvolvimento econômico. Diferentes doenças são prevalentes em peixes do Paquistão, mas as informações relacionadas aos agentes causadores não são bem conhecidas. Tendo em vista a importância dos patógenos bacterianos como agentes causadores de múltiplas doenças em peixes, o presente estudo foi conduzido para identificação, caracterização e análise de genes de virulência de isolados de Aeromonas spp. de peixes doentes. Foram coletadas 50 amostras de peixes com múltiplas indicações clínicas em diferentes fazendas do distrito de Kasur, Punjab, Paquistão. Para isolar Aeromonas spp., as amostras foram enriquecidas e inoculadas em meio de isolamento. Os isolados foram identificados e caracterizados por diferentes testes bioquímicos, kit Analytical Profile Index (API) 20E, e ensaios de reação em cadeia da polimerase (PCR). Todos os isolados foram selecionados para três genes de virulência putativos, incluindo aerolisina (aer), hemolisina (hyl) e enterotoxina citotônica termolábil (alt). Sete isolados de Aeromonas hydrophila foram recuperados e identificados com base no API 20E. Esses isolados foram posteriormente confirmados como A. hydrophila de acordo com ensaios de PCR. Três isolados indicaram a presença de genes de virulência (alt e hyl), enquanto o gene aerolisina (aer) não esteve presente em nenhum dos isolados de A. hydrophila. O presente estudo confirmou A. hydrophila como o agente causador da síndrome ulcerativa epizoótica e septicemia móvel por Aeromonas em fazendas de peixes, no distrito de Kasur, Punjab, Paquistão. Além disso, a detecção de dois genes de virulência (alt e hyl) em isolados de A. hydrophila é uma ameaça para os consumidores de peixes da área de estudo.
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OBJECTIVE: Florfenicol (FFC) is a broad-spectrum antibiotic approved by the U.S. Food and Drug Administration to treat both systemic and external bacterial infections in food fish. The objective of this study was to evaluate the effect of FFC-medicated feed on the gut microbiota of Zebrafish Danio danio to determine (1) if the therapeutic dose of FFC-medicated feed induces dysbiosis and (2) if fish with altered gut microbiota were more susceptible to subsequent infection by the common opportunistic fish pathogen Aeromonas hydrophila. METHODS: Zebrafish that were treated with regular and FFC-medicated feeds were artificially challenged with A. hydrophila at the end of the recommended 15-day antibiotic withdrawal period. The gut microbiota of the Zebrafish at different stages was analyzed using 16S ribosomal RNA gene sequencing. RESULT: Our results found that FFC-medicated feed induced disruption of the gut microbiota. Dysbiosis was observed in all treated groups, with a significant increase in bacterial diversity, and was characterized by a remarkable bloom of Proteobacteria and a drastic decline of Mycoplasma and Cetobacterium in treated animals but without noticeable clinical signs or mortalities. In addition, the increase of Proteobacteria was not significantly reduced after the recommended 15-day withdrawal period, and the Zebrafish treated with FFC-medicated feed exhibited a significantly higher mortality rate when they were subsequently challenged with A. hydrophila compared to the control (regular feed) groups. Interestingly, the most dramatic changes in the gut microbiome composition occurred at the transition time between the late stage of the medicated treatment and the beginning of the withdrawal period instead of the time during the Aeromonas infection. CONCLUSION: The administration of FFC-medicated feed at the recommended dose induced gut dysbiosis in Zebrafish, and fish did not recover to the baseline after the recommended withdrawal period. Our findings suggest that the use of antibiotics in fish elicits a response similar to those previously described in mammals and possibly makes the host more susceptible to subsequent infections of opportunistic pathogens. This study using a controlled model system suggests that antibiotics in aquaculture may have long-term effects on the general well-being of the fish.
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R-(+)-limonene is a monoterpene from plants of the genus Citrus with diverse biological properties. This research evaluated the effects of dietary supplementation with R-(+)-limonene on growth, metabolic parameters in plasma and liver, and the antioxidant and stress responses in silver catfish, Rhamdia quelen, challenged or not with Aeromonas hydrophila. Fish were fed for 67 days with different doses of R-(+)-limonene in the diet (control 0.0, L0.5, L1.0, and L2.0 mL/kg of diet). On the 60th day, a challenge with A. hydrophila was performed. R-(+)-limonene in the diet potentiated the productive performance of the fish. The metabolic and antioxidant responses indicate that R-(+)-limonene did not harm the health of the animals and made them more resistant to the bacterial challenge. Histological findings showed the hepatoprotective effect of dietary R-(+)-limonene against A. hydrophila. Igf1 mRNA levels were upregulated in the liver of fish fed with an L2.0 diet but downregulated with bacterial challenge. The expression levels of crh mRNA were higher in the brains of fish fed with the L2.0 diet. However, the L2.0 diet downregulated crh and hspa12a mRNA expression in the brains of infected fish. In conclusion, the results indicated that R-(+)-limonene can be considered a good dietary supplement for silver catfish.
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Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.
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N-Acetyl-glucosaminidases (GlcNAcases) are exoenzymes found in a wide range of living organisms, which have gained great attention in the treatment of disorders related to diabetes, Alzheimer's, Tay-Sachs', and Sandhoff's diseases; the control of phytopathogens; and the synthesis of bioactive GlcNAc-containing products. Aiming at future industrial applications, in this study, GlcNAcase production by marine Aeromonas caviae CHZ306 was enhanced first in shake flasks in terms of medium composition and then in bench-scale stirred-tank bioreactor in terms of physicochemical conditions. Stoichiometric balance between the bioavailability of carbon and nitrogen in the formulated culture medium, as well as the use of additional carbon and nitrogen sources, played a central role in improving the bioprocess, considerably increasing the enzyme productivity. The optimal cultivation medium was composed of colloidal α-chitin, corn steep liquor, peptone A, and mineral salts, in a 5.2 C:N ratio. Optimization of pH, temperature, colloidal α-chitin concentration, and kLa conditions further increased GlcNAcase productivity. Under optimized conditions in bioreactor (i.e., 34 °C, pH 8 and kLa 55.2 h-1), GlcNAcase activity achieved 173.4 U.L-1 after 12 h of cultivation, and productivity no less than 14.45 U.L-1.h-1 corresponding to a 370-fold enhancement compared to basal conditions.
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Aeromonas caviae , Aeromonas caviae/genética , Reatores Biológicos , Carbono , Quitina , Hexosaminidases , NitrogênioRESUMO
Brazil is one of the world's leading producers of Nile tilapia, Oreochromis niloticus. However, the industry faces a major challenge in terms of infectious diseases, as at least five new pathogens have been formally described in the last five years. Aeromonas species are Gram-negative anaerobic bacteria that are often described as fish pathogens causing Motile Aeromonas Septicemia (MAS). In late December 2022, an epidemic outbreak was reported in farmed Nile tilapia in the state of São Paulo, Brazil, characterized by clinical signs and gross pathology suggestive of MAS. The objective of this study was to isolate, identify, and characterize in vitro and in vivo the causative agent of this epidemic outbreak. The bacterial isolates were identified as Aeromonas veronii based on the homology of 16S rRNA (99.9%), gyrB (98.9%), and the rpoB gene (99.1%). A. veronii showed susceptibility only to florfenicol, while it was resistant to the other three antimicrobials tested, oxytetracycline, enrofloxacin, and amoxicillin. The lowest florfenicol concentration capable of inhibiting bacterial growth was ≤0.5 µg/mL. The phenotypic resistance of the A. veronii isolate observed for quinolones and tetracycline was genetically confirmed by the presence of the qnrS2 (colE plasmid) and tetA antibiotic-resistant genes, respectively. A. veronii isolate was highly pathogenic in juvenile Nile tilapia tested in vivo, showing a mortality rate ranging from 3 to 100% in the lowest (1.2 × 104) and highest (1.2 × 108) bacterial dose groups, respectively. To our knowledge, this study would constitute the first report of highly pathogenic and multidrug-resistant A. veronii associated with outbreaks and high mortality rates in tilapia farmed in commercial net cages in Brazil.
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Vitamin B12 is a micronutrient required by a variety of organisms for healthy cellular functioning. Despite the systemic effects observed in cases of B12 deficiency, relatively little is known about how vitamin B12 affects immune health, especially in amphibians, which are declining at unprecedented rates. In this study, we tested how supplementing an algae diet with B12 affects the innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We found that innate immunity, as measured by a bacterial killing assay, was significantly more robust in B12-supplemented tadpoles than control tadpoles, but no significant differences were found in natural antibody production or hematocrit between groups. Adaptive immunity, as measured by Aeromonas hydrophila-specific IgY antibodies, was significantly greater in tadpoles challenged with A. hydrophila and supplemented with B12 than in control tadpoles, those only challenged with A. hydrophila, and those only given B12. Our results suggest that vitamin B12 is an important factor in maintaining a functional immune system in tadpoles, which may also be true for all vertebrates.
Assuntos
Anuros , Vitamina B 12 , Animais , Larva/fisiologia , Vitamina B 12/farmacologia , Imunidade Inata , Dieta/veterináriaRESUMO
Aeromonas veronii is a Gram-negative bacterial species that causes disease in fish and is nowadays increasingly recurrent in enteric infections of humans. This study was performed to characterize newly sequenced isolates by comparing them with complete genomes deposited at the NCBI (National Center for Biotechnology Information). Nine isolates from fish, environments, and humans from the São Francisco Valley (Petrolina, Pernambuco, Brazil) were sequenced and compared with complete genomes available in public databases to gain insight into taxonomic assignment and to better understand virulence and resistance profiles of this species within the One Health context. One local genome and four NCBI genomes were misidentified as A. veronii. A total of 239 virulence genes were identified in the local genomes, with most encoding adhesion, motility, and secretion systems. In total, 60 genes involved with resistance to 22 classes of antibiotics were identified in the genomes, including mcr-7 and cphA. The results suggest that the use of methods such as ANI is essential to avoid misclassification of the genomes. The virulence content of A. veronii from local isolates is similar to those complete genomes deposited at the NCBI. Genes encoding colistin resistance are widespread in the species, requiring greater attention for surveillance systems.
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RESUMEN Las plantas purificadoras de agua que carecen de un adecuado sistema de control de calidad pueden generar problemas de salud pública. El objetivo de este estudio fue examinar la calidad microbiológica del agua proveniente de pequeñas plantas purificadoras de la ciudad de Puebla, así como, determinar la existencia de bacterias Aeromonas sp y Pseudomonas sp, y caracterizar si presentan un fenotipo patógeno oportunista. Se recolectaron 70 muestras de garrafones de agua de 25 establecimientos. La cuantificación bacteriana se realizó mediante el método de goteo en placa. Se comprobaron los géneros microbianos mediante análisis bioquímico. En las cepas que mostraron discrepancia se utilizó la identificación molecular con base a secuencias parciales del gen 16S rRNA para confirmar su especie y se les evaluaron sus características de patogenicidad: multirresistencia a antibióticos, producción de biopelícula y actividad hemolítica. El 40 % de las plantas purificadoras no cumplieron con la calidad microbiológica del agua para consumo humano. El 41.4 % de los garrafones de agua muestreados incumplió la normativa, presentando coliformes totales 35.7 %, Pseudomonas 30 %, Enterococcus faecalis 8.6 % y bacterias coliformes fecales el 5.7 %. Se obtuvieron 56 aislados, provenientes de los 29 garrafones contaminados; 10 de ellos se caracterizaron molecularmente, resultando 7 aislados relacionados con especies diferentes de P. aeruginosa y 3 con especies de Aeromonas. De los aislados de Pseudomonas, 5 presentaron resistencia a 2 familias de antibióticos y 2 mostraron multirresistencia. El 36 % de los 10 aislados produjeron hemólisis y biopelícula. Dos cepas de Aeromonas mostraron resistencia a Cefalosporina 3a generación pero no produjeron hemólisis. Los 10 aislados analizados fueron clasificados como no patógenos. Es necesario un seguimiento sanitario más estricto para lograr el cumplimiento de las normas nacionales e internacionales relacionadas con el consumo de agua purificada, para evitar dañar la salud de los consumidores.
ABSTRACT Water purification establishments that lack an adequate quality control system can cause public health problems. The objective of this study was to examine the microbiological quality of water from small purification establishments in the city of Puebla, as well as to determine the existence of Aeromonas sp and Pseudomonas sp bacteria, and to characterize whether they present an opportunistic pathogenic phenotype. 70 water jug samples were collected from 25 establishments. Bacterial quantification was performed using the drop plate method. Microbial genera were determined by biochemical analysis using the standard methodology. In the strains that showed discrepancy, molecular identification based on partial sequences of the 16S rRNA gene was used to confirm their species, and their pathogenic characteristics were evaluated: multiresistance to antibiotics, biofilm production, and hemolytic activity. The results showed that 40 % of the purification establishments did not comply with the microbiological quality of water for human consumption. Similarly, 41.4 % of the jugs of water sampled failed to comply with the regulations, presenting total coliforms 35.7 %, Pseudomonas 30 %, Enterococcus faecalis 8.6 % and fecal coliform bacteria 5.7 %. Likewise, 56 isolates were obtained from the 29 contaminated jugs, of which 10 were molecularly characterized, resulting in 4 different species for P. aeruginosa and 3 for Aeromonas. Of the 7 Pseudomonas isolates, 5 presented resistance to 2 families of antibiotics and 2 showed multiresistance. In total, 36 % of the 10 isolates produced hemolysis and biofilm. Two Aeromonas strains showed resistance to 3rd generation Cephalosporin but did not produce hemolysis. The 10 isolates analyzed were classified as non-pathogenic. A stricter sanitary monitoring is necessary to achieve compliance with national and international standards related to the consumption of purified water, to avoid harming the health of consumers.
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The association of quantum dots (QDs) to carbohydrate-binding proteins - lectins - has revealed novel biotechnological strategies for glycobiology studies. Herein, carboxyl-coated QDs were conjugated by adsorption to Cramoll, a glucose/mannose lectin obtained from Cratylia mollis seeds. Then, the conjugates were optically characterized and used to evaluate the surface carbohydrate profiles of four Aeromonas species isolated from the tambaqui fish (Colossoma macropomum). All the Aeromonas cells were labeled by the conjugate. Inhibition assays with methyl-α-D-mannopyranoside and mannan were performed to confirm the labeling specificity. Cramoll-QDs conjugates presented high brightness and showed similar absorption and emission profiles compared to bare QDs. According to the labeling pattern of Aeromonas spp. by the conjugate, results suggested that A. jandaei and A. dhakensis strains may harbor a higher content of more complex glucose/mannose surface glycans, with more available sites for Cramoll-QDs interaction, than A. hydrophila and A. caviae. Noteworthy, the Cramoll-QDs conjugates demonstrated to be potential tools for bacterial characterization based on superficial carbohydrate detection.