Showing papers in "Journal of Applied Microbiology in 2004"

Development of an extensive set of 16S rDNA‐targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real‐time PCR

Abstract: Aims: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR. Methods and Results: A linear range of quantification between 0·1–10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30–4500 to 1·9 × 106–6·0 × 106 target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification. Conclusions: Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples. Significance and Impact of the Study: To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.

Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat.

Abstract: A . K H A L I D , M . A R S H A D A N D Z . A . Z A H I R . 2004. Aims: Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants for improving the growth and yield of agricultural crops, however screening for the selection of effective PGPR strains is very critical. This study focuses on the screening of effective PGPR strains on the basis of their potential for in vitro auxin production and plant growth promoting activity under gnotobiotic conditions. Methods and Results: A large number of bacteria were isolated from the rhizosphere soil of wheat plants grown at different sites. Thirty isolates showing prolific growth on agar medium were selected and evaluated for their potential to produce auxins in vitro. Colorimetric analysis showed variable amount of auxins (ranging from 1AE 1t o 12AE 1m g l )1 ) produced by the rhizobacteria in vitro and amendment of the culture media with L-tryptophan (L-TRP), further stimulated auxin biosynthesis (ranging from 1AE 8t o 24AE 8m g l )1 ). HPLC analysis confirmed the presence of indole acetic acid (IAA) and indole acetamide (IAM) as the major auxins in the culture filtrates of these rhizobacteria. A series of laboratory experiments conducted on two cv. of wheat under gnotobiotic (axenic) conditions demonstrated increases in root elongation (up to 17AE3%), root dry weight (up to 13AE5%), shoot elongation (up to 37AE7%) and shoot dry weight (up to 36AE3%) of inoculated wheat seedlings. Linear positive correlation (r ¼ 0AE99) between in vitro auxin production and increase in growth parameters of inoculated seeds was found. Based upon auxin biosynthesis and growth-promoting activity, four isolates were selected and designated as plant growth-promoting rhizobacteria (PGPR). Auxin biosynthesis in sterilized vs nonsterilized soil inoculated with selected PGPR was also monitored that revealed superiority of the selected PGPR over indigenous microflora. Peatbased seed inoculation with selected PGPR isolates exhibited stimulatory effects on grain yields of tested wheat cv. in pot (up to 14AE7% increase over control) and field experiments (up to 27AE5% increase over control); however, the response varied with cv. and PGPR strains. Conclusions: It was concluded that the strain, which produced the highest amount of auxins in nonsterilized soil, also caused maximum increase in growth and yield of both the wheat cv. Significance and Impact of Study: This study suggested that potential for auxin biosynthesis by rhizobacteria could be used as a tool for the screening of effective PGPR strains.

Identification of early microbial colonizers in human dental biofilm

Abstract: J. LI, E.J. HELMERHORST, C.W. LEONE, R.F. TROXLER, T. YASKELL, A.D. HAFFAJEE, S.S. SOCRANSKY AND F.G. OPPENHEIM. 2004. Aims: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation. Methods and Results: AcheckerboardDNA-DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis (Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. Conclusion: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro-organisms. Significance and Impact of the Study: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

Abstract: AIMS Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.

Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua.

Abstract: Aims: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. Methods and Results: In laboratory media, MICs of 15, 75 and 35 mmol l−1 vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10–40 mmol l−1 vanillin inhibited respiration of E. coli and L. innocua. Addition of 50–70 mmol l−1 vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l−1 vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l−1 vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l−1 vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. Conclusions: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. Significance and Impact of the Study: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.

Quantitative evaluation of antifungal activity of metallic oxide powders (MgO, CaO and ZnO) by an indirect conductimetric assay

Abstract: Aims: To evaluate antifungal activities of MgO, CaO and ZnO powders quantitatively by indirect conductimetric assay. Methods and Results: Candida albicans NBRC1060, Saccharomyces cerevisiae NBRC1950, Aspergillus niger NBRC4067 and Rhizopus stolonifer NBRC4781 were used as test micro-organisms. The indirect conductimetric assay, in which the change in electrical conductivity of an alkaline solution (NaOH) is produced by absorption of CO2 from microbial metabolism, could offer a simple and rapid evaluation of the antifungal activity within 24–48 h. The conductivity curves obtained for MgO, CaO and ZnO were analysed using the growth inhibition kinetic model proposed by Takahashi for calorimetric evaluation, and the kinetic parameters and minimum inhibitory concentration ([I]100) could be determined. MgO and CaO powders exhibited the antimicrobial activities against all fungi used in this study and showed little differences between types of fungi. However, although ZnO powder inhibited fungal growth, the values of [I]100 were over 100 mg ml−1. Conclusions: Although a common method for evaluating antifungal activity requires over 5–7 days, the indirect assay could provide a rapid and quantitative evaluation of antifungal activity within approx. 2 days, and MgO and CaO were found to have antifungal activities. Significance and Impact of the Study: The indirect assay can be applicable for simple and rapid evaluation of the antimicrobial activity of insoluble or slightly soluble materials with high turbidity such as antibacterial ceramic powders. Moreover, these materials can be useful for controlling fungi in food processing and the environment.

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

Abstract: Aim: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. Methods and Results: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. Conclusions: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. Significance and Impact of the Study: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

Abstract: Aims: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. Methods and Results: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4′,6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were ≤2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50–90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were ≤2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. Conclusions: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. Significance and Impact of the Study: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.

Screening for novel laccase-producing microbes

Abstract: Aims: To discover novel laccases potential for industrial applications. Methods and Results: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60°C. Conclusions: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478. Significance and Impact of the Study: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases.

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification.

Abstract: K. SEN AND M. RODGERS. 2004. Aims: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. Methods and Results: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. Conclusions: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. Significance and Impact of the Study: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.

Inactivation of indicator micro‐organisms from various sources of faecal contamination in seawater and freshwater

Abstract: Aim: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. Methods and Results: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0·001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115–121 and 144–177 h at 20 and 14°C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0·001). Conclusions: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. Significance and Impact of the Study: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil.

Abstract: V . D U A R T E , S . H . D E B O E R , L . J . W A R D A N D A . M . R . D E O L I V E I R A . 2004. Aims: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. Methods and Results: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. Conclusions: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. Significance and Impact of the Study: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26.

Abstract: Aims: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. Methods and Results: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2 : 1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was l-Glu, d-Orn, l-Tyr, d-allo-Thr, d-Ala, d-Val, l-Pro, and l-Ile in a molar ratio of 3 : 1 : 2 : 1 : 1 : 2 : 1 : 1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13–14 (m/z 303, 316) and there was no methyl group. Conclusion: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. Significance and Impact of the Study: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.

Role of viability of probiotic strains in their persistence in the gut and in mucosal immune stimulation

Abstract: Fil: Maldonado Galdeano, Maria Carolina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tucuman. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquimica, Quimica y Farmacia; Argentina

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

Abstract: Aims: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. Methods and Results: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0·2 and 6·7 g l−1) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. Conclusions: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. Significance and Impact of the Study: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.

Modelling and predicting the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon

Abstract: B. G IMEN E Z A N D P. D A L G A A R D. 2003. Aims: To evaluate and model the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon. Methods and Results: Growth kinetics of L. monocytogenes, lactic acid bacteria (LAB), Enterobacteriaceae, enterococci and Photobacterium phosphoreum were determined in two series of challenge tests with sliced and vacuum-packed cold-smoked salmon (SVP-CSS). The product contained a high level of smoke components and at 2� C levels of L. monocytogenes increased <100-fold in 193 days. Without the addition of spoilage micro-organisms, L. monocytogenes reached ca 10 8 CFU g )1 at 5, 10, 17AE5 and 25� C. Inoculation with spoilage micro-organisms reduced this level to 10 2 -10 4 CFU g )1 . LAB dominated the spoilage microfora of SVP-CSS and competition between LAB and L. monocytogenes in SVP-CSS was appropriately described by a simple expansion of the Logistic model. This interaction model aided in predicting the growth of L. monocytogenes in naturally contaminated SVP- CSS when it was used in combination with expanded versions of existing secondary models for L. monocytogenes and LAB. Conclusions: Temperature, water activity/NaCl, simultaneous growth of LAB, smoke components and to a lesser extent lactate and pH control growth of L. monocytogenes in SVP-CSS. These factors must be included in mathematical models to predict growth of the pathogen in this product. Significance and Impact of the Study: The suggested predictive model can be used to support assessment and management of the human health risk due to L. monocytogenes in SVP-CSS.

Phylogenetic diversity of drinking water bacteria in a distribution system simulator

Abstract: Aims: To characterize the composition of microbial populations in a distribution system simulator (DSS) by direct sequence analysis of 16S rDNA clone libraries. Methods and Results: Bacterial populations were examined in chlorinated distribution water and chloraminated DSS feed and discharge water. Bacterial strains isolated from DSS discharge water on R2A medium were identified using 16S rDNA sequence analysis. The majority of the bacteria identified were α-proteobacteria, ranging from approx. 34% in the DSS discharge water to 94% of the DSS isolates. Species richness estimators Chao1 and ACE (abundance-based coverage estimators) indicated that the chlorinated distribution water sample was representative of the total population diversity, while the chloraminated DSS feed water sample was dominated by Hyphomicrobium sp. sequences. The DSS discharge water contained the greatest diversity of α-, β-, γ-proteobacteria, with 36% of the sequences being operational taxonomic units (OTUs, sequences with >97·0% homology). Conclusions: This work demonstrated the dominance of α-proteobacteria in distribution system water under two different disinfectant residuals. The shift from chlorine to monochloramine residual may have played a role in bacterial population dynamics. Significance and Impact of the Study: Accurate identification of bacteria present in treated drinking water is needed in order to better determine the risk of regrowth of potentially pathogenic organisms within distribution systems.

Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence

Abstract: Aims: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system Methods and Results: A pilot-scale domestic water system consisting of seven galvanized steel re-circulation loops and copper dead legs was constructed Culture techniques, confocal laser scanning microscopy after fluorescent in situ hybridization and viability staining with the BacLight® LIVE/DEAD kit were used for planktonic and biofilm flora monitoring Before starting the treatments, the system was highly contaminated with Legionella pneumophila and biofilm populations mainly consisted of β-proteobacteria In the water and the biofilm of the loops, continuous application of chlorine dioxide (0·5 mg l−1), or chlorine (2·5 mg l−1) were very effective in reducing the microbial flora, including L pneumophila Heterotrophic bacteria, although strongly reduced, were still detectable after ozone application (0·5 mg l−1), whereas with monochloramine (0·5 mg l−1) and copper–silver ionization (0·8/0·02 mg l−1), the contamination remained significantly higher Monochloramine and copper–silver did not remove the biofilm During copper–silver application, Legionella re-growth was observed Only chlorine dioxide led to detectable effects in the dead leg Amoebae could not be eliminated, and after interrupting the treatments, L pneumophila quickly recovered their initial levels, in all cases Conclusions: Chlorine dioxide, applied as a continuous treatment, was identified in this study as the most efficient for controlling L pneumophila in a domestic water system Chlorine dioxide showed a longer residual activity, leading to improved performance in the dead leg Amoebae resisted to all the treatments applied and probably acted as reservoirs for L pneumophila, allowing a quick re-colonization of the system once the treatments were interrupted Significance and Impact of the Study: Control of microbial contamination requires maintenance of a constant disinfectant residual throughout the water system Treatment strategies targeting free-living amoebae should lead to improved control of L pneumophila Such treatment strategies still have to be investigated

Use of microcosms to determine persistence of Escherichia coli in recreational coastal water and sediment and validation with in situ measurements

Abstract: D . L . C R A I G , H . J . F A L L O W F I E L D A N D N . J . C R O M A R . 2004. Aims: To determine the persistence of the faecal indicator organism Escherichia coli in recreational coastal water and sediment using laboratory-based microcosms and validation with in situ measurements. Methods and Results: Intact sediment cores were taken from three distinct coastal sites. Overlying estuarine water was inoculated with known concentrations of E. coli and decay rates from both overlying water and sediment were determined following enumeration by the membrane filtration method at fixed time intervals over a 28-day period. It was demonstrated that E. coli may persist in coastal sediment for >28 days when incubated at 10� C. Escherichia coli survival was found to have an inverse relationship with temperature in both water and sediment. In general the decay rate for E. coli was greater in water than in sediment. Small particle size and high organic carbon content were found to enhance E. coli survival in coastal sediments in the microcosms. Conclusions: Results of this microcosm study demonstrated the more prolonged survival of E. coli in coastal sediments compared with overlying water, which may imply an increased risk of exposure because of the possible resuspension of pathogenic micro-organisms during natural turbulence or human recreational activity. Significance and Impact of the Study: A more accurate estimate of exposure risk has been described which may subsequently be used in a quantitative microbial risk assessment for recreational coastal waters.

Production of exopolysaccharides by Antarctic marine bacterial isolates

Abstract: Aims: This study was undertaken to examine and characterize Antarctic marine bacterial isolates and the exopolysaccharides (EPS) they produce in laboratory culture. Methods and Results: Two EPS-producing bacterial strains CAM025 and CAM036 were isolated from particulate material sampled from seawater and sea ice in the southern ocean. Analyses of 16S rDNA sequences placed these isolates in the genus Pseudoalteromonas. In batch culture, both strains produced EPS. The yield of EPS produced by CAM025 was 30-fold higher at -2 and 10°C than at 20°C. Crude chemical analyses showed that these EPS were composed primarily of neutral sugars and uronic acids with sulphates. Gas chromatographic analysis of monosaccharides confirmed these gross compositional findings and molar ratios of monosaccharides revealed differences between the two EPS. Conclusions: The EPS produced by Antarctic bacterial isolates examined in this study appeared to be polyanionic and, therefore, 'sticky' with respect to cations such as trace metals. Significance and Impact of the Study: As the availability of iron as a trace metal is of critical importance in the southern ocean where it is know to limit primary production, the role of these bacterial EPS in the Antarctic marine environment has important ecological implications.

Microbiological characterization and probiotic potential of koko and koko sour water, African spontaneously fermented millet porridge and drink

Abstract: Aims: To identify and examine the diversity of predominant lactic acid bacteria (LAB) in koko and koko sour water (KSW) from different Ghanaian production sites with regard to pattern of fermentation (API 50 CHL), genotype, antimicrobial activity, and resistance to low pH and bile salts. Methods and Results: In total 215 LAB were isolated from koko and KSW. The isolates were identified using intergenic transcribed spacers (ITS)-PCR restriction fragment length polymorphism (RFLP), API 50 CHL, restriction enzyme analysis with pulsed-field gel electrophoresis (REA-PFGE) and sequencing of the 16S rRNA gene. The dominating micro-organisms in koko was found to be Weisella confusa and Lactobacillus fermentum, followed by Lact. salivarius and Pediococcus spp. Chemometric data analysis were used to link the LAB species to the different production stages and production sites. At intra-species level the isolates were found to have a great diversity. The isolates were investigated for antimicrobial activity using agar diffusion assays, and acid and bile tolerance. Most isolates showed low levels of antimicrobial activity towards the indicator strain Listeria innocua, but not towards the bacteriocin-sensitive Lact. sakei. Growth of all LAB isolates was unaffected by the presence of 0·3% (v/v) oxgall bile. The isolates were able to survive, but were not able to grow in growth medium adjusted to pH 2·5. Conclusions: The dominating LAB of koko and KSW were W. confusa and Lact. fermentum showing a pronounced taxonomic biodiversity at sub-species level between stages within the production as well as between production sites. Other species observed in KSW were Lact. salivarius, Ped. pentosaceus, Ped. acidilactici and Lact. paraplantarum. They occurred in levels of 108 CFU ml−1 in fresh KSW and showed uniform antimicrobial activity, and acid and bile tolerance. Significance and Impact of the Study: The present study gives a detailed picture of the taxonomy and diversity of LAB in an African-fermented millet product that may have potential as a probiotic product for the local population. The chemometric tools Principal Component Analysis and anova Partial Least Squares Regression were proven to be useful in the analysis of microbial groupings and associations with specific sites and stages in the production of koko and KSW.

Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel.

Abstract: Aims: This study investigated the in vitro effects of water activity (aw; 0·85–0·987) and temperature (10–40°C) on growth and ochratoxin A (OTA) production by two strains of Aspergillus carbonarius isolated from wine grapes from three different European countries and Israel on a synthetic grape juice medium representative of mid-veraison (total of eight strains). Methods and Results: The synthetic grape juice medium was modified with glycerol or glucose and experiments carried out for up to 56 days for growth and 25 days for OTA production. The lag phase prior to growth, growth rates and ochratoxin production were quantified. Statistical comparisons were made of all factors and multiple regression analysis used to obtain surface response curves of aw × temperature for the eight strains and optimum growth and OTA production by A. carbonarius. The lag phase increased from 20 days at marginal temperatures and water availabilities. Generally, most A. carbonarius strains grew optimally at 30–35°C, regardless of solute used to modify aw, with no growth at <15°C. The optimum aw for growth varied from 0·93 to 0·987 depending on the strain, with the widest aw tolerance at 25–30°C. There was no direct relationship among growth, environmental factors and country of origin of individual strains. Optimum conditions for OTA production varied with strain. Some strains produced optimal OTA at 15–20°C and 0·95–98 aw. The maximum OTA produced after 10 days was about 0·6–0·7 μg g−1, with a mean production over all eight strains of 0·2 μg g−1 at optimum environmental conditions. Conclusions: This work demonstrates that optimum conditions for OTA production are very different from those for growth. While growth rates differed significantly between strains, integration of the OTA production data suggests possible benefits for use of the information on a regional basis. Significance and Impact of the Study: Very little detailed information has previously been available on the ecology of A. carbonarius. This knowledge is critical in the development and prediction of the risk models of contamination of grapes and grape products by this species under fluctuating and interacting environmental parameters.

Strategies for the detection of Escherichia coli O157:H7 in foods

Abstract: This review assesses the various methods used to detect Escherichia coli O157:H7 in foods. As this organism has been involved in many outbreaks of disease, it is essential to develop a rapid, yet reliable, method of detection. Conventional methods such as culturing and biochemical tests are covered, followed by a discussion of immunological methods. Both enzymatic and nonenzymatic approaches are discussed, and commercially available kits based on these principles are described. PCR has allowed the rapid amplification of very small numbers of organisms and standard PCR along with multiplex and real-time PCR are discussed. Biosensors and microarrays can provide real-time detection and the current status of each of these is reviewed. It is believed that molecular beacons and integrated systems (lab-on-a-chip) can offer potential advantages for the detection of this pathogen and both are analysed. Drawbacks and advantages of each method described are considered throughout the article. 2. INTRODUCTION

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

Abstract: Aims: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. Methods and Results: Spores of B. subtilis were killed 80–99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, OxoneTM, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. Conclusions: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. Significance and Impact of the Study: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802.

Abstract: G. BLAIOTTA, D. ERCOLINI, C. PENNACCHIA, V. FUSCO, A. CASABURI, O. PEPE AND F. VILLANI. 2004. Aims: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin genecluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative(CNS) staphylococcal strains isolated from meat and dairy products.Methods and Results: Specificity and reliability of the PCR detection methods used were ascertained by usingnine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seAtoseE; seG, seH, seI, seM, seJ, seN andseO) and egc (containing the following sequence of genes: seO, seM, seI, uent1, uent2, seN and seG). Of 109 wildStaphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference andwild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicatethat the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv).Conclusions: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-typesalami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare.Significance and Impact of the Study: During this study it was shown that at least five different egc may exist inS. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.Keywords: coagulase-negative staphylococci, enterotoxin gene cluster (egc), food, seGv (seG variant), seIv (seIvariant), staphylococcal enterotoxin (se) genes, Staphylococcus aureus.

Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media

Abstract: Aims: To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions. Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps. Methods and Results: Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures. In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD+-isocitrate dehydrogenase (ICDH) and NADP+-ICDH were not detectable in the cell-free extract. Specifically, in C. echinulata, NAD+-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period. On the contrary, in M. isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD+- and NADP+-ICDH were not detectable after nitrogen depletion. In C. echinulata reserve lipid was not degraded after glucose had been exhausted. Supplementations of the medium with Fe3+, yeast extract or Mg2+ induced, however, reserve lipid breakdown and formation of lipid-free material. In M. isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis. Nevertheless, in multiple-limited media, in which Mg2+ or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed. In both strains, the quantity of γ-linolenic acid (GLA) in the reserve lipids [varying between 9 and 16% (w/w) in C. echinulata and 1·5–4·5% (w/w) in M. isabellina] was proportional to lipid-free biomass. Conclusions: Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific. While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes. As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis. Significance and Impact of the Study: Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds. Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media.

Cellular effects of monohydrochloride of l‐arginine, Nα‐lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus

Abstract: Aims: Here we study the effect of monohydrochloride of l-arginine, Nα-lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 μg ml−1, respectively Methods and Results: Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane Cell integrity was altered mainly in the outer membrane of S typhimurium, but there was no significant change in the cytoplasm However, in Staph aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis In S typhimurium the proportion of damaged cells after 24 h was 97% and in Staph aureus 56·3% LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7·7 and 3·34 μg ml−1 for Staph aureus and S typhimurium, respectively Membrane disruption was detected by measuring the proton flow across the membrane Conclusions: Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted Significance and Impact of the Study: This is the first time the cellular effects of LAE on bacterial cells were studied

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

Abstract: Aims: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. Methods and Results: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0·05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. Conclusions: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. Significance and Impact of the Study: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.

Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria.

Abstract: Aims: To investigate whether sublethal treatments of stationary-phase probiotic cultures enhance their survival during lethal treatments and to adapt these treatments to the fermenter-scale production of probiotic cultures. Methods and Results: Conditions for acid and heat pretreatments were screened for three Lactobacillus and two Bifidobacterium strains. Strains were sublethally treated both at laboratory scale and at fermenter scale in a strain-specific manner and exposed to a subsequent lethal treatment. At laboratory scale viability improvement was detected in each strain. However, improvement was more pronounced in the Lactobacillus than in the Bifidobacterium strains. At fermenter scale three strains were tested: for the two Lactobacillus strains a marked improvement in viability was obtained whereas for the Bifidobacterium strain the improvement was either minor or not detected. Conclusions: Development of treatments for viability enhancement of probiotic strains is feasible, but strain-specific optimization is necessary to obtain notable improvements. Significance and Impact of the Study: Strain-specific treatments were developed for the viability enhancement of stationary-phase probiotic cells both at laboratory and fermenter scale. These results can be utilised in the production of probiotic cultures with improved viability.

Antifungal effects of herbal essential oils alone and in combination with ketoconazole against Trichophyton spp.

Abstract: Aims: To determine the effects of herbal essential oils on Trichophyton spp. growth and to evaluate the effects of Pelargonium graveolens oil and its main components citronellol and geraniol combined with ketoconazole against Trichophyton spp. Methods and Results: Growth inhibition of six Trichophyton spp. by herbal essential oils was accessed and the combined effects of P. graveolens oil and its main components citronellol and geraniol were evaluated using a checkerboard microtitre assay against T. schoenleinii, T. erinacei and T. soudanense. The essential oil fraction of P. graveolens and its main components, geraniol and citronellol, exhibited strong synergism with ketoconazole against T. schoenleinii and T. soudanense, with fractional inhibitory concentration (FIC) indices in the range of 0·18–0·38. Conclusions: The antifungal effects of ketoconazole against Trichophyton spp. are enhanced significantly by administering it in combination with the essential oil fraction of P. graveolens or its main components, because of strong synergism, especially against T. soudanense and T. schoenleinii. Significance and Impact of the Study: The combination of ketoconazole and the essential oil fraction from P. graveolens or its main components for treatment of infections caused by Trichophyton species may reduce the minimum effective dose of ketoconazole, and thus minimize the side-effects of ketoconazole.