Regional distribution and pH sensitivity ofAzospirillum associated with wheat roots in Eastern Australia

In a survey ofAzospirillum spp. on the roots and associated soil of wheat grown in eastern Australia, azospirilla were isolated from approximately 40% of samples from areas of soil pH between 5.0 and 6.6. However, azospirilla isolates were rare in soil between pH 4.5 and 5.0 and absent below pH 4.5. Of 25 independent isolates, 17 wereA. brasiliense and eight wereA. lipoferum. No selection forA. brasiliense Nir^– strains by wheat roots was observed. Only one of six endorhizosphere isolates wereA. brasiliense Nir^–, compared with three of nine from unsterilized roots plus associated soil, and three of eight from soil. With a medium buffered with 0.05 M malate and 0.05 M phosphate, it was found that allAzospirillum isolates had a lower minimum pH for growth when supplied with fixed nitrogen than when grown under nitrogen-fixing conditions. Strains isolated from soils had a minimum pH for growth that was less than the pH of the soil from which they were isolated. However, a significant proportion of strains isolated from roots had a minimum pH for growth that was higher than the pH of the associated soil suggesting that the wheat roots provided an ecological niche protecting against soil acidity.     

A phylogenetic analysis of micro-organisms isolated from subsurface environments

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis— a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria— including the pseudomonads and Agrobacterium tumefaciens— or the low-G+C Gram-positive bacteria.     

Characterization and identification of bacteria isolated from micropropagated mint plants

Summary Bacterial isolates from contaminated mint shoot cultures were characterized and identified as a preliminary step in determining an elimination treatment. The 22 bacteria were characterized using biochemical and morphological tests and subjected to sensitivity tests with four antibiotics. The isolates were compared with known organisms and assigned to genera according to similarities in characteristics. Seven isolates were analyzed by fatty acid analysis carried out by a commercial laboratory. Six were classified asAgrobacterium radiobacter; eight asXanthomonas; one each asPseudomonas fluorescens, Micrococcus spp.,Corynebacterium spp., andCurtobacterium spp.; four could not be assigned to genera. Inhibition of growth of the bacteria by most antibiotics was best at pH 7.5. Minimal inhibitory concentration and minimal bactericidal concentrations of gentamicin, rifampicin, streptomycin sulfate, and Timentin varied with genotype.     

Diazotrophic bacteria associated with banana (Musa spp.).

Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobiumsp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates.     

Acetylene reduction by Rhodospirillaceae from the Aswan High Dam Lake

Potential nitrogen-fixing freshwater photosynthetic bacteria of 32 isolates from the Aswan High Dam Lake were investigated using the acetylene reduction technique to ascertain the magnitude of their contribution of fixed nitrogen to the ecosystem. The superior nitrogen-fixing Rhodospirillaceae wereRhodocyclus gelatinosus andRhodomicrobium vannielii followed byRhodobacter capsulatus, Rhodopseudomonas viridis andRhodopseudomonas palustris. High and moderate nitrogenase activities (534 to 1528 nmol ethylene produced/4 ml/h) were found in 56% of the isolates, while the remainder showed low activities (76 to 477 nmol ethylene produced/4 ml/h).     

Salmonella, Campylobacter and Enterococcus spp.: Their Antimicrobial Resistance Profiles and their Spatial Relationships in a Synoptic Study of the Upper Oconee River Basin

Rivers may serve as reservoirs for enteric organisms. Very little is known about the boundaries of microbial communities in moving bodies of water so this study was undertaken to find the limits of distribution of some bacteria, focusing on enteric organisms. The presence of Salmonella, Campylobacter, and Enterococcus spp. and the antimicrobial resistance phenotypes carried by these organisms was evaluated for the Upper Oconee River basin, a small river in the lower Piedmont of northeastern Georgia, USA. Samples were obtained from 83 sites during a 3-h period on a spring day (April 2005) in an approximately 30 × 20 km region. Campylobacter spp. was isolated at 12 sites. The Campylobacter isolates from three sites were resistant to tetracycline. Of the five short-variable region (SVR) subtypes of Campylobacter that were found, three were found at more than one site, two types were found twice, and one subtype was found three times. Enterococcus was isolated at 71 sites. E. casseliflavus was the most common species. Based on species identification and antimicrobial resistance patterns, 24 types of Enterococcus were found. Salmonella was isolated from 62 sites. Of the 19 Salmonella serovars that were isolated, serovar Muenchen accounted for about 20% of the isolates. The next three most common serovars isolated, Rubislaw, Hartford, and Give, accounted for about 44% of the river isolates. Antimicrobial resistance profiling offered limited differentiation of Salmonella isolates because only seven isolates were resistant to any antimicrobial. The sites at which Salmonella, Campylobacter, or Enterococcus were isolated did not correlate with each other or with the total coliform number or Escherichia coli count for the site. However, isolates of some of the same species and type occurred in clusters that were restricted to areas within 5 to 6 km.     

Isolation and characterization of heavy metals resistant bacteria from Lagos Lagoon

A total of 228 bacteria with an ability to resist toxic heavy metals were isolated from 8 selected sites of the Lagos Lagoon. The bacteria isolated wereStaphylocaccus sp.,Bacillus sp.,Pseudomonas sp.,Streptococcus sp.,Moraxella sp.,Escherichia coli, Proteus sp.,Klebsiella sp. andSalmonella sp. The heavy metals to which resistance was recorded were mercury, lead, zinc, cobalt, copper and chromium. The lagoon sites from which the highest number of resistant bacteria were isolated were Marina and Ebute-Ero. The heavy metal to which most bacteria were resistant was cobalt, while the least was chromium. The significance of the result is discussed in relation to the Nigerian environment and human health.     

Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera. Correspondence to: P. Kämpfer.     

Diversity of spore‐forming bacteria in cattle manure,slaughterhouse waste and samples from biogas plants

Aims: As biowaste intended for biogas production can contain pathogenic micro‐organisms, the recommended treatment is pasteurization at 70°C for 60 min. This reduces pathogens such as Salmonella spp., whereas spore‐forming bacteria (Bacillus spp. and Clostridium spp.) survive. Most spore‐forming bacteria are harmless, but some can cause diseases such as blackleg, botulism and anthrax. In this study, the effect of the biogas process on Bacillus spp. and Clostridium spp. was investigated. Methods and Results: We analysed 97 faecal samples, 20 slaughterhouse waste samples and 60 samples collected at different stages in the biogas process. Bacillus spp. and Clostridium spp. were quantified and subcultured. The isolates were identified by biochemical methods and by 16S rRNA gene sequencing. Phylogenetic trees were constructed from the sequences obtained from isolates from the samples. Clostridium botulinum/Clostridium spp. and Clostridium sordellii were found both before and after pasteurization, but not after digestion (AD). Some of the isolated strains probably represented new members of the genera Clostridium and Bacillus. Conclusion: After digestion, the numbers of clostridia decreased, but none of the pathogenic bacteria did, whereas Bacillus spp. remained constant during the process. Significance and Impact of the Study: Biogas is gaining in importance as an energy source and because the residues are used as fertilizers, we needed to study the prevalence of pathogenic bacteria in such material.     

Randomly-amplified microsatellite polymorphism for preliminary typing of lactic acid bacteria from Bryndza Cheese

A high-throughput, medium-discrimination method for preliminary typing and selecting non-identical isolates of lactic acid bacteria in cheeses was developed. RAMP, a PCR with one microsatellite-targeted and one random primer, was used for preliminary typing of 1119 isolates of lactic acid bacteria from Slovak Bryndza cheese. A total of 59 genotypes were identified based on RAMP profiles consisting of 12–23 DNA fragments of 150–3000 bp. For example, 18, 17, 13 and 7 different RAMP-types were identified in Lactobacillus brevis, L. plantarum, L. paracasei and L. fermentum, respectively. The method facilitated well reproducible, medium-discrimination typing of Lactobacillus spp. and Pediococcus spp. at a subspecies level and proved to be suitable for preliminary typing of lactic acid bacteria isolated from cheese.     

Antagonistic activity of bacteria isolated from crops cultivated in a rotation system and a monoculture against <Emphasis Type="Italic">Pythium debaryanum</Emphasis> and <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The effect of crop rotation and monocropping on the occurrence of bacteria with antagonistic activity toward Pythium debaryanum and Fusarium oxysporum was shown. Arthrobacter spp., fluorescent Pseudomonas spp. and actinomycetes were isolated from winter rape, sugar beet and winter barley rhizosphere and bulk soil from the plots of a long-term crop rotation experiment (18 years). The occurrence of mycoantagonistic isolates and their antibiosis level exhibited specificity for the site, crop and crop rotation. Mycoantagonistic activity was common among actinomycetes and fluorescent Pseudomonas spp. and less frequent among Arthrobacter spp. Antibiosis of fluorescent Pseudomonas spp. and Arthrobacter spp. was in general stronger against P. debaryanum than F. oxysporum. The highest percentage of antagonistic Pseudomonas spp. against P. debaryanum was in the plots of barley crop, while plots of winter rape showed higher frequency of antagonists against F. oxysporum. The highest antibiosis activity of Arthrobacter spp. against both pathogens occurred in isolates from barley and winter rape monoculture, and there were no F. oxysporum antagonists among these bacteria in sugar beet monoculture. Most of actinomycete isolates strongly inhibited growth of P. debaryanum and F. oxysporum. The percentage of mycoantagonistic actinomycetes and their antibiosis level were the highest in the 6-year crop rotation system.     

Screening for microbial trehalases: extracellular trehalases produced byFusarium species

Two hundred microorganisms comprising actinomycetes, bacteria, fungi and yeasts were screened for extracellular trehalases by their growth on trehalose in solid and liquid culture.Candida albicans, Gelasinospora retispora and four isolates belonging to the genusFusarium produced extracellular trehalases. The production of trehalase by theFusarium spp. was influenced by the nutrient composition of the medium and the carbon source; of the substrates examined starch produced the highest enzyme titre. Trehalase was an inducible enzyme and was repressed when theFusarium spp. were grown on glucose. The properties of the trehalases from two of theFusarium spp. (isolates MU-105 and TR-8) were typical of non-regulatory trehalases. Activity of several α-glucosidases, an amylase, an invertase and a cellobiase was also demonstrated when the two isolates were grown on trehalose.     

PCR amplification of four genes coding for aminoglycoside-modifying enzymes in bacteria of clinical isolates from Jordan University Hospital

Three species of Gram-negative G(–) bacteria were chosen for this study: Escherichia spp. (29 isolates), Pseudomonas spp. (16 isolates) andEnterobacter spp. (17 isolates), utilizing colony PCR to detect genes coding for aminoglycoside-modifying enzymes. The 62 isolates were purified and cultured on nutrient broth media supplemented with 50 g kanamycin/ml. Only 17 out of the 62 isolates were resistant to kanamycin and were subjected to colony PCR protocol using 20 l cell lysate and eight sets of primers with each isolate. Sizing of the amplified fragments was carried out in order to determine the specificity of PCR. The 17 isolates were shown to carry the rrs, aacC2, aacA-aphD and aphA3 genes.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Qualitative microbiological changes during decomposition of plant material in a sandy sierozem soil

The successive qualitative microbial changes during the decomposition of bajra stalk in a sandy sierozem soil were studied.Alternaria spp.,Aspergillus spp.,Cladosporium spp.,Fusarium spp.Gliocladium spp.,Mucor spp. andRhizopus spp. were most common fungi. The bacteria observed wereAchromobacter, Arthrobacter, Bacillus, Micrococcus, Pseudomonas andXanthomonas. Cellvibrio andCellulomonas were also observed.     

Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Mycoflora and aflatoxin production in pigeon pea stored in jute sacks and iron bins

The mycoflora, moisture content and aflatoxin contamination of pigeon pea (Cajanus cajan (L.) Millisp) stored in jute sacks and iron bins were determined at monthly intervals for a year. The predominant fungi on freshly harvested seeds wereAlternaria spp.,Botryodiplodia theobromae, Fusarium spp. andPhoma spp. These fungi gradually disappeared from stored seeds with time and by 5–6 months, most were not isolated. The fungi that succeeded the initially dominant ones were mainly members of the generaAspergillus, Penicillium andRhizopus. Population of these fungi increased up to the end of one year storage. Higher incidence of mycoflora andAspergillus flavus were recorded in jute-sack samples throughout the storage period. The moisture content of stored seeds was found to fluctuate with the prevailing weather condition, being low during the dry season and slightly high during the wet season. The stored seeds were free of aflatoxins for 3 and 5 months in jute sacks and iron bins respectively. The level of aflatoxins detected in jute-sack storage system was considerably higher than that occurring in the iron bin system. Of 196 isolates ofA. flavus screened, 48% were toxigenic in liquid culture (54% from jute sacks and 41% from iron bins).     

Biofilm formation in an ice cream plant

The sites of biofilm formation in an ice cream plant were investigated by sampling both the production line and the environment. Experiments were carried out twice within a 20-day period. First, stainless steel coupons were fixed to surfaces adjacent to food contact surfaces, the floor drains and the doormat. They were taken for the analysis of biofilm at three different production stages. Then, biofilm forming bacteria were␣enumerated and also presence of Listeria monocytogenes was monitored. Biofilm forming isolates were selected on the basis of colony morphology and Gram’s reaction; Gram negative cocci and rod, Gram positive cocci and spore forming isolates were identified. Most of the biofilm formations were seen on the conveyor belt of a packaging machine 8 h after the beginning of the production, 6.5 × 10³ cfu cm⁻². Most of the Gram negative bacteria identified belong to Enterobacteriaceae family such as Proteus, Enterobacter, Citrobacter, Shigella, Escherichia, Edwardsiella. The other Gram negative microflora included Aeromonas, Plesiomonas, Moraxella, Pseudomonas or Alcaligenes spp. were also isolated. Gram positive microflora of the ice cream plant included Staphyloccus, Bacillus, Listeria and lactic acid bacteria such as Streptococcus, Leuconostoc or Pediococcus spp. The results from this study highlighted the problems of spread of pathogens like Listeria and Shigella and spoilage bacteria. In the development of cleaning and disinfection procedures in ice cream plants, an awareness of these biofilm-forming bacteria is essential for the ice cream plants.     

Isolation of a newLegionella species from thermally altered waters

Legionella-like bacteria were isolated from thermally altered water in Pennsylvania and Minnesota. These organisms were isolated from tissues of infected guinea pigs after intraperitoneal inoculation of water concentrates. While the cultural and morphological characteristics indicated the isolates wereLegionella, they did not react with antibodies prepared against known species of the genus. Antisera prepared against one of the isolates reacted maximally with the other ten isolates. Fluorescent antibody analysis of water concentrates from geographically disparate sites indicated that the environmental distribution is broad and that concentrations of the Oak Ridge strain ofLegionella were similar to those of serogroups 1, 2, 3, and 4 ofLegionella pneumophila.     

Resistance to copper ions and antibiotics in marine heterotrophic bacteria in the coastal waters of Vietnam

Copper and antibiotic resistance was experimentally studied for the first time in marine heterotrophic bacteria that were isolated from microfoulings of copper and copper-alloy test plates in coastal waters of Vietnam. Resistance to copper ions and to at least one of the antibiotics tested was detected in 78.7% of the isolates. A total of 28 models of antibiotic resistance were found in the bacteria. All strains isolated from the foulings of the copper plates were resistant to seven or more antimicrobials. The microfouling communities of copper and copper-alloy plates were dominated by Bacillus spp. and Staphylococcus spp. Copper and antibiotic resistance was statistically independent of the taxon of the tested bacteria.