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Genome Announcements Jun 2018We report here the whole-genome sequencing results of two bacterial isolates, IMET J and IMET F, that inhibit (possibly due to denitrifying gene clusters) and promote...
Draft Genome Sequences of Cloacibacterium normanense IMET F, a Microalgal Growth-Promoting Bacterium, and Aeromonas jandaei IMET J, a Microalgal Growth-Inhibiting Bacterium.
We report here the whole-genome sequencing results of two bacterial isolates, IMET J and IMET F, that inhibit (possibly due to denitrifying gene clusters) and promote (possibly due to an ammonification system), respectively, the growth of the microalgal strains HTB1 and 1807.
PubMed: 29903817
DOI: 10.1128/genomeA.00503-18 -
Water Research Sep 2019The wide application of chlorine disinfectant for drinking water treatment has led to the appearance of chlorine-resistant bacteria, which pose a severe threat to public...
The wide application of chlorine disinfectant for drinking water treatment has led to the appearance of chlorine-resistant bacteria, which pose a severe threat to public health. This study was performed to explore the physiological-biochemical characteristics and environmental influence (pH, temperature, and turbidity) of seven strains of chlorine-resistant bacteria isolated from drinking water. Ozone disinfection was used to investigate the inactivation effect of bacteria and spores. The DNA concentration and cell surface structure variations of typical chlorine-resistant spores (Bacillus cereus spores) were also analysed by real-time qPCR, flow cytometry, and scanning electron microscopy to determine their inactivation mechanisms. The ozone resistance of bacteria (Aeromonas jandaei < Vogesella perlucida < Pelomonas < Bacillus cereus < Aeromonas sobria) was lower than that of spores (Bacillus alvei < Lysinibacillus fusiformis < Bacillus cereus) at an ozone concentration of 1.5 mg/L. More than 99.9% of Bacillus cereus spores were inactivated by increasing ozone concentration and treatment duration. Moreover, the DNA content of Bacillus cereus spores decreased sharply, but approximately 1/4 of the target genes remained. The spore structure exhibited shrinkage and folding after ozone treatment. Both cell structures and gene fragments were damaged by ozone disinfection. These results showed that ozone disinfection is a promising method for inactivating chlorine-resistant bacteria and spores in drinking water.
Topics: Chlorine; Disinfectants; Disinfection; Drinking Water; Ozone; Spores, Bacterial; Water Purification
PubMed: 31158616
DOI: 10.1016/j.watres.2019.05.014 -
Journal of Applied Microbiology Oct 2021This study aimed to evaluate the in vitro cytotoxicity and efficacy of synthetic host defence peptides (HDPs), alone or in combination with florfenicol (FFC),...
AIMS
This study aimed to evaluate the in vitro cytotoxicity and efficacy of synthetic host defence peptides (HDPs), alone or in combination with florfenicol (FFC), oxytetracycline (OTC) or thiamphenicol (TAP), against different pathogenic bacteria isolated from diseased fish.
METHODS AND RESULTS
Solid-phase synthesis, purification and characterization of several HDPs were performed manually, using the fluorenylmethyloxycarbonyl protecting group in different resins and via high-performance liquid chromatography-mass spectrometry, respectively. The in vitro cytotoxicity and antimicrobial activity of HDPs, FFC, OTC and TAP against Nile tilapia red blood cells (RBCs) and relevant fish pathogenic bacteria (Aeromonas, Citrobacter, Edwardsiella, Streptococcus, Lactococcus and Vibrio) was determined using the haemolysis assay and broth microdilution method, respectively. The checkerboard assay was used to evaluate the synergy between the most active HDPs and other antimicrobials against the tested strains. MUC 7 12-mer, FFC, OTC and TAP were not cytotoxic to Nile tilapia RBCs, in all tested concentrations. LL-37, (p-BthTX-I) and Hylin-a1 were not cytotoxic at concentrations up to 78·13, 19·53 and 9·77 μg ml , respectively. HDPs demonstrated potent antimicrobial activity (minimum inhibitory concentration ≤31·25 µg ml ) against Aeromonas jandaei (KR-12-a5), Citrobacter freundii (Kr-12-a5; (p-BthTX-I) ; LL-37; and Hylin a1), Streptococcus agalactiae (Hylin a1; (p-BthTX-I) and LL-37), Lactococcus garviae (Hylin a1), and Vibrio fluvialis (KR-12-a5). The combinations of (p-BthTX-I) with TAP and LL-37 with FFC showed synergistic activity against C. freundii (fractional inhibitory concentration index of 0·25 and 0·50, respectively).
CONCLUSIONS
Synthetic HDPs have the potential as a good treatment option for bacterial diseases in aquaculture.
SIGNIFICANCE AND IMPACT OF THE STUDY
The in vivo effectiveness of synthetic HDPs such as KR-12-a5; LL-37; (p-BthTX-I) and Hylin a1 can be tested alone or in combination with conventional antimicrobials as a treatment option to reduce the use of antimicrobials in aquaculture.
Topics: Aeromonas; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cichlids; Fish Diseases; Lactococcus; Microbial Sensitivity Tests; Vibrio
PubMed: 33742508
DOI: 10.1111/jam.15080 -
Microbial Drug Resistance (Larchmont,... Jul 2020Integrons are prokaryotic genetic elements known to carry and exchange antibiotic resistance gene cassettes through a site-specific recombinase called integrase. In this...
Integrons are prokaryotic genetic elements known to carry and exchange antibiotic resistance gene cassettes through a site-specific recombinase called integrase. In this work, 107 isolates from environmental origin, including fish, water, and sediments, were investigated for the presence of integrons. Using specific primers for Class 1, 2 and 3 integrases, only Class 1 and Class 2 integrons were detected. Detection of Class 2 integrases and their associated variable regions required two rounds of polymerase chain reaction (PCR). Sequencing of the 2 amplicons confirmed them as integrase-derived products. Class 1 integrons were detected in 26 out of 107 isolates. PCR amplification of the variable regions associated to these integrons revealed an outstanding homogeneity, 25 of them having variable regions with an identical A12-F-A2 cassette array and one integron carrying only the A16 cassette. To assess clone diversity, chromosomal DNA from isolates was subjected to enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), which discarded clonality in all instances. Class 2 integrons were surprisingly more prevalent than Class1 integrons, being detected in 60 out of 107 isolates. Forty-six of them showed a unique ERIC profile, while the remaining 14 strains displayed profiles that could be grouped in five different patterns. Cassette arrangements of all Class 2 variable regions were those described as the most prevalent (A1-2-A1). A rather startling result of this work is the sensitivity to trimethoprim, streptomycin, and streptothricin of most strains, despite the presence of the cognate resistance genes. To know the integron distribution in environmental species, a phylogenetic reconstruction was done using D/B or D/A gene sequences. Isolates bearing these elements corresponded to , , , , , , , , , and sp. This work revealed an unusual high incidence of Class 2 integrons and a low variability of cassette arrangements in environmental species.
Topics: Aeromonas; Anti-Bacterial Agents; DNA, Bacterial; Drug Resistance, Multiple, Bacterial; Integrases; Integrons; Microbial Sensitivity Tests; Polymerase Chain Reaction
PubMed: 31990611
DOI: 10.1089/mdr.2019.0250 -
Food Microbiology May 2015We aimed to document the risk of Aeromonas spp. in marine shrimp species cultured in inland low salinity ponds in Thailand. In 14 of 18 shrimp samples retrieved from...
We aimed to document the risk of Aeromonas spp. in marine shrimp species cultured in inland low salinity ponds in Thailand. In 14 of 18 shrimp samples retrieved from inland grow-up ponds, Aeromonas spp. were detected at ranges from 4667 to 1,500,000 CFU/g body weight. The phylogenetic tree constructed with the gyrB and cpn60 concatenated sequences indicated that the 87 isolates consisted of Aeromonas veronii (70%), Aeromonas aquariorum (18%), Aeromonas caviae (7%), Aeromonas jandaei (2%), and Aeromonas schubertii (2%). The potential virulence of the isolates was examined by phenotypic and PCR assays. Hemolytic activity and the extracellular activity of lipase, DNase, and gelatinase were observed in most isolates (94-99%). PCR revealed the presence of 9 genes related to virulence in the 87 isolates: act (75%), aer (74%), alt (30%), ast (1%), ascV (34%), aexT (24%), fla (92%), ela (34%), and lip (24%). The susceptibility profiles to 14 antimicrobial agents of isolates were typical for the genus, but resistance to cefotaxime, a third-generation cephalosporin, and imipenem were found in two A. aquariorum and in three A. veronii isolates, respectively. These resistances were confirmed by determining minimum inhibitory concentrations. Our results indicate that the microbiological risk posed by Aeromonas should be considered for marine shrimp species that are cultured in low-salinity ponds. These shrimps may also be a vehicle for the transfer of different genotypes of Aeromonas and antibiotic-resistant determinants to regions worldwide through trade.
Topics: Aeromonas; Animals; Anti-Bacterial Agents; Cefotaxime; Deoxyribonucleases; Drug Resistance, Microbial; Gelatinases; Hemolysis; Imipenem; Microbial Sensitivity Tests; Penaeidae; Penicillin Amidase; Phenotype; Phylogeny; Polymerase Chain Reaction; Salinity; Shellfish; Thailand; Virulence
PubMed: 25583334
DOI: 10.1016/j.fm.2014.11.003 -
FEBS Open Bio Jun 2018Understanding the role of specific amino acid residues in the molecular mechanism of a protein's function is one of the most challenging problems in modern biology. A...
Understanding the role of specific amino acid residues in the molecular mechanism of a protein's function is one of the most challenging problems in modern biology. A systematic bioinformatic analysis of protein families and superfamilies can help in the study of structure-function relationships and in the design of improved variants of enzymes/proteins, but represents a methodological challenge. The pyridoxal-5'-phosphate (PLP)-dependent enzymes are catalytically diverse and include the aspartate aminotransferase superfamily which implements a common structural framework known as type fold I. In this work, the recently developed bioinformatic online methods Mustguseal and Zebra were used to collect and study a large representative set of the aspartate aminotransferase superfamily with high structural, but low sequence similarity to l-threonine aldolase from (LTAaj), in order to identify conserved positions that provide general properties in the superfamily, and to reveal family-specific positions (FSPs) responsible for functional diversity. The roles of the identified residues in the catalytic mechanism and reaction specificity of LTAaj were then studied by experimental site-directed mutagenesis and molecular modelling. It was shown that FSPs determine reaction specificity by coordinating the PLP cofactor in the enzyme's active centre, thus influencing its activation and the tautomeric equilibrium of the intermediates, which can be used as hotspots to modulate the protein's functional properties. Mutagenesis at the selected FSPs in LTAaj led to a reduction in a native catalytic activity and increased the rate of promiscuous reactions. The results provide insight into the structural basis of catalytic promiscuity of the PLP-dependent enzymes and demonstrate the potential of bioinformatic analysis in studying structure-function relationship in protein superfamilies.
PubMed: 29928580
DOI: 10.1002/2211-5463.12441 -
Frontiers in Bioengineering and... 2019Most of biochemical and mutagenesis studies performed with L-threonine aldolases were done with respect to natural activity, the cleavage of L-threonine and sometimes...
Most of biochemical and mutagenesis studies performed with L-threonine aldolases were done with respect to natural activity, the cleavage of L-threonine and sometimes L-β-phenylserine. However, the properties of variants and the impact of mutations on the product synthesis are more interesting from an applications point of view. Here we performed site-directed mutagenesis of active site residues of L-threonine aldolase from to analyze their impact on the retro-aldol activity and on the aldol synthesis of L-β-phenylserine and L-α-alkyl-β-phenylserines. Consequently, reduced retro-aldol activity upon mutation of catalytically important residues led to increased conversions and diastereoselectivities in the synthetic direction. Thus, L-β-phenylserine can be produced with conversions up to 60% and .'s up to 80% () under kinetic control. Furthermorem, the donor specificity of L-threonine aldolase was increased upon mutation of active site residues, which enlarged the pocket size for an efficient binding and stabilization of donor molecules in the active site. This study broadens the knowledge about L-threonine aldolase catalyzed reactions and improves the synthetic protocols for the biocatalytic asymmetric synthesis of unnatural amino acids.
PubMed: 31192202
DOI: 10.3389/fbioe.2019.00119 -
The Journal of Antimicrobial... May 2018To investigate Aeromonas spp. isolates for the presence of the novel resistance gene mcr-3 or variants thereof and to characterize the positive isolates by whole genome...
Identification of novel variants of the colistin resistance gene mcr-3 in Aeromonas spp. from the national resistance monitoring programme GERM-Vet and from diagnostic submissions.
OBJECTIVES
To investigate Aeromonas spp. isolates for the presence of the novel resistance gene mcr-3 or variants thereof and to characterize the positive isolates by whole genome sequence analysis.
METHODS
A total of 479 unrelated Aeromonas isolates were investigated by PCR for the genes mcr-1, mcr-2 and mcr-3. Positive isolates were investigated for their colistin MICs. Species assignment was based on sequence analysis of 16s rRNA and gyrB and rpoB genes. The mcr-carrying contigs obtained by WGS were analysed for the genetic environments of the mcr genes.
RESULTS
Four (0.84%) Aeromonas isolates were positive in the mcr-3-specific PCR assay, whereas none of the isolates harboured mcr-1 or mcr-2. Each of the four mcr-3 genes encoded a novel variant, which showed amino acid identities of 95.0%-98.0% to the original Mcr-3 protein. These variants were designated Mcr-3.6 [Aeromonas allosaccharophila from golden orfe (Leuciscus idus)], Mcr-3.7 [Aeromonas media from turkey (Meleagris gallopavo)], Mcr-3.8 [Aeromonas jandaei from koi carp (Cyprinus carpio)] and Mcr-3.9 [Aeromonas hydrophila from koi carp]. The isolate harbouring the mcr-3.9 gene carried an additional mcr-3.8 gene and showed a distinctly higher colistin MIC of ≥128 mg/L than all other isolates. The genetic environments of the mcr-3 variant genes in all four isolates differed, but in part resembled the flanking regions of mcr-3.3 from Aeromonas veronii of chicken meat.
CONCLUSIONS
This study identified four novel Mcr-3 variants. The isolates carrying the respective genes dated back to 2005 suggesting that this gene has existed for more than 12 years.
Topics: Aeromonas; Animals; Anti-Bacterial Agents; Bacterial Proteins; Cluster Analysis; Colistin; DNA, Bacterial; DNA, Ribosomal; Drug Resistance, Bacterial; Fish Diseases; Fishes; Gram-Negative Bacterial Infections; Microbial Sensitivity Tests; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sequence Homology; Turkeys; Whole Genome Sequencing
PubMed: 29394397
DOI: 10.1093/jac/dkx538 -
Applied Microbiology and Biotechnology Nov 2019Streptococcus agalactiae is a major pathogen causing streptococcosis. To prevent and control this bacterial disease, antagonistic bacteria have become a new research...
Bacillus velezensis LF01: in vitro antimicrobial activity against fish pathogens, growth performance enhancement, and disease resistance against streptococcosis in Nile tilapia (Oreochromis niloticus).
Streptococcus agalactiae is a major pathogen causing streptococcosis. To prevent and control this bacterial disease, antagonistic bacteria have become a new research hotspot. This study evaluated the probiotic potential of Bacillus velezensis LF01 strain, which is antagonistic to S. agalactiae. The active compounds produced by LF01 showed antimicrobial activity against a broad spectrum of fish pathogens, including S. agalactiae, Streptococcus iniae, Aeromonas hydrophila, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei, and Vibrio harveyi. The antimicrobial compounds were heat stable, pH stable, UV stable, resistant to proteases, and could be stored for a long time. To evaluate the probiotic function of LF01 in Nile tilapia, juveniles were divided into three treatment groups: a control group, an interval feeding group, and a continuous feeding group. Tilapia fed with LF01-supplemented diets (1.0 × 10 CFU/g) showed significantly better growth performances than those of the control group (P < 0.05). Tilapia fed with LF01-supplemented diets significantly increased lysozyme (LZY) and superoxide dismutase (SOD) activities. The expression of three immune-related genes (C3, lyzc, and MHC-IIβ) was higher in the intestine, head kidney, and gill of tilapia from the continuous feeding group than in those from the control group (P < 0.05). Tilapia fed with LF01-supplemented diets showed remarkably improved survival rates after S. agalactiae infection, and analysis of their intestinal tract pathogens revealed that the abundance of Edwardsiella and Plesiomonas had significantly decreased compared with the control group. Our findings demonstrate that LF01 is an effective antagonist against various fish pathogens and has potential for controlling infections by Streptococcus spp. and other pathogens in tilapia.
Topics: Animals; Antibiosis; Bacillus; Biological Control Agents; Cichlids; Fish Diseases; High-Throughput Nucleotide Sequencing; Probiotics; Streptococcal Infections; Streptococcus agalactiae
PubMed: 31654082
DOI: 10.1007/s00253-019-10176-8