Role of rpoS in the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure-sensitive and took up more PI than the parent strains with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure-sensitive, was unaffected in membrane resealing implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

Variation in the sensitivity of Callosobruchus (Coleoptera: Bruchidae) acetylcholinesterase to the organophosphate insecticide malaoxon: effect of species, geographical strain and food type

BACKGROUND: Bruchid beetles, Callosobruchus species, are serious pests of economically important grain legumes; their activity in stores is often controlled by use of synthetic insecticides. Esterases are known to be involved in insecticide resistance in insects. However, there is dearth of information on esterase activity in the genus Callosobruchus. In this study we investigated the effect of species, geographical strain and food type on the variation of acetylcholinesterase (AChE) activity and its inhibition by malaoxon (malathion metabolite) using an in vitro spectrophotometric method. RESULT: AChE activity varied significantly among species and strains and also among legume type used for rearing them. Generally irrespective of species, strain or food type, the higher the AChE activity of a population, the higher its inhibition by malaoxon. C. chinensis had the highest AChE activity of the species studied and in the presence of malaoxon it had the lowest remaining AChE activity, while C. rhodesianus retained the highest activity. CONCLUSION: A firsthand knowledge of AChE activity in regional Callosobruchus in line with the prevailing food types should be of utmost importance to grain legume breeders, researchers on plant materials for bruchid control and pesticide manufacturer/applicators for a robust integrated management of these bruchids.

Use of virulence determinants and seropathotypes to distinguish high- and low-risk Escherichia coli O157 and non-O157 isolates from Europe

The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).

An evaluation of different growth functions for describing the profile of live weight with time (age) in meat and egg strains of chicken

A total of 86 profiles from meat and egg strains of chickens (male and female) were used in this study. Different flexible growth functions were evaluated with regard to their ability to describe the relationship between live weight and age and were compared with the Gompertz and logistic equations, which have a fixed point of inflection. Six growth functions were used: Gompertz, logistic, Lopez, Richards, France, and von Bertalanffy. A comparative analysis was carried out based on model behavior and statistical performance. The results of this study confirmed the initial concern about the limitation of a fixed point of inflection, such as in the Gompertz equation. Therefore, consideration of flexible growth functions as an alternatives to the simpler equations (with a fixed point of inflection) for describing the relationship between live weight and age are recommended for the following reasons: they are easy to fit, they very often give a closer fit to data points because of their flexibility and therefore a smaller RSS value, than the simpler models, and they encompasses simpler models for the addition of an extra parameter, which is especially important when the behavior of a particular data set is not defined previously.

Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

Comparison of Euryarchaea strains in the guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.

Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium

Aims: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance. Methods and Results: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied. Conclusions: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance. Significance and Impact of Study: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.

Isopimaric acid from Pinus nigra shows activity against multidrug-resistant and EMRSA strains of Staphylococcus aureus

The diterpene isopimaric acid was extracted from the immature cones of Pinus nigra (Arnold) using bioassay. guided fractionation of a crude hexane extract. Isopimaric acid was assayed against multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) were 32-64 mu g/mL and compared with a commercially obtained resin acid, abietic acid, with MICs of 64 mu g/mL. Resin acids are known to have antibacterial activity and are valued in traditional medicine for their antiseptic properties: These results show that isopimaric acid is active against MDR an MRSA strains of S. aureus which are becoming, increasingly resistant to antibiotics. Both compounds were evaluated for modulation activity in combination with antibiotics, but did not potentiate the activity of the antibiotics tested. However, the compounds were also assayed in combination with the efflux pump inhibitor reserpine, to ice if inhibition of the TetK or NorA efflux pump increased their activity. Interestingly, rather than a potentiation of activity by a reduction in MIC, a two to four-fold increase in MIC was seen. It may he that isopimaric acid and abietic acid are not substrates for these efflux pumps, but it is also possible that an antagonistic interaction with reserpine may render the antibiotics inactive. H-1-NMR of abietic acid and reserpine taken individually and in combination, revealed a shift in resonance of some peaks for both compounds when mixed together compared with the spectra of the compounds on their own. It is proposed that this may he due to complex formation between abietic acid and reserpine and that this complex formation is responsible for a reduction in activity and elevation of MIC. Copyright (c) 2005 John Wiley & Sons, Ltd.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

Screening human intestinal Bifidobacterium strains for growth, acidification, EPS production and viscosity potential in low-fat milk

Bifidobacterium strains of human origin were screened for their ability to grow in milk and produce exopolysaccharides (EPS). Bifidobacterium strains were grown in low-fat UHT milk and were evaluated for their growth, acidification properties, EPS production and ability to increase the viscosity of fermented milk. The strains that grew well in milk were strains of Bifidobacterium breve and Bifidobacterium longum and B. longum subsp. longum. Among the 22 strains, EPS was produced by Bifidobacterium bifidum ALM 35, B. breve NCIMB 8807 (UCC 2003), B. longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 at concentrations ranging from 25 to 140 . The molecular mass and the composition varied considerably, depending on the strain. Analysis of the correlation between the apparent viscosity of the fermented milk and pH indicated that the EPS produced during the acidification of milk possibly contributed to the viscosity of the milk products.

Production of angiotensin-I-converting enzyme (ACE) inhibitory activity in milk fermented with probiotic strains: Effects of calcium, pH and peptides on the ACE-inhibitory activity

Recently, probiotic fermented milk products have raised interest regarding their potential anti-hypertensive activity mainly due to the production of angiotensin-I-converting enzyme (ACE) inhibitory peptides. Ionic calcium released upon milk acidification during fermentation is also known to exert hypotensive activity. Thus, the main aim of this study was to screen probiotic strains for their ability to induce ACE-inhibitory activity upon fermentation of milk. The relationship of ACE-inhibitory activity percentage (ACEi%) with cell growth, pH, degree of hydrolysis and the concentration of ionic calcium released during the fermentation was also investigated. Compared with other lactic acid bacteria, Lactobacillus casei YIT 9029 and Bifidobacterium bifidum MF 20/5 were able to induce strong ACE-inhibitory activity. Furthermore, it was found that the ionic calcium released during milk fermentation could contribute to the ACE-inhibitory activity. These findings will contribute to the development of new probiotic dairy products with anti-hypertensive activity.

Effect of dairy-based protein sources and temperature on growth, acidification and exopolysaccharide production of Bifidobacterium strains in skim milk

The aim of the present study was to find out the best growing conditions for exopolysaccharide (EPS) producing bifidobacteria, which improve their functionality in yoghurt-like products. Two Bifidobacterium strains were used in this study, Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205. In the first part of the study the effect of casein hydrolysate, lactalbumin hydrolysate, whey protein concentrate and whey protein isolate, added at 1.5% w/v in skim milk, was evaluated in terms of cell growth and EPS production; skim milk supplemented with yeast extract served as the control. Among the various nitrogen sources, casein hydrolysate (CH) showed the highest cell growth and EPS production for both strains after 18 h incubation and therefore it was selected for subsequent work. Based on fermentation experiments using different levels of CH (from 0.5 to 2.5% w/v) it was deduced that 1.5% (w/v) CH resulted in the highest EPS production, yielding 102 and 285 mg L− 1 for B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486, respectively. The influence of temperature on growth and EPS production of both strains was further evaluated at 25, 30, 37 and 42 °C for up to 48 h in milk supplemented with 1.5% (w/v) CH. The temperature had a significant effect on growth, acidification and EPS production. The maximum growth and EPS production were recorded at 37 °C for both strains, whereas no EPS production was observed at 25 °C. Lower EPS production for both strains were observed at 42 °C, which is the common temperature used in yoghurt manufacturing compared to that at 37 °C. The results showed that the culture conditions have a clear effect on the growth, acidification and EPS production, and more specifically, that skim milk supplemented with 1.5% (w/v) CH could be used as a substrate for the growth of EPS-producing bifidobacteria, at 37 °C for 24 h, resulting in the production of a low fat yoghurt-like product with improved functionality.

Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid.