Ecological dynamics of marine luminous bacteria

Marine luminous bacteria are heterotrophic, Gram negative microorganisms which continuously emit light. Eight species of luminous bacteria have been described belonging to three different genera, viz. Photobacterium, Vibrio and Alteromonas. In this review we report on the distribution of luminous procaryotes in abiotic and biotic components of the marine environment and the interactions among them.     

Polyphasic approach to the characterisation of marine luminous bacteria

Fifteen luminous bacterial strains were isolated from the Tyrrhenian Sea coastal waters off northeastern Sicily and characterised by a combination of phenotypic and molecular tests in order to identify them and to determine their intraspecific genetic variability. Five luminous type strains, Vibrio splendidus NCIMB 1, V. harveyi NCIMB 1280, V. fischeri NCIMB 1281, V. orientalis NCIMB 2195 and Photobacterium leiognathi NCIMB 2193, were used as reference. On the basis of their phenotypic characters, the isolates were assigned to the family Vibrionaceae and all were related to the V. harveyi reference strain. Amplified 16S ribosomal DNA restriction analysis (ARDRA) enabled the strains to be subdivided into three groups, two of which exhibited the same restriction pattern as the two reference strains, V. harveyi and V. splendidus. Comparison of the full 16S rDNA sequence and of a 100-bp highly variable 16S rDNA region (selected as a 'signature' sequence for the luminous bacteria) confirmed ARDRA data and suggested that the strains of the third group could be considered a subspecies of V. harveyi or a tyrrhenian biovar, different from the other reference strains whose 16S rDNAs have already been sequenced. Random amplified polymorphic DNA (RAPD) fingerprinting and analysis of plasmid content suggested a high degree of intraspecific genetic variability within the largest ARDRA group. Data obtained suggest that the ARDRA method and the sequencing of the 16S rDNA signature region could be a powerful tool for a rapid identification of marine luminous bacteria.     

Isolation and characterization of lactic acid bacteria from lakes

Lactic acid bacteria (LAB) were isolated from lake-water samples collected at 7 lakes distributed in Yamanashi prefecture, Japan. Sampling was performed year round. 112 cultures were isolated and divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified eleven different bacterial groups (A to K), and the results showed that the isolates represented seven genera: Lactococcus, Leuconostoc, Enterococcus, Lactobacillus, Carnobacterium, Streptococcus, and Weissella. Lactococcus lactis subsp. lactis was the most abundant lactic acid bacteria found in these lakes. Furthermore, Lactococcus lactis subsp. lactis was also the most abundant lactic acid bacteria found throughout the year. Seasonal differences, numbers of isolates and the species of lactic acid bacteria were also recorded in this study.     

Isolation and characterization of antibiotic-resistant bacteria from pharmaceutical industrial wastewaters

Contamination of surface waters in underdeveloped countries is a great concern. Treated and untreated wastewaters have been discharged into rivers and streams, leading to possible waterborne infection outbreaks which may represent a significant dissemination mechanism of antibiotic resistance genes among pathogenic bacterial populations.The present study aims to determine the multi-drug resistance patterns among isolated and identified bacterial strains in a pharmaceutical wastewater effluent in north Tunisia. Fourteen isolates were obtained and seven of them were identified. These isolates belong to different genera namely, Pseudomonas, Acinetobacter, Exiguobacterium, Delftia and Morganella. Susceptibility patterns of these isolates were studied toward commonly used antibiotics in Tunisia. All the identified isolates were found to have 100% susceptibility against colistin sulfate and 100% resistance against amoxicillin. Among the 11 antibiotics tested, six patterns of multi-drug resistance were obtained. The potential of the examined wastewater effluent in spreading multi-drug resistance and the associated public health implications are discussed.     

Isolation and characterization of nitrogen-fixing moderate halophilic bacteria from saline soils of Egypt

Twenty out of 400 isolates of bacteria mainly from salt marshes and saline soils of Egypt were successfully grown on mannitol N-free medium. The nitrogen-fixing activity was then demonstrated for the twenty isolates in modified Stanier'S medium using the acetylene reduction assay. All of them possessed appreciable nitrogenase activity (acetylene reduction) under non-saline conditions; however, at 5% NaCl only 60% of the isolates exhibited a high rate of this activity and 25% were completely negative under these conditions. The bacterial isolates grew well in Stanier'S medium; nevertheless, growth of the majority of isolates was reduced by about 30-80% in the same medium containing 5% NaCl. Cellulolytic activity was detected in 60% of the twenty strains, amylolytic in 45%, and pectinolytic in 10% of the isolates. The bacterial isolates showed also enzymatic activity under saline conditions (5% NaCl). The preliminary identification indicated that six isolates were Gram positive spore-forming bacteria of the genus Bacillus, the others were Gram negative rods which remain to be identified.     

Antibiotic resistance patterns of enterococci isolated from coastal bathing waters.

Recreational water should be considered a risk for enterococcal infections in regions with high utilisation and long exposure periods. A total of 1113 enterococcal isolates was obtained from 1670 bathing water samples from 120 bathing areas of seven prefectures in northern Greece. Enterococcus avium, E. raffinosus and E. faecium were the most prevalent species. Single, double and multiple antibiotic resistance patterns were observed in 33.5% 31.0% and 22.8% of the isolates, respectively. Resistance to erythromycin occurred most frequently, in 57.3% isolates, many of which also exhibited resistance to ciprofloxacin and rifampicin as well as high-level resistance to kanamycin and streptomycin. The results suggest that bathing water may contribute to the dissemination of uncommon enterococcal species that exhibit resistance to several antibiotics which are used to treat community-acquired infections.     

Isolation and characterization of biocides resistant bacteria from dental unit water line biofilms

Six biocides resistant isolates were isolated from dental unit water lines (DUWL) in Pakistan. All isolates could tolerate 150 μg ml^–1 of biocides (5.25% sodium hypocholrite, 35% H₂O₂, 4% tween 20, 1% povidine iodine, 0.2% chlorohexidine gluconate, 1% ethylene di‐amino tetra acetic acid and 1% phenol) on l‐agar and 100 μg ml^–1 in l‐broth. The growth rate of all isolates was determined by generating growth curves at 37 °C for 48 h. The isolates were found to differ in growth rates with lag phase varying from (4–6 h) in biocides supplemented media compared to 2–4 h in biocides free medium. They have wide temperatures (24–42 °C) and pH (5–9) ranges. Traditional ways of identification of bacteria by phenotypic characteristics were accomplished by phenotypic and biochemical characterization. Heavy metals and antimicrobial susceptibility tests indicated that all isolates examined were resistant to trimethoprim, chloramphenicol while sensitive to HgCl₂ and Pb (NO₃)₂. Almost all isolates were opportunistic pathogens. The 16S rRNA‐encoding genes from these six isolates were sequenced to confirm the identity of these isolates. 5 different genera (Bacillus, Achromobacter, Pseudomonas and Klebsiella) of bacteria were identified by 16S rDNA genes amplified from genomic DNA of biocides resistant DUWL biofilm isolates. Analysis of 16S rDNA genes revealed a much more clear identification of microrganisms than culture methods. However, different species of the same genera can have the same 16S rRNA gene sequence but are different due to phenotypic differences or different clinical manifestations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Isolation,characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with α-Proteobacteria (Pseudovibrio sp.) and four with γ-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.     

Isolation and characterization of proteases from Bacteroides melaninogenicus.

We isolated two types of intracellular proteases from a strain of Bacteroides melaninogenicus. These enzymes were extracted from cells by ultrasonic treatment and were partially purified. These two enzymes (proteases I and II) differed in molecular weight, heat stability, sensitivity to reducing agents, Km value, and optimum pH for activity.     

Isolation, characterization and identification of a Paracoccus sp. 2-haloacid-degrading bacterium from the marine sponge Hymeniacidon perlevis

A 2-haloacid dehalogenase-producing bacterium, designated DEH99, was isolated from the marine sponge Hymeniacidon perlevis using a modified enrichment medium and a pH indicator method. DEH99 could degrade only half of the racemic mixture 2-chloropropionic acid (2-CPA) in the medium. The dehalogenase of DEH99 was further determined to be a (S)-2-haloacid dehalogenase, which can degrade 2-CPA, 2-bromopropionic acid (2-BPA), and iodoacetic acid. The gene encoding the (S)-2-haloacid dehalogenase was partially sequenced and classified into the Group II family. The 2-haloacid dehalogenase showed the highest sequence similarity (77% with 21% query coverage) to the haloacid dehalogenase (dhlB) gene of Xanthobacter autotrophicus. A phylogenetic analysis of the 16S rDNA sequence demonstrated that the isolate DEH99 is a member of the genus Paracoccus. To our knowledge, this is the first report detailing the isolation of a strain of genus Paracoccus having 2-haloacid dehalogenase activity from marine sponges.     

Isolation and characterization of flagellar preparations from Pseudomonas species.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used previously to characterize the flagellin of Pseudomonas aeruginosa. flFlagella from several other clinically important species of pseudomonads have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their molecular weights have been found to vary among species as follows: P. maltophilia B69 Fla, 33,000; P. stutzeri HEW, 55,000; P. aeruginosa M-2, 53,000. The flagella of P. cepacia strains were divided into two groups based on molecular weight. Type I had a molecular weight of 31,000. The molecular weight of type II was in the range of 44,000 to 46,000. Serologically, type I is a homologous group, whereas type II is a heterologous group. The two flagellin types of P. cepacia appear to be analogous to the two major flagellin types of P. aeruginosa. Characterization of P. cepacia strains by flagellin types may serve as a molecular epidemiological tool.     

Isolation and characterization of C-reactive protein from the dog.

Using calcium-dependent affinity chromatography on Sepharose-bearing, covalently-coupled pneumococcal C-polysaccharide, a protein was isolated from the serum of dogs that had undergone general anaesthesia and major surgery. This protein was confirmed as the canine analogue of C-reactive protein (CRP) in other species by virtue of its electron microscopic appearance, subunit composition and behaviour as an acute phase reactant. Dog CRP had an apparent molecular weight of approximately 100,000 and was composed of five subunits of approximately 20,000 MW each. Two of the five subunits in each molecule were glycosylated. Negatively stained preparations had the typical cyclic pentameric disc-like structure of proteins of the pentraxin family, and in some preparations had a tendency to form stacks. Serum from normal healthy dogs of various strains usually contained less than 5 mg/l of CRP but, following the stimulus of major surgery, an increase in the CRP concentration was first detected at 4 hr.     

Isolation and characterization of a protease from Bacteroides gingivalis.

A protease was purified from Bacteroides gingivalis ATCC 33277 culture fluid by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, and isoelectric focusing. The enzyme was active against benzoyl-L-arginine-p-nitroanilide, carbobenzoxy-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide azoalbumin, azocasein, azocoll, and p-tosyl-L-arginine methyl ester. The molecular weight of the enzyme was about 300,000 as determined by gel filtration. Its isoelectric point was 5.0. The maximum activity was found at pH 7.5, and the optimum temperature for activity was between 40 and 45 degrees C. The apparent Km value for benzoyl-L-arginine-p-nitroanilide was 2 mM. The enzyme was inhibited by sulfhydryl group-blocking reagents, tosyl-L-lysine chloromethyl ketone, and EDTA. Soybean trypsin inhibitor and diisopropylfluorophosphate were not inhibitory.     

Isolation, characterization and beneficial effects of rice associated plant growth promoting bacteria from Zanzibar soils

This study was undertaken to isolate and characterize plant growth promoting bacteria (PGPB) occurring in four soils of Zanzibar, Tanzania as well as to evaluate their potential use as biofertilizers for rice. A total of 12 PGPB strains were isolated from rice and studied for growth characteristics, carbon/nitrogen source utilization patterns using QTS-24 kits, phosphate solubilization, indole acetic acid (IAA) production, antibiotic resistance patterns and growth at different pH, temperature and salt concentrations. All the isolates were motile and gram negative except Z3-4. Acetylene reduction activity was detected in all isolates ranging from 5.9-76.4 nmole C2H2 reduced/h x mg protein while 9 isolates produced IAA ranged from 20-90.8 mg/l. Most of the isolates showed resistance against different environmental stresses like 10-40 degrees C temperature, 0.2-1 M salt concentration and 4-8.5 pH range. Only one isolate Z2-7 formed clear zones on Pikovskaia's medium showing its ability to solubilize phosphates. Z3-2 was used to develop fluorescent antibodies to check the cross reactivity of the isolates. Inoculation of these bacterial isolates resulted in higher plant biomass, root area, and total N and P contents on Tanzanian rice variety BKN PRAT3036B under controlled conditions. Bacillus sp. Z3-4 and Azospirillum sp. Z3-1 are effective strains and, after further testing under field conditions, can be used for inoculum production of rice in Tanzania. The plant growth promoting effects of these PGPRs suggest that these can be exploited to improve crop productivity of rice in Tanzania.     

Isolation and characterization of toxic fractions from Brucella abortus.

Two types of toxic fractions, protein-rich and carbohydrate-rich, were isolated from attenuated (strain 19) and virulent (strain 2308) Brucella abortus organisms. Polyacrylamide gel electrophoresis of the protein-rich fraction, in the presence and absence of sodium dodecyl sulfate, revealed qualitative and quantitative differences in the protein bands derived from the attenuated and virulent strains. Sodium dodecyl sulfate-gel electrophoresis indicated that the major differences between these protein fractions were in the molecular weight range from 14,000 to 40,000. Immunoelectrophoresis of these fractions from the attenuated and virulent strains revealed differences in the antigenic spectrum. Polypeptides in the carbohydrate-rich fraction could be visualized on polyacrylamide gels only when reacted with fluorescamine before electrophoresis. Immune sera did not precipitate the components of the carbohydrate-rich fraction. Intradermal injecttion of the protein and carbohydrate-rich fractions resulted in different types of skin lesions in guinea pigs, i.e., edematous/erythematous and necrotic lesions, respectively. Fractions derived from attenuated and virulent strains of B. abortus were equally toxic in the guinea pig skin test. The toxic activity of both types of fractions was susceptible to pronase and heat treatment.     

The use of luminous bacteria for determination of phagocytosis

A system has been developed for the isolation of large numbers of unfractionated mononuclear cells from single, well characterized normal individuals and for the separation by elutriation of these cells into populations of greater than 90% pure monocytes and greater than 99% pure lymphocytes. The total number of monocytes obtained from a single donor averaged about 550 million. After cryopreservation and thawing of these cells, the viability remained greater than 90%, 80% of original cells were recovered, and the ability to ingest antibody-coated targets was comparable to that of fresh monocytes. The cells remained sterile without the use of antibiotics and were suitable for long-term culture. The monocytes that were isolated and cryopreserved by these procedures functioned reproducibly as inhibitors of tumor cell growth and in an assay of responsiveness to monocyte migration inhibitory factor (MIF).     

Isolation and primary characterization of a new bacteriophage of obligate methylotrophic bacteria

Ø KISR 1, a new bacteriophage of obligate methylotrophic bacteria, was isolated from a local pilot plant. The phage belongs to the Podoviridae family of viruses characterized by a short non-contractile tail and double-stranded DNA. The size of phage genome was determined on the basis of electron micrographs and restriction enzyme analysis to be about 46 kb. The host range of Ø KISR 1 is restricted to three very closely related methanol-utilizing KISR strains belonging to the genus Methylophilus. The phage does not originate from the methylotrophic KISR strains which were tested for lysogeny.     

抗分枝杆菌海洋微生物的分离与鉴定

目的 从海洋微生物中发现具有抗分枝杆菌活性的细菌,并对其进行鉴定.方法 从印度洋热带地区的海底沉积物中分离海洋微生物.通过纸片法活性测定,发现了一株具有抗耻垢分枝杆菌活性的细菌.光学显微下观察细菌形态和菌落.测定细菌的理化特性,提取其基因组,荧光法测定基因组的Tm值,扩增其16S rDNA并进行测序.将序列提交NCBI...