Pomegranate peel extract reduced colonic damage and bacterial translocation in a mouse model of infectious colitis induced by Citrobacter rodentium.

The pomegranate fruit peel is a rich source of polyphenols including punicalins, punicalagins, and ellagic acids, but is considered an agricultural waste product. Pomegranate derived products have been reported to have a wide variety of health promoting benefits including antibacterial properties in vitro but there is limited evidence of their antibacterial properties in vivo. The purpose of this study was to test the in vivo antibacterial properties of a pomegranate peel extract (PPX) containing punicalin, punicalagin, and ellagic acid. C3H/He mice were orally pre-treated with water or PPX prior to infection with the mouse bacterial pathogen, Citrobacter rodentium (Cr) that mimics many aspects of human enteropathogenic Escherichia coli infections. Fecal excretion of Cr was monitored and mice were euthanized on day 12 post-infection to assess Cr colonization of the colon and spleen, histological changes, and gene expression. PPX-treatment reduced Cr infection induced weight loss and mortality that was observed in water-treated infected mice. However, Cr colonization of the colon and clearance was unaffected by PPX-treatment. Consistent with this, PPX treatment did not alter the potent Th1/Th17 pro-inflammatory response elicited by Cr infection. Significant colonization of the spleen was only seen in water-treated infected mice and was inversely correlated with the dose of PPX administered. PPX treatment decreased the extent of Cr-induced colon damage that correlated with decreased mortality and reduced colonization of the spleen. Thus, a pomegranate peel extract contains bioactive compounds that mitigate the deleterious effects of an in vivo infection with the model enteropathogenic bacteria, Cr.

Pomegranate peel extract alters the microbiome in mice and dysbiosis caused by Citrobacter rodentium infection.

Treatment of mice with a pomegranate peel extract (PPX) decreased the pathogenicity of Citrobacter rodentium (Cr) infections. Here, we investigate the effects of PPX on the microbiome of uninfected or Cr-infected C3H/HeNCr mice by 16S rRNA gene sequencing. Mice were treated with water or PPX for 14 days, feces were collected, and then, the mice were infected with Cr and feces collected again at day 6 postinfection. DNA was isolated from the fecal samples and subjected to 16S rRNA gene sequencing to determine the microbial composition. Differences in the composition of the microbiome were observed for untreated and PPX-treated mice with PPX mice having decreased diversity. PPX treatment decreased the Firmicutes/Bacteroidetes ratio by increasing Bacteroidetes and decreasing Firmicutes levels. The decrease in Firmicutes was driven by a large reduction in Lactobacillus. PPX treatment increased the abundance of Proteobacteria and Verrucomicrobiae and decreased Actinobacteria. The relative abundance of Cr reached 22% in water-treated but only 5% in PPX-treated infected mice. These results suggest that consumption of pomegranate polyphenols altered the microbiome, making it more resistant to displacement by infection with Cr, indicating that pomegranate polyphenols may mitigate the pathogenic effects of food-borne bacterial pathogens.

Inverted Regulation of Multidrug Efflux Pumps, Acid Resistance, and Porins in Benzoate-Evolved Escherichia coli K-12.

Benzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during the experimental evolution of Escherichia coli K-12. Transcriptomes of E. coli isolates evolved with benzoate showed the reversal of benzoate-dependent regulation, including the downregulation of multidrug efflux pump genes, the gene for the Gad acid resistance regulon, the nitrate reductase genes narHJ, and the gene for the acid-consuming hydrogenase Hyd-3. However, the benzoate-evolved strains had increased expression of OmpF and other large-hole porins that admit fermentable substrates and antibiotics. Candidate genes identified from benzoate-evolved strains were tested for their roles in benzoate tolerance and in chloramphenicol sensitivity. Benzoate or salicylate tolerance was increased by deletion of the Gad activator ariR or of the acid fitness island from slp to the end of the gadX gene encoding Gad regulators and the multidrug pump genes mdtEF Benzoate tolerance was also increased by deletion of multidrug component gene emrA, RpoS posttranscriptional regulator gene cspC, adenosine deaminase gene add, hydrogenase gene hyc (Hyd-3), and the RNA chaperone/DNA-binding regulator gene hfq Chloramphenicol resistance was decreased by mutations in genes for global regulators, such as RNA polymerase alpha subunit gene rpoA, the Mar activator gene rob, and hfq Deletion of lipopolysaccharide biosynthetic kinase gene rfaY decreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism, such as aroF (encoding tyrosine biosynthesis) and apt (encoding adenine phosphoribosyltransferase). Overall, benzoate or salicylate exposure selects for the loss of multidrug efflux pumps and of hydrogenases that generate a futile cycle of PMF and upregulates porins that admit fermentable nutrients and antibiotics.IMPORTANCE Benzoic acid is a common food preservative, and salicylic acid (2-hydroxybenzoic acid) is the active form of aspirin. At high concentrations, benzoic acid conducts a proton across the membrane, depleting the proton motive force. In the absence of antibiotics, benzoate exposure selects against proton-driven multidrug efflux pumps and upregulates porins that admit fermentable substrates but that also allow the entry of antibiotics. Thus, evolution with benzoate and related molecules, such as salicylates, requires a trade-off for antibiotic sensitivity, a trade-off that could help define a stable gut microbiome. Benzoate and salicylate are naturally occurring plant signal molecules that may modulate the microbiomes of plants and animal digestive tracts so as to favor fermenters and exclude drug-resistant pathogens.

Transcriptomic Analysis of the Swarm Motility Phenotype of Salmonella enterica Serovar Typhimurium Mutant Defective in Periplasmic Glucan Synthesis.

Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in Osmoregulated periplasmic glucans (OPG) synthesis are unable to exhibit motility on moist surfaces (swarming); however, their mobility in liquid (swim motility) remains unaffected. In order to understand the role of OPG in swarm motility, transcriptomic analysis was performed using cells growing on a moist agar surface. In opgGH deletion mutant, lack of OPG significantly altered transcription of 1039 genes out of total 4712 genes (22%). Introduction of a plasmid-borne copy of opgGH into opgGH deletion mutant restored normal expression of all but 30 genes, indicating a wide-range influence of OPG on gene expression under swarm motility condition. Major pathways that were differentially expressed in opgGH mutants were motility, virulence and invasion, and genes related to the secondary messenger molecule, cyclic di-GMP. These observations provide insights and help explain the pleiotropic nature of OPG mutants such as sub-optimal virulence and competitive organ colonization in mice, biofilm formation, and sensitivity towards detergent stress.

Swarm motility inhibitory and antioxidant activities of pomegranate peel processed under three drying conditions.

During processing of ready-to-eat fresh fruits, large amounts of peel and seeds are discarded as waste. Pomegranate (Punicagranatum) peels contain high amounts of bioactive compounds which inhibit migration of Salmonella on wet surfaces. The metabolic distribution of bioactives in pomegranate peel, inner membrane, and edible aril portion was investigated under three different drying conditions along with the anti-swarming activity against Citrobacter rodentium. Based on the multivariate analysis, 29 metabolites discriminated the pomegranate peel, inner membrane, and edible aril portion, as well as the three different drying methods. Punicalagins (∼38.6-50.3mg/g) were detected in higher quantities in all fractions as compared to ellagic acid (∼0.1-3.2mg/g) and punicalins (∼0-2.4mg/g). The bioactivity (antioxidant, anti-swarming) and phenolics content was significantly higher in peels than the edible aril portion. Natural anti-swarming agents from food waste may have promising potential for controlling food borne pathogens.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.

Development of an Acid-Resistant Salmonella Typhi Ty21a Attenuated Vector For Improved Oral Vaccine Delivery.

The licensed oral, live-attenuated bacterial vaccine for typhoid fever, Salmonella enterica serovar Typhi strain Ty21a, has also been utilized as a vaccine delivery platform for expression of diverse foreign antigens that stimulate protection against shigellosis, anthrax, plague, or human papilloma virus. However, Ty21a is acid-labile and, for effective oral immunization, stomach acidity has to be either neutralized with buffer or by-passed with Ty21a in an enteric-coated capsule (ECC). Several studies have shown that efficacy is reduced when Ty21a is administered in an ECC versus as a buffered liquid formulation, the former limiting exposure to GI tract lymphoid tissues. However, the ECC was selected as a more practical delivery format for both packaging/shipping and vaccine administration ease. We have sought to increase Ty21a acid-resistance to allow for removal from the ECC and immune enhancement. To improve Ty21a acid-resistance, glutamate-dependent acid resistance genes (GAD; responsible for Shigella spp. survival at very low pH) were cloned on a multi-copy plasmid (pGad) under a controllable arabinose-inducible promoter. pGad enhanced acid survival of Ty21a by 5 logs after 3 hours at pH 2.5, when cells were pre-grown in arabinose and under conditions that promote an acid-tolerance response (ATR). For genetically 100% stable expression, we inserted the gad genes into the Ty21a chromosome, using a method that allowed for subsequent removal of a selectable antibiotic resistance marker. Further, both bacterial growth curves and survival assays in cultured human monocytes/macrophages suggest that neither the genetic methods employed nor the resulting acid-resistance conferred by expression of the Gad proteins in Ty21a had any effect on the existing attenuation of this vaccine strain.

Understanding the host-adapted state of Citrobacter rodentium by transcriptomic analysis.

Citrobacter rodentium (Cr) is a mouse pathogen that mimics many aspects of enteropathogenic Escherichia coli infections including producing attaching and effacing (A/E) lesions. Host-adapted (HA) Cr cells that are shed at the peak of infection have been reported to be hyper-infective. The exact mechanism underlying this phenomenon has remained elusive since the pathogen loses its HA 'status' immediately upon subculturing in laboratory media. We sequenced the entire transcriptome of Cr directly from the feces of infected mice and analyzed the gene expression pattern. We observed that the entire transcriptional machinery as well as several transcriptional regulators to be differentially expressed when compared with the transcriptome of cells grown on laboratory media. Major adhesion and effector genes, tir and eae, were highly expressed in HA along with many genes located on all five loci of enterocyte effacement regions (LEE 1-5). Notable absent among the HA expressed genes were 19 fimbrial operons and non-fimbrial adhesions and several non-LEE encoded effectors. These results demonstrate that host-adapted Cr has a unique transcriptome that is associated with increased host transmission.

Hypervirulent-host-associated Citrobacter rodentium cells have poor acid tolerance.

Enhanced virulence or infectivity after passage through a mammalian host has been reported for a number of enteric food-borne pathogens. Citrobacter rodentium is a mouse pathogen that mimics many aspects of enterohemorrhagic Escherichia coli infection of humans and serves as a useful model for studying virulence mechanisms. Emergence of a hyperinfectious state after passage through mouse gastrointestinal tract was reported for C. rodentium. We wanted to investigate if increased acid tolerance could explain hypervirulence status of C. rodentium. Although we were able to observe hyperinfectious state of C. rodentium upon host passage, the cells were extremely acid sensitive. Growth under mildly acidic conditions (LB-MES, pH 5.5) induced acid tolerance of C. rodentium, but did not improve the organism's ability to establish infection. Growth under anaerobic environment on fecal components also did not induce hyperinfectious state. Thus, contrary to conventional anticipation, hypervirulent C. rodentium cells were found to be acid sensitive thereby revealing limitations of the role of mouse gastric acidity by itself in elucidating the hypervirulent phenotype.

Role of anionic charges of periplasmic glucans of Shigella flexneri in overcoming detergent stress.

Osmoregulated periplasmic glucans (OPGs) are synthesized by the members of the family Enterobacteriaceae when grown under low osmotic growth conditions. Enteropathogens such as Shigella flexneri spend considerable time outside the host environment such as irrigation waters where low nutrient low osmolarity conditions normally may exist. We recently demonstrated that OPGs of S. flexneri are required for optimal growth under low osmolarity low nutrient conditions. Based on homology of the OPG biosynthesis genes to those of Escherichia coli, the presumptive function of opgC and opgB genes is to add succinate and phosphoglycerol residues respectively on OPGs, rendering them anionic. Using lambda-red recombination procedure, we constructed opgB, opgC, and opgBC mutants of S. flexneri. The mutant strain defective in opgC and opgB genes synthesized neutral OPGs. The OPGs without any anionic charges were beneficial for the organism's growth in hypo-osmotic media. However, with the loss of anionic charges from OPGs, mutants were compromised in their ability to combat stress caused by anionic detergents in hypo-osmotic growth conditions. Cloned wild-type genes opgB, opgC, and opgBC, when mobilized to respective opg mutants, simultaneously restored anionic charges to OPGs and tolerance to detergents. The data indicate that anionic charges on the OPGs contribute towards overcoming the stress caused by anionic detergents such as sodium dodecyl sulfate and sodium deoxycholate.

Proteomic pleiotropy of OpgGH, an operon necessary for efficient growth of Salmonella enterica serovar typhimurium under low-osmotic conditions.

Salmonella enterica, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. Unfortunately for consumers, the bacteria can survive in water used to wash away contaminating bacteria. The ability to survive the low-osmotic conditions of the wash water is attributed to the OpgGH operon that leads to the production of osmotically regulated periplasmic glucans. Mutants lacking OpgGH grow slowly under low-osmotic conditions, but there are also unexpected traits such as abnormal flagellar motility and reduced virulence in mice. To get a broader understanding of these pleiotropic effects under low osmolarity, we examined the proteome of these mutants using high-throughput mass spectrometry. We identified approximately one-third of the proteins encoded by the genome and used label-free spectral counting to determine the relative amounts of proteins in wild-type cultures and mutants. Mutants had reduced amounts of proteins required for osmotic sensing, flagellar motility, purine and pyrimidine metabolism, oxidative energy production, and protein translation. By contrast, mutants had greater amounts of ABC transporters needed to balance cellular osmolarity. Hence, the effects of OpgGH reach across the proteome, and the data are consistent with the mutant phenotypes.

Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium SL1344 in mice virulence.

opgB gene of Salmonella enterica serovar Typhimurium was identified earlier in a genome-wide screen for mice virulence (Valentine et al. in Infect Immun 66:3378-3383, 1998). Although mutation in opgB resulted in avirulent Salmonella strain, how this gene contributes to pathogenesis remains unclear. Based on DNA homology, opgB is predicted to be responsible for adding phosphoglycerate residues to osmoregulated periplasmic glucans (OPGs) giving them anionic characteristics. In Escherichia coli, yet another gene, opgC, is also reported to contribute to anionic characteristics of OPGs by adding succinic acid residues. We constructed opgB, opgC, and opgBC double mutants of S. enterica serovar Typhimurium strain SL1344. As predicted opgBC mutant synthesized neutral OPGs that were devoid of any anionic substituents. However, opgB, opgC, and opgBC mutations had no significant impact on mice virulence as well as on competitive organ colonization. In low osmotic conditions, opgB, opgC, and opgBC mutants exhibited delay in growth initiation in the presence of sodium deoxycholate. Anionic substituents of OPGs from Salmonella although appear to be needed to overcome resistance of deoxycholate in hypoosmotic growth media, no evidence was found for their role in mice virulence.

Microbial genome analysis and comparisons: Web-based protocols and resources.

Fully annotated genome sequences of many microorganisms are publicly available as a resource. However, in-depth analysis of these genomes using specialized tools is required to derive meaningful information. We describe here the utility of three powerful publicly available genome databases and analysis tools. Protocols outlined here are particularly useful for performing pairwise genome comparisons between closely related microorganisms to identify similarities and unique features, for example to identify genes specific to a pathogenic strain of Escherichia coli compared to a nonpathogenic strain.

Arginine-dependent acid-resistance pathway in Shigella boydii.

Ability to survive the low pH of the human stomach is considered be an important virulent determinant. It was suggested that the unique acid tolerance of Shigella boydii 18 CDPH, the strain implicated in a 1998 outbreak, may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arginine-dependent acid-resistance (ADAR) pathway. This pathway was assumed to be absent in Shigella sp. Here, we have examined occurrence and efficacy of ADAR pathway in 21 S. boydii strains obtained from the American Type Culture Collection (ATCC) along with strains of S. flexneri (n = 7), S. sonnei (n = 4), and S. dysenteriae (n = 2). The eight S. boydii strains were able to induce ADAR to survive the acid challenge at pH 2.0; additional 8 strains could tolerate acid challenge at pH 2.5 but not at pH 2.0. The remaining five S. boydii strains were not able to induce ADAR pathway and could not survive acid challenge even at pH 2.5. ADAR pathway also appears to be present in all four Shigella sp. Shigella ADAR pathway was induced when cells were grown under partial oxygen pressure while its expression in E. coli required mere fermentative growth on glucose.

Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef.

We investigated the efficacy of bacteriophage-based detection technology to detect Escherichia coli O157:H7 from ground beef. The assay involved a short enrichment period of 8 h followed by capture of the pathogen on O157-specific immunomagnetic beads. The captured cells were treated with O157-specific lytic bacteriophage, CSLO157. Upon phage-induced lysis, the enzyme adenylate kinase, which was released from the lysed cells, was measured in terms of relative light units using luciferin-luciferase assay. The plaque forming efficiency (e.g., phage susceptibility) and ability to capture cells with immunomagnetic beads were examined using an array of 74 E. coli O157:H7 isolates obtained from various clinical and foodborne samples. Immunmagnetic beads successfully captured all 74 isolates; however, only 53 isolates showed susceptibility toward the bacteriophage. Susceptible isolates were further classified into two broad groups, moderately sensitive isolates, which generated phage titer ∼ 10(7)pfu/mL (group I, n=15), and highly susceptible isolates, which generated high phage titer ∼ 10(9)pfu/mL (group II, n=38). We selected 15 isolates (9 from group I and 6 from group II) and individually spiked beef samples (ca. 3-8 cells/25 g beef) to evaluate the bacteriophage-based detection system. Eight out of nine isolates from group I and all six isolates from group II were successfully detected. Pathogenic E. coli strains belonging to other serogroups (12 serogroups, 67 isolates) as well as nontarget microorganisms (n=18) were not lysed by the bacteriophage and hence were not detected. The method is high-throughput compatible, is rapid, and can provide live culture the following day by streaking an aliquot before phage lysis on conventional selective agar media.

Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri.

UNLABELLED: Osmoregulated periplasmic glucans (OPGs) of food- and water-borne enteropathogen Shigella flexneri were characterized. OPGs were composed of 100% glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2-linked and 2,6-linked glucose also present in high quantities. Most dominant backbone polymer chain length was seven glucose residues. Individual genes from the opg gene family comprising of a bicistronic operon opgGH, opgB, opgC and opgD were mutagenized to study their effect on OPGs synthesis, growth in hypo-osmotic media and ability to invade HeLa cells. Mutation in opgG and opgH abolished OPGs biosynthesis, and mutants experienced longer lag time to initiate growth in hypo-osmotic media. Longer lag times to initiate growth in hypo-osmotic media were also observed for opgC and opgD mutants but not for opgB mutant. All opg mutants were able to infect HeLa cells, and abolition of OPGs synthesis did not affect actin polymerization or plaque formation. Ability to synthesize OPGs was beneficial to bacteria in order to initiate growth under low osmolarity conditions, in vitro mammalian cell invasion assays, however, could not discriminate whether OPGs were required for basic aspect of Shigella virulence. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00203-009-0538-z) contains supplementary material, which is available to authorized users.

Motility revertants of opgGH mutants of Salmonella enterica serovar Typhimurium remain defective in mice virulence.

We recently demonstrated that osmoregulated periplasmic glucans (OPGs) of Salmonella enterica serovar Typhimurium are required for optimal mouse virulence. However, lack of OPGs also generated pleiotropic phenotypes such as reduced motility and slower growth rate under hypoosmotic growth conditions. Whether the observed suboptimal virulence of opg mutants was due to reduced motility was investigated by isolating fully motile revertants of opgGH mutants. Motility revertants remained defective in OPGs synthesis and restitution of motility did not restore mouse virulence. In Escherichia coli, inactivation of rcsB, rcsD, and rcsF lead to restoration of motility in opg mutants, while in Salmonella strains, inactivation of the Rcs pathway is known to attenuate virulence. DNA sequence analysis revealed that except for two silent mutations no other changes in the DNA sequences of Rcs pathway genes were detected in the motility-revertant strain. Moreover, transcripts of all the Rcs phosphorelay pathway genes were detected in opgGH mutants and revertant strain. The data suggest that Salmonella may have distinctive regulatory elements in addition to Rcs phosphorelay genes to rescue motility of opg mutants and affecting also mouse virulence.

Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash waters and colonization in mice.

Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host-microorganism interactions. Here, we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and biofilm formation in leafy-green vegetable wash water. The opgGH mutant strain, which was defective in OPG biosynthesis, initiated the growth at a slower rate in wash waters obtained from spinach, lettuce and green collard and severely impaired biofilm formation. The lack of OPG synthesis did not influence biofilm formation by the opgGH mutant in low-nutrient low-osmolarity laboratory media. In coculture experiments initiated with equal proportions of cells, the opgGH mutant was outnumbered by the wild-type strain under the planktonic as well as the biofilm growth conditions. The opgGH mutant strain poorly colonized mouse organs when introduced orally along with the wild-type strain. This is the first report demonstrating the role of OPGs of Salmonella in competitive colonization of biofilms, planktonic cultures and mouse organs.

Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice.

We purified osmoregulated periplasmic glucans (OPGs) from Salmonella enterica serovar Typhimurium and found them to be composed of 100 % glucose with 2-linked glucose as the most abundant residue, with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structural genes for OPG biosynthesis, opgG and opgH, form a bicistronic operon, and insertion of a kanamycin resistance gene cassette into this operon resulted in a strain devoid of OPGs. The opgGH mutant strain was impaired in motility and growth under low osmolarity conditions. The opgGH mutation also resulted in a 2 log increase in the LD50 in mice compared to the wild-type strain SL1344. Inability to synthesize OPGs had no significant impact on the organism's lipopolysaccharide pattern or its ability to survive antimicrobial peptides-, detergent-, pH- and nutrient-stress conditions. We observed that the opgGH-defective strain respired at a reduced rate under acidic growth conditions (pH 5.0) and had lower ATP levels compared to the wild-type strain. These data indicate that OPGs of S. Typhimurium contribute towards mouse virulence as well as growth and motility under low osmolarity growth conditions.

Characterization of Salmonella isolates from retail foods based on serotyping, pulse field gel electrophoresis, antibiotic resistance and other phenotypic properties.

Sixteen Salmonella strains isolated from a variety of foods during 2000 and 2003 by the Florida State Department of Agriculture were characterized by various genotypic and phenotypic tests. Among 16 isolates, 15 different serotypes were identified. Pulse-field gel electrophoresis (PFGE) fingerprinting profiles obtained using restriction endonucleases XbaI and BlnI revealed that 16 Salmonella isolates were genetically diverse with 16 unique PFGE patterns. The PFGE pattern of eight isolates matched with the CDC/FDA data base of previous outbreaks and clinical isolates indicating their potential to cause disease. With the exception of isolates obtained from alligator meat (tetracycline resistant) and orange juice (chloramphenicol and sulfisoxazole resistant), the remainder of the isolates were susceptible to the panel of 15 antimicrobials tested. Molecular subtyping was further complimented by a variety of phenotypic tests such as acid-tolerance, Caco-2 cell invasion and biofilm formation which have often been used as a gauge of virulence and infection potential of Salmonella isolates. The induced acid tolerance level of the isolate obtained from orange juice was not significantly different from the laboratory reference strain S. enterica serovar Typhimurium SL1344. Six isolates exhibited very low levels of constitutive acid-tolerance, of which four isolates failed to infect differentiated Caco-2 cells. Although all isolates formed biofilms, there was no clear relation between the ability to form biofilms, infect differentiated Caco-2 cells and induce acid-tolerance. This study indicated that different serotypes of Salmonella were present in a variety of retail foods and exhibited diverse phenotypic characteristics.