Showing papers in "Journal of Applied Microbiology in 2010"

Biofilm formation and the food industry, a focus on the bacterial outer surface

Abstract: The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food-processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.

Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Abstract: Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B amyloliquefaciens strain Assays using B amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect The results are supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested Significance and Impact of the Study: We elucidated the principal mechanism used by B amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits

Suppression of bacterial cell–cell signalling, biofilm formation and type III secretion system by citrus flavonoids

Abstract: Aim: This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Methods and Results: Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96-well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi-quantitative PCR. Differential responses for different flavonoids were observed for different cell–cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell–cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. Conclusion: The results of the study indicate a potential modulation of bacterial cell–cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer-mediated cell–cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Significance and Impact of the Study: Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell–cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.

Relevance of microbial coculture fermentations in biotechnology.

Abstract: The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro-organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.

The effect of Pediococcus acidilactici on the gut microbiota and immune status of on‐growing red tilapia (Oreochromis niloticus)

Abstract: Aim: To assess Pediococcus acidilactici as a dietary supplement for on-growing red tilapia (Oreochromis niloticus). Methods and Results: Tilapia were fed either a control diet or control diet supplemented with Ped. acidilactici at 10 7 CFU g -1 for 32 days. Ped. acidilactici colonized the intestinal tract and significantly affected the intestinal microbial communities. PCR-DGGE revealed direct antagonism of gastric Ped. acidilactici with an endogenous uncultured bacterium during a period of reverting to nonsupplemented feeding. Light microscopy revealed that gut integrity and leucocyte levels were unaffec ted by Ped. acidilactici; however, blood leucocyte levels and serum lysozyme activity were elevated after 14-days' feeding. No significant improvements in growth performance were observed at the end of the trial (day 32), but survival was significantly higher in the probiotic group. Conclusions: The study demonstrates that oral supplementation of Ped. acidilactici modulates intestinal bacterial communities in on-growing red tilapia and also stimulates some aspects of the nonspecific immune response. Significance and Impact of the study: To our knowledge this is the first study assessing the effects of probiotics on the gut microbiota of tilapia using culture-independent methods. Such methods are crucial to understand the mechanisms which underpin and mediate host benefits. © 2010 The Society for Applied Microbiology.

Culturable bacteria from Zn- and Cd-accumulating Salix caprea with differential effects on plant growth and heavy metal availability

Abstract: Aims: To characterize bacteria associated with Zn/Cd-accumulating Salix caprea regarding their potential to support heavy metal phytoextraction. Methods and Results: Three different media allowed the isolation of 44 rhizosphere strains and 44 endophytes, resistant to Zn/Cd and mostly affiliated with Proteobacteria, Actinobacteria and Bacteroidetes/Chlorobi. 1-Aminocyclopropane-1-carboxylic acid deaminase (ACCD), indole acetic acid and siderophore production were detected in 41, 23 and 50% of the rhizosphere isolates and in 9, 55 and 2% of the endophytes, respectively. Fifteen rhizosphere bacteria and five endophytes were further tested for the production of metal-mobilizing metabolites by extracting contaminated soil with filtrates from liquid cultures. Four Actinobacteria mobilized Zn and/or Cd. The other strains immobilized Cd or both metals. An ACCD- and siderophore-producing, Zn/Cd-immobilizing rhizosphere isolate (Burkholderia sp.) and a Zn/Cd-mobilizing Actinobacterium endophyte were inoculated onto S. caprea. The rhizosphere isolate reduced metal uptake in roots, whereas the endophyte enhanced metal accumulation in leaves. Plant growth was not promoted. Conclusions: Metal mobilization experiments predicted bacterial effects on S. caprea more reliably than standard tests for plant growth-promoting activities. Significance and Impact of the Study: Bacteria, particularly Actinobacteria, associated with heavy metal-accumulating Salix have the potential to increase metal uptake, which can be predicted by mobilization experiments and may be applicable in phytoremediation.

Bartonellosis, an increasingly recognized zoonosis

Abstract: Cat scratch disease is the most common zoonotic infection caused by Bartonella bacteria. Among the many mammals infected with Bartonella spp., cats represent a large reservoir for human infection, as they are the main reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Bartonella spp. are vector-borne bacteria, and transmission of B. henselae by cat fleas occurs mainly through infected flea faeces, although new potential vectors (ticks and biting flies) have been identified. Dogs are also infected with various Bartonella species and share with humans many of the clinical signs induced by these infections. Although the role of dogs as source of human infection is not yet clearly established, they represent epidemiological sentinels for human exposure. Present knowledge on the aetiology, clinical features and epidemiological characteristics of bartonellosis is presented.

Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenase.

Abstract: Microbial biodiversity in the soil plays a significant role in metabolism of complex molecules, helps in plant nutrition and offers countless new genes, biochemical pathways, antibiotics and other metabolites, useful molecules for agronomic productivity. Phosphorus being the second most important macro-nutrient required by the plants, next to nitrogen, its availability in soluble form in the soils is of great importance in agriculture. Microbes present in the soil employ different strategies to make use of unavailable forms of phosphate and in turn also help plants making phosphate available for plant use. Azotobacter, a free-living nitrogen fixer, is known to increase the fertility of the soil and in turn the productivity of different crops. The glucose dehydrogenase gene, the first enzyme in the direct oxidation pathway, contributes significantly to mineral phosphate solubilization ability in several Gram-negative bacteria. It is possible to enhance further the biofertilizer potential of plant growth-promoting rhizobacteria by introducing the genes involved mineral phosphate solubilization without affecting their ability to fix nitrogen or produce phytohormones for dual benefit to agricultural crops. Glucose dehydrogenases from Gram-negative bacteria can be engineered to improve their ability to use different substrates, function at higher temperatures and EDTA tolerance, etc., through site-directed mutagenesis.

Inactivation of Escherichia coli by citral

Abstract: Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 μl l−1 of citral at pH 4·0 for 24 h at 20°C caused the inactivation of more than 5 log10 cycles of cells starting at an inoculum size of 106 or 107 CFU ml−1, whereas increasing the cell concentration to 109 CFU ml−1 caused <1 log10 cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4·0 than pH 7·0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology

Abstract: The exploration of new source materials and the use of alternative isolation and identification methods have led to rapid expansion in the knowledge of diversity; in Lysobacter, 11 new species having been described since 2005, and in Stenotrophomonas with six new species since 2000. The new species of Lysobacter, isolated by dilution and direct plating on standard media, differ in several key phenotypic properties from those obtained by enrichment on complex polysaccharides in the original description of the genus. Revision of the definition of the genus will be required. Both culture-dependent and culture-independent methods to assess community structure, in a variety of host and nonhost environments, have established that some species of Lysobacter are a dominant component of the microflora, where previously their presence had not been suspected. Culture-independent studies have generally not added new information on the occurrence and distribution of Stenotrophomonas maltophilia and other members of the genus, which are readily isolated on standard media from source materials. Lysobacter enzymogenes and Sten. maltophilia produce similar antibiotics and share some enzyme activities which, subject to safety considerations, may make them attractive candidates for use in biological control of plant diseases and of nematodes.

The survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol

Abstract: Aims: Filoviruses are associated with high morbidity and lethality rates in humans, are capable of human-to-human transmission, via infected material such as blood, and are believed to have low infectious doses for humans. Filoviruses are able to infect via the respiratory route and are lethal at very low doses in experimental animal models, but there is minimal information on how well the filoviruses survive within aerosol particles. There is also little known about how well filoviruses survive in liquids or on solid surfaces which is important in management of patients or samples that have been exposed to filoviruses. Methods and Results: Filoviruses were tested for their ability to survive in different liquids and on different solid substrates at different temperatures. The decay rates of filoviruses in a dynamic aerosol were also determined. Conclusions: Our study has shown that Lake Victoria marburgvirus (MARV) and Zaire ebolavirus (ZEBOV) can survive for long periods in different liquid media and can also be recovered from plastic and glass surfaces at low temperatures for over 3 weeks. The decay rates of ZEBOV and Reston ebolavirus (REBOV) plus MARV within a dynamic aerosol were calculated. ZEBOV and MARV had similar decay rates, whilst REBOV showed significantly better survival within an aerosol. Significance and Impact of the Study: Data on the survival of two ebolaviruses are presented for the first time. Extended data on the survival of MARV are presented. Data from this study extend the knowledge on the survival of filoviruses under different conditions and provide a basis with which to inform risk assessments and manage exposure to filoviruses.

Antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food.

Abstract: Aim: To develop antibody–aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single-stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer-A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre-optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti-Listeria antibody, P66, was immobilized on streptavidin-coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647-conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 103 CFU ml−1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 102 CFU 25 g−1) ready-to-eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre-optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre-optic biosensor platform for the sensitive detection of L. monocytogenes from food products.

Filamentous fungal characterizations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract: Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV-absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI-TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI-TOF ICMS to identify fungi are required.

Enterococcus species distribution among human and animal hosts using multiplex PCR.

Abstract: Aims: This study evaluated the use of Enterococcus species differentiation as a tool for microbial source tracking (MST) in recreational waters. Methods and Results: Avian, mammalian and human faecal samples were screened for the occurrence of Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae and Enterococcus saccharolyticus using multiplex PCR. Host-specific patterns of Enterococcus species presence were observed only when data for multiple Enterococcus species were considered in aggregate. Conclusions: The results suggest that no single Enterococcus species is a reliable indicator of the host faecal source. However, Enterococcus species composite ‘fingerprints’ may offer auxiliary evidence for bacterial source identification. Significance and Impact of Study: This study presents novel information on the enterococci species assemblages present in avian and mammalian hosts proximate to the nearshore ocean. These data will aid the development of appropriate MST strategies, and the approach used in this study could potentially assist in the identification of faecal pollution sources.

Antimicrobial-resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended-spectrum beta-lactamases in wild boars.

Abstract: Aims: To determine the presence of antibiotic-resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia. Methods and Results: Rectal swabs or faeces collected during 2006–2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2m g l )1 of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, nE. coli = 242), 12% in wild ruminants and foxes (nE. coli = 42), while no resistant isolates were detected in brown bears (nE. coli = 16). In wild boars (Sus scrofa) (nE. coli = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, nrectal smears = 293) multiresistant isolates producing ESBL were recovered: one isolate with blaCTX-M-1 + blaTEM-1, three with blaCTX-M-1 and one with blaTEM-52b. The blaCTX-M-1 genes were carried on approx. 90 kb IncI1 conjugative plasmids. Conclusions: Antibiotic-resistant E. coli occured in populations of wild mammals in various prevalences. Significance and Impact of the Study: Wild mammals are reservoirs of antibiotic-resistant E. coli including ESBL-producing strains which were found in wild boars.

Functional and ultrastructural changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Cinnamomum verum essential oil

Abstract: Aims: To study cellular damage induced by Cinnamomum verum essential oil in Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Methods and Results: The effect of cinnamon bark essential oil on these two strains was evaluated by plate counts, potassium leakage, flow cytometry and transmission electron microscopy (TEM). Exposure to this oil induced alterations in the bacterial membrane of Ps. aeruginosa, which led to the collapse of membrane potential, as demonstrated by bis-oxonol staining, and loss of membrane-selective permeability, as indicated by efflux of K+ and propidium iodide accumulation. Thus, respiratory activity was inhibited, leading to cell death. In Staph. aureus, cells treated with the oil entered a viable but noncultivable (VNC) state. The oil initially caused a considerable decrease in the metabolic activity and in the replication capacity of these bacterial cells. The loss of membrane integrity appeared later, as indicated by bis-oxonol and Propidium iodide (PI) staining. Data provided by TEM showed various structural effects in response to cinnamon essential oil. In Ps. aeruginosa cells, coagulated cytoplasmic material was observed, and intracellular material was seen in the surrounding environment, while oil-treated Staph. aureus showed fibres extending from the cell surface. Conclusions: Cinnamon essential oil damages the cellular membrane of Ps. aeruginosa, which leads to cell death. There is evidence of VNC Staph. aureus after exposure to the oil. Significance and Impact of the Study: Cinnamon essential oil shows effective antimicrobial activity and health benefits and is therefore considered a potential food additive. To use this oil as a natural food preservative, especially in combination with other preservation methods, a thorough understanding of the mechanism through which this oil exerts its antibacterial action is required.

Antimicrobial activity of honey from the stingless bee Trigona carbonaria determined by agar diffusion, agar dilution, broth microdilution and time-kill methodology.

Abstract: Aims: The aim of this study was to determine the spectrum of antimicrobial activity of 11 samples of stingless bee honey compared to medicinal, table and artificial honeys. Methods and Results: Activity was assessed by agar diffusion, agar dilution, broth microdilution and time-kill viability assays. By agar dilution, minimum inhibitory concentration (MIC) ranges were 4% to >10% (w/v) for Gram-positive bacteria, 6% to >16% (w/v) for Gram-negative bacteria and 6% to >10% (w/v) for Candida spp. By broth microdilution, all organisms with the exception of Candida albicans and Candida glabrata were inhibited at ≤32% (w/v). Geometric MIC (w/v) means for stingless bee honeys ranged from 7·1% to 16·0% and were 11·7% for medicinal honey and 26·5% for table honey. Treatment of organisms with 20% (w/v) stingless bee honey for 60 min resulted in decreases of 1–3 log for Staphylococcus aureus, >3 log for Pseudomonas aeruginosa and <1 log for C. albicans. Similar treatment with each control honey resulted in decreases of <1 log for all organisms. Conclusions: Stingless bee honey has broad-spectrum antibacterial activity although activity against Candida was limited. Stingless bee honey samples varied in activity and the basis for this remains to be determined. Significance and Impact of the Study: Stingless bee honey had similar activity to medicinal honey and may therefore have a role as a medicinal agent.

Bioconversion of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk by probiotic bacteria and yeast.

Abstract: Aim: To study the role of b-glucosidase producing probiotic bacteria and yeast in the biotransformation of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk. Methods and Results: Five isolates of probiotic lactic acid bacteria (LAB), Lactobacillus acidophilus B4496, Lactobacillus bulgaricus CFR2028, Lactobacillus casei B1922, Lactobacillus plantarum B4495 and Lactobacillus fermentum B4655 with yeast Saccharomyces boulardii were used to ferment soymilk to obtain the bioactive isoflavones, genistein and daidzein. High-performance liquid chromatography was used to analyse the concentration of isoflavones. Bioactive aglycones genistein and daidzein after 24 and 48 h of fermentation ranged from 97AE49 to 98AE49% and 62AE71 to 92AE31% respectively with different combinations of LAB with yeast. Increase in bioavailability of minerals and vitamin B complex were also observed in fermented soymilk. Conclusions: LAB in combination with yeast S. boulardii has great potential for the enrichment of bioactive isoflavones, enhancing the viability of LAB strains, decreasing the antinutrient phytic acid and increasing the mineral bioavailability in soymilk fermentation. Significance and Impact of the Study: Fermentation of soymilk with probiotic organisms improves the bioavailability of isoflavones, assists in digestion of protein, provides more soluble calcium, enhances intestinal health and supports immune system. Increased isoflavone aglycone content in fermented soymilk improves the biological functionality of soymilk.

A five-gene stress survival islet (SSI-1) that contributes to the growth of Listeria monocytogenes in suboptimal conditions.

Abstract: Aims: The aim of this study was to examine the contribution of a five-gene islet (lmo0444 – lmo0448) to the growth of Listeria monocytogenes under suboptimal conditions. Methods and Results: Bioinformatics and PCR analyses revealed that a five-gene islet is present in c. half of all L. monocytogenes strains examined (66 in total). A deletion mutant that lacks the entire c. 8·7-kb islet was created in L. monocytogenes strain LO28. This mutant was impaired in growth at low pH and at high salt concentrations and demonstrated a decreased ability to survive and grow in a model food system (frankfurters). Transcriptional analysis revealed that the islet is self-regulated in that the product of lmo0445 regulates the expression of the other four genes. A role of the alternative stress sigma factor SigB in regulating the islet was also uncovered. Conclusions: The five-gene islet (herein designated as SSI-1; stress survival islet 1) contributes to the growth of L. monocytogenes under suboptimal conditions. Significance and Impact of the Study: SSI-1 may contribute to the survival of certain strains of L. monocytogenes in food environments.

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi.

Abstract: Aims: To study the potential biocontrol activity of bioflocs technology. Methods and Results: Glycerol-grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth-inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing-regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Conclusions: Bioflocs grown on glycerol as carbon source inhibit quorum sensing-regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. Significance and Impact of the Study: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.

Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions.

Abstract: Aims: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1-aminocyclopropane-1-carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. Methods and Results: In the first experiment, cucumber seedlings were exposed to 0·1, 50, 100 or 200 mmol l−1 NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l−1 NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l−1 salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase-producing strain Pseudomonas putida UW4 (AcdS+), its mutant unable to produce the enzyme (AcdS−), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l−1 salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro-organism on the photosynthetic efficiency were evaluated. The AcdS+ strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. Conclusions: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase-producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. Significance and Impact of the Study: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS+ increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.

Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma.

Abstract: Aims: Avermectins are major antiparasitic agents used commercially in animal health, agriculture and human infections. To improve the fermentation efficiency of avermectins, for the first time a plasma jet generated by a novel atmospheric pressure glow discharge (APGD) was employed to generate mutations in Streptomyces avermitilis. Methods and Results: The APGD plasma jet, driven by a radio frequency (RF) power supply with water-cooled and bare-metallic electrodes, was used as a new mutation method to treat the spores of S. avermitilis. The plasma jet yielded high total (over 30%) and positive (about 21%) mutation rates on S. avermitilis, and a mutated strain, designated as G1-1 with high productivity of avermectin B1a and genetic stability, was obtained. Conclusions: Because of the low jet temperature, the high concentrations of the chemically reactive species and the flexibility of its operation, the RF APGD plasma jet has a strong mutagenic effect on S. avermitilis. Significance and Impact of the Study: This is a proof-of-concept study for the use of an RF APGD plasma jet for inducing mutations in microbes. We have shown that the RF APGD plasma jet could be developed as a promising and convenient mutation tool for the fermentation industry and for use in biotechnology research.

Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus.

Abstract: Aims: Studies to date have shown rapid killing of bacterial cells when exposed to copper surfaces. The mechanistic action of copper on bacterial cells is so far unknown. Methods and Results: To investigate potential mechanisms involved, meticillin-resistant Staphylococcus aureus (MRSA) cells (107 CFU) were inoculated onto coupons of copper or stainless steel and stained with either the viability fluorophore 5-cyano-2,3-ditolyl tetrazolium (CTC), to detect respiration, or BacLight™ (SYTO9/propidium iodide), to determine cell wall integrity. Coupons were then observed in-situ using epifluorescence microscopy. In addition, DNA from cells inoculated onto either copper or stainless steel surfaces was isolated and analysed by agarose gel electrophoresis. An effect on cellular respiration with CTC reduction was evident but no effect on cell membrane integrity (BacLight™) was observed. Results from the DNA isolation indicated a copper-induced detrimental effect on MRSA genomic material as no bands were observed after exposure to copper surface. Conclusions: The results indicate that exposure to copper surfaces rapidly kills MRSA by compromising cellular respiration and damaging DNA, with little effect on cell membrane integrity. Significance and Impact of the study: This research provides a mechanistic explanation in support of previous suggestions that although copper surfaces do not affect membrane integrity of cells, there is still a rapid antimicrobial effect.

Advances in enteropathogen control in poultry production.

Abstract: Poultry meat has been associated frequently and consistently with the transmission of enteric pathogens, including Salmonella and Campylobacter. This association has resulted in the development of HACCP-based intervention strategies. These strategies (hurdles) begin with elite breeder flocks and filter down the production pyramid. These hurdles include those already established, such as biosecurity, vaccination, competitive exclusion, pre- and probiotics, feed and water control, and those more experimental, such as bacteriophage or immunoglobulin therapy. The reduction in enteropathogens entering the processing plant, which employs critical control points, further reduce the exposure of consumers to these organisms. The synergistic application of hurdles will result in an environment that is restrictive and detrimental to enteropathogen colonization and contamination.

Zoonotic cryptosporidiosis in the UK - challenges for control.

Abstract: The protozoan parasite Cryptosporidium infects all classes of vertebrates. Of the major human pathogenic species, Cryptosporidium parvum and Cryptosporidium hominis predominate in the UK. Cryptosporidium hominis is a human-adapted species, while C. parvum has many animal hosts and is particularly common in preweaned farmed ruminants. Evaluation of zoonotic risks has been provided mainly by descriptive and analytical epidemiological studies and enhanced recently by genetic typing of isolates. The robust nature of the transmissive oocyst stage, multiple transmission routes, lack of antiparasitic treatment options and vaccines, and resistance to chlorine disinfection present challenges for control. Subtyping C. parvum isolates has been used to link human cases and suspected sources of infection in sporadic cases and outbreaks. Although it is possible that all C. parvum isolates are potentially zoonotic, populations with and without farm animal linkage have been identified. New zoonotic risks have emerged in at least one outbreak, caused by the Cryptosporidium sp. rabbit genotype. This re-enforces the need to characterize infecting and contaminating isolates to ensure appropriate interventions. This study describes the risks of zoonotic cryptosporidiosis by detailing the hosts providing a potential reservoir, the risks of transmission to humans, outbreaks in animal-associated settings and guidance for control with special emphasis on the UK.

Impact of Aspergillus section Flavi community structure on the development of lethal levels of aflatoxins in Kenyan maize (Zea mays).

Abstract: Aims: To evaluate the potential role of fungal community structure in predisposing Kenyan maize to severe aflatoxin contamination by contrasting aflatoxin-producing fungi resident in the region with repeated outbreaks of lethal aflatoxicosis to those in regions without a history of aflatoxicosis. Methods and Results: Fungi belonging to Aspergillus section Flavi were isolated from maize samples from three Kenyan provinces between 2004 and 2006. Frequencies of identified strains and aflatoxin-producing abilities were assessed, and the data were analysed by statistical means. Most aflatoxin-producing fungi belonged to Aspergillus flavus. The two major morphotypes of A. flavus varied greatly between provinces, with the S strain dominant in both soil and maize within aflatoxicosis outbreak regions and the L strain dominant in nonoutbreak regions. Conclusions: Aspergillus community structure is an important factor in the development of aflatoxins in maize in Kenya and, as such, is a major contributor to the development of aflatoxicosis in the Eastern Province. Significance and Impact of the Study: Since 1982, deaths caused by aflatoxincontaminated maize have repeatedly occurred in the Eastern Province of Kenya. The current study characterized an unusual fungal community structure associated with the lethal contamination events. The results will be helpful in developing aflatoxin management practices to prevent future outbreaks in Kenya.

Assessment of adenovirus, hepatitis A virus and rotavirus presence in environmental samples in Florianopolis, South Brazil.

Abstract: Aims: To assess the presence of human adenovirus (HAdV), hepatitis A (HAV) virus and rotavirus A (RV-A) in environmental samples from the Southern region of Brazil and to provide viral contamination data for further epidemiological studies and governmental actions. Methods and Results: Water samples from various sources (seawater, lagoon brackish water, urban wastewater, drinking water sources-with and without chlorination and water derived from a polluted creek) and oysters of two growing areas were analysed by enzymatic amplification (nested PCR and RT-PCR), quantification of HAdV genome (qPCR) and viral viability assay by integrated cell culture-PCR (ICC-PCR). From June 2007 to May 2008 in a total of 84 water samples, 54 (64·2%) were positive for HAdV, 16 (19%) for RV-A and 7 (8·3%) for HAV. Viability assays showed nonpositive samples for HAV; though, infectious viruses were confirmed for RV-A (12·5%) and HAdV (88·8%). Oyster samples by PCR were positive for HAdV (87·5%) and RV-A (8·3%), but none for HAV. Quantitative PCR in oysters showed means loads in genomic copies (gc) of 9·1 × 104 gc g−1 (oyster farm south) and 1·5 × 105 gc g−1 (oyster farm north) and in waters ranging from 2·16 × 106 (lagoon water) to 1·33 × 107 gc l−1 (untreated drinking water). Conclusions: This study has shown a widespread distribution of the analysed viruses in this particular region with high loads of HAdV in the environment which suggests the relevance of evaluating these viruses as positive indicators of viral contamination of water. Significance and Impact of the Study: The environmental approach in this study provides data concerning the prevalence, viability and quantification of enteric viruses in environmental waters and oysters in the South region of Brazil and has indicated that their presence might pose a risk to population in contact with the environmental samples searched.

Reconsideration of the derivation of Most Probable Numbers, their standard deviations, confidence bounds and rarity values

Abstract: Aims: The purpose of this work was to derive a simple Excel spreadsheet and a set of standard tables of most probable number (MPN) values that can be applied by users of International Standard Methods to obtain the same output values for MPN, SD of the MPN, 95% confidence limits and test validity. With respect to the latter, it is considered that the Blodgett concept of ‘rarity’ is more valuable than the frequently used approach of improbability (vide de Man). Methods and Results: The paper describes the statistical procedures used in the work and the reasons for introducing a new set of conceptual and practical approaches to the determination of MPNs and their parameters. Examples of MPNs derived using these procedures are provided. The Excel spreadsheet can be downloaded from http://www.wiwiss.fu-berlin.de/institute/iso/mitarbeiter/wilrich/index.html. Conclusions: The application of the revised approach to the determination of MPN parameters permits those who wish to use tabulated values, and those who require access to a simple spreadsheet to determine values for nonstandard test protocols, to obtain the same output values for any specific set of multiple test results. The concept of ‘rarity’ is a more easily understood parameter to describe test result combinations that are not statistically valid. Provision of the SD of the log MPN value permits derivation of uncertainty parameters that have not previously been possible. Significance and Impact of the Study: A consistent approach for the derivation of MPNs and their parameters is essential for coherence between International Standard Methods. It is intended that future microbiology standard methods will be based on the procedures described in this paper.

Assessing the effect of different ultrasonic frequencies on bacterial viability using flow cytometry

Abstract: Aims This research investigated the effect of sonication at frequencies of 20, 40 and 580 kHz and approximately the same acoustic intensity on the viability and declumping of two micro-organisms (Escherichia coli and Klebsiella pneumonia). Methods and results Two analytical methods were employed; viable plate counts (CFU ml−1) and flow cytometry to identify and quantify both live/viable and dead bacteria in the bulk liquid. Flow cytometry results for E. coli and Kl. pneumonia indicated a high sensitivity to 20 and 40 kHz frequency with a continuous decrease in the viable cells and an increase in dead cells during experiments. In contrast, results using the higher frequency of 580 kHz indicate predominantly deagglomeration of bacterial clumps rather than cell membrane disruption (Joyce et al. 2003). Results indicate a good correlation between flow cytometry and viable plate count methodology. Conclusions Sonication has two different effects on bacteria (i) inactivation and (ii) declumping; however, the scale of these effects is dependent on intensity and frequency. Flow cytometry provides a method to distinguish between and quantify the effects through the observation of two subpopulations: (i) live/viable and (ii) dead bacterial cells. Significance and impact of the study Treatment using power ultrasound has been shown to have a significant impact on microbial activity. This is the first time a study has compared the influence of a range of different frequencies, but at similar power settings on the survival of bacteria in phosphate buffer saline (PBS). This work is of importance for applications where ultrasound has been considered for use in industry as a means of disinfection including the treatment and pretreatment of water and also for the sterilization of liquid foods.

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences.

Abstract: Aims: The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). Methods and Results: The bactericidal effect of PHMG on E. coli was investigated based on β-galactosidase activity assay, fluorescein-5-isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 μg ml−1) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 μg ml−1) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Conclusions: Dose-dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 μg ml−1) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. Significance and Impact of the Study: The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.