A microcalorimetric method for studying the biological effects of La(3+) on Escherichia coli

A microcalorimetric technique based on the bacterial heat-output was explored to evaluate the stimulatory effect of La(3+) on Escherichia coli. The power-time curves of the growth metabolism of E. coli and the effect of La(3+) on it were studied using an LKB-2277 BioActivity Monitor, stopped-flow method, at 37 degrees C. For evaluation of the results, the maximum power (P(max)), the growth rate constants (k) and the heat effects (Q(LOG), Q(STAT)) for the log phase, the stationary phase and the total heat effect (Q(T)) for E. coli were determined. The microcalorimetric method agreed with the conventional methods, such as cell numbers and biomass. La(3+) in the concentration ranges of 0-400 microg/ml has stimulatory effects on E. coli, while La(3+) ion of higher concentrations (>400 microg/ml) can inhibit the growth. This phenomenon is very similar to those observed from the in vitro cells and tissues from animals, plants and some microorganisms by other methods.     

Isolation and functional analysis of a 24-residue linear alpha-helical antimicrobial peptide from Korean blackish cicada, Cryptotympana dubia (Homoptera)

A new antimicrobial peptide, cryptonin, was isolated and characterized from the adult Korean blackish cicada, Cryptotympana dubia. It consists of 24 amino acid residues and has a molecular weight of 2,704 Da on mass spectroscopy. The predicted alpha-helical structure analysis and increased helix percent in 40% trifloroethanol of cryptonin suggests that it belongs to the typical linear alpha-helix forming peptide. Binding of the biotin-labeled cryptonin at the surface of E. coli cells and increased influx of propidium iodide in E. coli after cryptonin treatment indicates that it kills microbial cells by binding bacterial cell surfaces and disrupting the cell permeability. Cryptonin showed strong antibacterial (MIC 1.56-25 microg/ml) and antifungal (MIC 3.12-50 microg/ml) activities against tested bacteria and fungi including two antibiotic-resistant bacterial strains; methicilin-resistant S. aureus and vancomycin-resistant Enterococci (MIC 25 microg/ml, each).     

Detection of Escherichia coli in blood using flow cytometry

A rapid method for the detection of Escherichia coli in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coli/ml and from in vivo rabbit model studies at less than 100 E. coli/ml.     

Kenojeinin I, antimicrobial peptide isolated from the skin of the fermented skate, Raja kenojei

An antimicrobial peptide was purified from fermented skate skin extract using the solid-phase extraction and separation on HPLC reversed-phase chromatography. Amino acid sequence of the purified peptide (Peak A) having an antimicrobial activity revealed the presence of many cationic residues of the total 28 amino acids. Its molecular mass was found to be 3059 Da. This result was in excellent agreement with the theoretical molecular mass calculated from the amino acid sequence. The synthetic kenojeinin I had inhibitory effects on B. subtilis (MIC, 12 microg/ml), E. coli (28 microg/ml), and S. cerevisiae (12 microg/ml). These results indicate that fermented skate skin is potentially antimicrobial.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro.

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Cytoplasmic membrane polarization in Gram-positive and Gram-negative bacteria grown in the absence and presence of tetracycline

The ability of numerous diverse compounds and ions to cross the bacterial cytoplasmic membrane by diffusion and active transport is highly dependent on cytoplasmic membrane fluidity, which can be measured using fluorescent probes to estimate membrane polarization values. However, membrane polarization data are lacking for most bacterial species. The cytoplasmic membrane polarization values for Arthrobacter sp. ATCC 21908, Bacillus cereus NRC 3045, Pseudomonas fluorescens R2F, Pseudomonas putida NRC 2986 and Escherichia coli C600 bacterial cells were spectrofluorometrically measured over a temperature range from 10 to 50 degrees C, and in the absence and presence of 1 microg/ml tetracycline, using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to obtain new information on their membrane fluidity. At an assay temperature of 10 degrees C, E. coli cells grown in the absence of tetracycline exhibited the highest cytoplasmic membrane polarization value (least fluid membrane) of 0.446, followed by values of 0.392, 0.371, 0.344 and 0.293, respectively, for B. cereus, Arthrobacter sp., P. fluorescens and P. putida. At an assay temperature of 30 degrees C, the polarization values ranged from 0.357 to 0.288 for cells grown in the absence of tetracycline, regardless of the species. B. cereus grown in the presence of 1 microg/ml tetracycline had lower polarization values than when grown in the absence of this antibiotic at all assay temperatures. Regardless of the absence or presence of 1 microg/ml tetracycline in the growth medium, all bacterial species generally exhibited a more fluid membrane as the assay temperature increased from 10 to 50 degrees C. To our knowledge, these are some of the first cytoplasmic membrane polarization values reported for these Gram-negative and Gram-positive bacteria over a broad temperature range and also for cells grown in the presence of tetracycline.     

Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 microg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 microg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 mug/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.     

Fluoroquinolone resistance and gyrA and parC mutations of Escherichia coli isolated from chicken

Escherichia coli is a common inhabitant of the intestinal tracts of animals and humans. The intestines of animals also represent an ideal environment for the selection and transfer of antimicrobial resistance genes. The aim of this study was to investigate the resistance of E. coli isolated from chicken fecal samples to fluoroquinolones and to analyze the characterization of mutations in its gyrA and parC gene related resistance. One hundred and twenty-eight E. coil isolates showed a high resistance to ciprofloxacin (CIP; 60.2%), enrofloxacin (ENO; 73.4%) and norfloxacin (NOR; 60.2%). Missense mutation in gyrA was only found in the amino acid codons of Ser-83 or Asp-87. A high percentage of isolates (60.2%) showed mutations at both amino acid codons. Missense mutation in parC was found in the amino acid codon of Ser-80 or Glu-84, and seven isolates showed mutations at both amino acid codons. Isolates with a single mutation in gyrA showed minimal inhibitory concentrations (MIC) for CIP (相似文献   

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

AIMS: The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7. METHODS AND RESULTS: Lactoferrin and nisin separately or in combination were suspended in peptone yeast glucose broth and following inoculation with L. monocytogenes or E. coli O157:H7 growth inhibition of each pathogen was determined. At 1000 microg ml(-1) lactoferrin L. monocytogenes was effectively inhibited. However, E. coli O157:H7 initially was inhibited and then grew to cell density similar to the control. A combination of 500 microg ml(-1) of lactoferrin and 250 IU ml(-1) of nisin effectively inhibited the growth of E. coli O157:H7, whereas, 250 microg ml(-1) of lactoferrin and 10 IU ml(-1) of nisin were inhibitory to L. monocytogenes. CONCLUSIONS: The results suggest that lactoferrin and nisin act synergistically to inhibit the growth of L. monocytogenes and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against gram-positive and gram-negative pathogens are desirable to the food industry and consumers. This study demonstrates that lactoferrin and nisin work synergistically reducing the levels required independently inhibiting growth of two major foodborne pathogens. Previous reported results indicated a low level of antimicrobial activity; however, this work was not performed in low divalent cation concentration media. It has been suggested that nondivalent cation-limiting medium such as trypticase soy broth (TSB), can reduce or completely eliminate the inhibitory activity. Further knowledge of these interactions can increase the understanding of the antimicrobial activity of lactoferrin. This should make the use of these compounds by industry more attractive.     

Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ . In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coli cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coli growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.     

Effects of growth factors, hormones, bacterial lipopolysaccharides, and lipotechoic acids on the clonal growth of normal ureteral epithelial cells in serum-free culture.

In vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F-12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F-12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F-12* medium could be replaced by three orders of magnitude less IGF-1. Further clonal growth experiments demonstrated that PGE1 is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP-dependent mitogenic pathway. Seven gram-negative bacterial lipopolysaccharides (LPS) and three gram-positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived from Escherichia coli (strains 055:B5, 0128:B12, and 0127:B8), LPS from Klebsiella pneumoniae, and LPS from Pseudomonas aeruginosa all showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 micrograms/ml. LPS derived from E. coli strain (0111:B4) had no growth effects at the highest concentration tested (100 micrograms/ml). In contrast, LT derived from Streptococcus pyogenes, S. faecalis, Staphylococcus aureas, and Bacillus subtilis all markedly enhanced clonal growth at concentrations ranging from 1 microgram/ml less than [LT] less than 50 micrograms/ml. LT from Strep. pyogenes was inhibitory to clonal growth at 100 micrograms/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation-competent uroepithelial cells to growth inhibition by LPS produced by gram-negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth-stimulating activity of LTs produced by gram-positive bacteria may be due to their ability to bind to cell-associated fibronectin and to activate the fibronectin receptor as part of ligand receptor-induced mitogenic transmembrane signalling pathway.     

Survival of bacterial enteric pathogens in traditional fermented foods

The survival of strains of bacterial enteric pathogens was investigated in two traditional fermented foods (mahewu and sour porridge) and in unfermented porridge. The foods were inoculated with cell suspensions of Salmonella, Shigella, Campylobacter, Aeromonas species and pathogenic Escherichia coli which had a final concentration of 10⁶-10⁷ cfu/ml of food. None of the strains of Aeromonas and Campylobacter were detected in mahewu and sour porridge 20 min after inoculation. The salmonellas were not found 4 h after inoculation in either fermented foods but the shigellas and pathogenic E. coli strains were more tolerant to the low pH of the fermented foods. Some of the shigellas and pathogenic E. coli strains survived for 24 h after inoculation but showed a sharp decrease in numbers. All the strains of the enteric pathogens survived for 24 h in the unfermented porridge and increased in the numbers except for campylobacters, the numbers of which declined. These results suggest that the traditional fermented foods have bacteriostatic and bactericidal properties and are unlikely to play a major role in the transmission of bacterial enteric pathogens.     

A luminescent Escherichia coli biosensor for the high throughput detection of beta-lactams

A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of beta-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for beta-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various beta-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams. Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-beta-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of beta-lactam action.     

Microcalorimetric study of the action of Ce(III) ions on the growth of E. coli

A microcalorimetric technique based on bacterial heat output was used to evaluate the action of Ce(III) ions on the growth of Escherichia coli. The power-time curves of the growth metabolism of the bacteria were studied in the presence and absence of Ce(III) by means of a LKB-2277 Bioactivity Monitor, by a stopped-flow method at 37°C. For evaluation of the results, the maximum power, P _max, the growth rate constant k, and the heat effects Q _log, Q _stat, and Q _tot for the log phase, the stationary phase, and total heat output, respectively, were determined. For comparison, a spectrophotometer was used to estimate the number of cells in the liquid culture. The shape of the bacteria was examined by electron microscopy. We concluded that the presence of cerium ions at concentrations below 350 μg/mL have a stimulatory effect on the growth of E. coli, whereas concentrations at or above 400 μg/mL may have an inhibitory effect.     

Investigations on anti-Aspergillus properties of bacterial products

AIMS: To investigate the anti-Aspergillus properties of bacterial products. METHODS AND RESULTS: In the present study, 12 bacterial strains were screened for antifungal activity against Aspergilli. The culture supernatant and lysates of Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli (BL21, DH5alpha, HB101, XL Blue), Klebsiella pneumoniae, Streptomyces thermonitrificans, Streptococcus pneumoniae, Enterobacter aerogenes, Staphylococcus aureus and Salmonella typhi were examined for antifungal activity in protein concentration ranging from 1000.0 to 7.8 microg ml-1 using microbroth dilution assay. The lysate of Salm. typhi and E. coli BL21 exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 15.6-31.2 microg ml-1 by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, a concentration of 3.1 microg disc-1 of Salm. typhi lysate showed significant activity against Aspergilli. Escherichia coli BL21 exhibited similar activity at 6.2 microg disc-1. The work on identification of molecule endowed with antimycotic properties is in progress. CONCLUSION: The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that E. coli has been reported for anti-Aspergillus activity. It could be an important source of biologically active compounds useful for developing better new antifungal drugs/or probiotics.     

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.