bluuebunny/crossref_metadata_2025 huggingface dataset:
Tn5‐induced Xenorhabdus bovienii lecithinase mutants demonstrate reduced virulence for Galleria mellonella larvae
Authors: Pinyon R.A. , Linedale E.C. , Webster M.A. , Thomas C.J.
Published: March 2008

A derivative of Tn5 was used to construct a variety of stable insertion mutations in the entomopathogenic bacterium, Xenorhabdus bovienii T228/1. Mutants which had altered expression of Congo Red binding ability, ampicillin resistance, haemolytic activity and lecithinase were isolated. Isolates with altered lecithinase activity had either lost ability to produce this enzyme or showed reduced expression. The role of lecithinase in pathogenesis of X. bovienii T228/1 for Galleria mellonella larvae was examined by LD50 analysis. Maximum killing of G. mellonella was observed at 72 h post infection for both the wild‐type parent strain and a lecithinase mutant 34(45). However, the LD50 value for the wild‐type parent strain (8·7 cells per larva) was significantly less than that calculated for the lecithinase mutant (35·5 cells per larva). The data suggested that although lecithinase is a virulence factor produced by X. bovienii, lecithinase activity alone is not sufficient for killing of G. mellonella larvae.
Inhibition by 1,10‐phenanthroline of the breakdown of peptides by rumen bacteria and protozoa
Authors: Wallace R.J. , Newbold C.J. , McKain N.
Published: March 2008

The rate of peptide breakdown in the rumen frequently exceeds the rate at which the amino acids released can be used for microbial growth. The final step in this often wasteful process involves the cleavage of dipeptides. The main rumen bacterial species with high dipeptidase activity, Prevotella ruminicola, Fibrobacter succinogenes, Lachnospira multipara and Megasphaera elsdenii, had activities which were inhibited >95% by 1,10‐phenanthroline, a chelator of divalent metal ions and metalloprotease inhibitor. Dipeptidase activity in digesta taken from the rumen of sheep decreased by 33% in the presence of 1,10‐phenanthroline, while mixed bacteria from the same samples were inhibited by 80% and the activity of mixed protozoa decreased by only 15%. Thus a substantial amount of dipeptide breakdown appears to be due to ciliate protozoa in the mixed population. Extensive washing of the protozoa increased the sensitivity of protozoal dipeptidase activity to 1,10‐phenanthroline, suggesting that protozoa too have a metallo‐dipeptidase activity but that it is normally protected from inhibition by 1,10‐phenanthroline. Breakdown of the pentapeptide, Ala5, was also inhibited 27% by 1,10‐phenanthroline in the mixed population, and when Trypticase, a pancreatic casein hydrolysate containing a mixture of oligopeptides, dipeptides and amino acids, was incubated with rumen fluid, the production of ammonia and free amino groups was inhibited 71% by 1,10‐phenanthroline. It was concluded that metal ion chelation inhibits oligopeptidase and dipeptidase activities of rumen micro‐organisms and may be a means of controlling ammonia production from peptides in the rumen.
Biofilm formation by the Enterobacteriaceae: a comparison between Salmonella enteritidis, Escherichia coli and a nitrogen‐fixing strain of Klebsiella pneumoniae
Authors: Jones K. , Bradshaw S.B.
Published: March 2008

A simple laboratory reactor, which simulates biofilm formation in pipes, was used to compare biofilm formation by three members of the Enterobacteriaceae, namely, an environmental, nitrogen‐fixing strain of Klebsiella pneumoniae, a pathogen, Salmonella enteritidis, and a faecal indicator, Escherichia coli. All three attached to CVCP pipe surfaces in the reactor and formed substantial biofilm populations of over a million bacteria cm‐2 within 24 h. These populations increased by approximately 10‐fold over the next 48 h. Estimates of the numbers of metabolically active cells and the ratios of viable to direct counts showed that Kl. pneumoniae formed the densest and most metabolically active biofilms, followed by Salm. enteritidis and E. coli, respectively. Nitrogen fixation and polysaccharide production (EPS) by Kl. pneumoniae occurred only in mature biofilms and were of no selective advantage in the initiation of biofilms. Despite producing more EPS the rate of attachment of Salm. enteritidis was lower than for Kl. pneumoniae.
Computer‐aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water
Authors: Ferreira A.C. , Morais P.V. , Gomes C. , da Costa M.S.
Published: March 2008

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS‐PAGE of wholecell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating that populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.
Immunomagnetic separation of Erwinia carotovora subsp. atroseptica from potato peel extracts to improve detection sensitivity on a crystal violet pectate medium or by PCR
Authors: van der Wolf J.M. , Hyman L.J. , Jones D.A.C. , Grevesse C. , Van Beckhoven J.R.C.M. , Van Vuurde J.W.L. , Pérombelon M.C.M.
Published: March 2008

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of nontarget bacteria. A streptomycin‐resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medium supplemented with 100 μg ml−1 of streptomycin to determine the recovery level of the target bacterium. Recovery obtained with a polyclonal antiserum against Erw. carotovora subsp. atroseptica at a concentration of 6 μg IgG ml−1 was greater than that obtained with two monoclonal antibodies against lipopolysaccharides of Erw. carotovora subsp. atroseptica at a concentration of 10 μg IgG ml−1. A linear relationship was found between particle concentration ranging from 12 to 200 μg ml−1 and recovery level. When the Advanced Magnetics (AM) protein A and anti‐rabbit IgG particles in the AM separation system and the Dynal anti‐rabbit IgG particles in the Dynal separation system were examined, the highest recovery level per μg of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti‐rabbit particles (37%). Without IMS, detection of Erw. carotovora subsp. atroseptica in tuber peel extracts on a CVP‐medium without streptomycin was impossible when the ratio of Erw. carotovora subsp. carotovora to Erw. carotovora subsp. atroseptica was greater than 100 or when large numbers of other saprophytic bacteria were present, because of overcrowding. IMS, using the AM anti‐rabbit IgG particles, ensured that Erw. carotovora subsp. atroseptica could be enumerated in tuber peel extract consistently, to a detection level of 100 cells ml−1. Similarly, the IMS procedure lowered the detection level of Erw. carotovora subsp. atroseptica in a twofold diluted peel extract by PCR to ca 2·0 × 103 cells ml−1 or 50 cells per reaction tube. In contrast, positive results in PCR without IMS were obtained only when the peel extract was diluted 100 times and when the concentration of Erw. carotovora subsp. atroseptica was at least 105 cells ml−1.
Quantification of Staphylococcus aureus and Escherichia coli in the liquid medium by fluorimetry and its use in phagocytosis assay
Authors: Fang W.
Published: March 2008

A fluorimetric technique was compared with the plate counting method for quantification of viable cells of Staphylococcus aureus and Escherichia coli in the liquid medium. The fluorimetric assay measures the release of fluorogenic 4‐methylumbelliferone (4‐MU) from 4‐methylumbelliferyl phosphate by the bacterial phosphatases. The increase in fluorescence was dependent on the size of bacterial inocula. Setting the fluorescence threshold at the middle of the logarithmic growth phase resulted in good linear relationship between bacterial counts and fluorescence (r= 0·99 for both Staph. aureus and E. coli). There was also an excellent correlation between the fluorimetric assay and the plate counting method in quantifying viable bacteria in saline (r= 0·99). Both methods were further compared for evaluation of extracellular bacteria following phagocytosis. The fluorimetric technique, in general, gave a higher percentage of phagocytosis than the plate counting method with statistical significance for E. coli.
The use of an automated assay to assess phage survival after a biocidal treatment
Authors: Maillard J.‐Y. , Beggs T.S. , Day M.J. , Hudson R.A. , Russell A.D.
Published: March 2008

The coliphage K1‐5 has been used in an automated assay to monitor the viricidal activity of various disinfectants. This indirect assay based on the spectrophotometric reading of the lysis of the host cell (Escherichia coli D837) produced encouraging results and was faster than the overlay counting method (previously studied) which relies on plaque formation. However, differences in sensitivity towards some disinfectants were observed between the two methods.
Microbial biomass in a shallow, urban aquifer contaminated with aromatic hydrocarbons: analysis by phospholipid fatty acid content and composition
Authors: Franzmann P.D. , Patterson B.M. , Power T.R. , Nichols P.D. , Davis G.B.
Published: March 2008

The city of Perth contains a number of sites that have been contaminated with hydrocarbons due to leakage from petroleum underground storage tanks. Microbial biomass in groundwater and sediment cores from above and below the water table, and from within and outside a plume of hydrocarbon contamination, was examined using phospholipid fatty acid methyl ester analysis. Microbial numbers, calculated from the phospholipid content, ranged from 0·9 × 106 to 7·8 × 106‘Escherichia coli equivalent cells’ g−1 dry wt of sediment. Over 96% of the microbial biomass was attached to the sediment and the proportion of attached cells did not decrease within the plume of contaminants. The amount of biomass within aquifer samples seemed to be related more to the proximity of the rhizosphere to the shallow aquifer, and other unknown urban inputs, rather than to the effects of the plume of contaminants. Fatty acids common to many bacterial groups dominated within the plume, and as such the analyses gave limited insight into microbial community structure. For site assessment of intrinsic remediation of shallow aquifers in urban areas, estimates of microbial biomass may not provide information that is readily applicable to plume management.
Characterization of growth and product formation by a thermophilic streptomycete grown in a particulate rapemeal‐derived liquid medium
Authors: Brabban A.D. , Edwards C.
Published: March 2008

Streptomyces thermoviolaceus was grown in a glutamate salts medium and the applicability of DNA and a number of intracellular dehydrogenases as indicators for growth in a particulate repemeal salts medium were assessed. Only NADH dehydrogenase proved unsuitable but analysis of the rates of increase of activities of this enzyme during batch culture suggested that energy metabolism was an important factor in defining the onset of secondary metabolism and as a causal element of the biphasic pattern of growth exhibited by this streptomycete. Growth, substrate utilization and production of extracellular products could readily be analysed in the particulate rapemeal salts medium. Free sugars and carbohydrates were utilized predominantly in early exponential phase but thereafter the rapemeal proteins were metabolized as observed by rises in protease activities and levels of ammoniacal nitrogen. Growth in rapemeal‐derived media produced titres of the antibiotic granaticin which were at least as high (usually higher) as most of the growth substrates tested suggesting that rapemeal may have considerable potential for the exploitation of natural products. Particulate rapemeal medium also resulted in high yields of commercially important extracellular enzymes.
Rapid physicochemical detachment, separation and concentration of bacteria from beef surfaces
Authors: Rodrigues‐Szulc U.M. , Ventoura G. , Mackey B.M. , Payne M.J.
Published: March 2008

A simple, rapid, physicochemical treatment for the removal of viable bacteria from the surface of raw and cooked beef is described. The detachment method was linked to a differential centrifugation step which removed large amounts of particulate food matter and concentrated the detached bacteria. The method increased the numbers of bacteria released from beef surfaces and increased the numbers detected by at least one and a half orders of magnitude, when compared to the traditional ‘stomaching’ technique. This 1‐h separation and concentration method produced cleaner suspensions of bacteria and improved the sensitivity of detection by DEFT and direct plate count.
Cross‐reactivity of antibodies raised to Pseudomonas fluorescens protease with extracellular proteins produced by meat‐spoiling pseudomonads
Authors: Lundy F.T. , Magee A.C. , Blair I.S. , McDowell D.A.
Published: March 2008

The cross‐reactivity patterns of antibodies to Pseudomonas fluorescens protease with the extracellular proteins produced by a number of meat‐spoiling pseudomonads were studied. Immunoblotting studies showed that purified IgG to Ps. fluorescens protease cross‐reacted with extracellular proteins in the cell culture supernatant fluids of Pseudomonas spp., including Ps. fragi and Ps. lundensis. In the case of Ps. lundensis and Pseudomonas spp. 11390, the cross‐reactive moieties were of similar molecular weight to the Ps. fluorescens protease (46 kDa). However, in Ps. fragi the cross‐reactive moiety was a lower molecular weight protein (8 kDa). This may represent a fragment of the active enzyme. These results indicate the presence of common antigenic determinants among the proteases of meat spoiling pseudomonads.
Survival of parental and genetically modified derivatives of a soil isolated Pseudomonas fluorescens under nutrient‐limiting conditions
Authors: Clegg C.D. , van Elsas J.D. , Anderson J.M. , Lappin‐Scott H.M.
Published: March 2008

Wild type, a rifampicin‐resistant mutant and three genetically modified derivatives of the soil isolate Pseudomonas fluorescens R2f were starved in pure cultures for periods of up to 70 d. Cells were starved after harvesting at a point early in the stationary phase of the growth curve and all five strains demonstrated the ability to survive nutrient deprivation and resuscitate rapidly when growth nutrients became available. No difference in total counts and metabolic activity was detected between the strains. Plate counts were similar for all strains up to day 35. Wild type and the rifampicin‐resistant mutant strain showed greater recovery than the genetically modified strains on day 70. During the starvation period there was a significant decrease in cell lengths of all five strains, however, there was no significant difference between the strains. The shape of the starved cells varied with the growth phase at which they had been harvested. Cells taken from early stationary phase and starved produced predominantly rod‐shaped cells whereas those taken from early log phase and starved produced small round cells. In experiments when the rifampicin‐resistant mutant and the genetically modified strain Art‐3 were starved at early log phase the cells were significantly smaller than respective cultures not exposed to the nutrient limiting conditions, and there was no significant difference in the response of the two strains. None of the cultures produced ultramicrobacteria, and none of the cultures entered a non‐culturable state. Starvation at different cell densities did not affect the recoverability of the cells. The results of this study demonstrate that responses to starvation conditions by the genetically modified and parental strains are similar.
Extracellular material from Leuconostoc mesenteroides subsp. dextranicum mediates the aggregation of lactococcal cells: implications for the isolation of single strains
Authors: Gopal P.K. , Davey G.P. , Crow V.L. , Pillidge C.J. , Coolbear T. , Holland R.
Published: March 2008

Cultures of Lactococcus lactis subsp. cremoris, originally derived from mixed cheese starter cultures, were assessed as pure following single colony selection and subculturing, yet nevertheless gave rise, under stress conditions, to an isolate with the ability to ferment citrate. The isolate was characterized with respect to its citrate enzymology and lactose fermentation and was identified as Leuconostoc mesenteroides subsp. dextranicum and assigned the strain number 663. The extracellular material (ECM) from Leuc. mesenteroides subsp. dextranicum 663 was characterized and found to contain carbohydrate, protein and phosphate (30.9, 50.7 and 18.4%, by weight, respectively). Glucose was the most prominent sugar (39% by weight of total carbohydrate) with mannose, galactose and rhamnose being the other major monosaccharides. The ECM protein resolved into a large number of bands on SDS‐polyacrylamide gel electrophoresis, the most prominent having molecular masses of 40 and 49 kDa. The ECM from Leuc. mesenteroides subsp. dextranicum 663 caused aggregation of suspensions of lactococcal cells and may facilitate intergeneric interactions and/or co‐culture during cheese starter strain isolation.
Transmission of pea bacterial blight (Pseudomonas syringae pv. pisi) from seed to seedling: effects of inoculum dose, inoculation method, temperature and soil moisture
Authors: Roberts S.J. , Ridout M.S. , Peach L. , Brough J.
Published: March 2008

The effects of inoculum dose, inoculation method, temperature and soil water potential on transmission of bacterial blight (Pseudomonas syringaepv.pisi) from seed to seedling of pea (Pisum sativum) were examined in two separate experiments. Pea seed (cv. Solara), artificially inoculated with different concentrations of bacteria by dipping or vacuum infiltration, or naturally infected, was sown in pots which were maintained at different soil water potentials and temperatures. Dose received (mean number of bacteria per seed) and soil water potential had the greatest effects, with the proportion of plants infected and the mean number of lesions increasing with dose received and soil moisture. Inoculation method had little direct effect on transmission but greatly influenced the dose received. Temperature, in the range 5 to 18°C, had no effect on the number of lesions and little effect on the proportion of plants infected. Generalized linear models were derived relating the proportion of plants infected,p, and the mean number of lesions,m, to the dose received,d, and the soil water potential, ψ. The equations were:p‐ 1 ‐exp [‐0.064d0.24exp (58.55ψ)] andm= 0.24d0.25 exp (93.3ψ). The exponents for dose in these equations were considerably less than the theoretical value of one implied by a one‐hit model and suggested considerable heterogeneity in the one‐hit probability. Some possible explanations for this are considered.
Irish kefir‐like grains: their structure, microbial composition and fermentation kinetics
Authors: Rea M. C. , Lennartsson T. , Dillon P. , Drinan F.D. , Reville W.J. , Heapes M. , Cogan T.M.
Published: March 2008

The structure of six Irish kefir samples was studied in the electron microscope, and the microbial composition and fermentation kinetics during growth in 10% reconstituted skim milk at 21°C. The microbial composition of the six samples was similar; at the end of the fermentation the counts of lactococci, leuconostocs, lactobacilli, acetic acid bacteria and yeasts were 109, 108, 5 × 106, 105 and 106 ml−1 respectively; the levels of acetic acid bacteria and lactobacilli showed some intersample differences. Lactate was the major metabolite followed in order by ethanol, acetate and acetoin. The final concentrations of L‐lactate produced (66–90 mmol kg−1) were 10‐fold higher than those of D‐lactate. Acetate and ethanol concentrations varied from 4 to 14 and 2 to 40 mmol kg−‘1 respectively. The rates of citrate utilization and concentration of acetoin produced during growth differed between samples. Scanning electron microscopy showed not only variation between the interior and exterior of the sample but also large variation between different sections of the interiors and exteriors of the same sample. Long and short, and straight and curved rods and yeasts were seen in all samples, the curved rods observed in the interior, but lactococci were seen on the surface of only one sample. There were no gross differences in structure between samples.
Organic acid metabolism under different glucose concentrations of Leuconostoc oenos from wine
Authors: Sagui F.M. , Manca de Nadra M.C.
Published: March 2008

Leuconostoc oenos M isolated from wine did not grow in the absence of glucose and it was clearly stimulated by the presence of L‐malic and citric acids in synthetic medium with different glucose concentrations. In basal medium, D‐glucose and L‐malic and citric acids were simultaneously consumed. L‐Malic acid was metabolized at a higher rate than glucose and citric acid. When the organic acids were completely consumed only 50% of the glucose was utilized. In basal medium 1 mmol of D‐lactic acid was produced per mmol of glucose consumed and the amount of ethanol formed was higher with acetate present in the medium. L‐Malic acid was completely recovered as L‐lactic acid, and in the presence of L‐malic acid a carbon imbalance from glucose to D‐lactic acid was observed. In the presence of citric acid the amount of D‐lactic acid formed was directly proportional to glucose‐citrate utilization and acetic acid and ethanol were produced.
Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound
Authors: Langsrud S. , Sundheim G.
Published: March 2008

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Bac light bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation between the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcvs and Pseudomonas tested.
Isolation and characterization of a new extracellular polysaccharide from an Acetobacter species
Authors: MacCormick C.A. , Harris J.E. , Jay A.J. , Ridout M.J. , Colquhoun I.J. , Morris V.J.
Published: March 2008

An expolysaccharide (EPS)‐producing strain of the genus Acetobacter (named IFR 101) was isolated from a commercial vinegar acidifier. IFR 101 can be grown over the pH range 3.0–8.0 and the temperature range 12–35°C, and is ethanol‐tolerant to 10% v/v. Neutral sugar analysis of the purified EPS indicates the presence of the sugars mannose, galactose and glucose. Methylation studies and NMR data suggest that the EPS may either be a complex branched polysaccharide or a mixture of simpler linear and branched structures. Aqueous preparations of IFR 101 exhibit reversible shear‐thinning behaviour.
Significance of pre‐incubation temperature and inoculum concentration on subsequent growth of Listeria monocytogenes at 14°C
Authors: Gay M. , Cerf O. , Davey K.R.
Published: March 2008

The influence of the bacterial concentration of an inoculum (101 or 103 cfu ml‐1) of two strains of Listeria monocytogenes (Scott A : serotype 4b and V7: serotype 1) and one strain of L. innocua (Lin 11), and the time and temperature at which the inoculum was stored (cold storage: 4°C for 4 weeks, or without cold storage: ‐ 20°C before immediate transfer), and the temperature at which cells were pre‐incubated (30°C and 14°C) on subsequent growth in Richard's broth at 14°C was investigated. Richard's broth at a pH 5.9 was used to simulate potential growth in soft cheese (camembert type) and an incubation temperature of 14°C was used to simulate storage‐temperature ripening of cheese. Enumeration of the number of viable cells was by plate count method, except where viable cell numbers were less than 103 cfu ml‐1, when the MPN (Most Probable Number) technique was used. With cold storage and an inoculum of 103 cfu ml‐1 (high bacterial concentration) the pre‐incubation temperatures (30°C and 14°C) did not significantly influence the subsequent growth curve: there was no significant lag (less than 21 h) and cell numbers peaked in about 8.5 d. However, with cold storage and an inoculum of 101 cfu ml‐1 (low bacterial concentration) and a pre‐incubation temperature of 30°C a significant shift in the growth curve was observed over that pre‐incubated at 14°C, with the appearance of a lag of about 7.7 d. At a pre‐incubation temperature of 14°C with the low inoculum concentration, there was a measurable lag of about 1 d. Without cold storage and a pre‐incubation temperature of 30°C, there was a lag time of 2.3 d. Storage conditions, pre‐incubation temperature and inoculum concentration therefore appear to influence the subsequent growth curve. Importantly, however, the growth curves for cultures from inocula, pre‐incubated at either 30°C or 14°C, appeared to involve two distinct values of the exponential growth rate (k): the initial portion of the growth curve described by a low value of k and the subsequent portion by a consistently and significantly greater value. The appearance of two distinct growth phases was reproduced in further data determined for all the studied strains of the microorganism. Further study to explain these unexpected and reproducible findings is being conducted.
Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil
Authors: Brandi G. , Sisti M. , Schiavano G. F. , Salvaggio L. , Albano A.
Published: March 2008

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic (Aer. hydrophila and Aer. caviae) and one invasive (Aer. sobria) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas, the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re‐growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria, while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas‐associated human infections.
Arginine catabolism in wine lactic acid bacteria: is it via the arginine deiminase pathway or the arginase‐urease pathway?
Authors: Liu S.‐Q. , Pritchard G.G. , Hardman M.J. , Pilone G.J.
Published: March 2008

The wine lactic acid bacteria Leuconostoc oenos OENO and Lactobacillus buchneri CUC‐3 catabolize L‐arginine to ornithine and ammonia as major end‐products, with 1 mole of arginine converted into 2 moles of ammonia and 1 mole of ornithine. Some citrulline was also excreted into the medium. The excreted citrulline was reassimilated and catabolized by the lactobacillus strain, though not by the leuconostoc. Urea was not detected during arginine degradation. The activities of all three enzymes of the arginine deiminase pathway (arginine deiminase, ornithine transcarbamylase and carbamate kinase) increased significantly over time in the presence of arginine. On the other hand, arginase and urease activities were undetectable in cell extracts of cultures grown in the presence of arginine. The results show that the arginine deiminase pathway, and not the arginase‐urease pathway, is the route for arginine degradation in wine lactic acid bacteria.
Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant
Authors: Duan K. , Harvey M.L. , Liu C.‐Q. , Dunn N.W.
Published: March 2008

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nis R. as well as nis K and the recently reported nis F, nis E and nis G, but does not encode nis I. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nis R, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.
Indices for performance evaluation of predictive models in food microbiology
Authors: Ross T.
Published: March 2008

Two complementary measures are proposed as simple indices of the performance of models in predictive food microbiology. The indices assess the level of confidence one can have in the predictions of the model and whether the model displays any bias which could lead to ‘fail‐dangerous’predictions. The use of the indices is demonstrated using data collated from independent and published literature. This analysis supports previous reports that evaluation of predictive models by comparison to published microbial growth rate data may be inappropriate because of limitations in that data. The indices may fail to reveal some forms of systematic deviation between observed and predicted behaviour. It is concluded, however, that the indices provide an objective and readily interpreted summary of model performance and may serve as a first step towards the development of an objective and useful definition of the term ‘validated model’in predictive food microbiology.
Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences
Authors: Marshall D.G. , Coleman D.C. , Sullivan D.J. , Xia H. , O'Moráin C.A. , Smyth C.J.
Published: March 2008

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori, and their relative discriminatory abilities were assessed. Four probes, (GACA)4, (GT)8, (GTG)5 and (GGAT)4, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer‐assisted similarity analysis of (GGAT)4‐generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori.
The effect of immobilization on rhamnolipid production by Pseudomonas fluorescens
Authors: Wilson N.G. , Bradley G.
Published: March 2008

Pseudomonas fluorescens in free suspension and immobilized to a commercially available biosupport (Biofix) and a biosorbent (Drizit), were used as bioremediation agents in an aqueous system with petrol (Slovene diesel) as the carbon source. Analysis of cellular growth and estimation of rhamnolipid production was carried out on the free suspension of the free and immobilized systems over 5 d. An increase in growth and rhamnolipid production was seen in the immobilized systems in comparison to the free system. EDTA was shown to be an inhibitor of rhamnolipid production. Its addition to the aqueous suspensions of all systems resulted in a fall in production of the surface‐active agent in all cases, with no corresponding decrease in growth. This indicates the bacteria can rely on contact between the cell and the oil droplet for hydrocarbon transport into the cell. The data from this study indicated that immobilization resulted in a combination of increased contact between cell and hydrocarbon droplets and enhanced levels of rhamnolipid production.
Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea
Authors: Walker R. , Emslie K.A. , Allan E.J.
Published: March 2008

Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo. Cell‐free filtrates showed activity against B. cinerea growing on Potato Dextrose Agar (PDA) in a media‐dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB). Cell‐free filtrates of CDB‐grown cultures also significantly reduced conidial germination of B. cinerea. An assays based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA. The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter. The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves. Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps. antimicrobica were applied to the leaf tissue with the conidia. In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps. antimicrobica. These tests indicate that Ps. antimicrobica would be a suitable biocontrol agent for the control of B. cinerea.
Characterization of proteolytic activities of rumen bacterial isolates from forage‐fed cattle
Authors: Attwood G.T. , Reilly K.
Published: March 2008

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell‐bound activity, which included a mixture of serine and cysteine‐type proteinases which had high activity against leucine p‐nitroanilide (LPNA). The Eubacterium strains had a mainly cell‐associated activity with serine and metallo‐type proteinases which showed high activity against the chymotrypsin substrate, N‐succinyl alanine alanine phenylalanine proline p‐nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell‐bound mixture of cysteine and metallo‐proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio‐like strain, B316, had a cell‐bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella‐like strain, C21a, had a mixture of cysteine, serine and metallo‐proteinase activities which were cell‐bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N‐succinyl phenylalanine p‐nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.
Biocides incorporated into plasticized polyvinylchloride reduce adhesion of Pseudomonas fluorescens BL146 and substratum hydrophobicity
Authors: Jones C.R. , Handley P.S. , Robson G.D. , Eastwood I.M. , Greenhalgh M.
Published: March 2008

A quantitative adhesion assay was developed to monitor attachment of Pseudomonas fluorescens BL146 to discs of plasticized polyvinylchloride (pPVC) with and without incorporated biocides. Adherent cells were quantified by radiolabelling with DL‐[4,5‐3H]leucine. Adhesion reached a maximum after 6 h incubation at an initial cell concentration of 5 x 107 cells ml‐1. The adhesion assay was used to compare bacterial attachment to pPVC containing the biocides 10,10‐oxybisphenoxyarsine (OBPA), 2‐n‐octyl‐4‐isothiazolin‐3‐one (OIT), 2,3,5,6‐tetrachloro‐4‐(methylsulphonyl)pyridine (TCMP) and N‐trichloromethylthiophthalimide (NCMP) at 0, 250, 750 and 2250 ppm. All four biocides reduced adhesion with increasing concentration, with statistically significant reductions in adhesion (< 53%) occurring with OBPA, OIT and TCMP at 2250 ppm. Significant reductions in adhesion to pPVC containing OBPA were found whether adhering cells were viable or non‐viable. The hydrophobicity of the pPVC surfaces was quantified by the measurement of water contact angles using the Wilhelmy plate technique. A trend of reduced hydrophobicity was observed with increasing biocide concentration. Incorporation of all four biocides at 2250 ppm caused statistically significant reductions in contact angle from 104.7° to a minimum of 93.5°. Incorporation of biocides into pPVC therefore concurrently reduces both bacterial adhesion and surface hydrophobicity.
Microbiological contamination of ovine carcasses associated with the presence of wool and faecal material
Authors: Biss M.E. , Hathaway S.C.
Published: March 2008

The microbiological contamination of ovine hind legs at sites which were visibly clean (control carcasses), sites immediately adjacent to and below visually contaminated sites, and sites contaminated with visible faecal material or wool were determined by excision sampling immediately after pelting and immediately after a pre‐evisceration wash. The mean aerobic plate count (APC) andEscherichia colicount (EC) at clean sites immediately after pelting ranged from log10cm‐23.98 to 4.44 and log10cm‐20.96 to 1.51, respectively. These levels of contamination were significantly lower than those on sites contaminated with faecal material (log10cm‐26.00 and 3.00, respectively) or wool (log10cm‐25.44 and 2.45, respectively). The presence of faecal material or wool on the carcass was not associated with increased bacterial numbers on visually clean areas of the carcass. This indicates that the presence of faecal material or wool alone cannot be used as an indicator of the hygienic status of the carcass as a whole, particularly in the role of on‐line monitoring parameters for HACCP systems. Pre‐evisceration washing of carcasses had very little effect on the uncontaminated areas of the carcasses, but reduced the mean APC and EC at the site of visible contaminants. There was little evidence of redistribution of bacteria to immediately adjacent but visually clean sites. However, the residual levels of both APCs and ECs directly at sites of faecal contamination after washing were still significantly higher than at visually clean sites. Application of HACCP principles to ovine slaughter and dressing suggests that visible faecal material should be removed by trimming, whereas pre‐evisceration washes can have a practical and microbiologically validated role in the removal of wool. There was generally a good correlation between APCs and ECs at the uncontaminated sites prior to pre‐evisceration washing, suggesting that in some situations APCs can act as a useful indicator of both general carcass hygiene and the presence of faecal indicators.
Response of Leuconostoc œnos to environmental changes
Authors: Garbay S. , Lonvaud‐Funel A.
Published: March 2008

Malolactic fermentation, a spontaneous step in vinification, is frequently controlled by use of malolactic bacterial starters of Leuconostoc œnos. Strains isolated from fermenting wines and then submitted to industrial processes to obtain freeze‐dried preparations lose their natural adaptation to survive and to grow in wine. However, culture conditions influence their adaptability as well as their survival after exposure to stress. As the plasma membrane was considered as the primary target for the deleterious effects of the environment, the changes of its components after cell heating and incubation in hostile conditions were studied. The ratio of phospholipids to proteins of the membrane decreased significantly when cells were submitted to any kind of stress. Phospholipids decreased while the protein content increased up to fivefold. Only some membrane proteins were over‐synthesized compared to control cells. Seventeen protein bands were separated by gel electrophoresis of which three in particular were always over‐expressed after the cells were shocked. Some also appeared when the cells entered the stationary phase. These results show that Leuc. œnos responded to environmental stresses by changing its membrane composition. Stress proteins appeared whatever the nature of the shock. This phenomenon may be involved in the acquisition of resistance of Leuc. œnos to harsh conditions in wine.
Microtitre plate riboprobe system for detection of ultrasonicated Campylobacter jejuni genomic DNA
Authors: Lamoureux M. , Fliss I. , Messier S. , Blais B.W. , Holley R.A. , Simard R.E.
Published: March 2008

Microtitre plate nucleic acid probe hybridization systems were developed for the detection of thermophilic Campylobacter and Campylobacter jejuni. Specific RNA probes obtained by in vitro transcription of DNA templates synthesized by polymerase chain reaction using two sets of specific primers incorporating bacteriophage T7 promoter sequences were immobilized on a microtitre plate. The hybridizations were carried out on samples of genomic DNA sheared by ultrasonication. Optimum conditions for the ultrasonic treatment were determined in order to obtain the highest degree of hybridization with immobilized RNA probe. Finally, detection of RNA‐DNA hybrids in the wells was accomplished by an immunoenzymatic assay using a monoclonal anti‐RNA‐DNA hybrid antibody. This rapid, simple hybridization and immunoenzymatic assay system will facilitate the detection of Campylobacter in foods and clinical samples.
Heat‐stable serotyping antigens expressed by strains of Campylobacter jejuni are probably capsular and not long‐chain lipopolysaccharide
Authors: Chart H. , Frost J.A. , Oza A. , Thwaites R. , Gillanders S. , Rowe B.
Published: March 2008

The role of lipopolysaccharide (LPS) in the serotyping of Campylobacter jejuni based on heat‐stable antigens was examined using SDS‐PAGE and a silver stain for carbohydrate. None of the 32 type strains of Camp. jejuni expressed long‐chain LPS. Rabbit antibodies, prepared to 10 selected strains of Camp. jejuni, reacted with surface‐exposed carbohydrate antigens, which were not LPS. This study suggests that the heat‐stable antigens of Camp. jejuni, which form the basis for the established Penner serotyping scheme, are probably capsular and not LPS.
The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing
Authors: Fenlon D.R. , Wilson J. , Donachie W.
Published: March 2008

Listeria monocytogenes was isolated in low numbers from a variety of environmental samples associated with the primary production of food, including vegetation, faeces and meat. The organism was rarely detected on growing grass and vegetables prior to processing. The excretion of L. monocytogenes by farm animals was linked to their diet, with animals fed entirely on hay or manufactured diets not excreting detectable levels of Listeria (i.e. absence in 25 g). However, animals fed on silage, which is frequently contaminated with L. monocytogenes, commonly excreted the organism. Transport of live animals over long distances (> 100 km) significantly increased the level of excretion of Listeria, but the contamination of carcasses of sheep and cattle was not high. Pigs and poultry faeces were free of Listeria prior to slaughter and pig carcasses were not found to have Listeria present. Frozen and chilled chicken did show detectable levels reflecting the greater potential for contamination during poultry processing. Samples of minced beef were tested and 21 of 23 samples were positive for L. monocytogenes, demonstrating that processing significantly increases the level of contamination compared to whole carcasses. Multilocus enzyme electrophoresis of a representative selection of the isolates showed that there was a wide range of electrophoretic types present in the primary production environment, relatively few of which have been linked to cases of human listeriosis. However, these types do arise on farms and occasional contamination of food raw material by potentially virulent strains may be sufficient to allow adaptable strains to become established in the processing environment and thus be responsible for more widespread contamination of the food available to the consumer.
Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum
Authors: Kelly W.J. , Asmundson R.V. , Huang C.M.
Published: March 2008

A protease‐sensitive antibacterial substance, produced by a strain of Lactobacillus plantarum isolated from fermented corn, was classified as a bacteriocin and designated plantaricin KW30. The bacteriocin was stable to heat, pH and treatment with surfactants, and unaffected by α‐amylase, lipase or lysozyme. Plantaricin KW30 exhibited a bactericidal and non‐bacteriolytic mode of action against indicator cells, and inhibitory activity was limited to other lactobacilli. Maximum production was in MRS broth, and coincided with the onset of stationary phase under conditions of low pH and high cell numbers. In a complex medium bacteriocin production was enhanced by the presence of sodium acetate and Tween 80. Curing experiments gave derivatives that no longer produced the bacteriocin but retained immunity to it. These Bac derivatives showed the same plasmid pattern as the parent strain suggesting a chromosomal location for the genes for bacteriocin production.