Neem (Azadirachta indica) extract as an antibacterial agent against fish pathogenic bacteria

Aquaneem, an emulsified product prepared from the neem (A. indica) kernel was tested against four pathogenic bacteria of fish (i.e. Aeromonas hydrophila, Pseudomonas fluorescens, Escherichia coli and Myxobacteria spp.) to test its efficacy as an antibacterial agent. Growth inhibitory property of the product at 10, 15 and 20 ppm has been noticed and recorded. The percentage reduction of bacterial cell population was noted to be maximum on 9th day at 20 ppm concentration (i.e. 70.14%, 74.15% and 61.75% for A. hydrophila, P. fluorescens and E. coli respectively) with the only exception of myxobacteria which showed maximum reduction percentage (63.90%) on 15th day. Among all the bacteria tested A. hydrophila, P. fluorescens and Myxobacteria spp. exhibited maximum sensitivity to Aquaneem in terms of percentage reduction of bacterial cell population in comparison to E. coli.     

Secretion of an Aeromonas hydrophila aerolysin by a mutant strain of Escherichia coli

Abstract Expression of the Aeromonas hydrophila AH2 aerolysin was analysed in 2 mutant derivatives of Escherichia coli 5K that overproduce E. coli haemolysin, encoded by the multicopy plasmid pANN202–312. When plasmid pHPC3–700 carrying the A. hydrophila aerolysin genes was transformed into one of the mutants, Hha-2T, the transformants produced external aerolysin. Neither the parental 5K strain or the other mutant, Hha-1T, showed extracellular aerolysin activity. For strain Hha-2T, the kinetics of external aerolysin production was similar to that previously reported for A. hydrophila AH2. No cell lysis or release of other proteins to the culture medium could be detected for the period of time that strain Hha-2T exported aerolysin into the external medium.     

Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.     

Inhibition of frog antimicrobial peptides by extracellular products of the bacterial pathogen Aeromonas hydrophila

Aims:  To determine whether the extracellular products (ECPs) from Aeromonas hydrophila , a frog bacterial pathogen that is resistant to skin antimicrobial peptides of three different frog species Xenopus laevis , Litoria aurea and Litoria raniformis , can modulate the activity of these peptides.
Methods and Results:  ECPs were collected from cultures of Klebsiella pneumoniae , a pathogen susceptible to skin antimicrobial peptides of all three tested frog species, and from cultures of Aer .  hydrophila . They were tested for protease activity and for inhibition of the antimicrobial activity of natural peptide mixtures and single peptides of all three frog species against Escherichia coli ATCC 25922. ECPs from cultures of Aer .  hydrophila grown for 16, 24 and 36 h showed protease activity and inhibited the antibacterial activity of all peptides against E .  coli ATCC 25922. In contrast, the ECPs from cultures of Kl .  pneumoniae neither had protease activity nor inhibited the activity of any peptides.
Conclusion:  The proteolytic ECPs of Aer .  hydrophila have the ability to inhibit the skin antimicrobial peptides of frogs.
Significance and Impact of the Study:  The results of this study provide new information on the association of ECPs with the resistance of Aer .  hydrophila to frog antimicrobial peptides.     

The cell division genes (ftsE and X) of Aeromonas hydrophila and their relationship with opsonophagocytosis

A transposon mutant from Aeromonas hydrophila AH-3 was obtained which was highly resistant to opsonophagocytosis. The mutation was identified in the ftsE gene and we characterised the operon ftsY, E and X from this bacterium. These genes, as in enteric bacteria, are neighbours to rpoH. The A. hydrophilia ftsE and X genes were fully able to complement Escherichia coli ftsE mutants, and also complement the opsonophagocytosis-resistant phenotype of the A. hydrophila mutant strain. This phenotype seems to be related to the filamentous phenotype at 37 degrees C exhibited by the A. hydrophila ftsE mutant.     

Cloning, expression, and mapping of the Aeromonas hydrophila aerolysin gene determinant in Escherichia coli K-12.

DNA sequences corresponding to the aerolysin gene (aer) of Aeromonas hydrophila AH2 DNA were identified by screening a cosmid gene library for hemolytic and cytotoxic activities. A plasmid containing a 5.8-kilobase EcoRI fragment of A. hydrophila DNA was required for full expression of the hemolytic and cytotoxic phenotype in Escherichia coli K-12. Deletion analysis and transposon mutagenesis allowed us to localize the gene product to 1.4 kilobases of Aeromonas DNA and define flanking DNA regions affecting aerolysin production. The reduced hemolytic activity with plasmids lacking these flanking regions is associated with a temporal delay in the appearance of hemolytic activity and is not a result of a loss of transport functions. The aerolysin gene product was detected as a 54,000-dalton protein in E. coli maxicells harboring aer plasmids and by immunoblotting E. coli whole cells carrying aer plasmids. We suggest that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively.     

Occurrence of conjugated R-plasmids in bacteria of the Aeromonas genus isolated from purified urban sewage water]

The aim of the study was to determine a participation of Aeromonas sp., having conjugation R plasmids, in a population of the bacteria present in purified urban sewage . In 1 ml of sewage 1.8 x 10(3) - 50 x 10(3) of Aeromonas sp., were found. The isolated strains belonged to the following genera: A. hydrophila, A. caviae, A. sorbia. All tested strains were sensitive to gentamicin, tetracycline, kanamycin and nalidixic acid. Among 186 strains of Aeromonas sp., two belonging to A. caviae genus transferred resistance to streptomycin to A. hydrophila and E. coli recipients on the other hand two strains of A. caviae transferred resistance to streptomycin exclusively to A. hydrophila recipient.     

Cloning, characterisation and expression of Aeromonas hydrophila major adhesin

Aeromonas hydrophila, an important pathogen in fish, is believed to cause diseases by adhesive and enterotoxic mechanisms. The adhesion is a prerequisite for successful invasion. In this study, the gene of a 43 kDa major adhesin (designated as AHA1) was cloned and expressed. Nucleotide sequence analysis of AHA1 revealed an open reading frame encoding a polypeptide of 373 amino acids with a 20-amino-acid putative signal peptide (molecular weight 40,737 Da). The amino acid sequences of Aha1p showed a very high homology with the other two outer membrane proteins of A. hydrophila. Using the T-5 expression system, this major adhesin Aha1p was expressed in Escherichia coli. The purified recombinant adhesin could competitively inhibit A. hydrophila from invading fish epithelial cells in vitro. Western-blot analysis showed that this major adhesin is a very conserved antigen among various strains of Aeromonas. When used to immunise blue gourami, the recombinant adhesin could confer significant protection to fish against experimental A. hydrophila challenge.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

Evaluation of the antimicrobial activity of methylglyoxal bis(guanylhydrazone) analogues, the inhibitors for polyamine biosynthetic pathway

Metabolic and antiproliferative effects of methylglyoxal bis(butylamidinohydrazone) (MGBB) and methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP), inhibitors for polyamine biosynthetic pathway, on Escherichia coli, Shigella sonnei, Aeromonas sobria, Aeromonas hydrophila and Vibrio cholerae were investigated. MGBB at the concentration of 100 mumol/l depleted intracellular putrescine and spermidine concentrations of E. coli to 25 and 20% of the controls, respectively, while MGBCP depressed their concentrations to 38 and 24%, respectively. In these polyamine-depleted E. coli cells the syntheses of RNA, DNA and protein decreased to 13, 54 and 29% of the control, respectively, with MGBB and to 23, 71 and 55%, respectively, with MGBCP. The minimum inhibitory concentrations (MIC) of MGBB for the growth of A. sobria, E. coli, A. hydrophila, V. cholerae and Sh. sonnei were estimated to be 50, 160, 240, 285 and 320 mumol/l, respectively, whereas those of MGBCP were slightly higher for respective bacteria.     

Molecular cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli

The polyhydroxyalkanoate synthesis operon was cloned from Aeromonas hydrophila CGMCC 0911. Heterogeneous expression of the cloned polyhydroxyalkanoate synthesis operon in Escherichia coli resulted in accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) consisting of 13.9 mol % 3-hydroxyhexanoate up to 29.2 wt % of cell dry weight when grown in lauric acid. The cell dry weight of recombinant E. coli harboring the polyhydroxyalkanoate synthesis operon was improved to 1.7 g L (-1), which was much higher than that of 0.3 g L (-1) of the wild type E. coli. Coexpression of acyl-CoA dehydrogenase gene (yafH) from E. coli and Vitreoscilla hemoglobin gene (vgb) from Vitreoscilla together with the whole A. hydrophila CGMCC 0911 polyhydroxyalkanoate synthesis operon facilitated cell growth and polyhydroxyalkanoate accumulation in E. coli. When yafH was coexpressed together with the polyhydroxyalkanoate synthesis operon, the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) content was increased from 29.2 to 52.1 wt %, and the cell dry weight was also increased slightly from 1.70 to 1.86 g L (-1). Coexpression of vgb gene could further enhance the cell dry weight to 2.0 g L(-1) and the polyhydroxyalkanoate content to 60.7 wt %.     

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA.

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.     

Influence of air quality on the composition of microbial pathogens in fresh rainwater

In this study, the microbiological quality of fresh rainwater was assessed from 50 rain events under tropical weather conditions for a year. The levels of four major opportunistic waterborne pathogens, namely, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Aeromonas hydrophila, in rainwater samples were quantified by using a robust and sensitive quantitative PCR (qPCR) method. Of the 50 rainwater samples, 25 were found to be positive for at least one pathogen: 21 for E. coli, 16 for P. aeruginosa, 6 for K. pneumoniae, and 1 for A. hydrophila. In addition to the microbiological assessment of rainwater samples, we also studied the influence of prevailing air quality on the microbial quality of rainwater over the sampling period. A significant change in the diversity and relative abundance of the basic microbial indicator organisms in rainwater was observed during a major regional air pollution episode in Southeast Asia due to biomass-burning emissions.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate.

A structural gene which codes for an extracellular lipase (EC 3.1.1.3) in Aeromonas hydrophila H3, which was isolated from a female hospitalized patient, was cloned in Escherichia coli by using pBR322 as a vector. Lipase purified from both A. hydrophila culture supernatant and the periplasmic fluids of E. coli containing the lip determinant in the original clone (plasmid pLA2) showed an M(r) of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the M(r) determined by Sephacryl S-200 chromatography. Regarding substrate specificity, the optimum chain lengths for the acyl moiety were C6 for ester hydrolysis and C6 and C8 for triacylglycerol hydrolysis. Sequence analysis showed a major open reading frame of 2,052 bp, which predicts a polypeptide with an M(r) of 71,804. The polypeptide was found to contain an amino acid sequence (V-H-F-L-G-H-S-L-G-A) which is highly preserved among lipases.     

Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp

Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.     

Evaluation of the antimicrobial activity of methylglyoxal bis(guanylhydrazone) analogues, the inhibitors for polyamine biosynthetic pathway

Metabolic and antiproliferative effects of methylglyoxal bis(butylamidinohydrazone) (MGBB) and methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP), inhibitors for polyamine biosynthetic pathway, on Escherichia coli, Shigella sonnei, Aeromonas sobria, Aeromonas hydrophila and Vibrio cholerae were investigated. MGBB at the concentration of 100 μmol/1 depleted intracellular putrescine and spermidine concentrations of E. coli to 25 and 20% of the controls, respectively, while MGBCP depressed their concentrations to 38 and 24%, respectively. In these polyamine-depleted E. coli cells the syntheses of RNA, DNA and protein decreased to 13, 54 and 29% of the control, respectively, with MGBB and to 23, 71 and 55%, respectively, with MGBCP. The minimum inhibitory concentrations (MIC) of MGBB for the growth of A. sobria, E. coli, A. hydrophila, V. cholerae and Sh. sonnei were estimated to be 50, 160, 240, 285 and 320 μmol/1, respectively, whereas those of MGBCP were slightly higher for respective bacteria.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila

A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.     

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.