Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C₅ and C₆ sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216^T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 10³ colonies per μg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C₅ and C₆ sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.     

Sensitivity of an Immunomagnetic-Separation-Based Test for Detecting Escherichia coli O26 in Bovine Feces

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED₈₀) was 1.0 × 10⁴ CFU g⁻¹ feces (95% confidence interval, 4.7 × 10³ to 2.4 × 10⁴). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED₈₀ for each strain and fecal pat combination ranged from 4.2 × 10² to 4.8 × 10⁵ CFU g⁻¹. These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Quantitative Detection of the Free-Living Amoeba Hartmannella vermiformis in Surface Water by Using Real-Time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 × 10⁻¹ and 1.14 × 10⁴ cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 ± 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (≥98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.     

Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of Escherichia coli O157:H7

The influence of nutrients in wastewater from dairy lagoons on the survival of Escherichia coli O157:H7 was monitored. Initially, the survival of E. coli O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of E. coli O157:H7 or E. coli ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of E. coli O157:H7 strains was monitored in 300 μl of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater. E. coli O157:H7 and E. coli ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of E. coli O157:H7 and E. coli ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of E. coli O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components, E. coli O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.     

Mutation of Candida tropicalis by Irradiation with a He-Ne Laser To Increase Its Ability To Degrade Phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 × 10⁷ cells ml⁻¹ were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter⁻¹ phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter⁻¹. The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Recognition of Individual Genes in Diverse Microorganisms by Cycling Primed In Situ Amplification

Cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) was developed to recognize individual genes in a single bacterial cell. In CPRINS, the amplicon was long single-stranded DNA and thus retained within the permeabilized microbial cells. FISH with a multiply labeled fluorescent probe set enabled significant reduction in nonspecific background while maintaining high fluorescence signals of target bacteria. The ampicillin resistance gene in Escherichia coli, chloramphenicol acetyltransferase gene in different gram-negative strains, and RNA polymerase sigma factor (rpoD) gene in Aeromonas spp. could be detected under identical permeabilization conditions. After concentration of environmental freshwater samples onto polycarbonate filters and subsequent coating of filters in gelatin, no decrease in bacterial cell numbers was observed with extensive permeabilization. The detection rates of bacterioplankton in river and pond water samples by CPRINS-FISH with a universal 16S rRNA gene primer and probe set ranged from 65 to 76% of total cell counts (mean, 71%). The concentrations of cells detected by CPRINS-FISH targeting of the rpoD genes of Aeromonas sobria and A. hydrophila in the water samples varied between 2.1 × 10³ and 9.0 × 10³ cells ml⁻¹ and between undetectable and 5.1 × 10² cells ml⁻¹, respectively. These results demonstrate that CPRINS-FISH provides a high sensitivity for microscopic detection of bacteria carrying a specific gene in natural aquatic samples.     

Enhanced Surface Colonization by Escherichia coli O157:H7 in Biofilms Formed by an Acinetobacter calcoaceticus Isolate from Meat-Processing Environments

A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 × 10⁶ μm³) in a dynamic cocultured biofilm than in a monoculture (3.0 × 10³ μm³), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.Shiga toxin-producing Escherichia coli (STEC) is a food-borne human pathogen responsible for severe gastrointestinal disease (16, 17). Processing, handling, and preparation of food may lead to cross-contamination of food and uncontaminated surface areas of the food chain with pathogens from contaminated surfaces (8). Though most processing plants ensure and maintain good manufacturing practices with elaborated sanitary operations, persisting microorganisms may survive well after cleaning and disinfection procedures (1, 9, 12-14), possibly in the form of biofilms (11). A review of the underlying problems caused by biofilms in the food industry was presented by Carpentier and Cerf (4). Several studies have shown that E. coli, including STEC strains, has the capacity to attach to and form biofilms on various surface materials (5, 18). However, such studies have mainly used monocultures without considering the possible influence of resident organisms from food-processing environments on the surface colonization of E. coli. One recent study showed that resident microflora increased E. coli O157:H7 colonization on solid surfaces under static conditions (10). To our knowledge, no studies have investigated the influence of meat industry resident bacteria on surface colonization by E. coli under dynamic-flow conditions.The aim of this study was to investigate how a biofilm-forming isolate of Acinetobacter calcoaceticus influences surface colonization by E. coli O157:H7. This study focused on the spatial distribution of cells during biofilm formation under static and dynamic growth conditions.Here we used an A. calcoaceticus strain (MF3627) isolated from a clean and disinfected meat-processing environment, as well as Shiga toxin-negative E. coli O157:H7 (ATCC 43888) harboring the plasmid pGFP-uv (Clontech Laboratories, Palo Alto, CA). For static growth conditions, mono- and coculture biofilms were harvested at 25°C in Lab-Tek II chamber slide systems (VWR, Oslo, Norway) consisting of miniature polystyrene medium chambers with a sealed cover glass as the growth surface. For dynamic growth conditions, mono- and coculture biofilms were grown at 25°C in three-channel flow cells with individual channel dimensions of 1 by 4 by 40 mm and a sealed glass coverslip substratum (Knittel Glass, Germany). A 1/10 dilution of Luria-Bertani broth was continuously pumped through the sterile flow cell channels at a rate of 0.5 ml/min. In two of the channels, A. calcoaceticus and E. coli were inoculated individually, while the third channel was reserved for the inoculation of a mixed culture of A. calcoaceticus and E. coli (1:1). The flow cell channels and Lab-Tek chambers were stained with SYTO 61. Horizontal-plane images of the biofilms were acquired using a Leica SP5 AOBS laser scanning confocal microscope (Leica Microsystems, Lysaker, Norway). Three independent biofilm experiments were performed for each biofilm growth condition. Three-dimensional projections were performed with IMARIS software (Bitplane, Zürich, Switzerland). The structural quantification of biofilms (biovolume in cubic micrometers) was performed using the PHLIP Matlab program (http://www.phlip.org/phlip-ml/).Under static growth conditions, E. coli O157:H7 formed a homogeneous flat biofilm yielding biovolumes ranging between 3.3 × 10⁵ and 5.4 × 10⁵ μm³ after 24 and 72 h of biofilm growth. Although the E. coli biovolume revealed no significant differences in monoculture or when cocultured with A. calcoaceticus, microscopic analysis revealed how E. coli cells were gradually covered by a carpet of A. calcoaceticus cells after 72 h of biofilm growth (for visualization, see the supplemental material). A. calcoaceticus monospecies biofilms were heterogeneous, highly structured, and channeled under both static and dynamic conditions (Fig. ​(Fig.11 A), yielding a biovolume of 1.46 × 10⁶ μm³ after 72 h of biofilm growth (Fig. ​(Fig.2).2). E. coli O157:H7 did not form monospecies biofilms under dynamic-flow conditions (Fig. ​(Fig.1A),1A), with biovolume values below 3.5 × 10⁴ μm³ after 72 h (Fig. ​(Fig.2).2). The presence of A. calcoaceticus had a significant impact on E. coli O157:H7 surface colonization with a 400-fold increase in the total biovolume of E. coli O157:H7 from 3.0 × 10³ μm³ to 1.2 × 10⁶ μm³ between 24 and 48 h (Fig. ​(Fig.2),2), as observed from the increase in E. coli O157:H7 biomass between A. calcoaceticus cell clusters (Fig. 1A and B). After 72 h of development, E. coli O157:H7 cell clusters were partially covered by A. calcoaceticus cells. The poor settlement and subsequent poor colonization of E. coli O157:H7 under dynamic-flow conditions could have been attributed to shear forces, which made it difficult for E. coli O157:H7 cells to establish colonies on the substratum. The observed spatial distribution of A. calcoaceticus cells at the liquid-biofilm interface may offer E. coli O157:H7 cells better protection from shear stress and could potentially provide additional protection against disinfectants, as has been observed in other multispecies biofilm studies (2, 21). Whether E. coli cells had increased resistance to antimicrobial agents in our experimental setup as a result of being at the bottom layers of mixed-species biofilms will be the subject of further investigations. Biofilm formation of meat industry surface bacteria can enhance E. coli surface colonization and thereby increase the risk of persistence of and food contamination by potential pathogens. The occurrence of Acinetobacter in food-processing environments is well documented (1, 9, 15), and it has also been isolated from spoiled food products (3, 6, 7). Furthermore, a recent study showed that A. calcoaceticus biofilms are able to interact and coaggregate with other bacteria (19). Cleaning and disinfection procedures used in food industries should thus take into account the risks involved in ignoring the presence of resident flora biofilms.Open in a separate windowFIG. 1.Structural development of A. calcoaceticus and E. coli in mono- and dual-species biofilms under dynamic conditions. (A) Representative biofilms of A. calcoaceticus and pGFP-uv-tagged E. coli O157:H7 grown in flow cells using Luria-Bertani broth as a growth medium at 25°C. The spatial structures in the developing biofilms were studied by laser scanning confocal microscopy. (B) Vertical sections (in the x-z plane) representing the spatial distribution of pGFP-uv-tagged E. coli O157:H7 in the presence of A. calcoaceticus under dynamic-flow conditions after 24, 48, and 72 h of growth. The lower side of each section corresponds to the substratum. Green cells represent pGFP-uv-tagged E. coli O157:H7, red cells represent SYTO 61-stained A. calcoaceticus cells, and yellow cells represent GFP-tagged E. coli O157:H7 marked with SYTO 61.Open in a separate windowFIG. 2.Biovolume of A. calcoaceticus and E. coli O157:H7 biofilm development after 24, 48, and 72 h of growth under dynamic conditions. A. calcoaceticus in monospecies biofilms is represented by the symbol □, A. calcoaceticus in dual-species biofilms is represented by the symbol ▪, E. coli O157:H7 in mono-species biofilms is represented by the symbol Δ, and E. coli O157:H7 in dual-species biofilms is represented by the symbol ▴. Mean values of at least 30 individual images ± the standard errors from three independent experiments are shown.Cleaning and disinfection procedures are employed by the food industry to ensure clean and hygienic surfaces for food production. However, due to the ubiquitous nature of biofilms and their potential to resist antimicrobial treatments (21), new strategies based on preventive actions to reduce the incidence of biofilm formation on food-processing surfaces should be employed (20). In light of the results obtained in this study, combining curative actions with preventive actions based on the use of surface materials with antiadhesive or antifouling surfaces could enhance the hygienic standards of food-processing surfaces.In conclusion, we have shown that under both static and dynamic growth conditions, E. coli cells were found embedded and covered by A. calcoaceticus cells in mixed-species biofilms. Moreover, the presence of an A. calcoaceticus biofilm structure favored E. coli O157:H7 colonization and biofilm formation under dynamic-flow conditions. These results offer new insights into the spatial distribution of pathogenic bacteria and resident flora during cocultured biofilm formation. Conditions allowing active biofilm formation of resident microflora may provide increased opportunities for pathogens to thrive in food-processing environments. The hazardous influences of resident biofilms should therefore not be ignored, since improper cleaning procedures in food-processing environments could potentially increase the risk of food contamination by spoilage and pathogenic bacteria.      

Determination of the Hydrogenase Status of Individual Legume Nodules by a Methylene Blue Reduction Assay

We adapted a method for the rapid screening of colonies of free-living Rhizobium japonicum for hydrogenase activity to determine the hydrogenase status of individual soybean nodules. Crude bacteroid suspensions from nodules containing strains known to be hydrogen uptake positive (Hup⁺) caused a localized decolorization of filter paper disks, whereas suspensions from nodules arising from inoculation with hydrogen uptake-negative (Hup⁻) mutants or strains did not decolorize the disks. The reliability of the method was demonstrated by its successful application to 29 slow-growing rhizobia. The Hup phenotype on methylene blue filters agreed with that determined amperometrically with either methylene blue or oxygen as the electron acceptor.     

Effect of Surface-Active Pseudomonas spp. on Leaf Wettability

Different strains of Pseudomonas putida and P. fluorescens isolated from the rhizosphere and phyllosphere were tested for surface activity in droplet cultures on polystyrene. Droplets of 6 of the 12 wild types tested spread over the surface during incubation, and these strains were considered surface active; strains not showing this reaction were considered non-surface active. Similar reactions were observed on pieces of wheat leaves. Supernatants from centrifuged broth cultures behaved like droplets of suspensions in broth; exposure to 100°C destroyed the activity. Average contact angles of the supernatants of surface-active and non-surface-active strains on polystyrene were 24° and 72°, respectively. The minimal surface tension of supernatants of the surface-active strains was about 46 mN/m, whereas that of the non-surface-active strains was 64 mN/m (estimations from Zisman plots). After 6 days of incubation, wheat flag leaves sprayed with a dilute suspension of a surface-active strain of P. putida (WCS 358RR) showed a significant increase in leaf wettability, which was determined by contact angle measurements. Increasing the initial concentration of bacteria and the amount of nutrients in the inoculum sprayed on leaves reduced the contact angles from 138° on leaves treated with antibiotics (control) to 43° on leaves treated with surface-active bacteria. A closely related strain with no surface activity on polystyrene did not affect leaf wettability, although it was present in densities similar to those of the surface-active strain. Nutrients alone could occasionally also increase leaf wettability, apparently by stimulating naturally occurring surface-active bacteria. When estimating densities of Pseudomonas spp. underneath droplets with low contact angles, it appeared that populations on leaves treated with a surface-active strain could vary from about 10⁴ to 10⁶ CFU cm⁻², suggesting that the surface effect may be prolonged after a decline of the population. The possible ecological implications are discussed.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth,constitute a new genomovar of Acinetobacter calcoaceticus

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006^T, as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus–baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S–23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA–DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.     

Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7

A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10¹⁰, 10⁹, and 10⁸ PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10⁹ PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.     

Bdellovibrio and Like Organisms Enhanced Growth and Survival of Penaeus monodon and Altered Bacterial Community Structures in Its Rearing Water

In this study, a 96-h laboratory reduction test was conducted with strain BDHSH06 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF011103","term_id":"120561011","term_text":"EF011103"}}EF011103) as the test strain for Bdellovibrio and like organisms (BALOs) and 20 susceptible marine bacterial strains forming microcosms as the targets. The results showed that BDHSH06 reduced the levels of approximately 50% of prey bacterial strains within 96 h in the seawater microcosms. An 85-day black tiger shrimp (Penaeus monodon) rearing experiment was performed. The shrimp survival rate, body length, and weight in the test tanks were 48.1% ± 1.2%, 99.8 ± 10.0 mm, and 6.36 ± 1.50 g, respectively, which were values significantly (P < 0.05) higher than those for the control, viz., 31.0% ± 2.1%, 86.0 ± 11.1 mm, and 4.21 ± 1.56 g, respectively. With the addition of BDHSH06, total bacterial and Vibrio numbers were significantly reduced (P < 0.05) by 1.3 to 4.5 log CFU · ml⁻¹ and CFU · g⁻¹ in both water and shrimp intestines, respectively, compared to those in the control. The effect of BDHSH06 on bacterial community structures in the rearing water was also examined using PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of rearing water samples from the control and test tanks revealed that the amounts of 44% of the bacterial species were reduced when BDHSH06 was added to the rearing water over the 85-day rearing period, and among these, approximately 57.1% were nonculturable. The results of this study demonstrated that BDHSH06 can be used as a biocontrol/probiotic agent in P. monodon culture.     

Microflora of Drainage from Ice in Fishing Vessel Fishholds

The microbial load in ice-melt drainage collected from fishholds of fishing vessels stowing lizard fish, black croakers, cuttle fish, or nemipterids was very high, ranging from 2.1 × 10⁷ to 2.2 × 10⁹/ml for bacteria and 6.3 × 10³ to 7.2 × 10⁴/ml for yeasts and molds. Analysis of 100 colonies each randomly isolated from drainage samples of cuttle fish and lizard fish showed that the occurrence of bacterial genera as a percentage of the total was Moraxella-Acinetobacter, 61 to 62%; Pseudomonas, 19 to 21%; Alcaligens, 5 to 10%; Flavobacterium, 1 to 4%; Micrococcus, 1 to 4%; Bacillus, Vibrio 0 to 2%; Corynebacterium, 1 to 2%; and others, 1 to 2%. The organisms demonstrated versatile hydrolytic activities to a wide range of biological substrates including casein, gelatin, starch, DNA, and RNA. The possible connection between these bacteria and the deterioration of fish quality are discussed.