Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.     

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).     

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.     

Occurrence of both subspecies of Photobacterium damselae in mullets collected in the river Magra (Italy)

The natural reservoirs and biological characteristics of pathogenic populations of both subspecies of Photobacterium damselae in aquatic habitants remain unclear because of difficulties in obtaining pathogenic strains from the environment. In the present study, we assessed the occurrence of Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae , considered to be the causative agent of past epizootic outbreaks in mullets collected in the river Magra, Italy. Two hundred and seventy-eight mullets were collected during a period of two years (2008-2009) and analyzed using multiplex PCR. During this period, 57% of fishes were positive for Photobacterium damselae subsp. piscicida and 37% for Photobacterium damselae subsp. damselae, with an higher presence in summer months although none of PCR-positive mullets showed clinical signs of disease. Our results indicate that the two micro-organisms are widespread in the population of mullets studied, and this could be a possible cause for outbreaks in favourable environmental conditions.     

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.     

半滑舌鳎病原菌(发光杆菌杀鱼亚种)的分离与鉴定

2006年夏,山东青岛某渔场养殖半滑舌鳎(Cynoglossus semilaevis Gunther)大量死亡。症状主要表现为体表溃烂,鳍基部出血等,解剖可见胆囊发黑,肾脏发黄。从患病半滑舌鳎胆囊分离出优势菌并命名为WY06。人工感染试验证实WY06对半滑舌鳎及模式动物斑马鱼都具有很强的致病性,其半数致死量分别为5.5×103cfu/克鱼(5.2×105cfu/条鱼)和1.9×103cfu/克鱼(8.9×102cfu/条鱼)。该病原菌革兰氏染色阴性,菌体呈杆状。综合该菌在形态、生理生化特征及16S rDNA同源性等方面的结果,确认WY06为美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp.piscicida)。该菌对头孢呋肟、菌必治等抗生素敏感。美人鱼发光杆菌杀鱼亚种在美国、日本、欧洲的海水养殖中为常见的病原菌,但作为鱼类病原菌在中国属首次报道。     

Antimicrobial activity of hydrophobic xanthones from Cudrania cochinchinensis against Bacillus subtilis and methicillin-resistant Staphylococcus aureus

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against B. subtilis (MIC = 1.56 microg/ml). Four xanthones, gerontoxanthone I, toxyloxanthone C, cudraxanthone S, and 1,3,7-trihydroxy-2-prenylxanthone, showed weak antibacterial activity against the bacterium (MICs = 3.13-6.25 microg/ml). These compounds also exhibited similar MIC values against methicillin-sensitive S. aureus, MRSAs, and Micrococcus luteus.     

Assessment of a magnetic bead-EIA based kit for rapid diagnosis of fish pasteurellosis.

The accuracy of the magnetic beads-EIA based BIONOR AQUAEIA-Pp kit for the rapid diagnosis of pasteurellosis was evaluated. The kit reacted with all the Photobacterium damselae subsp. piscicida strains included in this study, with a detection limit of 10(4) bacteria/ml. However, non-specific reactions were observed with isolates of Ph. damselae subsp. damselae or Ph. histaminum when the bacterial concentration was high (10(9)-10(10) bacteria/ml). Similar findings in specificity and sensitivity were observed when the kit was applied to experimentally infected fish tissues. However, since those bacterial species are not usually found in the fish species susceptible to pasteurellosis, the AQUAEIA kit appears applicable for a rapid screening of the disease. In addition, when the kit was utilized to analyze cultured populations of seabream, it allowed the detection of the pathogen, not only in individuals affected by the disease, but also in asymptomatic carrier fish. Furthermore, the positive detection of Ph. damselae subsp. piscicida in broodstock gonads, seminal, and ovaric fluids, and also in eggs indicated the possibility of vertical transmission of pasteurellosis.     

Antibacterial and antifungal activity of cicerfuran and related 2-arylbenzofurans and stilbenes

Cicerfuran, 2-(2-methoxy-4,5-methylenedioxyphenyl)-6-hydroxybenzofuran, is an antifungal phytoalexin previously isolated from the roots of chickpea, Cicer spp. The synthesis of cicerfuran, five 2-arylbenzofuran analogues and nine stilbene intermediates was reported recently. The antimicrobial activities of these compounds were evaluated against two species of bacteria, Bacillus subtilis and Pseudomonas syringae, and four species of filamentous fungi, Aspergillus niger, Botrytis cinerea, Cladosporium herbarum and Monilinia aucupariae. Stilbenes with a free hydroxyl group were active against both bacteria and fungi with MICs in the range 25-100microg/ml. Cicerfuran was the only 2-arylbenzofuran that showed antimicrobial activity with MICs as low as 25microg/ml. Some aspects of the structure-activity relationship are discussed.     

Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata L.) did not correlate with disease resistance to Photobacterium damselae

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabream's immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.     

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.     

Antimicrobial activity of isoprenoid-substituted xanthones from Cudrania cochinchinensis against vancomycin-resistant enterococci.

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against vancomycin-resistant enterococci (VRE). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against five VRE strains (VanA, VanB and VanC) (MICs = 1.56 microg/ml). Four xanthones, 1,3,7-trihydroxy-2-prenylxanthone, gerontoxanthone I, alvaxanthone and isoalvaxanthone, showed weaker antibacterial activity against these VREs (MICs = 3.13-6.25 microg/ml). .     

Antimicrobial constituents from the stem bark of Feronia limonia.

The stem bark of Feronia limonia (Fam. Rutaceae) yielded (-)-(2S)-5,3'-dihydroxy-4'-methoxy-6",6"-dimethylchromeno-(7,8,2",3")-flavanone along with several known compounds including an alkaloid, five coumarins, a flavanone, a lignan, three sterols and a triterpene. The structures of these compounds were determined by spectroscopic methods, mainly 1D and 2D NMR. The antimicrobial screening of compounds by a microdilution technique resulted in MICs in the range 25-100 microg/ml. Other biological activities of the known compounds are also discussed.     

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae

AIMS: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). METHODS AND RESULTS: Seventy-one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as production of urease, which are used to differentiate P. damselae subsp. damselae from P. damselae subsp. piscicida. Genetic characterization was conducted on a selection of 33 strains, including two reference strains. Dice coefficient (Sd) and the unweighted pair group method with average linkage (UPGMA) were used for numerical analysis of banding patterns. AFLP type was defined on the basis of 100% similarity in the dendrogram obtained, yielding 24 distinct AFLP profiles. At 70% similarity, 13 clusters were defined, thus confirming the great variability observed for the phenotypic traits. CONCLUSIONS: The AFLP variability shown by the isolates was high enough to discriminate between different strains which colonize the same fish. However, closely related AFLP types were usually derived from strains isolated at the same fish farm, indicating an epidemiological relationship. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has confirmed that the AFLP technique allows discrimination of individual strains within P. damselae subsp. damselae for epidemiological studies, and that this subspecies exhibits greater variability than that described for subspecies piscicida.     

Simple and rapid detection of Photobacterium damselae ssp. piscicida by a PCR technique and plating method

AIMS: To detect Photobacterium damselae ssp. piscicida using the PCR technique and plating method. METHODS AND RESULTS: Two strains of P. damselae ssp. piscicida were isolated from cultured cobia (Rachycentron canadum) at two different fish farms in Taiwan. A pair of primers was designed to detect the capsular polysaccharide gene of P. damselae ssp. piscicida by PCR. Reference strains of different genus and different clinical strains were used for this study. The expected product (410 bp) was obtained from both P. damselae ssp. piscicida and P. damselae ssp. damselae, and they were differentiated by culturing on thiosulphate citrate bile salts-sucrose agar (TCBS-1). Photobacterium damselae ssp. damselae grew on TCBS-1 producing green colonies whereas P. damselae ssp. piscicida did not grow. CONCLUSIONS: The methods used are cost and labour effective when compared with the other methods and commercially available kits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an integrated set of methods to identify the species P. damselae and to differentiate P. damselae ssp. piscicida from P. damselae ssp. damselae.     

Antibacterial activities of naturally occurring compounds against Mycobacterium avium subsp. paratuberculosis

The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of Mycobacterium avium subsp. paratuberculosis (a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method. M. avium subsp. paratuberculosis was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 microg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37 degrees C. Six of the compounds were found to inhibit the growth of M. avium subsp. paratuberculosis. The most effective compound was trans-cinnamaldehyde, with a MIC of 25.9 microg/ml, followed by cinnamon oil (26.2 microg/ml), oregano oil (68.2 microg/ml), carvacrol (72.2 microg/ml), 2,5-dihydroxybenzaldehyde (74 microg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 microg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or the aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three M. avium subsp. paratuberculosis strains studied. Possible mechanisms of the antimicrobial effects are discussed.     

The effect of growth medium salinity of Photobacterium damselae subsp. piscicida on the immune response of hybrid bass (Morone saxatilis x M. chrysops)

Photobacterium damselae subsp. piscicida (P. damselae) was grown on various media and the effect of media salinity on certain immune responses of hybrid bass was studied. In Israel, pasteurellosis outbreaks have not been reported at water salinities below 1.38 per thousand. During vaccination experiments the salinity of the medium on which P. damselae is grown, was shown to affect stimulation of the immune system. No correlation was found between antibody response and protection. Bacterial envelopes separated by electrophoresis and subjected to western blot analysis revealed an antibody response against some protein bands. Band sequencing was performed to identify the protein stimulating the immune response. Sequence identity of 80% was seen in 10-amino-acid overlap of the 36-kDa band with a specific gene of alkalophilic Bacillus firmus. A preparation of P. damselae grown in a 2.5% NaCl medium at 25 degrees C is the most effective vaccine against pasteurellosis, providing hybrid bass with quite good protection.     

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.     

Trypanocidal and antimicrobial activities of Moquinia kingii.

A chloroform crude extract (aerial part) and two compounds, apigenin (1) and cynaropicrin (2), isolated from Moquinia kingii were evaluated against Trypanosoma cruzi trypomastigotes in vitro. Antimicrobial activity was also screened using twenty-two strains including gram-positive and gram-negative bacteria and the yeasts Candida albicans and C. tropicalis. The chloroform crude extract, fractions and isolated compounds from M. kingii were active for both activities. The IC50 values for trypanocidal activity obtained for cynaropicrin and apigenin were 93.5 microg/ml and 181 microg/ml, respectively, while the minimum inhibitory concentrations (MICs) varied from 100 microg/ml to 2500 microg/ml, against the strains of bacteria and yeasts evaluated.