Showing papers in "Journal of Applied Microbiology in 1998"

Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract.

Abstract: An in vitro methodology which mimics in vivo human upper gastrointestinal transit was developed. The transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species was determined by exposing washed cell suspensions at 37°C to a simulated gastric juice (pH 2·0), containing pepsin (0·3% w/v) and sodium chloride (0·5% w/v), and a simulated small intestinal juice (pH 8·0), containing pancreatin USP (1 g l−1) and sodium chloride (5 g l−1), and monitoring changes in total viable count periodically. The methodology was also employed to determine the effect of adding milk proteins (1 g l−1), hog gastric mucin (1 g l−1) and soyabean trypsin-chymotrypsin inhibitor [SBTCI] (1 g l−1) on transit tolerance. The majority (14 of 15) of isolates lost >90% viability during simulated gastric transit. Only one isolate, Lactobacillus fermentum KLD, was considered intrinsically resistant. The addition of milk proteins, singly and in combination, generally improved gastric transit tolerance. In this regard, two isolates, Lact. casei 212.3 and Bifidobacterium infantis 25962, exhibited 100% gastric transit tolerance in the presence of milk proteins. In general, the addition of hog gastric mucin did not influence simulated gastric transit tolerance of lactobacilli but tended to increase that of bifidobacteria. However, it increased that of Lact. casei 242 and Lact. salivarius 43338 but diminished that of B. bifidum 2715 and B. animalis Bo. Selected bile salts-resistant isolates were intrinsically tolerant to simulated small intestinal transit. Only Lact. casei F19 and B. adolescentis 15703T showed significant reduction in viability after 240 min. In general, the addition of milk proteins and SBTCI did not affect simulated small intestinal transit tolerance. However, they significantly improved the intrinsic resistance of Lact. casei F19 but diminished that of B. breve 15700T. It is concluded that, whereas the majority of bile salts-resistant lactobacilli and bifidobacteria may be intrinsically sensitive to gastric transit, they are intrinsically resistant to small intestinal transit. In addition, it is postulated that milk proteins and mucin may function as both buffering agents and inhibitors of digestive protease activity in vivo, thereby protecting ingested bacterial strains during upper gastrointestinal transit.

A new method for determining the minimum inhibitory concentration of essential oils

Abstract: A new microdilution method has been developed for determining the minimum inhibitory concentration (MIC) of oil-based compounds. The redox dye resazurin was used to determine the MIC of a sample of the essential oil of Melaleuca alternifolia (tea tree) for a range of Gram-positive and -negative bacteria. Use of 0.15% (w/v) agar as a stabilizer overcame the problem of adequate contact between the oil and the test bacteria and obviated the need to employ a chemical emulsifier. A rapid version of the assay was also developed for use as a screening method. A comparison of visual and photometric reading of the microtitre plates showed that results could be assessed without instrumentation; moreover, if the rapid assay format was used, rigorous asepsis was not necessary. Accuracy of the resazurin method was confirmed by plate counting from microwells and MIC values were compared with results obtained using an agar dilution assay. The MIC results obtained by the resazurin method were slightly lower than those obtained by agar dilution.

The normal Lactobacillus flora of healthy human rectal and oral mucosa

Abstract: The Lactobacillus flora of the rectal and oral mucosa was sampled from 42 healthy volunteers. Species identification was carried out by numerically comparing API 50CH fermentation patterns with type strains, using an SJ-similarity cut-off level of 79%. For the largest groups, identity was further confirmed by DNA-DNA hybridizations against the type strain of the species. Seventeen lactobacilli clusters were defined, of which most were found both on rectal and oral mucosa. The largest taxa were Lactobacillus plantarum, Lact. rhamnosus and Lact. paracasei ssp. paracasei, which were isolated from 52%, 26% and 17% of the individuals, respectively. Most isolates were tested for their capacity to adhere to the human colonic cell line HT-29 in the absence and presence of methyl-alpha-D-mannoside. Mannose-sensitive adherence to HT-29 cells was encountered in two-thirds of the Lact. plantarum isolates, but infrequently among isolates of other taxa. The results suggest that Lact. plantarum is a major colonizer of the human gastrointestinal mucosa, and that its capacity to adhere to mannose-containing receptors may be of some ecological importance.

Influence of hydrodynamics and nutrients on biofilm structure

Abstract: Hydrodynamic conditions control two interlinked parameters; mass transfer and drag, and will, therefore, significantly influence many of the processes involved in biofilm development. The goal of this research was to determine the effect of flow velocity and nutrients on biofilm structure. Biofilms were grown in square glass capillary flow cells under laminar and turbulent flows. Biofilms were observed microscopically under flow conditions using image analysis. Mixed species bacterial biofilms were grown with glucose (40 mg/l) as the limiting nutrient. Biofilms grown under laminar conditions were patchy and consisted of roughly circular cell clusters separated by interstitial voids. Biofilms in the turbulent flow cell were also patchy but these biofilms consisted of patches of ripples and elongated 'streamers' which oscillated in the flow. To assess the influence of changing nutrient conditions on biofilm structure the glucose concentration was increased from 40 to 400 mg/l on an established 21 day old biofilm growing in turbulent flow. The cell clusters grew rapidly and the thickness of the biofilm increased from 30 μ to 130 μ within 17 h. The ripples disappeared after 10 hours. After 5 d the glucose concentration was reduced back to 40 mg/l. There was a loss of biomass and patches of ripples were re-established within a further 2 d.

Solar disinfection of drinking water contained in transparent plastic bottles : characterizing the bacterial inactivation process

Abstract: series of experiments is reported to identify and characterize the inactivation process in operation when drinking water, heavily contaminated with a Kenyan isolate of Escherichia coli, is stored in transparent plastic bottles that are then exposed to sunlight. The roles of optical and thermal inactivation mechanisms are studied in detail by simulating conditions of optical irradiance, water turbidity and temperature, which were recorded during a series of solar disinfection measurements carried out in the Kenyan Rift Valley. Optical inactivation effects are observed even in highly turbid water (200 ntu) and at low irradiances of only 10 mW cm 2 . Thermal inactivation is found to be important only at water temperatures above 45 °C, at which point strong synergy between optical and thermal inactivation processes is observed. The results confirm that, where strong sunshine is available, solar disinfection of drinking water is an effective, low cost method for improving water quality and may be of particular use to refugee camps in disaster areas. Strategies for improving bacterial inactivation are discussed.

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum

Abstract: Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 degrees C, but loses 50% of its activity after 60 min at 100 degrees C and 75% of its activity after autoclaving (121 degrees C, 15 min). Plantaricin 423 remains active after incubation at pH 1-10 and is inactivated when treated with pepsin, papain, alpha-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3.5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1.0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.

Production of conjugated linoleic acid by dairy starter cultures

Abstract: Nineteen different strains of lactobacilli, lactococci, streptococci and propionibacteria commonly used as dairy starter cultures were tested for their ability to produce conjugated linoleic acid (CLA) from free linoleic acid in vitro. Two strains of Propionibacterium freudenreichii ssp. freudenreichii and one strain of P. freudenreichii ssp. sheramnii were found to be capable of converting free linoleic acid to extracellular CLA. The highest level of CLA formed in the media was 265 micrograms ml-1. Of the different isomers, cis- and trans-9,11-octadecadienoic acid represented more than 70% of the total CLA formed. The inhibitory effect of linoleic acid on the growth of the bacteria and its conversion to CLA in different media by propionibacteria are discussed.

Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins.

Abstract: Until recently, all clostridia producing neurotoxins able to cause paralysis symptomatic of botulism were deemed to be Clostridium botulinum Defining Cl botulinum on the basis of this single phenotypic trait has resulted in the species encompassing metabolically very diverse organisms, and four distinct phenotypic groups are recognized within this taxon (designated groups I-IV) Nucleic acid hybridization and 16S ribosomal RNA sequencing studies have revealed the presence of four phylogenetically distinct lineages within the species, which correlate with these phenotypic divisions In addition to marked phenotypic and genotypic heterogeneity between groups, the taxonomy of the species is further complicated by the existence of strains which are closely related, if not genetically identifiable, to members of each Cl botulinum group, but are non-toxigenic Furthermore, strains of species other than Cl botulinum (viz Cl baratii, Cl butyricum) have been found which express botulinum neurotoxin (BoNT) Great advances have been made in recent years in elucidating the nucleotide sequences of genes encoding the various BoNT antigenic types (A through to G) Genealogical trees derived from BoNTs show marked discordance with those depicting 'natural' relationships inferred from 16S rRNA and phenotypic clusters, and strong evidence exists for BoNT gene transfer between some groups of Cl botulinum (eg groups I and II), and with non-botulinum species Botulinum neurotoxin is produced by Cl botulinum as a non-covalently bound progenitor toxin complex of two or more protein components Information on the evolutionary histories of the various non-toxic progenitor proteins is currently limited, although there is evidence of gene recombination In particular, chimera-like or mosaic non-toxic-non-haemagglutinins (NTNH) genes in group I Cl botulinum have been described, and it is now apparent that the phylogeny of the NTNHs is not going to 'mirror' that of botulinal neurotoxins, although their genes are physically contiguous In this article, the current state of knowledge of the phylogenetics of the species Cl botulinum and its neurotoxins is reviewed, and a view is presented that a nomenclature based rigidly on BoNT production is no longer tenable

Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis

Abstract: Bacterial biofilm formation on contact lenses (CLs), and CL storage cases may be a risk factor for CL-associated corneal infection and may explain the persistence of organisms in CL storage cases. This study evaluated biofilm formation on, and microbial contamination of, CLs and CL storage cases from patients with microbial keratitis. Contact lenses and CL storage cases from 20 wearers with microbial keratitis were sampled microbiologically and visualized using scanning electron microscopy (SEM). Culture results from the cornea were also noted. Bacterial biofilm was present more frequently (P < 0.05) on CL storage case surfaces (17/20) compared with CL surfaces (11/20) and biofilm density was significantly greater on case surfaces (P < 0.05). There was no association between poor compliance and microbial contamination of the CL storage case, nor between poor compliance and biofilm formation or density on the CL or CL storage case. Biofilm formation occurred equally frequently with hydrogen peroxide and chlorine release care systems. Microbial keratitis in CL wearers is frequently associated with bacterial biofilm in the CL storage case. Despite the use of current CL disinfection systems, the CL storage case is a favourable environment for proliferation of certain organisms. Biofilm on CLs may prolong the retention time of organisms at the ocular surface and increase their potential pathogenicity.

The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens

Abstract: Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes, Flavimonas oryzihabitans, and three species of Pseudomonas. The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0.125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10(3) to 10(6) cfu ml-1. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens. The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0.1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0.1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.

Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis.

Abstract: Four Streptococcus thermophilus strains (Strep. thermophilus BTC, Strep. thermophilus LY03, Strep. thermophilus 480 and Strep. thermophilus Sfi20) have been examined for their exopolysaccharide production capacity. All strains produced a polymer composed of the neutral sugars glucose and galactose, but in different ratios. It was clearly shown that the biosynthesis of exopolysaccharides from Strep. thermophilus LY03 is growth-associated and hence displays primary metabolite kinetics. The monomer ratio of the exopolysaccharide synthesized did not vary throughout the fermentation cycle. The production kinetics and exopolysaccharide yields were strongly dependent on the fermentation conditions. Physical factors such as temperature, pH and oxygen tension as well as chemical factors (medium composition, initial lactose concentration, carbon/nitrogen levels) were of utmost importance.

Dynamics of indigenous and inoculated yeast populations and their effect on the sensory character of Riesling and Chardonnay wines

Abstract: To study the impact of yeast populations on wine flavour and to better understand yeast growth dynamics, wines were produced by the (i) indigenous microflora, (ii) vigorous yeast starter EC1118 and (iii) slowly fermenting yeast Assmannshausen. Sensory analysis revealed that wines differed depending on the fermentation type. However, these yeast-related differences did not exceed the varietal character. Both added starter cultures clearly dominated the Saccharomyces population from the middle of fermentation onwards. The starter cultures differed in their repression of indigenous non-Saccharomyces yeast. EC1118 limited growth of non-Saccharomyces yeasts more strongly than Assmannshausen. Sulphite addition further repressed growth of non-Saccharomyces yeasts. On completion, more than one Saccharomyces strain was present in each fermentation, with the largest variety in the non-inoculated and the smallest in the EC1118-inoculated fermentation. Results from the two genetic assays, karyotyping, and PCR using δ-primers were not fully equivalent, limiting the usefulness of δ-PCR in studies of native Saccharomyces yeasts.

The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves

Abstract: The epidemiology of clinical cases of campylobacter in temperate climates shows a striking seasonality. In the search for a seasonal environmental reservoir changes in the carriage rate and population size of campylobacters in bovine hosts with time have been measured. Most probable number (MPN) methodology was used to enumerate thermophilic campylobacters in samples taken from the small intestines of beef cattle at slaughter and the fresh faeces of four dairy herds and new-born calves. Statistical analyses revealed significant evidence for seasonal periodicity in the data from dairy herds (P = 0·044). Not only was there a departure from constancy within a 12-month interval but these data revealed a true seasonality, that is, the same periodicity in numbers from one year to the next. Each herd had two peaks per year, in approximately spring and autumn. Peaks coincided in herds on neighbouring farms but those on farms in the north preceded those on farms in the south by 2 and 1 months, respectively (P = 0·0057). Intestinal carriage by beef cattle at slaughter was 89·4% (n = 360) with an average MPN campylobacters per gram fresh weight (MPN gfw−1) of 6·1 × 102. Average MPN gfw−1 in faeces from the dairy herds and calves were 69·9 (S.D. 3) and 3·3 × 104 (S.D. 1·7 × 102). There was no evidence of seasonal periodicity in the size of the campylobacter population in beef cattle at slaughter. Calves were campylobacter free at birth but became colonized within a few days.

Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus

Abstract: Carvacrol, a natural plant constituent occurring in oregano and thyme, was investigated for its bactericidal effect towards the food-borne pathogen Bacillus cereus. Carvacrol showed a dose-related growth inhibition of B. cereus. At concentrations of 0·75 mmol l−1 and above, total inhibition of the growth was observed. Below this concentration, carvacrol extended the lag-phase, reduced the specific growth rate and reduced the maximum population density. Incubation for 40 min in the presence of 0·75–3 mmol l−1 carvacrol decreased the number of viable cells of B. cereus exponentially. Spores were found to be approximately 2·3-fold less sensitive to carvacrol than vegetative cells. Bacillus cereus cells showed reduced susceptibility towards carvacrol at pH 7·0 compared with different values between pH 4·5 and 8·5. The culture and exposure temperatures had a significant influence on the survival of vegetative cells. The highest death rate of cells was observed at an exposure temperature of 30 °C. Membrane fluidity was found to be an important factor influencing the bactericidal activity of carvacrol.

Inter-species differences in maximum specific growth rates and cell yields of bifidobacteria cultured on oligosaccharides and other simple carbohydrate sources

Abstract: M.J. HOPKINS, J.H. CUMMINGS AND G.T. MACFARLANE. 1998.The abilities of seven bifidobacterial isolates (Bifidobacterium adolescentis, B. bifidum (two strains), B. catenulatum, B. infantis, B. longum, B. pseudolongum) to utilize 15 different carbohydrate sources (eight oligosaccharide products, and a variety of monosaccharides and disaccharides) were studied, with regard to maximum specific growth rates and production of bacterial cell mass. Results showed that substrate utilization was highly variable and that considerable interspecies and interstrain differences existed. Galactooligosaccharides and oligofructose, with a low degree of polymerization, supported best growth of the test micro-organisms. In contrast, xylooligosaccharides and pyrodextrins were almost invariably poor bifidobacterial substrates. In many species, maximum specific growth rates and bacterial cell yields were higher on oligosaccharides compared to their monosaccharide constituents, particularly with respect to fructooligosaccharides. Bifidobacterium pseudolongum, B. longum and B. catenulatum were the most nutritionally versatile isolates studied in relation to the range of oligosaccharide products utilized, and the extent to which bacteria could grow on these substrates.

Bacteriophage and associated polysaccharide depolymerases: Novel tools for study of bacterial biofilms

Abstract: Bacteriophage for three representative strains of Gram-negative biofilm bacteria have proved to be of widespread occurrence. Lytic bacteriophage have been isolated from local sewage for the bacterium 1.15, an exopolysaccharide (EPS)-producing pseudomonad found originally as a component of biofilms in a local river, and for two Enterobacter agglomerans strains from industrial biofilms. Representative examples of all three bacteriophage possess a relatively low burst size and on solid media, exhibit very large plaques surrounded by a wide halo (5-20 mm) indicative of polysaccharide depolymerase action. The bacteriophage are thus similar to other viruses for EPS-producing bacteria in inducing the synthesis of enzymes degrading the polymers which occlude the bacterial cell surface. In each preparation, the polysaccharase activity was associated both with sedimented phage particles and with the supernate of bacterial lysates. The enzymes have been partially purified and used to prepare polysaccharide digests in which the major products from each polysaccharide are the presumed repeat units of the polymers or oligomers of these. The soluble phage enzymes each degrade their substrate by acting as endo-glycanohydrolases. The phage and their associated enzymes thus provide very useful highly specific tools for studies of biofilms incorporating the bacterial host strains. Their potential applications in studies on bacterial biofilms are discussed.

Pyrrolnitrin from Burkholderia cepacia: antibiotic activity against fungi and novel activities against streptomycetes

Abstract: A bacterial strain identified as Burkholderia cepacia NB-1 was isolated from water ponds in the botanical garden in Tubingen, Germany, and was found to produce a broad spectrum phenylpyrrole antimicrobial substance active against filamentous fungi, yeasts and Gram-positive bacteria. In batch culture containing glycerol and L-glutamic acid, the isolate NB-1 produced the antibiotic optimally late in the growth phase and accumulated a main portion in their cells. Isolation and purification of the antibiotic from Burkholderia (Pseudomonas) cepacia NB-1 by acetone extraction, gel filtration on Sephadex LH-20 and preparative HPLC yielded 0·54 mg l−1 of a pure substance. Spectroscopic data (HPLC, MS and NMR) confirmed that the compound was pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chloro-phenyl) pyrrole]. Pyrrolnitrin has an inhibitory effect on the electron transport system, as demonstrated by isolated mitochondria from Neurospora crassa 74 A. This inhibition was relieved by N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), indicating that pyrrolnitrin blocked the electron transfer between the dehydrogenases and the cytochrome components of the respiratory chain. Among Gram-positive bacteria, pyrrolnitrin was most active against certain Streptomyces species, especially S. antibioticus, which has not previously been described in the literature. In the presence of pyrrolnitrin, aerial mycelium and spore formation of Strep. antibioticus was suppressed, although growth continued via substrate mycelium. The new findings of inhibition of streptomycetes and their secondary metabolism by pyrrolnitrin may contribute to the fact that Pseudomonas species predominate in soil and compete even with antibiotic-producing Streptomyces.

Sensitivity of food pathogens to garlic (Allium sativum)

Abstract: The inhibitory activity of garlic (Allium sativum) against Staphylococcus aureus, Salmonella typhi, Escherichia coli and Listeria monocytogenes was measured by the 'turbidity' method. Minimum inhibitory concentration (MIC) of garlic at 80% inhibition level was calculated for these bacteria. All bacterial pathogenic strains tested were inhibited by garlic; E. coli was most sensitive and Listeria monocytogenes was least sensitive. Therefore, garlic has potential for the preservation of processed foods.

Reduction of faecal coliform, coliform and heterotrophic plate count bacteria in the household kitchen and bathroom by disinfection with hypochlorite cleaners

Abstract: Fourteen sites evenly divided between the household kitchen and bathroom were monitored on a weekly basis for numbers of faecal coliforms, total coliforms and heterotrophic plate count bacteria. The first 10 weeks comprised the control period, hypochlorite cleaning products were introduced into the household during the second 10 weeks, and a strict cleaning regimen using hypochlorite products was implemented during the last 10 weeks. The kitchen was more heavily contaminated than the bathroom, with the toilet seat being the least contaminated site. The highest concentrations of all three classes of bacteria were found on sites that were moist environments and/or were frequently touched; these included the sponge/dishcloth, the kitchen sink drain area, the bath sink drain area, and the kitchen faucet handle(s). The implementation of a cleaning regimen with common household hypochlorite products resulted in the significant reduction of all three classes of bacteria at these four sites and other household sites.

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4

Abstract: A protease-sensitive antibacterial substance produced by Bacillus coagulans I4 strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus, Leuconostoc, Oenococcus, Listeria and Pediococcus. Coagulin was stable at 60 degrees C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by alpha-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3-4 kDa by SDS-PAGE. The B. coagulans I4 strain harbours a plasmid, pI4, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI4, whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.

Soil: the environmental source of Escherichia coli and Enterococci in Guam's streams

Abstract: We have previously documented that faecal indicator bacteria (Escherichia coli, faecal coliform, enterococci) recommended by the U.S. Environmental Protection Agency (USEPA) to establish recreational water quality standards are naturally found in high concentrations in the surface and subsurface of soils in Hawaii. Rain, the source of all streams in Hawaii, washes the soil sources of faecal bacteria into all the streams of Hawaii, at concentrations which consistently exceed the USEPA recreational water quality standards. The objective of this study was to test the hypothesis that faecal bacteria are able to establish themselves in the soil environments of tropical islands by conducting the same study in Guam, a tropical pacific island with warmer temperatures and higher humidity than Hawaii. The same methods and study design used in Hawaii was used in Guam. The results of the study conducted in Guam revealed that all streams contain consistently high concentrations of faecal coliform, E. coli, and enterocci which exceeded the old USEPA recreational water quality standard of 200 faecal coliform/100 ml as well as the new water quality standards of 126 E. coli/100 ml or 33 enterococci/100 ml. These same faecal indicator bacteria were recovered in high concentrations in surface and subsurface (18-36 cm depth) soil samples in Guam. Limited coastal water analysis showed that most coastal marine waters contain low concentrations of faecal bacteria but coastal waters impacted by stream run-off showed elevated levels of faecal bacteria. The results of this study support the hypothesis that environmental conditions in the tropical areas of the world can support the growth and establishment of populations of faecal bacteria in the soil. Thus, soil becomes an environmental, non-faecal source of faecal indicator bacteria. These results indicate that USEPA water quality standards may not be directly applicable to tropical island environments.

Selective effects of sulfur dioxide and yeast starter culture addition on indigenous yeast populations and sensory characteristics of wine

Abstract: Riesling musts, with or without sulfur dioxide added, were fermented either with or without the addition of yeast. Uninoculated fermentations took much longer to finish than inoculated musts. There were no significant differences in growth of non-Saccharomyces yeasts in uninoculated musts with less than 50 mg l−1 SO2 added. The starter culture was completely dominant over indigenous Saccharomyces cerevisiae and strongly inhibitory to non-Saccharomyces. Alcohol and acetaldehyde were greater in the inoculated treatments ; titratable acidity and acetic acid were greater in the uninoculated fermentations. There were no statistically significant differences among any treatments in final pH, ammonia content, or colour (A420). Uninoculated fermentations had higher sensory scores (P > 0·95) for ‘spicy’, ‘apple’, ‘melon’, ‘pear’, and ‘H2S’, while inoculated wines had higher scores (P > 0·95) for ‘paper’, ‘oxidized’, and ‘sweaty’. Sulfite treatment produced an assortment of significant sensory differences in the finished uninoculated wines, but in inoculated wines the additions of SO2 to the must had no significant effect on indigenous yeast populations or on flavour.

Selection of lactobacilli for chicken probiotic adjuncts

Abstract: During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.

Isolation and partial characterization of antifungal metabolites of Bacillus pumilus.

Abstract: C. MUNIMBAZI AND L.B. BULLERMAN. 1998. Antifungal metabolites produced by Bacillus pumilus in Potato Dextrose Broth (PDB) were isolated from culture supernatant fluid by precipitation with ammonium sulphate. The antifungal metabolites inhibited mycelial growth of many species of Aspergillus, Penicillium and Fusarium. They also inhibited production of aflatoxins, cyclopiazonic acid, ochratoxin A and patulin. The metabolites were heat-stable and remained active after sterilization at 121 °C for 15 min. Their activity was stable over a wide range of pH (2-10). The metabolites were resistant to hydrolysis by various proteases, peptidases and other enzymes. They were also resistant to denaturation by many protein-denaturing detergents except Nonidet P-40. The metabolites were soluble in water and relatively polar organic solvents. Chromatographic bioassay revealed that a crude precipitate of the metabolites contained only one compound with antifungal activity. The active compound did not form a fluorescent derivative with fluorescamine suggesting that the compound is either a cyclic polypeptide or a non-peptidic compound.

Assessment of bacterial viability status by flow cytometry and single cell sorting

Abstract: Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g., enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by: active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.

Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment

Abstract: The inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (Mano-Sonication, MS) and a combined MS/heat treatment (Mano-Thermo-Sonication) was investigated. The sporicidal effect of MS treatments depended on static pressure, amplitude of ultrasonic waves and treatment temperature. At 70 degrees C, pressure increments up to 500 kPa caused progressively more inactivation. An MS treatment at 500 kPa and 117 microns of amplitude for 12 min inactivated approximately 99% of the B. subtilis spore population. Over 500 kPa, further increments in pressure did not increase the percentage of inactivation. In the range 90-150 microns, an exponential relationship was observed between the amplitude of ultrasonic waves under pressure and the number of survivors. While an MS treatment (20 kHz, 300 kPa, 70 degrees C, 12 min) at 90 microns inactivated 75% of the B. subtilis spore population, the same treatment at 150 microns inactivated 99.9% of this population. The MS treatments at temperatures higher than 70 degrees C (MTS) led to more spore inactivation. In the range 70-90 degrees C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 microns, 6 min) had a synergistic effect on spore inactivation. The inactivating effect of ultrasound was due neither to titanium particles eroded from the sonication tip, nor to free radicals released during ultrasonic treatment. The MS treatments sensitized spores of B. subtilis to lysozyme.

Evaluation of the preservative properties of Thymus vulgaris essential oil in topically applied formulations under a challenge test

Abstract: The preservative properties of thyme essential oil (3%) with a known composition were evaluated in two types of final formulations, suitable for use as pharmaceutical or cosmetic vehicles, by means of a standard challenge test proposed by the latest European Pharmacopoeia. The required preservation efficacy criteria were satisfied against the bacterial strains, against the yeast in one of the formulations, but not against the mould strain involved in this study. Interactions between the essential oil compounds and other factors present in the final formulation might have influenced the activity of this essential oil, leading to an incomplete satisfaction of the criteria.

Continuous culture selection of bifidobacteria and lactobacilli from human faecal samples using fructooligosaccharide as selective substrate

Abstract: The human large intestine contains a large and diverse population of bacteria Certain genera, namely Bifidobacterium and Lactobacillus, are thought to exert health-promoting effects Prebiotics such as fructooligosaccharides (FOS) have been shown to stimulate the growth of endogenous bifidobacteria In this study, changes of lactic acid producing bacteria in continuous culture fermentors (semi-defined, anaerobic medium containing 5 g 1(-1) FOS, dilution rate of 01 h-1, pH 55) were followed over a 21 d period after inoculation with blended human faeces from four healthy adults Samples were also taken every 3 d for influent/effluent FOS, short chain fatty acid (SCFA), lactate and microbiological analyses Results showed that SCFA concentrations decreased abruptly 1 d after inoculation while lactate concentrations increased Classical methods of enumeration using selective media showed that the proportion of total culturable count represented by bifidobacteria and lactobacilli increased from 119% on day 1 to 981% on day 21 However, molecular methods using genus-specific 16S rRNA oligonucleotide probes indicated that the bifidobacterial population maintained a level between 10 and 20% of total 16S rRNA during the first 6 d and disappeared rapidly when the maximum concentration of lactate was reached Lactobacilli, which were initially present in low numbers, increased until day 9 and remained at high levels (20-42% of total 16S rRNA) to day 21, with the exception of day 18 Although FOS has usually been regarded as a selective substrate for bifidobacteria, these observations suggest that: (1) lactobacilli are also able to use FOS, (2) lactobacilli can out-compete bifidobacteria in continuous culture at pH 52-54 when FOS is the primary carbon and energy source, and (3) bifidobacteria can grow faster on FOS than lactobacilli under controlled conditions