Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA

Ghatak, Sandeep; Agarwal, R. K.; Bhilegaonkar, K.N.

doi:10.1111/j.1472-765x.2006.02104.x

Cited by 32 publications

(24 citation statements)

References 17 publications

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“…Aeromonas hydrophila isolates were identiϐied in advance by morphological, tinctorial and biochemical characteristics (Quinn et al 1994) and by PCR for conϐirmation of genus and species, using the methods described by Ghatak et al (2007). The cultures were subcultured on Tryptone Soy Agar (TSA), and then used for molecular characterization.…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

2012

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Pesq. Vet. Bras. 32(8) Multiple factors can be involved in the virulence processes of Aeromonas hydrophila. The objective of the present paper was to verify the presence of aerolysin, hidrolipase, elastase and lipase virulence genes through the polymerase chain reaction (PCR) in A. hydrophila isolates obtained from ϐish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia ϐingerlings. One hundred and fourteen isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using speciϐic primers described in the literature. For in vivo tests Nile tilapia ϐingerlings were used. From the PCR tests, negative isolates for all genes tested were selected, positive isolates for two genes (aerolysin and elastase) and positive for the four genes tested. These were inoculated at a concentration of 10 8 UFC/ml into the tilapias, considered as treatments; another group of animals was used as control (with inoculation of saline solution). In all, 12 distinct standards regarding the presence of virulence factors in isolates from A. hydrophila, were observed. Of the 114 isolates analyzed, 100 (87.72%) presented at least one of the virulence factors under study. The virulence factors were widely distributed among the A. hydrophila isolates. Aerolysin was the most frequent virulence factor present in the isolates analyzed. A. hydrophila led to the mortality of the Nile tilapia ϐingerlings, regardless of the absence or quantity of virulence genes tested.INDEX TERMS: Aeromonas hydrophila, virulence factors, aerolysin, elastase, hidrolipase, lipase, Oreochromis niloticus.

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Section: Introductionmentioning

confidence: 99%

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

2012

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“…However, these are not always conclusive, since some Aeromonas species display heterogeneous biochemical properties; compared to molecular methods, the correct identification rate with biochemical tests has been shown to be very low (4,5). Molecular species identification has been exploited by the 16S rRNA gene, either by restriction fragment length polymorphism (RFLP) (6)(7)(8)(9) or direct sequencing (10,11). However, due to insufficient interspecies sequence variation and heterogeneity among copies of ribosomal RNA operons in the same bacteria (10,12,13), this gene may not be an optimal target.…”

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Identification of Clinical Aeromonas Species by rpoB and gyrB Sequencing and Development of a Multiplex PCR Method for Detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media

et al. 2015

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Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.A eromonas spp. are highly adapted to aquatic environments and have been described as pathogenic to humans and animals. The genus Aeromonas consists of more than 20 valid species, of which A. hydrophila, A. caviae (synonymous with A. punctata), A. media, A. veronii bv. sobria, and A. veronii bv. veronii are of particular clinical significance, because they can cause gastroenteritis, wound and soft tissue infections, and septicemia (1). Aeromonas spp. may produce an array of virulence factors (e.g., cytolytic toxins with hemolytic activity and enterotoxins). Recent reviews suggested that only a subset of Aeromonas spp. are truly pathogenic and may be transmitted by hitherto unknown routes, and they proposed that further epidemiological and molecular studies are needed (2, 3). Aeromonas species identification has traditionally been performed by a combination of different biochemical tests. However, these are not always conclusive, since some Aeromonas species display heterogeneous biochemical properties; compared to molecular methods, the correct identification rate with biochemical tests has been shown to be very low (4, 5). Molecular species identification has been exploited by the 16S rRNA gene, either by restriction fragment length polymorphism (RFLP) (6-9) or direct sequencing (10, 11). However, due to insufficient interspecies sequence variation and heterogeneity among copies of ribosomal RNA operons in the same bacteria (10, 12, 13), this gene may not be an optimal target. A number of studies have shown that the sequences of several different housekeeping genes are able to differentiate this tight taxonomic group of organisms. These genes include an RNA polymerase B subunit (rpoB), an RNA polymerase D subunit (rpoD), and a DNA gyrase B subunit (gyrB) (10, 14-16). The objective of the present study was to identify species of Aeromonas by partial gyrB and rpoB sequencing in order to develop a multiplex PCR (mPCR) that targets the four most prevalent and clinically relevant species identified by sequence analysis on a Danish strain collection.The strain collection used in this study was composed of 51 Aeromonas spp. collected from diarrheagenic patients during the period of 2005 to 2010. Each stool specimen was grown on enteric medium (Statens Serum Institut, Hillerød, Denmark) (17), and Aeromonas was identified by its distinct colony morphology, while further delineation of clinically relevant species was performed manually according to their biochemical characteristics, including Voges-Proskauer test results and lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, glucose (gas), esculin, and acid production fr...

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“…A proteína codificada por esse gene pertence à família de sistemas de efluxo da Superfamília dos Facilitadores Majoritários (MFS) (Prosite 2016). Os sistemas de efluxo em bactérias protegem as células dos antibióticos por transportar ativamente os compostos para fora do citoplasma e/ou periplasma e limitar assim o seu acúmulo em seu local de ação (Lynch 2006 (Quinn et al 1994), e identificados através de restrição enzimática (Ghatak et al 2007).…”

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Gene floR e a resistência ao florfenicol em isolados de Aeromonas spp. autóctones de organismos aquáticos

et al. 2018

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ABSTRACT.-Silva N.C.S.L., Nogueira J.F., Gouveia J.J.S., Costa M. The floR gene is described in related literature as responsible for resistance to florfenicol, which is a widely used antimicrobial agent in aquaculture. This gene has been reported in many species of bacteria, including the genus Aeromonas. These bacteria cause high mortality in fish farming bringing economic losses. It is important that studies of this gene and possible mutations that can lead to changes in the structure and function of the protein. The aim of this study was to characterize the floR gene in isolates of Aeromonas spp. and check if the presence of this gene is associated with resistance to florfenicol in Aeromonas spp. obtained from the San Francisco Valley. PCR (Polymerase Chain Reaction) were also performed to verify the presence of the floR gene in 27 isolates of Aeromonas spp. Positive samples for the presence of the gene were sequenced and analyzed for the presence of polymorphisms using alignments. Different haplotypes detected were used for analysis with the SIFT and PolyPhen programs for prediction of changes in protein function. The structural modeling of protein encoded by the floR gene was performed using the Modeller software, and the models were evaluated by Procheck, Verify3D and Whatif. The similarity of the dimensional structure of reference protein with the dimensional structures of the proteins encoded by the different haplotypes was compared by TM-align. Bacterial resistance to florfenicol was assessed by the microdilution test, which was also performed in the presence of carbonyl cyanide m-chlorophenyl hydrazone to verify the effect of inhibiting the efflux pump. 14 isolates were positive for the presence of floR gene and 10 were sequenced and allowed the identification of three polymorphisms in the floR gene, which led to construction of three different haplotypes (TAA TTA and CTG). The analyzes carried out with the SIFT and PolyPhen programs showed that the TTA and TAA haplotypes could probably change the protein structure-function. Proteins modeled for the three haplotypes were found to have substantially the same structural conformation with each other. All isolates presenting the gene were resistant to florfenicol and those who did not have were sensitive. The test in the presence of carbonyl cyanide m-chlorophenylhydrazone was conducted for three isolates, representing each single haplotype and was observed inhibition of bacterial growth at all concentrations independent of the haplotype. The results of this study show that resistance to flofenicol in Aeromonas spp. may be explained by the presence of floR gene and that this gene is associated with an efflux pump. Mutations observed in floR gene do not appear to be involved with chenges in structure and function of the protein encoded by gene.

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Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA

Cited by 32 publications

References 17 publications

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

Identification of Clinical Aeromonas Species by rpoB and gyrB Sequencing and Development of a Multiplex PCR Method for Detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media

Gene floR e a resistência ao florfenicol em isolados de Aeromonas spp. autóctones de organismos aquáticos

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