Survival of Bacillus subtilis AF 1 in the bacterized peanut rhizosphere and its influence on native microflora and seedling growth

A mutant of Bacillus subtilis AF 1 (an antifungal strain with plant growth-promoting activity, resistant to 100 g streptomycin/ml) was isolated from pigeon pea rhizosphere and, when used in peanut bacterization, decreased fungal and bacterial numbers and increased the actinomycete population in the rhizosphere. Over 28 days, AF 1 declined by 0.9 log c.f.u. in sterilized soil and by 2.6 log c.f.u. in native soil. From the seedlings raised with peanut seeds bacterized with AF 1 at 6.5 log c.f.u./seed, 4.1 and 4.7 log c.f.u. of AF 1 were recovered from the rhizosphere and total underground parts, respectively. Bacterization with AF 1 had no significant growth-promoting or -inhibiting effect on the peanut.     

Apparatus for the electrical characterisation of conductive fluids

Non-invasive and fully automated conductimetric measurements of electrolyte and bacterial samples were achieved in a closed volume test cell, comprising a magnetic field coil and detector. By monitoring field induced currents in sample electrolytes the magnitude of the sample current was shown to vary as the inverse of the sample impedance. The impedance characteristic was shown to be that of an LCR resonant circuit. This characteristic is primarily a function of the applied frequency and the solution/cell properties being dependent on the solution conductivity and dielectric permittivity at any given concentration. Small changes in sample dielectric permittivity in the presence of a large background conductivity are shown to be significant.The apparatus described can provide fixed or swept frequency conductivity measurements in the range 1 kHz to 2.25 MHz with a lower conductivity sensitivity of 0.9 × 10⁻³ Scm⁻¹. Bulk impedimetric characteristics of cell suspensions are derived by a two stage measurement.     

The effect of udder infection on the bacterial flora of the bulk milk of ten dairy herds

The significance of udder infection as a factor increasing the bacterial count of herd bulk milk was measured monthly for one year in ten dairy herds in Southern England. Staphylococcus aureus or mastitis streptococci were detected in 86% of samples, usually in numbers between 1000 and 10 000 c.f.u./ml of milk. However, in 8 and 2% of samples respectively > 20 000 or 100 000 c.f.u. of mastitis pathogens/ml of milk were detected. This occurred most commonly in the herds with a high incidence of Streptococcus uberis mastitis. The total bacterial counts of the herds' milks varied between 13 960 and 46 230 c.f.u./ml in the winter and between 6510 and 63 000 c.f.u./ml in the summer. No correlation was found between bacteriological quality of herd milk and the cleanliness of the milking machine and pipeline as assessed by plant rinses.     

The effect of udder infection on the bacterial flora of the bulk milk of ten dairy herds

The significance of udder infection as a factor increasing the bacterial count of herd bulk milk was measured monthly for one year in ten dairy herds in Southern England. Staphylococcus aureus or mastitis streptococci were detected in 86% of samples, usually in numbers between 1000 and 10 000 c.f.u./ml of milk. However, in 8 and 2% of samples respectively greater than 20 000 or 100 000 c.f.u. of mastitis pathogens/ml of milk were detected. This occurred most commonly in the herds with a high incidence of Streptococcus uberis mastitis. The total bacterial counts of the herds' milks varied between 13 960 and 46 230 c.f.u./ml in the winter and between 6510 and 63 000 c.f.u./ml in the summer. No correlation was found between bacteriological quality of herd milk and the cleanliness of the milking machine and pipeline as assessed by plant rinses.     

Endophytic bacterial flora in the stem tissue of a tropical maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) genotype: isolation,identification and enumeration

The genotypic diversity of indigenous bacterial endophytes within stem of tropical maize (Zea mays L.) was determined in field and greenhouse experiments. Strains were isolated from stem tissues of a tropical maize cultivar (PEHM-1) by trituration and surface disinfestation and their population dynamics was determined. Endophytes were found in most of the growing season at populations ranging from 1.36–6.12 × 10⁵ colony-forming units per gram fresh weight (c.f.u./gm fw) of stem. Analysis of these bacterial endophytes using Gas Chromatography—Fatty Acid Methyl Ester (GC-FAME) led to the identification of Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens as the relatively more predominant group of bacterial species residing in maize stem. When the maize seedlings grown in a greenhouse were inoculated with these four isolates individually, their population densities decreased (1.6–3.1 × 10⁵ c.f.u./gm fw of stem) as compared to the field-grown maize (1.8–3.8 × 10⁵ c.f.u./gm fw of stem). The highest persistence, however, was recovered in the case of B. subtilis with a population density of 3.1 × 10⁵ c.f.u./gm fw of stem tissue on 28 days after emergence (DAE). This is the first report on population dynamics of bacterial endophytes from tropical maize and the results establish that symptomless populations of bacteria exist in the maize stem.     

Studies on biologically active complexes of cobalt(II) and nickel(II) with dithiooxamide-derived ligands.

A number of cobalt(II) and nickel(II) complexes with novel dithiooxamide ligands derived from precursors such as benzonitrile, benzosulfonamide, benzaldehyde, benzoic acid, and phenol have been synthesized and characterized on the basis of their elemental analysis molar conductance, magnetic moment, H NMR and electronic and IR spectral data. These ligands and their complexes have been screened for their antibacterial activities against bacterial species Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Tuberculi bacilli. The studies indicate that in comparison to the ligands their metal complexes are more potent and antibacterial.     

Ampicillin resistance and penicillin-binding proteins of Haemophilus influenzae

Penicillin-binding protein (PBP) alterations have been associated with non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. influenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 microgram ml-1 for the susceptible isolates at an inoculum of 100,000 c.f.u. when tested by broth dilution, and was 0.5 microgram ml-1 for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 micrograms ml-1 when tested by broth dilution, and from 1.5 to 16 micrograms ml-1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in vivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillin-susceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two of the resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of bacterial antagonists on lettuce: active biocontrol of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms

The aim of this study was to assess the biocontrol efficacy against Rhizoctonia solani of three bacterial antagonists introduced into naturally Rhizoctonia-infested lettuce fields and to analyse their impact on the indigenous plant-associated bacteria and fungi. Lettuce seedlings were inoculated with bacterial suspensions of two endophytic strains, Serratia plymuthica 3Re4-18 and Pseudomonas trivialis 3Re2-7, and with the rhizobacterium Pseudomonas fluorescens L13-6-12 7 days before and 5 days after planting in the field. Similar statistically significant biocontrol effects were observed for all applied bacterial antagonists compared with the uninoculated control. Single-strand conformation polymorphism analysis of 16S rRNA gene or ITS1 fragments revealed a highly diverse rhizosphere and a less diverse endophytic microbial community for lettuce. Representatives of several bacterial (Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Bacteriodetes), fungal (Ascomycetes, Basidiomycetes) and protist (Oomycetes) groups were present inside or on lettuce plants. Surprisingly, given that lettuce is a vegetable that is eaten raw, species of genera such as Flavobacterium, Burkholderia, Staphylococcus, Cladosporium and Aspergillus, which contain potentially human pathogenic strains, were identified. Analysis of the indigenous bacterial and endophytic fungal populations revealed only negligible, short-term effects resulting from the bacterial treatments, and that they were more influenced by field site, plant growth stage and microenvironment.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Immunomodulation by Candida albicans: crucial role of organ colonization and chronic infection with an attenuated agerminative strain of C. albicans for establishment of anti-infectious protection

Intravenous inoculation of an attenuated agerminative strain of Candida albicans (PCA-2) of low virulence, but not of two other species of Candida of low virulence (C. parapsilosis and C. viswanathii) into CD2F1 mice conferred protection against the highly virulent microbes C. albicans CA-6, Staphylococcus aureus and Aspergillus fumigatus. To provide protection, a definite inoculum size (10(6) cells per mouse) resulting in organ colonization and establishment of a long-lasting chronic infection with PCA-2 was needed. An inoculum of 10(5) cells gave rise to transient kidney colonization whereas inocula greater than 10(6) cells led to acute septicaemia and eventual death. Chronic infection of mice following inoculation of 10(6) PCA-2 cells was accompanied by detectable mannoprotein antigen levels in the serum (30-70 ng ml-1) while specific antibodies did not appear until 14 d after inoculation, at which time low antimannan antibody was present (ELISA titre 1:40-1:80). Chronic infection was characterized by the presence in the kidneys of 2-3 x 10(6) c.f.u. of PCA-2 for at least 40 d after inoculation. Pharmacological modulation of the host through administration of either an anti-Candida drug, amphotericin B, or an immunosuppressive agent, cyclophosphamide, strongly supported the premise that the anti-infectious state conferred by PCA-2 'immunization' correlated with the maintenance of a sufficient number of PCA-2 in vivo. Protection was 'switched on' when 2-3 x 10(5) cells were present in the kidneys. It was maximal at a kidney count of 2-3 x 10(6) c.f.u. of PCA-2, and promptly declined when the number of PCA-2 cells in the kidney fell below 2 x 10(5). Mice chronically infected with PCA-2 had splenic macrophages with pronounced candidacidal activity in vitro. Modulation of the growth of PCA-2 in vivo, which determined activation or deactivation of the protective state, was paralleled by a similar modulation in macrophage activation, showing that in all cases resistance to virulent organisms persisted as long as macrophage activation was present. The results demonstrate that a critical in vivo antigenic load is crucial for the occurrence of resistance to infection and suggests that macrophages could be involved in this protection.     

Microbial Diversity in Ngari,Hentak and Tungtap,Fermented Fish Products of North-East India

Ngari, hentak and tungtap are traditional fermented fish products of North-East India. Eighteen samples of ngari, hentak and tungtap were collected and were analysed for microbial load. Lactic acid bacteria, endospore-forming rods, yeasts and aerobic mesophilic counts ranged from 4.0 to 7.2, 3.3–4.6, <1–3.5 and 4.3–7.3 log c.f.u./g, respectively. Lactic acid bacteria were identified as Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Enterococcus faecium, Lactobacillus fructosus, Lactobacillus amylophilus, Lactobacillus coryniformis subsp. torquens and Lactobacillus plantarum. Endospore-forming rods were identified as Bacillus subtilis and Bacillus pumilus, aerobic coccal strains were identified as Micrococcus. Yeasts were identified as species of Candida and Saccharomycopsis. Pathogenic contaminants were detected in all samples, however, none of the sample contained more than 10² c.f.u./g of Bacillus cereus, 10³ c.f.u./g of Staphylococcus aureus and enterobacteriaceae population, respectively. Enzymatic and antimicrobial activities of the isolates were tested. None of the strains produced biogenic amines in the method applied. Most strains of LAB had a high degree of hydrophobicity, indicating their ‘probiotic’ characters. This study has demonstrated the microbial diversity within the species of lactic acid bacteria, Bacillus and yeasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Some Are More Equal - A Comparative Study on Swab Uptake and Release of Bacterial Suspensions

Objectives

Swabs are widely used to collect samples for microbiological analyses from various clinical settings. They vary by material, size, and structure of the tip. This study investigates the uptake and release capacities for liquid and bacteria.

Methods

Five swabs were analyzed for their uptake and release capacities of Staphylococcus aureus and Staphylococcus epidermidis suspensions. Two approaches were investigated providing volume-restricted and unrestricted amounts of bacterial suspensions to mimic various clinical situations. Volume and bacterial uptake and release were measured in milligrams and by counting colony forming units (CFU), respectively.

Results

Volume uptake and release in the unrestricted setting varied highly significant between 239.6 mg and 88.7 mg (p<0.001) and between 65.2 mg and 2.2 mg (p<0.001), respectively. In the volume-restricted setting the complete volume was absorbed by all swabs, volume release could only be detected for flocked swabs (2.7 mg; p<0.001). Highest amount of CFU release was detected for the MWE Dryswab in the unrestricted setting for both S. aureus and S. epidermidis with 1544 CFU and 553 CFU, respectively, lowest release for the Sarstedt neutral swab with 32 CFU and 17 CFU, respectively (p<0.001). In the volume-restricted setting MWE Σ-Swab released the highest bacterial amount with 135 CFU S. aureus and 55 CFU S. epidermidis, respectively, the lowest amount was released by Mast Mastaswab with 2 CFU S. aureus and 1 CFU S. epidermidis, respectively (p<0.001). Within the range of the utilized bacterial concentrations, uptake/release ratios were identical for the particular swab types and independent of the bacterial species.

Conclusions

The influence of the swab type on subsequent diagnostic results is often underestimated. Uptake and release of the investigated bacteria vary significantly between different swab types and sampling conditions. For best diagnostic outcome swabs should be chosen according to the examined situation and the swab performance profile.     

Effect of CaCO<Subscript>3</Subscript> particles and suspended bacteria on biofilm components and activity in the model recirculating cooling water system

Biofilms are a serious problem in industrial recirculating cooling water systems. Biofilm formation and properties are affected by many factors, such as inorganic particles and suspended bacteria. In this research a laboratory model recirculating cooling water system was applied to investigate the effects of CaCO₃ concentration and suspended bacterial count on extracellular polymeric substances (EPS) content and dehydrogenase activity (DHA) in the attached biofilms. In addition, nutrient level was also the key factor when investigating the effect of suspended bacterial count. The results showed that EPS content and DHA first increased and then decreased with the increase of CaCO₃ concentration from 0 to 200 mg/l. At the low nutrient level, with the increase of suspended bacterial count from 4.04 to 5.78 log₁₀ c.f.u./ml, biofilm EPS content decreased firstly and then increased. However, biofilm DHA always gradually increased. At the medium nutrient level, biofilm EPS content increased firstly and then decreased and DHA always gradually decreased when suspended bacterial count ranged from 4.04 to 5.78 log₁₀ c.f.u./ml. At the high nutrient level, biofilm EPS content and DHA both showed the increasing trend with the increase of suspended bacterial count. This work provides the basis and reference for management strategies in actual recirculating cooling water systems.     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.     

Augmentation of antibiotic activity by low-frequency electric and electromagnetic fields examining Staphylococcus aureus in broth media

Systemic treatment of biomaterial‐associated bacterial infections with high doses of antibiotics is an established therapeutic concept. The purpose of this in vitro study was to determine the influence of magnetic, electromagnetic, and electric fields on gentamicin‐based, antibiotic therapy. It has been previously reported that these fields are successful in the treatment of bone healing and reducing osteitis in infected tibia‐pseudarthroses. Four separate experimental setups were used to expose bacterial cultures of Staphylococcus aureus both in Mueller‐Hinton broth (MHB) and on Mueller‐Hinton agar (MHA), in the presence of gentamicin, to (1) a low‐frequency magnetic field (MF) 20 Hz, 5 mT; (2) a low‐frequency MF combined with an additional alternating electric field (MF + EF) 20 Hz, 5 mT, 470 mV/cm; (3) a sinusoidal alternating electric field (EF AC) 20 Hz, 470 mV/cm; and (4) a direct current electric field (EF DC) 588 mV/cm. No significant difference between samples and controls was detected on MHA. However, in MHB each of the four fields applied showed a significant growth reduction of planktonically grown Staphylococcus aureus in the presence of gentamicin between 32% and 91% within 24 h of the experiment. The best results were obtained by a direct current EF, decreasing colony‐forming units (CFU)/ml more than 91%. The application of electromagnetic fields in the area of implant and bone infections could offer new perspectives in antibiotic treatment and antimicrobial chemotherapy. Bioelectromagnetics 32:367–377, 2011. © 2011 Wiley‐Liss, Inc.     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water.

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.     

The effect of the extremely weak field on the cultures of bacteria Escherichia coli and Staphylococcus aureus

The effect of extremely weak human electromagnetic field on the suspensions of Escherichia coli and Staphylococcus aureus in a physiological solution at 25 degrees C was studied. The results obtained indicate that the human field induces changes in the physical properties of liquid water in physiological solution and the induced structural and dynamic rearrangements of extracellular water are tranferred to bacterial cells and change their functional activity.     

Antimicrobial activity of Mitracarpus scaber extract and isolated constituents

The antimicrobial activity of a methanol extract and isolated constituents of Mitracarpus scaber, a species used in folk medicine by West African native people, was evaluated against Staphylococcus aureus and Candida albicans strains. The mitracarpus methanol extract possesses both antibacterial and antimycotic activities (minimum inhibitory concentration-MIC 31.25 and 62.50 microg ml-, respectively). This extract was subsequently fractioned and monitored by bioassays leading to the isolation of seven compounds screened for antibacterial and antimycotic activities. Among these compounds, gallic acid and 3,4,5-trimethoxybenzoic acid inhibited the growth of Staph. aureus (MIC 3.90 and 0.97 microg ml-). 4-Methoxyacetophenone and 3,4,5-trimethoxyacetophenone effectively inhibited C. albicans (MIC 1.95 microg ml-). The other compounds (kaempferol-3-O-rutinoside, rutin and psoralen) which were also isolated showed low antibacterial and antimycotic activities (125-500 microg ml-).     

PCR-DGGE analysis for the identification of microbial populations from Argentinean dry fermented sausages

Different PCR-DGGE protocols were evaluated to monitor fermentation process and to investigate bacterial communities developed in two artisanal Argentinean fermented sausages. Bacterial universal primers frequently used in PCR-denaturing gradient gel electrophoresis (DGGE) were evaluated. Lactic acid bacteria (LAB) and staphylococci species isolated from Tucumán sausages were used to determine the experimental conditions for PCR amplification and DGGE differentiation. Total microbial DNA extracted directly from both fermented sausages was subjected to DGGE analysis. PCR-DGGE results were different for each set of primers used. Primers Bact-0124f(GC)-Uni-0515r and V1f(GC)-V1r showed to be efficient to differentiate LAB and Staphylococcus cultures while the set V3f(GC)-Uni-0515r allowed to demonstrate the succession of different Lactobacillus and Staphylococcus species during ripening process. An intense band corresponding to Lactobacillus sakei was observed to be present in both samples. Staphylococcus saprophyticus was only observed in Tucumán sausage while a band identified as Brochothrix thermophacta was detected in Córdoba sausage. PCR-DGGE analysis of different 16S rDNA amplicons was able to discriminate between LAB and Gram-positive, coagulase-negative cocci, resulting an effective tool to establish the microbiota developed in artisanal dry sausages.