Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Classificação dos graus de lesões de aerossaculite em perus associadas com enterobactérias / [Classification of the levels of the airsacculitis injuries in turkeys associated with Enterobacteriaceae]

Foram padronizados os graus de lesões dos sacos aéreos em perus com aerossaculite, associadas com a presença de isolados de enterobactérias nesses órgãos. Um total de 110 amostras de sacos aéreos de perus machos com aerossaculite foi coletado para o estudo. Durante o processo de abate, as amostras foram coletadas por meio de swabs e submetidas a três métodos de armazenamento (imediato, congelado ou pré-incubado após congelamento) para posterior comparação das suas eficiências de isolamento. Os gêneros da família Enterobacteriaceae foram identificados pelas séries bioquímicas EPM, MILi e citrato de Simmons. O crescimento bacteriano ocorreu em 43,64% das amostras. Neste estudo, quatro padrões de lesões de aerossaculite foram identificados de acordo com as características patológicas dos sacos aéreos. Os principais gêneros de enterobactérias identificadas foram: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganella, Kluyvera, Salmonella e Klebsiella. Foi observado que os graus padronizados como 3 e 4 apresentaram maior variedade de gêneros bacterianos. O armazenamento imediato apresentou maior porcentagem de positividade, 41,82%, no entanto o pré-incubado após congelamento se apresentou mais eficaz em relação à quantidade de colônias.(AU)

The degrees of air sac lesions in turkeys with airsacculitis were standardized, associated with the presence of Enterobacteriaceae isolated from these organs. A total of 110 samples of air sacs from male turkeys with airsacculitis were collected and analyzed. During the slaughtering process, the sample collection was done using swabs and submitted to three storage methods (immediate, frozen, or pre incubated after freezing) for further comparison of their isolated efficiency. The bacterial genera of the family Enterobacteriaceae were identified biochemical series EPM, MILi and Simmons citrate. Bacterial growth occurred in 43.64% of samples. In this study, four patterns of aerossaculitis lesions were identified according to the pathological characteristics of air sacs. The frequencies of the Enterobacteriaceae isolated identified in the samples were: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganell, Kluyvera, Salmonella and Klebsiella. Otherwise, it was observed that the levels already standardized as level three and four showed higher variety of genus. The immediate storage showed higher percentage of positivity at 41.82%, however, the pre incubated after freezing showed more efficiency in relation to the quantity of colonies.(AU)

Classificação dos graus de lesões de aerossaculite em perus associadas com enterobactérias / [Classification of the levels of the airsacculitis injuries in turkeys associated with Enterobacteriaceae]

Foram padronizados os graus de lesões dos sacos aéreos em perus com aerossaculite, associadas com a presença de isolados de enterobactérias nesses órgãos. Um total de 110 amostras de sacos aéreos de perus machos com aerossaculite foi coletado para o estudo. Durante o processo de abate, as amostras foram coletadas por meio de swabs e submetidas a três métodos de armazenamento (imediato, congelado ou pré-incubado após congelamento) para posterior comparação das suas eficiências de isolamento. Os gêneros da família Enterobacteriaceae foram identificados pelas séries bioquímicas EPM, MILi e citrato de Simmons. O crescimento bacteriano ocorreu em 43,64% das amostras. Neste estudo, quatro padrões de lesões de aerossaculite foram identificados de acordo com as características patológicas dos sacos aéreos. Os principais gêneros de enterobactérias identificadas foram: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganella, Kluyvera, Salmonella e Klebsiella. Foi observado que os graus padronizados como 3 e 4 apresentaram maior variedade de gêneros bacterianos. O armazenamento imediato apresentou maior porcentagem de positividade, 41,82%, no entanto o pré-incubado após congelamento se apresentou mais eficaz em relação à quantidade de colônias.(AU)

The degrees of air sac lesions in turkeys with airsacculitis were standardized, associated with the presence of Enterobacteriaceae isolated from these organs. A total of 110 samples of air sacs from male turkeys with airsacculitis were collected and analyzed. During the slaughtering process, the sample collection was done using swabs and submitted to three storage methods (immediate, frozen, or pre incubated after freezing) for further comparison of their isolated efficiency. The bacterial genera of the family Enterobacteriaceae were identified biochemical series EPM, MILi and Simmons citrate. Bacterial growth occurred in 43.64% of samples. In this study, four patterns of aerossaculitis lesions were identified according to the pathological characteristics of air sacs. The frequencies of the Enterobacteriaceae isolated identified in the samples were: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganell, Kluyvera, Salmonella and Klebsiella. Otherwise, it was observed that the levels already standardized as level three and four showed higher variety of genus. The immediate storage showed higher percentage of positivity at 41.82%, however, the pre incubated after freezing showed more efficiency in relation to the quantity of colonies.(AU)

Potential immune regulatory role of miR-146a upon Aeromonas hydrophila and Edwardsiella piscicida infections in zebrafish.

This study was aimed to understand the expression of miR-146a in zebrafish (Danio rerio) and its role in regulating immune responses during Aeromonas hydrophila and Edwardsiella piscicida infections. The miR-146a expression was observed from the 1-h post fertilization (hpf) stage and gradually increased up to the early larval stage of zebrafish. The ubiquitous expression of miR-146a was detected in all tested tissues, with the highest level in gills. The expression of miR-146a was significantly increased in larvae when exposed to E. piscicida infection at 24 and 48 h post exposure (hpe). Intraperitoneally (i.p.) injected A. hydrophila and E. piscicida into adult zebrafish showed significant upregulation of miR-146a in gills. Furthermore, immune-related genes, toll-like receptor, tlr-4, transducing signaling pathway molecules, traf-6 and myd88 (bacteria-infected larvae and adults), transcription factor relA and mcp-1b (bacteria-infected adults), pro-inflammatory, il-6 (A. hydrophila-exposed larvae) and mmp-9 (bacteria-exposed larvae) were significantly repressed. In contrast, il-1ß, tnf-α, cxcl-18b, and ccl-34a.4 were induced in both bacteria-challenged larvae and adults. Based on the results, it is suggested that endogenous miR-146a could act as an infection inducible miRNA in zebrafish upon A. hydrophila and E. piscicida infections; also, it could potentially regulate the immune responses in zebrafish.

Isolamento e determinação de sensibilidade e resistência a antimicrobianos de cepas bacterianas presentes na cloaca de Trachemys scripta elegans (Wied, 1839) criadas em cativeiro em Petrolina, PE / Isolation and determination of antimicrobials sensitivity and resistance of bacterial strains present in the cloaca Trachemys scripta elegans (Wied, 1839) raised in captivity in Petrolina/PE, Brazil

Este estudo isolou e determinou o perfil de sensibilidade e de resistência a antimicrobianos de cepas bacterianas isoladas da cloaca de Trachemys scripta elegans (T. s. elegans) criadas em cativeiro. Após 120 dias de adaptação, amostras de swab cloacal obtidas de 20 animais adultos foram cultivadas e, após a identificação dos patógenos através de testes bioquímicos, submetidas ao teste de suscetibilidade a nove antimicrobianos. Enterobacter aerogenes (85%); Shigella spp. (10%) e Edwadsiella spp. (5%) foram isolados e identificados. Os isolados de E. aerogenes foram sensíveis à gentamicina (86%), enrofloxacina (79%), estreptomicina (50%), sulfazotrim (36%) e ampicilina (29%) e resistentes a penicilina (100%), eritromicina (93%), cefalexina (86%) ampicilina (71%) e sulfazotrim (64%). Isolados de Shigella spp. apresentaram sensibilidade à gentamicina (100%), enrofloxacina (50%), doxicilina (50%), estreptomicina (50%), ampicilina (50%), penicilina (50%) e sulfazotrim (50%) e resistência a doxicilina (50%), estreptomicina (50%), ampicilina (50%), penicilina (100%), cefalexina (50%) e sulfazotrim (50%), enquanto que os de Edwardsiella spp. foram sensíveis apenas à gentamicina (100%) e altamente resistentes (100%) aos demais antimicrobianos. Os resultados sugerem a participação de T. s. elegans na cadeia epidemiológica, como reservatório de patógenos importantes, como E. aerogenes, Shigella spp. e Edwardisiella spp., tornando importante a adoção de medidas preventivas pelo risco zoonótico que apresentam e corretas de tratamento e de controle em cativeiros e domicílios, assim como de estudos que enfoquem as características de sensibilidade e de resistência antimicrobiana dos isolados cloacais, pois a multirresistência a drogas pode ser transmitida aos humanos e comprometer o tratamento de indivíduos com doenças graves.(AU)

This study isolated and determined the profile of susceptibility and antimicrobials resistance of bacterial strains isolated from the cloaca Trachemys scripta elegans (T. s. elegans) raised in captivity. After 120 days of adaptation, cloacal swab samples obtained from 20 adults animals were grown and, after the pathogens identification through biochemical tests, submitted to the test of susceptibility to nine antimicrobials. Enterobacter aerogenes (85%); Shigella spp. (10%) and Edwadsiella spp. (5%) were isolated and identified. Isolates from E. aerogenes were sensitive to gentamicin (86%), enrofloxacin (79%), streptomycin (50%), sulfazotrim (36%) and ampicillin (29%) and resistant to penicillin (100%), erythromycin (93%), cephalexin (86%), ampicillin (71%) and sulfazotrim (64%). Isolates from Shigella spp. showed sensitivity to gentamicin (100%), enrofloxacin (50%), doxycycline (50%), streptomycin (50%), ampicillin (50%), penicillin (50%) and sulfazotrim (50%) and resistance to doxycycline (50 %), streptomycin (50%), ampicillin (50%), penicillin (100%), cephalexin (50%) and sulfazotrim (50%), while the Edwardsiella spp. were sensitive only to gentamicin (100%) and were highly resistant (100%) to other antibiotics. The results suggest the participation of T. s. elegans in the epidemiological chain, as reservoir of important pathogens, such as E. aerogenes, Shigella spp. and Edwardisiella spp., making it important to adopt preventive measures for zoonotic risk that present and correct treatment and control in captivity and households, as well as studies that address the sensitivity characteristics and antimicrobial resistance of isolates from cloaca, as it multidrug resistance to drugs can be transmitted to humans and compromise the treatment of patients with serious diseases.(AU)

Isolamento e determinação de sensibilidade e resistência a antimicrobianos de cepas bacterianas presentes na cloaca de Trachemys scripta elegans (Wied, 1839) criadas em cativeiro em Petrolina, PE / Isolation and determination of antimicrobials sensitivity and resistance of bacterial strains present in the cloaca Trachemys scripta elegans (Wied, 1839) raised in captivity in Petrolina/PE, Brazil

Este estudo isolou e determinou o perfil de sensibilidade e de resistência a antimicrobianos de cepas bacterianas isoladas da cloaca de Trachemys scripta elegans (T. s. elegans) criadas em cativeiro. Após 120 dias de adaptação, amostras de swab cloacal obtidas de 20 animais adultos foram cultivadas e, após a identificação dos patógenos através de testes bioquímicos, submetidas ao teste de suscetibilidade a nove antimicrobianos. Enterobacter aerogenes (85%); Shigella spp. (10%) e Edwadsiella spp. (5%) foram isolados e identificados. Os isolados de E. aerogenes foram sensíveis à gentamicina (86%), enrofloxacina (79%), estreptomicina (50%), sulfazotrim (36%) e ampicilina (29%) e resistentes a penicilina (100%), eritromicina (93%), cefalexina (86%) ampicilina (71%) e sulfazotrim (64%). Isolados de Shigella spp. apresentaram sensibilidade à gentamicina (100%), enrofloxacina (50%), doxicilina (50%), estreptomicina (50%), ampicilina (50%), penicilina (50%) e sulfazotrim (50%) e resistência a doxicilina (50%), estreptomicina (50%), ampicilina (50%), penicilina (100%), cefalexina (50%) e sulfazotrim (50%), enquanto que os de Edwardsiella spp. foram sensíveis apenas à gentamicina (100%) e altamente resistentes (100%) aos demais antimicrobianos. Os resultados sugerem a participação de T. s. elegans na cadeia epidemiológica, como reservatório de patógenos importantes, como E. aerogenes, Shigella spp. e Edwardisiella spp., tornando importante a adoção de medidas preventivas pelo risco zoonótico que apresentam e corretas de tratamento e de controle em cativeiros e domicílios, assim como de estudos que enfoquem as características de sensibilidade e de resistência antimicrobiana dos isolados cloacais, pois a multirresistência a drogas pode ser transmitida aos humanos e comprometer o tratamento de indivíduos com doenças graves.(AU)

This study isolated and determined the profile of susceptibility and antimicrobials resistance of bacterial strains isolated from the cloaca Trachemys scripta elegans (T. s. elegans) raised in captivity. After 120 days of adaptation, cloacal swab samples obtained from 20 adults animals were grown and, after the pathogens identification through biochemical tests, submitted to the test of susceptibility to nine antimicrobials. Enterobacter aerogenes (85%); Shigella spp. (10%) and Edwadsiella spp. (5%) were isolated and identified. Isolates from E. aerogenes were sensitive to gentamicin (86%), enrofloxacin (79%), streptomycin (50%), sulfazotrim (36%) and ampicillin (29%) and resistant to penicillin (100%), erythromycin (93%), cephalexin (86%), ampicillin (71%) and sulfazotrim (64%). Isolates from Shigella spp. showed sensitivity to gentamicin (100%), enrofloxacin (50%), doxycycline (50%), streptomycin (50%), ampicillin (50%), penicillin (50%) and sulfazotrim (50%) and resistance to doxycycline (50 %), streptomycin (50%), ampicillin (50%), penicillin (100%), cephalexin (50%) and sulfazotrim (50%), while the Edwardsiella spp. were sensitive only to gentamicin (100%) and were highly resistant (100%) to other antibiotics. The results suggest the participation of T. s. elegans in the epidemiological chain, as reservoir of important pathogens, such as E. aerogenes, Shigella spp. and Edwardisiella spp., making it important to adopt preventive measures for zoonotic risk that present and correct treatment and control in captivity and households, as well as studies that address the sensitivity characteristics and antimicrobial resistance of isolates from cloaca, as it multidrug resistance to drugs can be transmitted to humans and compromise the treatment of patients with serious diseases.(AU)

Occurrence of heavy metals and antibiotic resistance in bacteria from internal organs of american bullfrog (Rana catesbeiana) raised in Malaysia

A total of 40 bacteria have been successfully isolated from internal organs of the American bullfrog (Rana catesbeiana) raised in Malaysia, namely, eight isolates of Aeromonas spp., 21 of Edwardsiella spp., six of Flavobacterium spp. and five of Vibrio spp. In terms of antibiotic susceptibility testing, each isolate was tested against 21 antibiotics, resulting in 482 (57.3 percent) cases of sensitivity and 61 (7.3 percent) cases of partial sensitivity. Meanwhile, 297 (35.4 percent) bacterial isolates were registered as resistant. The multiple antibiotic resistance (MAR) index of each bacterial species indicated that bacteria from raised bullfrogs have been exposed to tested antibiotics with results ranging from 0.27 to 0.39. Additionally, high percentages of heavy metal resistance among these isolates were observed, with values ranging from 85.0 to 100.0 percent. The current results provided us information on bacterial levels of locally farmed bullfrogs exposed to copper, cadmium, chromium as well as 21 types of antibiotics.(AU)