Showing papers in "Letters in Applied Microbiology in 2004"

Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface.

Abstract: S. STEPANOVIC´ ,I.C´ IRKOVIC´ ,L.RANINANDM.SVABIC´ -VLAHOVIC´ . 2004. Aims: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plasticsurface.Methods: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth(TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates.Results: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However,the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenesstrains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSBwas the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the leastquantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI,followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB.Conclusions: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes producemore biofilm in nutrient-rich medium.Keywords: biofilm, growth medium, Listeria monocytogenes, plastic, Salmonella spp.

The growth profile, thermotolerance and biofilm formation of Enterobacter sakazakii grown in infant formula milk

Abstract: Aims: To study the growth, thermotolerance and biofilm formation of the emergent pathogen Enterobacter sakazakii in infant formula milk (IFM). Methods and Results: The temperature range, death kinetics and biofilm formation of E. sakazakii were determined using impedance microbiology and conventional methods. In IFM the organism grew as low as 6°C and optimally at 37–43°C. In faecal coliform tests, 23% of strains (n = 70) produced gas from lauryl sulphate broth (LSB) at 44°C after 48 h incubation. Three strains failed to grow in LSB at any of the temperatures. The D-value of cells suspended in IFM was determined between 54 and 62°C. The resultant z-value was 5·7°C. The organism was able to adhere and grow on latex, polycarbonate, silicon and to a lesser extent stainless steel. Conclusions: Enterobacter sakazakii was able to grow at refrigeration temperatures and on infant-feeding equipment. The thermotolerance of the organism was similar to other Enterobacteriaceae and should be killed during standard pasteurization treatment. Significance and Impact of the Study: Enterobacter sakazakii has been associated with infant meningitis through consumption of contaminated IFM. Enterobacter sakazakii is able to grow in IFM during storage at refrigeration temperatures and attach to infant-feeding equipment, which may become reservoirs of infection.

Antimicrobial activities of eucalyptus leaf extracts and flavonoids from Eucalyptus maculata.

Abstract: Aims: We investigated the antimicrobial activities of eucalyptus leaf extracts to find effective antibacterial agents. Methods and Results: The antimicrobial activities of leaf extracts from 26 species of eucalyptus were measured. Extracts of Eucalyptus globulus, E. maculata and E. viminalis significantly inhibited the growth of six Gram-positive bacteria (Staphylococcus aureus, MRSA, Bacillus cereus, Enterococcus faecalis, Alicyclobacillus acidoterrestris, Propionibacterium acnes), and of a fungus (Trichophyton mentagrophytes), but they did not show strong antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas putida). 2′,6′-dihydroxy-3′-methyl-4′-methoxy-dihydrochalcone, eucalyptin and 8-desmethyl-eucalyptin, isolated from E. maculata extracts, exhibited potent antimicrobial activities against seven micro-organisms with minimum inhibitory concentrations (MIC) ranging from 1·0 to 31 mg l−1. Conclusions: The eucalyptus extracts and three compounds from E. maculata were found to be effective against micro-organisms that cause food poisoning, acne and athlete's foot. Significance and Impact of the Study: This study shows potential uses of extracts from E. globulus, E. maculata and E. viminalis, and antimicrobial compounds isolated from E. maculata.

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

Abstract: R . M . L A R A G I O N E , A . N A R B A D , M . J . G A S S O N A N D M . J . W O O D W A R D . 2004. Aims: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. Methods and Results: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1 · 10 9 CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nal r ) and E. coli O78:K80 (EC34195, nal r ). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1 · 10 9 CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. Conclusions: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. Significance and Impact of the Study: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.

Surface alteration of Saccharomyces cerevisiae induced by thymol and eugenol

Abstract: Aims: This study aims to bring some information about the mechanism of the fungicidal action of thymol and eugenol; phenolic major components of thyme and clove essential oils respectively. Saccharomyces cerevisiae was used as yeast model. Methods and Results: Treatment of yeast cells with these components led to their lysis as shown by the release of substances absorbing at 260 nm. In addition, scanning electron microscope observations revealed that the surface of the treated cells was significantly damaged. Conclusions: Antifungal activity of thymol and eugenol involve alteration of both membrane and cell wall of the yeast. Significance and Impact of the Study: This work is a preliminary contribution aiming to develop a new generation of efficient and natural antifungal agents.

Levels of zoonotic agents in British livestock manures.

Abstract: To determine the prevalence and levels of zoonotic agents in livestock wastes.A proportionally weighted survey was undertaken and livestock waste samples analysed quantitatively for Escherichia coli O157, pathogenic Listeria, Salmonella, Campylobacter, Giardia and Cryptosporidium. A significant proportion of wastes contained at least one zoonotic agent. Relationships were found between dry matter content and the presence and levels of some zoonotic agents.British livestock wastes contain measurable levels of the zoonotic agents that cause most cases of gastroenteritis in the UK.Animal wastes are disposed of by spreading to agricultural land used for the production of crops and livestock grazing. As British wastes are contaminated with significant levels of zoonotic agents, the practice may represent a way for pathogens to travel further up the food chain.

Bacteriocin-like substance production by Bacillus licheniformis strain P40

Abstract: Aims: To investigate the production of bacteriocin-like compounds by Bacillus spp. isolated from the Amazon basin. Methods and Results: An antimicrobial substance produced by Bacillus licheniformis strain P40 was inhibitory to a broad range of indicator strains, such as Listeria monocytogenes, Bacillus cereus and clinical isolates of Streptococcus spp. The compound was stable at 100°C, but lost its activity when treated at 121°C/103·5 kPa for 15 min. It was resistant to the proteolytic action of trypsin and papain but sensitive to pronase E and was stable within a wide range of pH (3–11). The substance was bactericidal and bacteriolytic to L. monocytogenes. Conclusions: An antibacterial peptide produced by Bacillus licheniformis was characterized, presenting a broad spectrum of activity against pathogenic and spoilage organisms. Significance and Impact of the Study: The identification of a substance active against important pathogens addresses an important aspect of food safety.

Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots.

Abstract: L. CAO, Z. QIU, J. YOU, H. TAN AND S. ZHOU. 2004. Aims: To isolate endophytic Streptomyces strains from tomato and examine their antimicrobial activity. Methods: Endophytic Streptomyces strains were isolated using surface-sterilization methods and identified by morphological characteristics. Antimicrobial activities were measured by the agar plate sensitivity method. Antifungal activity in vivo was measured by seedling mortality in infested soils. Results: Twenty-one per cent of endophytic streptomycete isolates produced antibacterial metabolites and 41% produced antifungal metabolites in S medium. Sixty-five per cent of the most frequently isolated strains inhibited the growth of Rhizoctonia solani by the antibiosis assay but only 32% produced metabolites active against R. solani in S medium. Growth promotion and enhanced disease resistance of seedlings inoculated with Streptomyces sp. strain S30 were observed in tomato but not in cucumber seedlings. Conclusions: Endophytic Streptomyces spp. strains were successfully isolated using stringent methods and strain S30 promoted growth and enhanced resistance to R. solani in tomato seedlings. Significance and Impact of the Study: Endophyticstreptomycetesshowingantifungalactivityinvitroandinvivo may indicate the potential for their use as biocontrol agents particularly of R. solani disease of tomato seedlings.

Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract

Abstract: Aims: To identify lactic acid bacteria (LAB) of porcine intestinal origin with anti-Salmonella activity. Methods and Results: Samples were obtained from pig faeces and caeca and screened for the presence of anti-Salmonella LAB. The 11 most promising isolates were identified as belonging to the genera Lactobacillus and Pediococcus. The LAB exhibited large variation in their ability to survive in simulated gastric juice at pH 1·85. While Lactobacillus johnsonii species survived at levels of 80% for up to 30 min, Lactobacillus pentosus species declined to <0·001% in that time. All isolates tolerated porcine bile at a concentration of 0·3% (w/v), with some isolates capable of growth in the presence of up to 5% (w/v) bile. The ability of the LAB isolates to prevent Salmonella invasion of intestinal epithelial HT-29 cells varied, with reductions of between 30% (Lact. pentosus) and 80% (Lactobacillus murinus spp.) observed. Conclusions: LAB of porcine origin were observed to survive simulated passage through the GIT and inhibit growth of Salmonella and its invasion of the intestinal epithelium. Significance and Impact of the Study: The data demonstrate that some porcine intestinal LAB isolates may offer potential as probiotics for the reduction of Salmonella carriage in pigs.

Characterization of a protease of a feather‐degrading Microbacterium species

Abstract: AIMS: To characterize a new feather-degrading bacterium. METHODS AND RESULTS: The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence. The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C. Complete feather degradation was achieved during cultivation. The keratinase was partially purified by gel filtration chromatography. It was optimally active at pH 7.0 and 55 degrees C. The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+). SIGNIFICANCE AND IMPACT OF THE STUDY: A new Microbacterium sp. strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase. This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis.

Comparative acid stress response of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium after habituation at different pH conditions.

Abstract: Aims: The aim of the study was to evaluate the effect of habituation at different pH conditions on the acid resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium, and to identify potential differences between the adaptive responses of the three pathogens. Methods: Stationary phase cells of L. monocytogenes, E. coli O157:H7 and S. Typhimurium, grown in glucose-free media, were exposed to pH 3·5 broth directly or after habituation for 90 min at various pH conditions from 4·0 to 6·0. Survivors at pH 3·5 were determined by plating on tryptic soy agar and incubating at 30°C for 48 h. The kinetics (death rate) of the pathogens at pH 3·5 was calculated by fitting the data to an exponential model. Results: Habituation to acidic environments provided protection of the pathogens against lethal acid conditions. This acid protection, however, was found to be pH dependent. For example, for E. coli O157:H7 an increased acid resistance was observed after habituation at a pH range from 4·0 to 5·5, while the maximum acid tolerance was induced at pH 5·0. Furthermore, the effect of low pH habituation was different among pathogens. For L. monocytogenes, E. coli O157:H7 and S. Typhimurium, the pH range within which habituation resulted to increased acid resistance was 5·0–6·0, 4·0–5·5 and 4·0–5·0, respectively, while the maximum acid tolerance was induced after habituation at pH 5·5, 5·0 and 4·5, respectively. Significance: Acid stress conditions are common within current food processing technologies. The information on adaptive responses of L. monocytogenes, E. coli O157:H7 and S. Typhimurium after habituation to different pH environments provided in the present study, could lead to a more realistic evaluation of food safety concerns and to a better selection of processes in order to avoid adaptation phenomena and to minimize the potential for food safety risks.

Fate of Escherichia coli originating from livestock faeces deposited directly onto pasture.

Abstract: Aims: To determine the fate of Escherichia coli deposited onto grassland via faeces, from naturally colonized cattle, sheep or pigs. Methods and Results: Groups of cattle, sheep and pigs were penned outdoors on grass during November, and removed after 14 days. Escherichia coli populations in the ground declined over 134 days from initial average levels of 5·34, 4·31 and 4·96 log10 CFU g−1 in cattle, sheep and pig pens, respectively. The maximum Escherichia coli survival time was up to 162 days (190 days taking sampling interval and deposition time into account), but numbers varied significantly amongst the 20 replicates taken each day. Escherichia coli originating from cattle and sheep had average decimal reduction times (D-values) of 38 and 36 days, respectively; E. coli originating from pigs declined significantly faster (average D-value of 26 days). Significance and Impact of the Study: Escherichia coli from livestock faeces can survive on grass for at least 5–6 months, affording opportunity for pathogenic biotypes to contaminate animals, plants or water.

Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract

Abstract: Aims: The purpose of the present study was to examine whether sublethal concentrations of Helichrysum italicum extract could affect some of the cariogenic properties of Streptococcus mutans. Methods and Results: We studied the antibacterial activity of H. italicum (ethanolic extract) against oral streptococci (Strep. mutans ATCC 35668, Strep. salivarius ATCC 13419 and Strep. sanguis ATCC 10556) and its influence on cell-surface hydrophobicity, in vitro sucrose-dependent adherence to glass surface and cellular aggregation of Strep. mutans. The results indicate that all streptococci were susceptible to ethanolic extract with minimum inhibitory concentration (MIC) values of 31·25–62·50 μg ml−1. Sub-MIC concentrations of H. italicum (7·81–31·25 μg ml−1) reduced the hydrophobicity and the adherence (almost 90%) to glass surface of Strep. mutans. The aggregation in the presence of dextran T2000 was also affected. Conclusion: The inhibitory activity of H. italicum extract on Strep. mutans is worthy of further study. Significance and Impact of the Study: There is considerable interest in the use of natural compounds as alternative methods to control undesirable micro-organisms.

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis

Abstract: Aims: To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). Methods and Results: The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. Conclusions: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. Significance and Impact of the Study: This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.

Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage.

Abstract: Aims: Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. Methods and Results: Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler® and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3′-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. Conclusions: Sewage was found to contain up to 1·8 × 106 norovirus cDNA copies per 100 ml and effluent contained up to 1·7 × 106 copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. Significance and Impact of the Study: The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods.

Incubation time and water activity effects on ochratoxin A production by Aspergillus section Nigri strains isolated from grapes.

Abstract: Aims: The objective of this study was to determine the temporal ochratoxin A (OTA) accumulation profile of Aspergillus section Nigri at different water activity (aw) levels. Methods and Results: Two Aspergillus carbonarius and two Aspergillus niger aggregate strains isolated from grapes were tested in vitro for OTA accumulation at 25°C on synthetic nutrient medium, over periods of 20 days at different aw levels. Results were modelled by a multiple linear regression and response surface predictive models were obtained. High levels of aw favoured OTA production by these moulds. Maximum amounts of OTA were found at the earlier growth states (5 days for A. carbonarius and 7–13 days for A. niger aggregate). Conclusions: Provided that A. section Nigri, and mainly A. carbonarius, play the main role in OTA presence in grapes, it would be critical to adjust the harvest and processing time to significantly reduce the chances for OTA accumulation. Significance and Impact of the Study: Ochratoxin A production by A. section Nigri has been shown for the first time to occur optimally after as little as 5 days on a grape-like medium.

Antimicrobial activity of ultrasound-assisted solvent-extracted spices.

Abstract: C. THONGSON, P.M. DAVIDSON, W. MAHAKARNCHANAKUL AND J. WEISS. 2004. Aims: The objective of this research was to determine the antimicrobial activity of conventional and high-intensity ultrasound-assisted (HI-US) solvent-extracted Thai spices, including ginger (Zingiber officinale Rose), fingerroot (Bosenbergia pandurata Holtt) and turmeric (Curouma longa Linn). Methods and Results: Extracts were obtained using hexane, isopropanol and a 7 : 3 isopropanol : hexane mixture as solvents with and without HI-US. The antimicrobial activity of the extracts was assayed against four strains each of Listeria monocytogenes and Salmonella Typhimurium DT 104 using an agar dilution assay. Application of HI-US did not alter antibacterial activity against S. Typhimurium, but antilisterial activity of some HI-US spice extracts decreased. Solvent type affected antimicrobial efficacy of extracts with hexane producing the least antimicrobial activity. Fingerroot extracted with isopropanol‐hexane and without HI-US had the best antilisterial effect while HI-US‐isopropanol fingerroot extract had the greatest antimicrobial efficacy against S. Typhimurium. Conclusions: Application of HI-US reduced time of extraction to 5 min, compared with the 24 h required for conventional extraction and maintained antimicrobial activity against Salmonella but slightly reduced activity against Listeria. Significance and Impact of the Study: HI-US in combination with proper solvent selection may offer a new tool to optimize extraction of spice essential oil for use as antimicrobial agents, and reduce processing time and costs.

Survey of mycoflora and ochratoxin A in dried vine fruits from Argentina markets

Abstract: Aims: The aims of this work were to identify the mycoflora and to evaluate the natural occurrence of OA in dried vine fruits. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. Materials and Methods: Fifty samples of dried vine fruits were obtained from Mendoza and San Juan provinces. The surface disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18) and dichloran Rose Bengal chloramphenicol agar (DRBC). Results: Statistical analysis demonstrated that the species A. niger var. niger and Aspergillus niger var. awamori were isolated in higher frequency from black dried vine fruits from DRBC and DG18 media (P < 0·01). OA was found in 74% of the dried vine fruits samples. Sixty-two strains (28%) of Aspergillus section Nigri, were OA producers. In the species A. carbonarius the highest percentages of ochratoxigenic strains were detected (82·6%). Conclusions: The presence of ochratoxigenic strains of Nigri section in dried vine fruits suggests that they may be an important source of OA in this substrate. Dried vine fruits can also be an important source of OA people who consume large amounts. Significance and Impact of the Study: The dried vine fruits contamination with Aspergillus section Nigri and OA was significant.

Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry.

Abstract: Aims: To investigate the antibacterial effect of five essential oils (EO) extracted from aromatic plants (Cymbopogon citratus, Ocimumbasilicum, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale) of Cameroon against strains of Listeria monocytogenes, L. innocua and Staphylococcus aureus. The ability of selected EO to permeabilize the cytoplasmic membrane of L. innocua was also examined. Methods and Results: The antibacterial activity of the EO determined by the agar diffusion method showed that T. vulgaris had the highest activity followed by O. gratissimum and C. citratus. Lowest activity was recorded from Z. officinale and O. basilicum. Significant differences in sensitivity between strains of Listeria and S. aureus were observed. Flow cytometry of L. innocua stained with carboxy-fluorescein diacetate showed that the fluorescence intensity of cells exposed to EO decreased faster than nonexposed cells, indicating that EO permeabilized the cytoplasmic membrane with the leakage of carboxy-fluorescein. Conclusions: Almost all the EO tested showed antibacterial activity to a different extent. The antibacterial effect was due to permeabilization of the cytoplasmic membrane. Significance and Impact of the Study: This study has identified the preservative potential of the EO examined. The use of sensitive method, such as flow cytometry, is advantageous for quick generation of data on the antibacterial effect of EO.

Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis

Abstract: Aims: The chemical composition of ethanol extracts from a Brazilian (Et-Bra) and a Bulgarian (Et-Blg) propolis, and their activity against the protozoan Trypanosoma cruzi, several fungi and bacteria species were determined. Methods and Results: The chemical composition was determined by high temperature high resolution gas chromatography coupled to mass spectrometry. Microbiological activity was assayed in vitro against T. cruzi, Candida albicans, Sporothrix schenckii, Paracoccidioides brasiliensis, Neisseria meningitidis, Streptococcus pneumoniae and Staphylococcus aureus. Conclusions: Et-Bra and Et-Blg, although with totally distinct compositions, were active against T. cruzi and the three species of fungi. Et-Blg was more effective than Et-Bra against bacteria, particularly N. meningitidis and Strep. pneumoniae. Significance and Impact of the Study: Although with different classes of components, both propolis extracts showed microbicidal activity. For the bactericidal activity it was possible to establish a positive correlation with the high content of flavonoids of the Bulgarian extract.

Comparison of the biotypes of Yersinia enterocolitica isolated from pigs, cattle and sheep at slaughter and from humans with yersiniosis in Great Britain during 1999-2000.

Abstract: Aims: To investigate the relationship between livestock carriage of Yersinia enterocolitica and human disease. The biotypes/serotypes of strains recovered from the faeces of pigs, cattle and sheep at slaughter during a national survey in Great Britain in 1999–2000, were compared with those of strains isolated from human cases of yersiniosis during the same period. Methods and Results: The faecal carriage of Y. enterocolitica by cattle, sheep and pigs at slaughter was 6·3, 10·7 and 26·1%, respectively. Yersinia enterocolitica biotype (BT) 1a was the most frequently isolated biotype from livestock (58%) and was the predominant biotype (53%) isolated from human cases over the same period. The main recognized pathogenic Y. enterocolitica biotype isolated from livestock was BT3 (O:5,27) (35% of sheep, 22% of pigs and 4% of cattle) but this biotype was not detected in any of the human isolates investigated. The major pathogenic biotypes of strains isolated from humans were BT3 (O:9) (24%) and BT4 (O:3) (19%) whereas of the veterinary isolates investigated, only pigs (11%) carried BT3 (O:9) strains. Conclusions: Because of significant overlaps in phenotypes of the veterinary and human strains it is not possible to comment on the correlation between host and pathogenicity, especially of biotype 1a. Significance and Impact of the Study: The data suggest that further investigations using methods with greater discriminatory power are required. However the data also suggests that pigs may be the primary reservoir for human pathogenic Y. enterocolitica infection.

Updated perspectives on emerging vibrios associated with human infections

Abstract: This review describes the ecological, clinical and epidemiological features of emerging vibrios and discusses what laboratory methods are being used for the detection of pathogenic vibrios in clinical, environmental and food samples. After selecting articles illustrative of the current scientific research on pathogenic vibrios, the review focuses on the need for better insight into the risk factors of emerging infections to establish adequate prevention procedures.

Production of leucinostatins and nematicidal activity of Australian isolates of Paecilomyces lilacinus (Thom) Samson.

Abstract: Aims: To evaluate the relationship between leucinostatin production by Paecilomyces lilacinus isolates and their biological activities. Methods and Results: The nematicidal, parasitic and enzymatic activity of Australian P. lilacinus isolates were investigated. Nematicidal activities of culture filtrates were measured by mortality and inhibition of reproduction of Caenorhabditis elegans, whereas egg-parasitic activity was measured by colonization on Meloidogyne javanica. Enzymatic activities (protease and chitinase) were assayed on solid media. The results suggest that leucinostatins in P. lilacinus are indicators of nematicidal activity, whereas chitinase activity might be related to parasitism. Conclusions: Nematicidal activity of culture filtrates of Paecilomyces lilacinus strains related to their ability to produce leucinostatins. Significance and Impact of the Study: This is the first study describing the leucinostatins as nematicides.

Rapid and quantitative detection of the microbial spoilage in chicken meat by diffuse reflectance spectroscopy (600-1100 nm).

Abstract: Aims: To evaluate the feasibility of visible and short-wavelength near-infrared (SW-NIR) diffuse reflectance spectroscopy (600–1100 nm) to quantify the microbial loads in chicken meat and to develop a rapid methodology for monitoring the onset of spoilage. Methods and Results: Twenty-four prepackaged fresh chicken breast muscle samples were prepared and stored at 21°C for 24 h. Visible and SW-NIR was used to detect and quantify the microbial loads in chicken breast muscle at time intervals of 0, 2, 4, 6, 8, 10, 12 and 24 h. Spectra were collected in the diffuse reflectance mode (600–1100 nm). Total aerobic plate count (APC) of each sample was determined by the spread plate method at 32°C for 48 h. Principal component analysis (PCA) and partial least squares (PLS) based prediction models were developed. PCA analysis showed clear segregation of samples held 8 h or longer compared with 0-h control. An optimum PLS model required eight latent variables for chicken muscle (R = 0·91, SEP = 0·48 log CFU g−1). Conclusions: Visible and SW-NIR combined with PCA is capable of perceiving the change of the microbial loads in chicken muscle once the APC increases slightly above 1 log cycle. Accurate quantification of the bacterial loads in chicken muscle can be calculated from the PLS-based prediction method. Significance and the Impact of the Study: Visible and SW-NIR spectroscopy is a technique with a considerable potential for monitoring food safety and food spoilage. Visible and SW-NIR can acquire a metabolic snapshot and quantify the microbial loads of food samples rapidly, accurately, and noninvasively. This method would allow for more expeditious applications of quality control in food industries.

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

Abstract: Aims: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. Methods and Results: Surface-sterilized and thinly sliced (1–2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30°C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99·2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99·5% similarity to Enterobacter asburiae ATCC 35953. Conclusion: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. Significance and Impact of the Study: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production.

Abstract: J.-H. RYU, H. KIM, J.F. FRANK AND L.R. BEUCHAT. 2004. Aims: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. Methods and Results: Three curli-deficient strains (43895), 43894) and E0018)) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4� C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22� C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. Conclusions: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. Significance and Impact of the Study: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.

Isolation and characterization of psychrophilic yeasts producing cold-adapted pectinolytic enzymes.

Abstract: Aims: The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold-active pectinolytic enzymes produced by the isolated psychrophilic yeasts. Methods and Results: Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic–psychrophilic yeast (PPY) strains PPY-3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY-3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY-5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5°C, and showed the activities of several cold-active pectinolytic enzymes. Conclusion: The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. Significance and Impact of the Study: It is possible that the cold-active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5°C.

Occurrence and dominance of yeast species in sourdough

Abstract: Aims: The aim of this work is to identify the dominant yeast species in homemade sourdoughs. Methods and Results: PCR/restriction fragment length polymorphism analysis of internal transcribed spacer regions was used for the identification of isolates and the data were confirmed with phenotypic tests. The strains belonging to Saccharomyces cerevisiae were identified to strain level by analysis of inter-δ regions. Conclusion: This work shows that the dominant species in homemade sourdoughs can differ from each other. Saccharomyces cerevisiae was found to be the dominant species, followed by the Candida milleri, C. humilis, S. exiguus and Issatchenkia orientalis. The inter-δ regions of S. cerevisiae strains showed high polymorphism. Significance and Impact of the Study: Occurrence of single, non-Saccharomyces species and S. cerevisiae polymorphism in the yeast populations of sourdough samples.

Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA.

Abstract: M. TAMMINEN, T. JOUTSJOKI, M. SJOBLOM, M. JOUTSEN, A. PALVA, E.-L. RYHANEN AND V. JOUTSJOKI. 2004 Aims: The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR-ELISA). Methods and Results: Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR-ELISA. Oligonucleotide probes used for hybridization in PCR-ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour-developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum-related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum. Conclusions: Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR-ELISA. Significance and Impact of the Study: PCR-ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.