LPS Structure and PhoQ Activity Are Important for Salmonella Typhimurium Virulence in the Gallleria mellonella Infection Model

The larvae of the wax moth, Galleria  mellonella , have been used experimentally to host a range of bacterial and fungal pathogens. In this study we evaluated the suitability of G . mellonella as an alternative animal model of Salmonella infection. Using a range of inoculum doses we established that the LD₅₀ of Salmonella Typhimurium strain NCTC 12023 was 3.6 × 10³ bacteria per larva. Further, a set of isogenic mutant strains depleted of known virulence factors was tested to identify determinants essential for S . Typhimurium pathogenesis. Mutants depleted of one or both of the type III secretion systems encoded by Salmonella Pathogenicity Islands 1 and 2 showed no virulence defect. In contrast, we observed reduced pathogenic potential of a phoQ mutant indicating an important role for the PhoPQ two-component signal transduction system. Lipopolysaccharide (LPS) structure was also shown to influence Salmonella virulence in G . mellonella . A waaL (rfaL) mutant, which lacks the entire O-antigen (OAg), was virtually avirulent, while a wzz _ST/wzz _fepE double mutant expressing only a very short OAg was highly attenuated for virulence. Furthermore, shortly after infection both LPS mutant strains showed decreased replication when compared to the wild type in a flow cytometry-based competitive index assay. In this study we successfully established a G . mellonella model of S . Typhimurium infection. By identifying PhoQ and LPS OAg length as key determinants of virulence in the wax moth larvae we proved that there is an overlap between this and other animal model systems, thus confirming that the G . mellonella infection model is suitable for assessing aspects of Salmonella virulence function.     

The Base Excision Repair System of Salmonella enterica serovar Typhimurium Counteracts DNA Damage by Host Nitric Oxide

Intracellular pathogens must withstand nitric oxide (NO·) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO·. Consequently, the level of NO· stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO· sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO· fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO·–dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO·. These observations demonstrate that host-derived NO· damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity.     

MomL,a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C₆-HSL is ∼2.9 × 10⁵ s⁻¹ M⁻¹. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.     

Equisetin as potential quorum sensing inhibitor of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Objective

To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa.

Results

QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes.

Conclusions

Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

     

Cold Plasma Inactivation of Bacterial Biofilms and Reduction of Quorum Sensing Regulated Virulence Factors

The main objectives of this work were to investigate the effect of atmospheric cold plasma (ACP) against a range of microbial biofilms commonly implicated in foodborne and healthcare associated human infections and against P. aeruginosa quorum sensing (QS)-regulated virulence factors, such as pyocyanin, elastase (Las B) and biofilm formation capacity post-ACP treatment. The effect of processing factors, namely treatment time and mode of plasma exposure on antimicrobial activity of ACP were also examined. Antibiofilm activity was assessed for E. coli, L. monocytogenes and S. aureus in terms of reduction of culturability and retention of metabolic activity using colony count and XTT assays, respectively. All samples were treated ‘inpack’ using sealed polypropylene containers with a high voltage dielectric barrier discharge ACP generated at 80 kV for 0, 60, 120 and 300 s and a post treatment storage time of 24 h. According to colony counts, ACP treatment for 60 s reduced populations of E. coli to undetectable levels, whereas 300 s was necessary to significantly reduce populations of L. monocytogenes and S. aureus biofilms. The results obtained from XTT assay indicated possible induction of viable but non culturable state of bacteria. With respect to P. aeruginosa QS-related virulence factors, the production of pyocyanin was significantly inhibited after short treatment times, but reduction of elastase was notable only after 300 s and no reduction in actual biofilm formation was achieved post-ACP treatment. Importantly, reduction of virulence factors was associated with reduction of the cytotoxic effects of the bacterial supernatant on CHO-K1 cells, regardless of mode and duration of treatment. The results of this study point to ACP technology as an effective strategy for inactivation of established biofilms and may play an important role in attenuation of virulence of pathogenic bacteria. Further investigation is warranted to propose direct evidence for the inhibition of QS and mechanisms by which this may occur.     

PCR Detection of Salmonella enterica Serotype Montevideo in and on Raw Tomatoes Using Primers Derived from hilA

Salmonellae have been some of the most frequently reported etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of primers derived from hilA and sirA, positive regulators of Salmonella invasive genes, were developed to identify Salmonella enterica serotype Montevideo on and in tomatoes. Based on examination of 83 Salmonella strains and 22 non-Salmonella strains, we concluded that a pair of hilA primers detects Salmonella specifically. The detection limits of the PCR assay were 10¹ and 10⁰ CFU/ml after enrichment at 37°C for 6 and 9 h, respectively. When the assay was validated by detecting S. enterica serotype Montevideo in and on artificially inoculated tomatoes, 10² and 10¹ CFU/g were detected, respectively, after enrichment for 6 h at 37°C. Our results suggest that the hilA-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonellae in or on fresh produce.     

Poultry Body Temperature Contributes to Invasion Control through Reduced Expression of Salmonella Pathogenicity Island 1 Genes in Salmonella enterica Serovars Typhimurium and Enteritidis

Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) are foodborne pathogens, and outbreaks are often associated with poultry products. Chickens are typically asymptomatic when colonized by these serovars; however, the factors contributing to this observation are uncharacterized. Whereas symptomatic mammals have a body temperature between 37°C and 39°C, chickens have a body temperature of 41°C to 42°C. Here, in vivo experiments using chicks demonstrated that numbers of viable S. Typhimurium or S. Enteritidis bacteria within the liver and spleen organ sites were ≥4 orders of magnitude lower than those within the ceca. When similar doses of S. Typhimurium or S. Enteritidis were given to C3H/HeN mice, the ratio of the intestinal concentration to the liver/spleen concentration was 1:1. In the avian host, this suggested poor survival within these tissues or a reduced capacity to traverse the host epithelial layer and reach liver/spleen sites or both. Salmonella pathogenicity island 1 (SPI-1) promotes localization to liver/spleen tissues through invasion of the epithelial cell layer. Following in vitro growth at 42°C, SPI-1 genes sipC, invF, and hilA and the SPI-1 rtsA activator were downregulated compared to expression at 37°C. Overexpression of the hilA activators fur, fliZ, and hilD was capable of inducing hilA-lacZ at 37°C but not at 42°C despite the presence of similar levels of protein at the two temperatures. In contrast, overexpression of either hilC or rtsA was capable of inducing hilA and sipC at 42°C. These data indicate that physiological parameters of the poultry host, such as body temperature, have a role in modulating expression of virulence.     

lac Repressor Is an Antivirulence Factor of Salmonella enterica: Its Role in the Evolution of Virulence in Salmonella

The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of LacI causes a remarkable reduction in the virulence of Salmonella enterica. LacI also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that LacI interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that LacI is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.     

Health Risk from the Use of Roof-Harvested Rainwater in Southeast Queensland,Australia, as Potable or Nonpotable Water,Determined Using Quantitative Microbial Risk Assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analyzed for the presence and numbers of zoonotic bacterial and protozoal pathogens using binary PCR and quantitative PCR (qPCR). Quantitative microbial risk assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from roof-harvested rainwater used as potable or nonpotable water. Of the 214 samples tested, 10.7%, 9.8%, 5.6%, and 0.4% were positive for the Salmonella invA, Giardia lamblia β-giardin, Legionella pneumophila mip, and Campylobacter jejuni mapA genes, respectively. Cryptosporidium parvum oocyst wall protein (COWP) could not be detected. The estimated numbers of Salmonella, G. lamblia, and L. pneumophila organisms ranged from 6.5 × 10¹ to 3.8 × 10² cells, 0.6 × 10° to 3.6 × 10° cysts, and 6.0 × 10¹ to 1.7 × 10² cells per 1,000 ml of water, respectively. Six risk scenarios were considered for exposure to Salmonella spp., G. lamblia, and L. pneumophila. For Salmonella spp. and G. lamblia, these scenarios were (i) liquid ingestion due to drinking of rainwater on a daily basis, (ii) accidental liquid ingestion due to hosing twice a week, (iii) aerosol ingestion due to showering on a daily basis, and (iv) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were (i) aerosol inhalation due to showering on a daily basis and (ii) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeded this threshold value and indicated that if undisinfected rainwater is ingested by drinking, then the incidences of the gastrointestinal diseases salmonellosis and giardiasis are expected to range from 9.8 × 10° to 5.4 × 10¹ (with a mean of 1.2 × 10¹ from Monte Carlo analysis) and from 1.0 × 10¹ to 6.5 × 10¹ cases (with a mean of 1.6 × 10¹ from Monte Carlo analysis) per 10,000 persons per year, respectively, in urban SEQ. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for use as potable water.Roof-harvested rainwater has received significant attention as a potential alternative source of potable and nonpotable water in regions where water is scarce (37). To encourage the use of roof-harvested rainwater, governmental bodies of many countries, such as Australia, Denmark, Germany, India, and New Zealand, are providing subsidies to residents to encourage the use of rainwater for domestic purposes. The use of rainwater is quite common in Australia, particularly in rural and remote areas, where reticulated mains or town water is not available. Recent water scarcity in several capital cities prompted the use of rainwater as an alternative source. For instance, the Queensland State Government initiated the “Home Water Wise Rebate Scheme,” which provides subsidies to Southeast Queensland (SEQ) residents who use rainwater as nonpotable water for domestic purposes (49). Over 260,000 householders were granted subsidies up to December 2008, when the scheme was concluded.There is a general community feeling that roof-harvested rainwater is safe to drink, and this is partially supported by limited epidemiological evidence (26). Some studies have reported that roof-harvested rainwater quality is generally acceptable for use as potable water (13, 29). In contrast, the presence of potential pathogens, such as Aeromonas spp. Campylobacter spp., Campylobacter jejuni, Salmonella spp., Legionella pneumophila, Giardia spp., Giardia lamblia, and Cryptosporidium spp., in roof-harvested rainwater samples has been reported (2, 9, 34, 45, 47, 48). Such pathogens can cause gastrointestinal illness in humans, with nausea, vomiting, and/or diarrhea occurring within 12 to 72 h (Salmonella enterica serovar Typhimurium) to 9 to 15 days (Giardia lamblia) after ingestion of contaminated water. L. pneumophila can cause the respiratory infection pneumonia, and the fatality rate can be 50% in immunocompromised patients (57).Direct routine monitoring of the microbiological quality of source water for all possible pathogens is not economically, technologically, or practically feasible. Consequently, traditional fecal indicators, such as fecal coliforms, Escherichia coli, and enterococci, have long been used to determine the presence of pathogens. Most studies assess the quality of roof-harvested rainwater based on the numbers of these fecal indicators (13, 30). However, the major limitation in using fecal bacteria as indicators is their poor correlation with the presence of pathogenic microorganisms in water (2, 30). An alternative is the measurement of pathogens using traditional culture-based methods. However, there are several limitations of such methods, including the underestimation of the bacterial number due to the presence of injured or stressed cells (10) and the fact that certain microorganisms in environmental waters can be viable but not culturable (39). Culture-based methods are also generally laborious and costly. Recent advances in molecular techniques such as PCR technology enable rapid, specific, and sensitive detection of many pathogens. Advances in PCR methodology also enable the quantification of potential pathogens in source water that are otherwise difficult and/or laborious to culture using traditional microbiological methods. In view of this, we used binary PCR (presence/absence)- and quantitative PCR (qPCR)-based assays to first detect and then quantify zoonotic pathogens in samples from roof-harvested rainwater in SEQ residential houses.The aims of the research study were 2-fold: (i) to quantify the number and frequency of occurrence of Salmonella, G. lamblia, and L. pneumophila organisms in a range of domestic water tanks in SEQ by using qPCR-based methods and (ii) to apply quantitative microbial risk assessment (QMRA) analysis in order to estimate the risk of infection from exposure to these pathogens found in roof-harvested rainwater. The uniqueness of this study stems from the fact that instead of measuring fecal indicators, the pathogens that are capable of causing illness were quantified and this information was combined with QMRA to assess the human health risk of using roof-harvested rainwater as potable or nonpotable water.     

Phenotypic and Genotypic Characterisation of Burkholderia cenocepacia J2315 Mutants Affected in Homoserine Lactone and Diffusible Signal Factor-Based Quorum Sensing Systems Suggests Interplay between Both Types of Systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid (“Burkholderia diffusible signal factor”, BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (ΔcepIΔBCAM0581, ΔcciIΔBCAM0581 and ΔcepIΔcciI) mutants and a triple (ΔcepIΔcciIΔBCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The ΔBCAM0581 mutant and the ΔcepIΔBCAM0581 and ΔcciIΔBCAM0581 double mutants produced significantly less AHL compared to the WT strain and the ΔcepI and ΔcciI single mutant, respectively. The expression of cepI and cciI in ΔBCAM0581, was approximately 3-fold and 7-fold (p<0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the ΔBCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system.     

Detection of Salmonella spp. Using a Generic and Differential FRET-PCR

To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a T _m of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella.     

Simultaneous Detection of Salmonella Strains and Escherichia coli O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in ≤4 h after enrichment.     

In vitro virulence characteristics of rare serovars of <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolated from sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis> L.)

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.     

Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.     

Genetic Mechanisms Underlying the Pathogenicity of Cold-Stressed Salmonella enterica Serovar Typhimurium in Cultured Intestinal Epithelial Cells

Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H₂O₂, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-stressed Salmonella. Further, a prophage gene, STM2699, induced in cold-stressed Salmonella and a spectrin gene, SPTAN1, induced in Salmonella-infected intestinal epithelial cells were found to have a significant contribution in increased adhesion and invasion of cold-stressed Salmonella in epithelial cells.     

Synergistic activity of sub-inhibitory concentrations of curcumin with ceftazidime and ciprofloxacin against Pseudomonas aeruginosa quorum sensing related genes and virulence traits

Quorum sensing (QS) system in Pseudomonas aeruginosa may be an important target for pharmacological intervention. The present study aimed to investigate the synergetic activity of sub-MIC concentrations of curcumin (C) with ceftazidime (CAZ) and ciprofloxacin (CIP) against P. aeroginusa QS system. We determined the MIC and synergistic activity of C, CAZ and CIP against P. aeroginusa PAO1 using broth microdilution and checkerboard titration methods. The activity of sub-MIC (1/4 and 1/16 MIC) concentrations of C on the QS signal molecules was assessed using a reporter strain assay. The influence of sub-MIC of C, CAZ and CIP alone and in combination on motility and biofilm formation was also determined and confirmed by RT-PCR to test the expression of QS regulatory genes lasI, lasR, rhlI and rhlR. The addition of C decreased the MIC of CAZ and CIP. Curcumin showed synergistic effects with CAZ and additive activity with CIP. Treated PAO1 cultures in the presence of C showed significant reduction of signals C12-HSL and C4-HSL (P?<?0.05). Sub-MIC concentrations (1/4 and 1/16 MIC) of C, CAZ and CIP alone and in combination significantly reduced swarming and twitching motilities and biofilm formation. Expression of QS regulatory genes lasI, lasR, rhlI, and rhlR using 1/4 MIC of C, CAZ and CIP alone and in combination was repressed significantly relative to untreated PAO1. Our results indicate that a combination of the sub-MIC concentration of C and CAZ exhibited synergism against P. aeroginusa QS system. This combination could lead to the development of a new combined therapy against P. aeruginosa.