Bacterial production of histamine in some tropical fish

Quantitative and qualitative distribution of histamine-forming bacteria associated with the fish Rastrelliger kanagurta, Sardinella longiceps, Sillago sihama and Liza subviridis, were investigated. These bacteria constituted a significant portion of the total bacterial population of fish and the values obtained in the present study were higher than those previously reported. The order of quantitative abundance of histamine-forming bacteria in the fish examined was: S. longiceps greater than R. kanagurta greater than S. sihama greater than L. subviridis. The bacterial genera isolated were Vibrio sp., Bacillus sp., Pseudomonas sp., Aeromonas sp. and Micrococcus sp., and among them Vibrio was dominant. Growth of the isolates (Vibrio sp., V. fischeri and Bacillus sp.) at different temperatures, pH and sodium chloride concentrations indicated them to be mesophilic, euryhaline and tolerant to acidic and alkaline pH. Bacillus sp. produced more histamine in R. kanagurta, while V. fisheri produced more histamine in S. longiceps.     

3种贝类原虫多重荧光定量PCR方法的建立

目的:建立能够同时检测单孢子虫、派琴虫和折光马尔太虫的三重荧光定量PCR方法。方法:根据基因库中单孢子虫、派琴虫和折光马尔太虫的基因序列,设计3对特异性引物和3条用不同荧光基团标记的TaqMan探针,对反应条件和试剂浓度进行优化,建立能够同时检测单孢子虫、派琴虫和折光马尔太虫的三重荧光定量PCR方法。结果:该方法对单孢子虫、派琴虫和折光马尔太虫的检测敏感性分别达到40、400和40个模板拷贝数;此外抗干扰能力强,对单孢子虫、派琴虫和折光马尔太虫不同模板浓度进行组合,仍可有效地同时检测这3种原虫,对嗜水气单胞菌、荧光假单胞菌、副溶血弧菌、溶藻弧菌、河弧菌和拟态弧菌等病原体的检测结果均为阴性。结论:建立的单孢子虫、派琴虫和折光马尔太虫多重荧光定量PCR具有特异、敏感、快速、定量和重复性好等优点,可用于临床上单孢子虫、派琴虫和折光马尔太虫感染的检测。     

Anaerobic Fish Spoilage by Bacteria II. Kinetics of Bacterial Growth and Substrate Conversions in Herring Extracts

The time course of the conversions of chemical components in herring extracts during anaerobic growth of Proteus sp., str. NTHC 153, Aeromonas sp., str. NTHC 154, and Enterobacter sp., str. NTHC 151 (Strøm & Larsen 1979) has been studied. When the Proteus sp. or the Aeromonas sp. were inoculated into the herring extracts and incubated at 15°C under anaerobic conditions, the sugar components (i.e. mainly ribose, free and bound) were the first substrates utilized. These compounds were converted to acetate and CO₂ by the use of trimethylamine oxide (TMAO) as an external hydrogen acceptor. Growth of bacteria ceased when all TMAO was reduced to trimethylamine (TMA). By adding an extra amount of TMAO to the herring extracts an increased growth of the Proteus sp. and the Aeromonas sp. ensued. The increased growth occurred concomitantly with a further conversion of TMAO to TMA and of lactate to acetate and CO₂. The Enterobacter sp., which did not utilize lactate, did not give an increased growth in herring extracts enriched with TMAO.     

A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems

A marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and alpha-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae.     

Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Differential toxicities of mercury to bacteria and bacteriophages in sea and in lake water.

Mixtures of anionic HgCl3-/HgCl4(2)-complexes were less toxic to terrestrial bacteria (Erwinia herbicola, Agrobacterium tumefaciens), to marine bacteria (Acinetobacter sp., Aeromonas sp.), and to bacteriophages (phi 11 M 15 of Staphylococcus aureus and P1 of Escherichia coli) than were equivalent concentrations of Hg as cationic Hg2+. The toxicity of 1 ppm Hg to A. tumefaciens. Aeromonas sp., and phi 11 M 15 was less in seawater than in lake water. Inasmuch as the Hg-Cl species are formed in environments of high chloride concentration, it was postulated that the lower toxicity of Hg in seawater was a result of the formation of HgCl3-/HgCl4(2)-complexes.     

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.     

几株产活性物质的海洋细菌的分离与初步鉴定

从海泥、海水及海洋动物中分离到数十株细菌 ,对其产生活性物质的菌株进一步筛选 ,得到产蛋白酶、淀粉酶和抑菌活性等生物活性物质的几株海洋细菌 ,将其中的活性高的四株菌纯化后进行菌种鉴定。通过对革兰氏染色、个体及群体形态观察、糖类发酵、硝酸盐还原等生理特性的研究 ,依据《伯杰氏细菌鉴定手册》确定它们的分类地位 ,它们分别应归属为欧文氏菌属 (Erwiniasp .)、气单孢菌属 (Aeromonassp .)、微球菌属 ( (Micrococoussp .)、和假单孢菌属 ( (Pseudomonassp .)。     

Enzymatic characterization of Vibrionaceae strains isolated from environment and cold-blooded animals.

Enzymatic profiles were determined by the API ZYM system for 15 strains of non 01 Vibrio cholerae, 4 strains of V. metschnikovii, 9 strains of V. anguillarum, 6 strains of Plesiomonas shigelloides and 115 strains motile Aeromonas sp. All of the tested strains produced alkaline phosphatase, leucine aminopeptidase and did not possess alpha-fucosidase and alpha-mannosidase. Some differences in enzymatic activities among the tested Vibrionaceae strains were noted. The strains of non 01 V. cholerae, V. metschnikovii, V. anguillarum and P. shigelloides did not produce trypsin, whereas all of the tested Vibrio sp. strains appeared to be positive for this enzyme. Only the strains of P. shigelloides produced BI-Phospho-hydrolase. The lack of acid phosphatase activity was observed among the strains of V. anguillarum.     

Differential media for quantitative recovery of waterborne Aeromonas hydrophila.

Because of the ubiquity of Aeromonas spp., their prevalence in drinking water, and the increasing number of reports on Aeromonas sp.-related infections, a standard method for routine and quantitative recovery had to be defined. On the basis of a survey of 10 media for recovery analysis and subsequent differentiation assays in mixed cultures, we conclude that ampicillin-dextrin agar performed the best for the recovery of Aeromonas spp. in drinking water and the differentiation by simple criteria of that genus from other common waterborne bacteria.     

Screening and identification of polyhydroxyalkanoates producing bacteria and biochemical characterization of their possible application

Polyhydroxyalkanoates (PHAs) accumulating bacteria were isolated under various selective conditions such as pH, salt concentrations and types of heavy metal. Fifty strains of bacterial isolates were found to belong to Bacillus, Proteus, Pseudomonas, Aeromonas, Alcaligenes and Chromobacterium, based on phenotypical features and genotypic investigation. Only twenty five bacterial isolates were selected and observed for the production of PHAs. Interestingly, bacteria belonging to Firmucutes Bacillus sp. produced a high amount of PHAs. The maximum PHAs were accumulated by B. licheniformis PHA 007 at 68.80% of dry cell weight (DCW). Pseudomonas sp., Aeromonas sp., Alcaligenes sp. and Chromobacterium sp. were recorded to produce a moderate amount of PHAs, varying from 10.00-44.32% of DCW. The enzymatic activity was preliminarily analyzed by the ratio of the clear zone diameter to colony diameter. Bacillus gave the highest ratio of hydrolysis zone which corresponds to the highest hydrolytic enzyme activities. Bacillus licheniformis PHA 007 had the highest lipase and protease activity at 2.1 and 5.1, respectively. However, the highest amylase activity was observed in Bacillus sp. PHA 023 at 1.4. Determination of metabolic characteristics was also investigated to check for their ability to consume a wide range of substrates. Bacillus, Aeromonas sp. and Alcaligenes sp. had great ability to utilize a variety of substrates. To decrease high PHA cost, different sources of cheap substrates were tested for the production of PHAs. Bacillus cereus PHA 008 gave the maximal yield of PHA production (64.09% of DCW) when cultivated in anaerobically treated POME. In addition, the accumulation of PHA copolymers such as 3-hydroxyvalerate and 3-hydroxyhexanoate was also observed in Bacillus and Pseudomomas sp. strain 012 and 045, respectively. Eight of the nine isolates accumulated a significant amount of PHAs when inexpensive carbon sources were used as substrates. Here it varied from 1.69% of DCW by B. licheniformis PHA 007 to 64.09% of DCW by B. cereus PHA 008.     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce.

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay(1)

PCR primers specific for the chiA gene were designed by alignment and selection of highly conserved regions of chiA sequences from Serratia marcescens, Alteromonas sp., Bacillus circulans and Aeromonas caviae. These primers were used to amplify a 225 bp fragment of the chiA gene from Vibrio harveyi to produce a chiA gene probe. The chiA PCR primers and probe were used to detect the presence of the chiA gene in an assemblage of 53 reference strains and gave consistent results. Selected chiA fragments amplified by PCR were cloned and sequenced from nine known strains and from Chesapeake Bay isolates 6d and 11d. This confirmed the specificity and utility of the primers for detection of chiA-positive environmental strains. Over 1000 bacterial isolates from Chesapeake Bay water samples were tested for the presence of the chiA gene which was found to be present in 5-41% (average 21%) of the culturable bacterial community. The approach developed in this study was valuable for isolation and enumeration of chiA-positive bacteria in environmental samples.     

Characterization of a galactose binding serum lectin from the Indian catfish, Clarias batrachus: possible involvement of fish lectins in differential recognition of pathogens

A lectin with molecular mass around 200 kDa was isolated from the serum of the Indian catfish Clarias batrachus. The bioactivity of this serum lectin was Ca2+ and pH dependent. The lectin appeared to be specific for alpha-methyl galactose and sialoglycoproteins like porcine and bovine submaxillary mucin and could agglutinate human, rabbit, mice, rat and chicken erythrocytes. This fish lectin was able to specifically agglutinate different gram negative bacteria. When it was checked against different strains of the fish pathogen Aeromonas sp., it significantly altered the viability and pathogenicity of the bacteria. Binding of the lectin to Aeromonas sp., resulted in a dose dependent increase in the bactericidal activity of fish macrophages. However, when the lectin was checked against different gram positive bacteria it could not agglutinate or affect the viability of those strains and also failed to bring about any significant change in the bactericidal potential of fish macrophages. The lectin was able to induce the proliferation of head kidney lymphocytes of Clarias and helped in the release of 'IL-1' like cytokines from head kidney macrophages.     

Comparison of different media for the enumeration of Aeromonas sp. in freshwaters

I. KERSTERS, N. SMEYERS AND W. VERSTRAETE. 1996. Ampicillin-dextrin agar (ADA), m-Aeromonas agar (mA), starch glutamate ampicillin penicillin C-glucose agar (SGAP-10C), trehalose agar (TRE) and ampicillin bile salts inositol xylose agar (MIX) were evaluated for the isolation of Aeromonas sp. from aquatic environments. Recovery of pure cultures was excellent on all media, except for Aer. sobria on SGAP-10C and MIX agars. Recovery of Aeromonas sp. from freshwaters was comparable on ADA, mA, SGAP-10C and TRE. The most selective media were SGAP-10C and ADA, which yielded an average reduction factor of more than 2.9 log. The ability to differentiate Aeromonas sp. from the background microbiota present in freshwaters was best on ADA. The present findings indicate that ADA is the most adequate medium for the selective isolation of Aeromonas sp. from freshwaters.     

DNA probes specific for Aeromonas hydrophila (HG1)

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.     

A preliminary study of parasites and diseases of perch in an intensive culture system

The recent development of intensive rearing of European perch Perca fluviatilis in warm water effluents revealed important pathological problems. This study gives a preliminary overview of parasites and diseases encountered in experimental perch farming. No virus has been recorded since the beginning of the experiments in 1993. By contrast, mass mortality caused by bacterial disease alone or associated with other pathogens were frequently observed. The main identified bacterial species were: Aeromonas sp., A. veronii, A. hydrophila, Streptococcus sp., Staphylococcus sp., Vibrio fluviatilis and Enterobacter agglomerans. Protozoans were the most common parasites observed on cultured P. fluviatilis. High mortality rate due to Ichthyobodo necator and Trichodina sp. may take place at different stages of perch rearing. In contrast, the infestation by Ambiphrya sp. and Heteropolaria sp. was size- or age-related; these two ciliates infested perch weighing < 1 g and > 20 g, respectively. In spite of the presence of other parasites such as Ichthyophthirius multifiliis, Hexamita spp., Gyrodactylus sp. and Argulus sp. in the rearing system P. fluviatilis does not appear to be affected. Further abnormalities or diseases inherent to intensive culture systems are also described.     

Anaerobic Fish Spoilage by Bacteria I. Biochemical Changes in Herring Extracts

Water extracts of herring fillets were used as laboratory model substrates for the study of anaerobic bacterial spoilage of fish stored in bulk. The organisms studied ( Enterobacter sp., str. NTHC 151, Proteus sp., str. NTHC 153, and Aeromonas sp., str. NTHC 154) were selected for by an enrichment method designed to isolate the bacteria having the highest anaerobic growth yields in these herring extracts. The extracts were then inoculated with pure cultures of the bacteria and incubated anaerobically at 15°C. Chemical analyses of the extracts were carried out at the start and at the end of the incubation period. The compounds determined accounted for 88% of total carbon and 93% of total nitrogen of the sterile extracts. Ribose (free and bound), free hexoses and lactate were the main substrates for the growth of the bacteria. These compounds were converted to acetate and CO₂; hydrogen liberated by these processes was used for the quantitative reduction of trimethylamine oxide to trimethylamine. Fermentation balances support the contention that the biochemical changes observed represent the quantitative dominating chemical events in the herring extracts as a result of the development of the spoilage bacteria.     

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.