Showing papers in "Letters in Applied Microbiology in 2002"

High organic loading influences the physical characteristics of aerobic sludge granules.

Abstract: Aims: The effect of high organic loading rate (OLR) on the physical characteristics of aerobic granules was studied. Methods and Results: Two column-type sequential aerobic sludge blanket reactors were fed with either glucose or acetate as the main carbon source, and the OLR was gradually raised from 6 to 9, 12 and 15 kg chemical oxygen demand (COD) m−3 d−1. Glucose-fed granules could sustain the maximum OLR tested. At a low OLR, these granules exhibited a loose fluffy morphology dominated by filamentous bacteria. At higher OLRs, these granules became irregularly shaped, with folds, crevices and depressions. In contrast, acetate-fed granules had a compact spherical morphology at OLRs of 6 and 9 kg COD m−3 d−1, with better settling and strength characteristics than glucose-fed granules at similar OLRs. However, acetate-fed granules could not sustain high OLRs and disintegrated when the OLR reached 9 kg COD m−3 d−1. Conclusions: The compact regular microstructure of the acetate-fed granules appeared to limit mass transfer of nutrients at an OLR of 9 kg COD m−3 d−1. The looser filamentous microstructure of the glucose-fed granules and the subsequent irregular morphology delayed the onset of diffusion limitation and allowed significantly higher OLRs to be attained. Significance and Impact of the Study: High organic loading rates are possible with aerobic granules. This research would be helpful in the development of aerobic granule-based systems for high-strength wastewaters.

Fungicidal activity of essential oils of Cinnamomum zeylanicum (L.) and Syzygium aromaticum (L.) Merr et L.M. Perry against crown rot and anthracnose pathogens isolated from banana.

Abstract: Aims: To develop a post-harvest treatment system against post-harvest fungal pathogens of banana using natural products. Methods and Results:Colletotrichum musae was isolated and identified as the causative agent responsible for anthracnose peel blemishes while three fungi, namely Lasiodiplodia theobromae, C. musae and Fusarium proliferatum, were identified as causative agents responsible for crown rot. During the liquid bioassay, cinnamon [Cinnamomum zeylanicum (L.)] leaf, bark and clove [Syzygium aromaticum (L.)] oils were tested against the anthracnose and crown rot pathogens. The test oils were fungistatic and fungicidal against the test pathogens within a range of 0·03−0·11% (v/v). Conclusions: Cinnamon and clove essential oils could be used as antifungal agents to manage post harvest fungal diseases of banana. Significance and Impact of the Study: Cinnamon and clove essential oil could be used as alternative post-harvest treatments on banana. Banana treated with essential oil is chemically safe and acceptable to consumers. Benomyl (Benlate), which is currently used to manage fungal pathogens, can cause adverse health effects and could be replaced with volatile essential oils.

Antimicrobial effect of essential oils on the seafood spoilage micro‐organism Photobacterium phosphoreum in liquid media and fish products

Abstract: Aims: To evaluate the antimicrobial effect of nine essential oils (EO) on P. phosphoreum and determine the effect of oregano oil on the shelf-life of modified atmosphere-packed (MAP) cod fillets. Methods and Results: The antimicrobial effect of EO was studied in a liquid medium and in product storage trials. Oils of oregano and cinnamon had strongest antimicrobial activity, followed by lemongrass, thyme, clove, bay, marjoram, sage and basil oils. Oregano oil (0·05%, v/w) reduced growth of P. phosphoreum in naturally contaminated MAP cod fillets and extended shelf-life from 11–12 d to 21–26 d at 2°C. Conclusions: Oregano oil reduced the growth of P. phosphoreum and extended the shelf-life of MAP cod fillets. Significance and Impact of the Study: Mild and natural preservation using EO can extend the shelf-life of MAP seafood through inhibiting the specific spoilage organism P. phosphoreum.

Fungal chitosan production and its characterization.

Abstract: Aims: The objective of this investigation was to evaluate the chitosans produced by several species of fungi. Methods and Results: Representatives of four species of filamentous fungi, Aspergillus niger , Rhizopus oryzae , Lentinus edodes and Pleurotus sajo-caju , and two yeast strains, Zygosaccharomyces rouxii TISTR5058 and Candida albicans TISTR5239, were investigated for their ability to produce chitosan in complex media. Fungal chitosan was produced at 10–140 mg g –1 cell dry weight, had a degree of deacetylation of 84–90% and a molecular weight of 2·7 × 10 4 –1·9 × 10 5 Da with a viscosity of 3·1–6·2 centipoises (cP). Conclusions: Rhizopus oryzae TISTR3189 was found to be the producer of the highest amounts of chitosan. Significance and Impact of the Study: Commercial chitosan could be obtained from Rhizopus mycelia and would have potential applications for medical and agricultural uses.

Aggregation of immobilized activated sludge cells into aerobically grown microbial granules for the aerobic biodegradation of phenol

Abstract: H . - L . J I A N G , J . - H . T A Y A N D S . T . - L . T A Y . 2002. Aims: The aim of this study is to evaluate the utility of aerobically grown microbial granules for the biological treatment of phenol-containing wastewater. Methods and Results: A column-type sequential aerobic sludge blanket reactor was inoculated with activated sludge and fed with phenol as the sole carbon source, at a rate of 1AE5 g phenol l )1 d )1 . Aerobically grown microbial granules first appeared on day 9 of reactor operation and quickly grew to displace the seed flocs as the dominant form of biomass in the reactor. These granules were compact and regular in appearance, and consisted of bacterial rods and cocci and fungi embedded in an extracellular polymeric matrix. The granules had a mean size of 0AE52 mm, a sludge volume index of 40 ml g )1 and a specific oxygen utilization rate of 110 mg oxygen g VSS )1 h )1 (VSS stands for volatile suspended solids). Specific phenol degradation rates increased with phenol concentration from 0 to 500 mg phenol l )1 , peaked at 1AE4 g phenol g VSS )1 d )1 , and declined with further increases in phenol concentration as substrate inhibition effects became important. Conclusions: Aerobically grown microbial granules were successfully cultivated in a reactor maintained at a loading rate of 1AE5 g phenol l )1 d )1 . The granules exhibited a high tolerance towards phenol. Significant rates of phenol degradation were attained at phenol concentrations as high as 2 g l )1 . Significance and Impact of the Study: This is the first study to demonstrate the ability of aerobically grown microbial granules to degrade phenol. These granules appear to represent an excellent immobilization strategy for microorganisms to biologically remove phenol and other toxic chemicals in high-strength industrial wastewaters.

Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut melon and kiwifruit at 4° and 8°C

Abstract: AIM: To establish whether or not carvacrol and cinnamic acid delay microbial spoilage of fresh-cut fruit. METHODS AND RESULTS: Dipping of fresh-cut kiwifruit in carvacrol solutions at 5-15 mM reduced total viable counts from 6.6 to < 2 log cfu g-1 for 21 d at 4 degrees C; however, undesirable colour and odour changes were also observed. Treatment with 1 mM of carvacrol or cinnamic acid reduced viable counts on kiwifruit by 4 and 1.5 log cfu g-1 for 5 d at 4 degrees C and 8 degrees C, respectively. Treatment of fresh-cut honeydew melon with 1 mM of carvacrol or cinnamic acid extended the lag phase of the microbial flora from less than 1 d in the untreated controls to 3 d at 8 degrees C and 5 d at 4 degrees C. Viable counts on the treated melon were 6 log cfu g-1 lower on Day 3 at 8 degrees C and 4 log cfu g-1 lower on Day 5 at 4 degrees C, compared with the untreated controls. IMPACT OF THE STUDY: Treatment with 1 mM of carvacrol or cinnamic acid delays spoilage of fresh-cut kiwifruit and honeydew melon at chill temperatures without adverse sensory consequences.

Removal of heavy metals by an Aspergillus terreus strain immobilized in a polyurethane matrix

Abstract: M . A . D I A S , I . C . A . L A C E R D A , P . F . P I M E N T E L , H . F . D E C A S T R O A N D C . A . R O S A . 2002. Aims: The aim was to investigate the biosorption of chromium, nickel and iron from metallurgical effluents, produced by a steel foundry, using a strain of Aspergillus terreus immobilized in polyurethane foam. Methods and Results: A. terreus UFMG-F01 was immobilized in polyurethane foam and subjected to biosorption tests with metallurgical effluents. Maximal metal uptake values of 164AE 5m g g )1 iron, 96AE 5m g g )1 chromium and 19AE 6m g g )1 nickel were attained in a culture medium containing 100% of effluent stream supplemented with 1% of glucose, after 6 d of incubation. Conclusions: Microbial populations in metal-polluted environments include fungi that have adapted to otherwise toxic concentrations of heavy metals and have become metal resistant. In this work, a strain of A. terreus was successfully used as a metal biosorbent for the treatment of metallurgical effluents. Significance and Impact of the Study: A. terreus UFMG-F01 was shown to have good biosorption properties with respect to heavy metals. The low cost and simplicity of this technique make its use ideal for the treatment of effluents from steel foundries.

Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone.

Abstract: Aims: (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(furanone) of the marine alga Delisea pulchra was synthesized, and its inhibition of swarming motility and biofilm formation of Bacillus subtilis was investigated. Methods and Results: Furanone was found to inhibit both the growth of B. subtilis and its swarming motility in a concentration-dependent way. In addition, as shown by confocal scanning laser microscopy, furanone inhibited the biofilm formation of B. subtilis. At 40 μg ml−1, furanone decreased the biofilm thickness by 25%, decreased the number of water channels, and reduced the percentage of live cells by 63%. Conclusions, Significance and Impact of the Study: Natural furanone has potential for controlling the multicellular behaviour of Gram-positive bacteria.

Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus

Abstract: Aims: An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. Methods and Results: An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0·25% corn steep liquor, 1% glucose, 0·5% tryptone, 1% sodium citrate, 0·02% MgSO4.7H2O and 0·65% Na2CO3 at 35°C and pH 9·6, agitation at 250 rev min–1 and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26·2% recovery of enzyme with 36·6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11·5 and temperature of 55–60°C. The Km and kcat values observed with synthetic substrates at 37°C and pH 8·0 were 1·1 mmol l–1 and 624 s–1 for Glu-Gly-Ala-Phe-pNA and 3·7 mmol l–1 and 826 s–1 for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. Conclusions: Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. Significance and Impact of the Study: The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.

Mycelial growth and exo-biopolymer production by submerged culture of various edible mushrooms under different media

Abstract: Aims: The effect of synthetic media on the submerged mycelial growth and exo-biopolymer production in various edible mushrooms was investigated in shake flask culture. Methods and Results: Among 19 mushrooms examined, the relatively high yield in mycelial biomass and exo-biopolymer production was achieved in potato malt peptone (PMP) medium. In particular, Ganoderma lucidum NO. 1 and Phellinus linteus KCTC 6190 showed favourable growth in PMP medium with exo-biopolymer concentration of 1170 and 1520 mg l−1, respectively. Conclusions: Enhanced exo-biopolymer production was achieved from Ganoderma lucidum NO. 1 and Phellinus linteus KCTC 6190 in a 5L batch fermentor, indicating approximately 5000 and 2410 mg l−1, respectively. Significance and Impact of the Study: The exo-biopolymer production and mycelial growth from various mushrooms were found to be strongly controlled by different complex media.

Bacteriocin production by spray‐dried lactic acid bacteria

Abstract: Aims: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. Methods and Results: Bacterial cell concentrates were spray-dried and stored at 4∞C and 18∞C and 0AE3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. Conclusions: A higher survival rate was obtained when survival was performed at 4∞C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. Significance and Impact of Study: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.

Long-term survival of Escherichia coli O157 on pasture following an outbreak associated with sheep at a scout camp.

Abstract: Aims: To monitor the decay of E. coli O157 in soil (loamy sand) on a scout campsite following an outbreak in humans. Methods and Results: Samples of soil and sheep faeces were collected from the campsite and tested for the presence of E. coli O157 by immunomagnetic separation (IMS) after enrichment in buffered peptone water + vancomycin at 42°C for 6 h. Enumeration of target was carried out by direct plating onto sorbitol MacConkey agar plates supplemented with cefixime and tellurite (CTSMAC) incubated at 37°C for 24 h. Low numbers (< 100 g−1) were estimated by the most probable number (3-tube MPN) technique. Conclusions: Survival was observed for 15 weeks. Significance and Impact of the Study: A number of laboratory studies have followed the decay of E. coli O157 in soil, animal faeces and water. This study follows (for the first time) the decay of the organism in soil after an outbreak associated with sheep. It demonstrates the long-term persistence of the organism in the environment and the results will be potentially important in performing risk assessments for both human and animal infection.

Microbial status and rumen enzyme profile of crossbred calves fed on different microbial feed additives.

Abstract: AIMS To test various microbial cultures as cattle feed additives. METHODS AND RESULTS Four groups of newly born crossbred calves (average body weight 23.5 kg) were reared on green berseem and calf starter which was devoid of cereal grains. Milk was fed up to 8 weeks of age, starting with one tenths and gradually reducing to one twentieths of the body weight. One hundred millilitres of microbial feed additive or 100 g fermented feed was fed to the animals of group 2 (curd containing lactic acid bacteria at 10(8) cfu x ml(-1)), group 3 (Saccharomyces cerevisiae NCDC-49 at 10(6) cfu x ml(-1)) and group 4 (Lactobacillus acidophilus-15 at 10(8) cfu x ml(-1)). Group 1 served as control. The incidence and duration of diarrhoea was lower in the animals of probiotic fed groups as compared to control group. Out of three microbial feed additives, yeast feeding showed maximum suppression of diarrhoea followed by Lactobacillus and curd. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY There was no effect of probiotic feeding on the log number of cells of lactic acid bacteria, yeast and coliform bacteria in the faeces and rumen liquor at any age. The activities of carboxymethylcellulase, xylanase, beta-glucosidase, alpha-glucosidase, alpha-amylase, protease, urease and pH of the rumen liquor remained unaffected by probiotic feeding at all ages tested in this experiment.

Production of red pigment by submerged culture of Paecilomyces sinclairii.

Abstract: Aims: From a survey of submerged culture of edible mushrooms, a high pigment-producing fungus Paecilomyces sinclairii was selected and its optimal culture conditions investigated. Methods and Results: The optimal culture conditions for pigment production were as follows: inoculum age, 3 d; temperature, 25 °C; initial pH, 6·0; carbon source, 1·5% (w/v) soluble starch; nitrogen source, 1·5% (w/v) meat peptone. Although addition of 10 mmol l−1 CaCl2 to the culture medium slightly increased pigment production, most of the bio-elements examined had no notable or detrimental effect on pigment production. Conclusions: Under the optimal conditions obtained in the flask culture tested, a ninefold increase in pigment production (4·4 g l−1) was achieved using a 5-l batch fermenter. Paecilomyces sinclairii secreted water-soluble red pigment into the culture medium. The pigment colour was strongly dependent on the pH of the solution: red at pH 3–4, violet at pH 5–9 and pink at pH 10–12. Significance and Impact of the Study: The high concentration of pigment (4·4 g l−1) produced by P. sinclairii demonstrates the possibility of commercial production of pigment by this strain, considering its relatively high production yield and light stability.

Preparation of microparticulate β-glucan from Saccharomyces cerevisiae for use in immune potentiation

Abstract: Aims: To develop a method for the preparation of an immunologically active, homogeneous, nonaggregated, microparticulate β-glucan-containing material from the budding yeast Saccharomyces cerevisiae. Methods and Results: Using a combination of sonication and spray-drying, a homogeneous preparation of 1–2-µ diameter β-glucan-containing particles was made from alkali- and acid-insoluble yeast cell wall material. This microparticulate β-glucan remained in suspension longer and, following oral administration at 0·1 mg kg−1 for 14 d, enhanced phagocytosis of mouse peritoneal macrophages significantly better than did aggregated β-glucan particles. Conclusions: A new sonication and spray-drying method can be employed to overcome the problem of aggregation of β-glucan microparticles in aqueous media. Significance and Impact of the Study: A microparticulate form of β-glucan that remains in suspension longer for pharmaceutical applications and has superior immune potentiation characteristics has been developed.

Specific layers in aerobically grown microbial granules

Abstract: Aims: To determine the optimal size of aerobically grown granules for wastewater treatment by measuring specific layers within the granules. Methods and Results: A variety of biological layers were detected by oligonucleotide probes, specific fluorochromes, and fluorescent microspheres. The channels in the granule matrix penetrated to depths of 900 μm. A layer of obligate anaerobic bacteria was detected at a depth of 800 μm below the granule surface. Dead cells were also observed in the granule interior. Conclusions: Aerobically grown granules contained layers of aerobic and anaerobic micro-organisms. Significance and Impact of the Study: The optimal diameter of the aerobic granule is less than 1600 μm. This is twice the distance from the granule surface to the anaerobic layer. This approach can be used to optimize the thickness of other microbial aggregates such as flocs, colonies and biofilms.

Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine

Abstract: G . - J . E . N Y C H A S , E . Z . P A N A G O U , M . L . P A R K E R , K . W . W A L D R O N A N D C . C . T A S S O U . 2002. Aims: To establish the site of microbial growth on naturally black fermented table olives, and to monitor the population dynamics of yeasts and selected micro-organisms together with the changes in organic acid profile and pH in the cover brine during fermentation. Methods and Results: During fermentation, the numbers of Enterobacteriaceae and Pseudomonas spp. in the brine decreased whilst lactic acid bacteria and yeast populations increased. Scanning electron microscopy showed that a yeast-rich biofilm developed on the epicuticular wax of the olive skin during fermentation. Yeasts also predominated in the stomatal openings, but bacteria were more numerous in intercellular spaces in the sub-stomatal flesh. Citric, malic and tartaric acids were the major organic acids accumulating in the brine during fermentation. Conclusions: Micro-organisms associated with the skin, stomata and flesh in fermenting black olives may experience different local conditions to those prevailing in the cover brine. Significant and Impact of the Study: These are the first observations of the microorganisms associated with the fruit of naturally fermented black olives and of the accumulation of specific organic acids during fermentation.

Antibacterial activity of isoflavonoids isolated from Erythrina variegata against methicillin-resistant Staphylococcus aureus.

Abstract: Aims: To screen 16 isoflavonoids isolated from Erythrina variegata (Leguminosae) for their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Methods and Results: The roots of E. variegata were macerated with acetone. The chloroform-soluble fraction of the residue was subjected to repeated silica gel column chromatography followed by elution with various solvents. Structures of the isolated compounds were determined by extensive spectroscopic studies. Each compound was dissolved in dimethyl sulphoxide and added to agar plates (final concentration 1·56–100 µg ml−1) and suspensions of MRSA spotted onto the agar plates to determine the minimum inhibitory concentration (MIC). Repeated silica gel chromatography yielded 16 compounds and spectroscopic studies revealed that all were isoflavonoids. Whilst 14 compounds showed antibacterial activity in this concentration range, the MIC values varied significantly among them. Of the active compounds, 3,9-dihydroxy-2,10-di(γ,γ-dimethylallyl)-6a,11a-dehydropterocarpan (erycristagallin) and 9-hydroxy-3-methoxy-2-γ,γ-dimethylallylpterocarpan (orientanol B) exhibited the highest activity with MIC values of 3·13–6·25 µg ml−1. Conclusions: Erycristagallin and orientanol B showed the highest anti-MRSA activity (3·13–6·25 μg ml−1). Significance and Impact of the Study: Erycristagallin and orientanol B could be leading compounds for phytotherapeutic agents against MRSA infections.

Identification and characterization of Saccharomyces cerevisiae and Saccharomyces paradoxus strains isolated from Croatian vineyards.

Abstract: The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential. A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 °C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins. Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards. Significance and Impact of the Study: this study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.

Series of incidents of Listeria monocytogenes non-invasive febrile gastroenteritis involving ready-to-eat meats

Abstract: Aims: A series of cases and outbreaks of febrile noninvasive gastrointestinal disease involving 31 identified cases was investigated in terms of the numbers and types of Listeria monocytogenes present in the suspect foods (ready-to-eat meats) and clinical samples from cases. Methods and Results: Foods and faecal samples involved in the incidents were tested for the presence and number of L. monocytogenes . Isolates were typed by macrorestriction analysis using pulsed-field gel electrophoresis. The foods contained high levels of L. monocytogenes , in one case 1·8 × 10 7 g −1 . Faecal samples contained L. monocytogenes for up to 15 d after the contaminated food was consumed. All isolates from the food and faecal samples were of serotype 1/2 and were indistinguishable from one another by macrorestriction typing. Conclusions: It is likely that the meats were contaminated either during their manufacture after they had been cooked or by underprocessing. The long shelf lives on these products would have allowed the contaminating L. monocytogenes to grow to the high numbers measured in this study, causing food poisoning as described. Significance and Impact of the Study: Outbreaks of febrile noninvasive listeriosis are relatively rare. This report adds ready-to-eat meats to the range of foods that have acted as vehicles for such outbreaks.

PCR-ELISA detection of Escherichia coli in milk.

Abstract: Aims: The purpose of this study was to develop a reliable molecular procedure for the detection of Escherichia coli in milk. Methods and Results: Robust and expeditious DNA extraction and PCR techniques were evaluated using Enzyme-Linked Immunosorbent Assay (ELISA) detection of biotin-labelled amplicons to facilitate optimal detection of E. coli DNA. Conclusions: It was found that 5 E. coli colony-forming units (cfu) could be detected per PCR reaction using the PCR-ELISA system, equating to a sensitivity of detection of 100 E. coli cfu ml)1 pasteurized milk. Significance and Impact of the Study: This approach should facilitate evaluation of milk contamination and enable rapid detection of E. coli mastitis, leading to correct deployment of relevant antibiotic therapy and improved animal welfare.

Development of a high-volume aerosol collection system for the identification of air-borne micro-organisms.

Abstract: Aims: A high-volume aerosol collector was developed to efficiently capture airborne bacteria in order to assess levels of diversity in the air. Methods and Results: Particulate matter was collected on a device designed to filter 1·4 × 106 litres of air in a 24 h period on a 1-lm pore size polyester membrane. Methods were optimized for extraction of genomic DNA from the air filter concentrate. Preparation times of 90 s with 0·5-0·05 mm diameter zirconia/silica beads yielded the highest concentration genomic DNA that was able to support PCR. A 24-h air sample was taken in Salt Lake City, Utah and the microbial composition was determined by the amplification and sequence analysis of 16S ribosomal DNA fragments. Conclusions: Sequence analysis revealed a large diversity in the type of microbial species present including clones matching the sequence of Clostridium botulinum. The primary components of the aerosol sample included many different spore-forming bacteria as well as more fragile members of the Proteobacteria division. Significance and Impact of the Study: The high-volume air collection and genomic DNA recovery system allows for the rapid detection of both cultivable as well as culture-resistant organisms in the environment.

The M. tuberculosis antigen 85 complex and mycolyltransferase activity.

Abstract: Aims: The antigen 85 complex (Ag85) from Mycobacterium tuberculosis consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2) which play a key role in the pathogenesis of tuberculosis and also exhibit cell wall mycolyltransferase activity. A related protein with similarity to the Ag85 complex was recently annotated in the M. tuberculosis genome as FbpC1. An investigation was carried out to determine whether FbpC1 may also possess mycolyltransferase activity, a characteristic feature of the Ag85 complex. Methods and Results: Heterologous expression of FbpA, FbpC1 and FbpC2 was performed in Escherichia coli. Recombinant proteins were purified under non-denaturating conditions and used in an in vitro mycolyltransferase assay. Conclusions: In contrast to FbpA and FbpC2, recombinant FbpC1 did not possess in vitro mycolyltransferase activity and was not recognized by two monoclonal antibodies to the native Ag85. Significance and Impact of the Study: Mycolyltransferase activity is restricted to FbpA, FbpbB and FbpC2 only; the actual function of FbpC1 remains to be established.

Occurrence and strain diversity of thermophilic campylobacters in cattle of different age groups in dairy herds

Abstract: Aims: To investigate the occurrence and numbers of thermophilic campylobacters excreted by cattle in dairy herds, and to assess the strain diversity within herds. Methods and Results: Faecal samples from 15 animals at each of 24 cattle farms were cultured quantitatively for thermophilic campylobacters and 23% of animals and 83% of farms were positive for Campylobacter jejuni. Young animals had a higher prevalence and higher faecal concentration than older animals. Serotyping and pulsed field gel electrophoresis (PFGE) of isolates showed that the most common serotypes were 2, 4-complex and 11. Serotype 2 was especially prevalent among calves (68% of the positive calves). In eight of the 20 positive herds, all isolates had the same sero- and PFGE type while, in the other herds, two to five different types were isolated. Conclusions: Significant differences were found between age groups in relation to the prevalence and numbers of excreted campylobacters, serotype distribution and strain diversity. The relatively few different strains in each herd indicate that transmission between animals is common. Significance and Impact of the Study: The high prevalence on cattle farms of the human pathogen C. jejuni and the wide distribution of serotype 2, the most common serotype among Danish patients, indicate that cattle might be an important reservoir for human infections. The ability of this serotype to colonize calves in high numbers further indicates that serotype 2 strains may have an advantage over other serotypes.

Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497

Abstract: Aims:The aim of this study was to determine the keratinolytic ability of a range of bacteria and subsequently, to characterize the keratinase showing the greatest biotechnological potential. Methods and Results: Nine bacteria, reported to produce extracellular proteases, were screened for production of keratinases. Of these, Lysobacter NCIMB 9497 exhibited the highest keratinolytic activity. The keratinase from this strain (Mr 148 kDa) was purified and characterized. Optimum activity occurred at 50°C; no inhibition was demonstrated by phenylmethylsulphonyl fluoride (PMSF), but inhibition by EDTA was demonstrated. Conclusions: This study indicates that keratinase is a metalloprotease with a high degree of keratinolytic activity and stability. Significance and Impact of the Study: This is the first detailed report of a metalloprotease with keratinolytic activity. The novel reaction mechanism, degree of keratinolytic activity and stability indicate considerable biotechnological potential in the leather industry, and in the processing of poultry waste.

Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida

Abstract: Aims: Propolis is known for its activity against micro-organisms and different in vitro assays have been used to evaluate this activity, frequently with contradictory results. Methods and Results: Brazilian propolis from the state of Sao Paulo was extracted by maceration using different concentrations of ethanol and water. The resultant extracts were analysed by chromatographic methods. Several microbiological methods were compared to determine which one best evaluated the activity of the propolis extracts against species of Candida, with average minimal inhibitory concentration values between 6 and 12 mg ml−1. Conclusions: Agar dilution in plates showed the clearest results. These were in agreement with the chromatographic analyses, which also identified the active substances. Significance and Impact of the Study: Although the active substances identified in this sample are typical of Brazilian propolis, their activity against Candida had not been recognized previously, demonstrating the importance of standardizing the correct combination of microbiological and chromatographic analyses.

Influence of the culture medium on antibiotic susceptibility testing of food‐associated lactic acid bacteria with the agar overlay disc diffusion method

Abstract: Aims: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. Method: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). Results: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. Conclusions, Significance and Impact of the Study: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains.

Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697.

Abstract: Aims: To characterize the β-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial β-fructofuranosidases. Methods and Results: The β-fructofuranosidase of B. infantis ATCC 15697 was purified 46·8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme was a monomeric protein (Mr 70 kDa) with β-fructofuranosidase and invertase activities. The isoelectric point was pH 4·3, the optimum pH 6·0 and pKas (4·5 and 7·2) of two active groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37 °C and activities were unstable at 55 °C. β-fructofuranosidase activity was more efficient than that of invertase with Vm/Km ratios of 0·65 and 0·025 × 10−3 l min−1 mg−1, respectively. The enzyme catalyses the hydrolysis of fructo-oligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. Conclusions: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has β-fructofuranosidase and invertase activities. This protein was different from the β-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). Significance and Impact of the Study: A better knowledge of bacterial β-fructofuranosidases, especially from bifidobacteria, has been gained.

Implementation of real‐time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry

Abstract: A . E Y I G O R , K . T A Y F U N C A R L I A N D C . B . U N A L . 2002. Aims: The present study describes the implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. Methods and Results: Real-time PCR with Salmonella invA-specific primers and a standard bacteriological method was applied to detect Salmonella in tetrathionate enrichment cultures of 492 intestinal homogenates and 27 drag swabs from 47 poultry flocks. The number of positive individual samples by real-time PCR and culture method was 65 (12AE5%) and 35 (6AE8%), respectively. The number of Salmonella-positive flocks was 13 (27AE7%) by both methods. PCR detection required 25 min for up to 32 samples. Melting curve analysis revealed the Tm for Salmonella-specific PCR product as 87 a 1∞C. Conclusions: Implementation of real-time PCR to tetrathionate broth enrichment step reduces the Salmonella detection time to 18 h and 25 min. Isolation of Salmonella should be carried out with PCR to determine the serovar. Significance and Impact of the Study: Real-time PCR is a powerful tool in rapid and accurate Salmonella monitoring in poultry companies, together with standard bacteriology.

Study on the mechanisms of the antibacterial action of some plant alpha,beta-unsaturated aldehydes.

Abstract: Aims: In this paper the mechanisms involved in the antibacterial effect of six 2E-alkenals [(E)-2-hexenal, (E)-2-eptenal, (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal and (E,E)-2,4-decadienal] were investigated Methods and Results: We measured the release of carboxyfluorescein (CF) trapped in liposomes of phosphatidylcholine (PC) following exposure to the aldehydes mentioned above, in comparison with that elicited by hexanal and nonanal; the modifications of the thermotropic behaviour of liposomes of dimyristoylphosphatidylcholine (DMPC) induced by (E,E)-2,4-decadienal (the aldehyde endowed with the highest microbicidal activity) were evaluated by means of differential scanning calorimetry With the exception of hexanal, all aldehydes tested caused rapid CF leakage from PC liposomes The effectiveness order correlates well with the chain length and the presence of the α,β-double bond Furthermore (E,E)-2,4-decadienal is able to interact with and cross DMPC bilayers Conclusions: The present findings suggest that the 2E-alkenals tested elicit, very likely, a gross perturbation of the lipidic fraction of plasmatic membranes and are able to penetrate into bacterial cells Significance and Impact of the Study: These data represent an interesting background for a rational employment of the plant 2E-alkenals tested as antimicrobial agents