Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

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 fish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia fingerlings. One hundred and fourteen isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using specific primers described in the literature. For in vivo tests Nile tilapia fingerlings 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 fingerlings, regardless of the absence or quantity of virulence genes tested.

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Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652006000400002 ◽

2006 ◽

Vol 48 (4) ◽

pp. 185-188 ◽

Cited By ~ 15

Author(s):

Edilene Santo ◽

Claudia Macedo ◽

José Moacir Marin

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Virulence Genes ◽

Tertiary Care ◽

Uropathogenic Escherichia Coli ◽

University Hospital ◽

Adhesive Systems ◽

Specific Primers ◽

Polymerase Chain ◽

The Difference

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0%, 76.0%, 24.0%, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0%, 19.0% and 11.0% for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.

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Prevalence of virulence factors in Escherichia coli strains isolated from the genital tract of healthy cows

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/s0102-09352007000300003 ◽

2007 ◽

Vol 59 (3) ◽

pp. 564-568 ◽

Cited By ~ 2

Author(s):

D. Resende ◽

E. Santo ◽

C. Macedo ◽

J.M. Marin

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Genital Tract ◽

Virulence Genes ◽

Clinical Signs ◽

Chain Reaction ◽

Specific Primers ◽

E Coli ◽

Bacterial Pathogenicity ◽

Polymerase Chain

The prevalence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin, was determined in Escherichia coli isolates from healthy cow’s genital tract not showing clinical signs of infection. The presence of fimbriae expression genes (pap, sfa, afa) was assayed using specific primers in a polymerase chain reaction; none were detected in any of the isolates. Yet, a prevalence of 90.4%, 69.8%, and 28.5% of virulence factors for colicin, hemolysin and aerobactin respectively, was detected in the isolates. Analysis of the bacterial pathogenicity of isolates from the bovine genital tract may contribute towards the understanding of E. coli behavior.

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Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4469-4477.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4469-4477 ◽

Cited By ~ 66

Author(s):

H. B. Yu ◽

Y. L. Zhang ◽

Y. L. Lau ◽

F. Yao ◽

S. Vilches ◽

...

Keyword(s):

Virulence Factors ◽

Aeromonas Hydrophila ◽

Virulence Genes ◽

Physical Map ◽

Lethal Dose ◽

Gene Clusters ◽

Genomic Islands ◽

Infection Model ◽

Genomic Subtraction

ABSTRACT Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.

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Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1

Antibiotics ◽

10.3390/antibiotics10060631 ◽

2021 ◽

Vol 10 (6) ◽

pp. 631

Author(s):

Mengfan Peng ◽

Wentao Tong ◽

Zhen Zhao ◽

Ling Xiao ◽

Zhaoyue Wang ◽

...

Keyword(s):

Virulence Factors ◽

Bacillus Licheniformis ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Carassius Auratus ◽

Quorum Quenching ◽

Inhibition Rate ◽

Sequence Identity

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B.subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B.subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 ± 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A.hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4–24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 ± 4.14% at 96 h, which was significantly lower than the average mortality of 78 ± 2.57% of the Carassius auratus injected with 108 CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

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Identification and Characterization of Benomyl-Resistant and -Sensitive Populations of Colletotrichum gloeosporioides from Statice (Limonium spp.)

Phytopathology ◽

10.1094/phyto-96-0542 ◽

2006 ◽

Vol 96 (5) ◽

pp. 542-548 ◽

Cited By ~ 40

Author(s):

Marcel Maymon ◽

Aida Zveibil ◽

Shimon Pivonia ◽

Dror Minz ◽

Stanley Freeman

Keyword(s):

Colletotrichum Gloeosporioides ◽

Sequence Analyses ◽

Specific Primers ◽

Tubulin Genes ◽

Colletotrichum Spp ◽

Polymerase Chain ◽

Species Specific ◽

Identification And Characterization ◽

Propagation Material

Sixty-four isolates of Colletotrichum gloeosporioides were isolated from infected Limonium spp. cultivated in 12 different locations in Israel. All isolates were identified as belonging to the C. gloeosporioides complex by species-specific primers. Of these isolates, 46 were resistant to benomyl at 10 μg/ml and 18 were sensitive to this concentration of fungicide. Based on arbitrarily primed polymerase chain reaction of all isolates and internal transcribed spacer-1 sequence analyses of 12 selected isolates, the benomyl-resistant and -sensitive populations belong to two distinct genotypes. Sequence analyses of the β-tubulin genes, TUB1 and TUB2, of five sensitive and five resistant representative isolates of C. gloeosporioides from Limonium spp. revealed that the benomyl-resistant isolates had an alanine substitute instead of a glutamic acid at position 198 in TUB2. All data suggest that the resistant and sensitive genotypes are two independent and separate populations. Because all Limonium plant propagation material is imported from various geographic regions worldwide, and benomyl is not applied to this crop or for the control of Colletotrichum spp. in Israel, it is presumed that plants are bearing quiescent infections from the points of origin prior to arrival.

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Virulence-Associated Genes and Antimicrobial Resistance of Aeromonas hydrophila Isolates from Animal, Food, and Human Sources in Brazil

BioMed Research International ◽

10.1155/2020/1052607 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Emily Moraes Roges ◽

Verônica Dias Gonçalves ◽

Maira Duarte Cardoso ◽

Marcia Lima Festivo ◽

Salvatore Siciliano ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Aeromonas Hydrophila ◽

Pathogenic Bacteria ◽

Virulence Genes ◽

Animal Health ◽

Diffusion Method ◽

Aquatic Environments ◽

Disk Diffusion Method ◽

Polymerase Chain ◽

Human Source

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.

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Ascophyllum nodosum in the diet of tilapia (Oreochromis niloticus) and its effect after inoculation of Aeromonas hydrophila

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2014000500003 ◽

2014 ◽

Vol 34 (5) ◽

pp. 403-408 ◽

Cited By ~ 2

Author(s):

Samira T.L. Oliveira ◽

Gisele Veneroni-Gouveia ◽

Augusto C. Santos ◽

Silia M.N. Sousa ◽

Marcelo L. Veiga ◽

...

Keyword(s):

Aeromonas Hydrophila ◽

Nile Tilapia ◽

Saline Solution ◽

Control Diet ◽

Brown Seaweed ◽

Ascophyllum Nodosum ◽

Control Group ◽

Performance Parameters ◽

Sterile Saline ◽

Bacterial Inoculum

The objective of this study was to evaluate the effect of Ascophyllum nodosum brown seaweed meal (FAM) on the health of Nile tilapia submitted to inoculation with Aeromonas hydrophila. The experiment was conducted for a period of 40 days using 120 Nile tilapia fingerlings, with age of 40 days, distributed in 20 tanks. A diet including Ascophyllum nodosum seaweed meal at 20g.kg-1 and a control diet (without FAM) were provided which constituted the treatments. Thirty days after beginning the experiment, A. hydrophila was inoculated by bacterial inoculum diluted in sterile saline solution at a concentration of 10(6) CFU ml-1. Except for the width, which was greater for the treatment with the provision of FAM (P<0.05), there was no influence on the performance parameters of the fingerlings, but the occurrence of lesions in animals inoculated with A. hydrophila and fed with FAM was lower and they also exhibited a decline in the lesions in a shorter period of time than the control group. FAM prevents hepatopancreatic congestion in infected animals. Ascophyllum nodosum brown seaweed meal reduced the number of lesions in fish in a shorter time when compared to the control group.

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Kinetic characterization of high-activity mutants of human butyrylcholinesterase for the cocaine metabolite norcocaine

Biochemical Journal ◽

10.1042/bj20131100 ◽

2013 ◽

Vol 457 (1) ◽

pp. 197-206 ◽

Cited By ~ 26

Author(s):

Max Zhan ◽

Shurong Hou ◽

Chang-Guo Zhan ◽

Fang Zheng

Keyword(s):

High Activity ◽

Kinetic Modelling ◽

Kinetic Characterization ◽

In Vivo Tests ◽

Human Butyrylcholinesterase

Catalytic parameters of butyrylcholinesterase and its mutants against norcocaine have been characterized in comparison with those against cocaine, indicating that the mutants can efficiently metabolize norcocaine, in addition to cocaine. Further in vivo tests and kinetic modelling support the indication.

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a Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Blood ◽

10.1182/blood.v124.21.2983.2983 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2983-2983

Author(s):

Silvia Gimondi ◽

Alessandra Cavanè ◽

Antonio Vendramin ◽

Giulia Biancon ◽

Paolo Corradini ◽

...

Keyword(s):

B Cell ◽

Multiplex Pcr ◽

Residual Disease ◽

Chain Reaction ◽

B Cell Malignancies ◽

Specific Primers ◽

Namalwa Cell ◽

Polymerase Chain ◽

Minimal Residual

Abstract Background: Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM). Methods: To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure. Results: The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time. Conclusions: IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

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