Preliminary assessment on potentials of probiotic B. subtilis RX7 and B. methylotrophicus C14 strains as an immune modulator in Salmonella-challenged weaned pigs.

A total of 40 crossbred weaned piglets (28 days old; [Landrace × Yorkshire] × Duroc) were used for preliminary assessment on potentials of Bacillus-based probiotics as an immune modulator in a Salmonella Typhimurium challenge model in a 3-week experiment. Pigs were randomly allotted to four experimental diets according to their initial body weight (9.21 ± 1.1 kg) and sex (10 pigs per treatment; 5 barrows and 5 gilts). The dietary treatments were basal diet (CON), basal diet + oral administration of Salmonella enterica ser. Typhimurium at the dosage of 1 mL containing 1 × 1011 cfu/mL of viable cell concentrations at day 21 (SC), SC + Bacillus subtilis (BS), and SC+ Bacillus methylotrophicus (BM). After 12 h of Salmonella challenge, the red blood cell (RBC), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations were reduced (P < 0.05) whereas haptoglobin and cortisol levels were greater (P < 0.05) in SC compared with CON. However, the concentrations of RBC, IgG, and IgM were increased whereas haptoglobin and cortisol levels were reduced in BS and BM compared with SC. The probiotic-treated groups showed reduced (P < 0.05) IgM levels and increased (P < 0.05) WBC and cortisol levels compared with CON. The supplementation of probiotics showed increased (P < 0.05) fecal Lactobacillus counts and reduced Escherichia coli and Salmonella counts in piglets though there was no biological relevance compared with SC. Thus, in our preliminary study, Bacillus-based probiotic has shown some positive immunomodulatory effects in Salmonella-challenged pigs which provided a base for further studies.

Ecotoxicological risk assessment of the "Acid Black 210" dye.

The "Acid Black 210" dye is one of the most used black dyes by the leather industry. This compound contains three azo groups in its chemical structure, and has been quoted as a non-regulated dye with toxicological concern, since it could generate carcinogenic aromatic amines. The objective of this study was to perform the ecotoxicological risk assessment of this dye through testing its toxicity in vitro and in vivo with the Ames test, the Comet assay, the Daphnia similis test, and the zebrafish embryo acute toxicity test. Moreover, we evaluated the presence of this dye in environmental samples related with a tannery industry. All the tests performed were negative, with the exception of the Ames test with the Salmonella typhimurium TA98 strain, which resulted in a low mutagenic potency. Due to the low concentrations of the "Acid Black 210" dye found in tannery effluents, and the high concentrations where any toxic activity is occasionally described, we concluded that this dye is safe from the ecotoxicological point of view in the areas evaluated and in the light of the current knowledge.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

Bacteria cells use osmoregulatory proteins as emergency valves to respond to changes in the osmotic pressure of their external environment. The existence of these emergency valves has been known since the 1960s, but they have never been used as the basis of a viability assay to tell dead bacteria cells apart from live ones. In this paper, we show that osmoregulation provides a much faster, label-free assessment of cell viability compared with traditional approaches that rely on cell multiplication (growth) to reach a detectable threshold. The cells are confined in an evaporating droplet that serves as a dynamic microenvironment. Evaporation-induced increase in ionic concentration is reflected in a proportional increase of the droplet's osmotic pressure, which in turn, stimulates the osmoregulatory response from the cells. By monitoring the time-varying electrical conductance of evaporating droplets, bacterial cells are identified within a few minutes compared with several hours in growth-based methods. To show the versatility of the proposed method, we show detection of WT and genetically modified nonhalotolerant cells (Salmonella typhimurium) and dead vs. live differentiation of nonhalotolerant (such as Escherichia coli DH5α) and halotolerant cells (such as Staphylococcus epidermidis). Unlike the growth-based techniques, the assay time of the proposed method is independent of cell concentration or the bacteria type. The proposed label-free approach paves the road toward realization of a new class of real time, array-formatted electrical sensors compatible with droplet microfluidics for laboratory on a chip applications.

Genome-scale co-expression network comparison across Escherichia coli and Salmonella enterica serovar Typhimurium reveals significant conservation at the regulon level of local regulators despite their dissimilar lifestyles.

Availability of genome-wide gene expression datasets provides the opportunity to study gene expression across different organisms under a plethora of experimental conditions. In our previous work, we developed an algorithm called COMODO (COnserved MODules across Organisms) that identifies conserved expression modules between two species. In the present study, we expanded COMODO to detect the co-expression conservation across three organisms by adapting the statistics behind it. We applied COMODO to study expression conservation/divergence between Escherichia coli, Salmonella enterica, and Bacillus subtilis. We observed that some parts of the regulatory interaction networks were conserved between E. coli and S. enterica especially in the regulon of local regulators. However, such conservation was not observed between the regulatory interaction networks of B. subtilis and the two other species. We found co-expression conservation on a number of genes involved in quorum sensing, but almost no conservation for genes involved in pathogenicity across E. coli and S. enterica which could partially explain their different lifestyles. We concluded that despite their different lifestyles, no significant rewiring have occurred at the level of local regulons involved for instance, and notable conservation can be detected in signaling pathways and stress sensing in the phylogenetically close species S. enterica and E. coli. Moreover, conservation of local regulons seems to depend on the evolutionary time of divergence across species disappearing at larger distances as shown by the comparison with B. subtilis. Global regulons follow a different trend and show major rewiring even at the limited evolutionary distance that separates E. coli and S. enterica.

Quantitative assessment of cytosolic Salmonella in epithelial cells.

Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

In vitro assessment of the susceptibility of planktonic and attached cells of foodborne pathogens to bacteriophage p22-mediated salmonella lysates.

This study was designed to evaluate the lytic activity of bacteriophage P22 against Salmonella Typhimurium ATCC 19585 (Salmonella Typhimurium P22(-)) at various multiplicities of infections (MOIs), the susceptibility of preattached Salmonella cells against bacteriophage P22, and the effect of P22-mediated bacterial lysates (extracellular DNA) on the attachment ability of Listeria monocytogenes ATCC 7644 and enterohemorrhagic Escherichia coli ATCC 700927 to surfaces. The numbers of attached Salmonella Typhimurium P22(-) cells were effectively reduced to below the detection limit (1 log CFU/ml) at the fixed inoculum levels of 3 × 10(-) CFU/ml (MOI = 3.12) and 3 × 10(3) CFU/ml (MOI = 4.12) by bacteriophage P22. The attached Salmonella Typhimurium P22(-) cells remained more than 2 log CFU/ml, with increasing inoculum levels from 3 × 10(4) to 3 × 10(7) CFU/ml infected with 4 × 10(8) PFU/ml of P22. The number of preattached Salmonella Typhimurium P22(-) cells was noticeably reduced by 2.72 log in the presence of P22. The highest specific attachment ability values for Salmonella Typhimurium P22(-), Salmonella Typhimurium ATCC 23555 carrying P22 prophage (Salmonella Typhimurium P22(+)), L. monocytogenes, and enterohemorrhagic E. coli were 2.09, 1.06, 1.86, and 1.08, respectively, in the bacteriophage-mediated cell-free supernatants (CFS) containing high amounts of extracellular DNA. These results suggest that bacteriophages could potentially be used to effectively eliminate planktonic and preattached Salmonella Typhimurium P22(-) cells with increasing MOI. However, further research is needed to understand the role of bacteriophage-induced lysates in bacterial attachment, which can provide useful information for the therapeutic use of bacteriophage in the food system.

Use of quantitative microbial risk assessment when investigating foodborne illness outbreaks: the example of a monophasic Salmonella Typhimurium 4,5,12:i:- outbreak implicating beef burgers.

A major community outbreak of salmonellosis occurred in France in October 2010. Classical epidemiological investigations led to the identification of beef burgers as the cause of the outbreak and the presence of the emerging monophasic Salmonella Typhimurium 4,5,12:i:-. The objective of this study was to understand the events that led to this large outbreak, that is to say, what are the contributing factors associated with consumer exposure to Salmonella. To this end, intensive microbiological investigations on several beef burgers were conducted and a risk assessment model was built. The microbiological results confirm the presence of Salmonella in all analysed frozen burgers at high levels of contamination above 1000 MPN/g. These results in frozen burgers combined with a model of thermal destruction were used to estimate the dose ingested by the exposed persons. Most people that consumed cooked beef burgers were exposed from 1.6 to 3.1 log10 (MPN). The number of sick people predicted with a dose-response relationship for Salmonella is consistent with the observed number of salmonellosis cases. The very high initial contamination level in frozen beef burgers is the primary cause of this large outbreak rather than bad cooking practices. Intensive investigations, modelling of the initial contamination and quantitative exposure and risk assessments are complementary to epidemiological investigation. They can be valuable elements for the assessment of missing information or the identification of the primary causes of outbreaks.

Incorporating parameter uncertainty into Quantitative Microbial Risk Assessment (QMRA).

Modern statistical models and computational methods can now incorporate uncertainty of the parameters used in Quantitative Microbial Risk Assessments (QMRA). Many QMRAs use Monte Carlo methods, but work from fixed estimates for means, variances and other parameters. We illustrate the ease of estimating all parameters contemporaneously with the risk assessment, incorporating all the parameter uncertainty arising from the experiments from which these parameters are estimated. A Bayesian approach is adopted, using Markov Chain Monte Carlo Gibbs sampling (MCMC) via the freely available software, WinBUGS. The method and its ease of implementation are illustrated by a case study that involves incorporating three disparate datasets into an MCMC framework. The probabilities of infection when the uncertainty associated with parameter estimation is incorporated into a QMRA are shown to be considerably more variable over various dose ranges than the analogous probabilities obtained when constants from the literature are simply 'plugged' in as is done in most QMRAs. Neglecting these sources of uncertainty may lead to erroneous decisions for public health and risk management.

Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

Plasmid-encoded functions compensate for the biological cost of AmpC overexpression in a clinical isolate of Salmonella typhimurium.

OBJECTIVES: In a previous study with a Salmonella typhimurium strain containing cloned ampC-ampR from Enterobacter cloacae, it was suggested that ampC expression must be kept at low levels by AmpR to maintain normal growth and virulent phenotype. The purpose of this study was to determine whether findings obtained with a laboratory model can be extended to a virulent clinical isolate of S. typhimurium expressing the plasmid-encoded bla(CMY-7). METHODS: Disc induction assays were carried out to investigate inducibility of bla(CMY-7). Primer extension and sequence analyses were carried out to map the transcriptional start site of bla(CMY-7) and determine the relative expression. Growth and invasion potential of Salmonella strains were monitored by optical density, viable counts and cell invasion assays. RESULTS: Sequence analysis confirmed the absence of ampR upstream of bla(CMY-7) therefore confirming the negative results observed using the disc induction assay. Primer extension analysis mapped the start site of bla(CMY-7) transcription within an ISEcp1-like element. The relative expression of bla(CMY-7) was approximately 965-fold higher than the expression of a wild-type Citrobacter freundii chromosomal ampC and approximately 4.1-fold higher than ampC expression from a derepressed mutant of C. freundii. Growth and the capacity to invade mammalian cells were not compromised for either the clinical isolate or the S. typhimurium transconjugant containing bla(CMY-7). However, a Salmonella transformant containing bla(CMY-7) exhibited a compromised phenotype with respect to growth and invasion of mammalian cells. CONCLUSION: These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.

Fitness cost of fluoroquinolone resistance in Salmonella enterica serovar Typhimurium.

High-level fluoroquinolone (FQ) resistance is still infrequent in salmonellae, compared with other pathogenic enterobacteria. Data provided in this work support the hypothesis that the mechanisms that confer high-level FQ resistance on salmonellae have a prohibitive fitness cost and may thus limit the emergence of highly resistant clones. In vitro mutants that were highly resistant to ciprofloxacin (MIC = 8 and 16 micro g ml(-1)) showed generation times 1.4- and 2-fold longer than their parent strains and were unable to colonize the gut of chickens. Electron microscopy showed an altered morphology for one of these mutants grown to stationary phase. Mutants selected in vivo and exhibiting intermediate resistance to ciprofloxacin (MIC = 2 micro g ml(-1)) also showed growth defects on solid media but had normal generation times in liquid culture and colonized the gut of chickens. After in vitro or in vivo passage in the absence of antibiotic selective pressure, partial reversals of the fitness cost were observed, which were associated with slight decreases in resistance to quinolones and other unrelated antibiotics, but were not linked to the loss of gyrA mutations.

Effectiveness of SYTOX Green stain for bacterial viability assessment.

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.

Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC.

Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.

Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol.

The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population. A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented. The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater. The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts. The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.