Publications by authors named "Arvind A Bhagwat"

39 Publications

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

Nutr Res 2020 01 16;73:27-37. Epub 2019 Nov 16.

Environmental Microbial & Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705. Electronic address:

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.
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http://dx.doi.org/10.1016/j.nutres.2019.11.001DOI Listing
January 2020

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

Food Sci Nutr 2019 Aug 7;7(8):2565-2576. Epub 2019 Jul 7.

Diet Genomics and Immunology Lab Beltsville Human Nutrition Research Center Agricultural Research Service, Department of Agriculture Beltsville Maryland.

Treatment of mice with a pomegranate peel extract (PPX) decreased the pathogenicity of () infections. Here, we investigate the effects of PPX on the microbiome of uninfected or -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 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 . PPX treatment increased the abundance of Proteobacteria and Verrucomicrobiae and decreased Actinobacteria. The relative abundance of 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 , indicating that pomegranate polyphenols may mitigate the pathogenic effects of food-borne bacterial pathogens.
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http://dx.doi.org/10.1002/fsn3.1106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694437PMC
August 2019

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

Appl Environ Microbiol 2019 08 1;85(16). Epub 2019 Aug 1.

Department of Biology, Kenyon College, Gambier, Ohio, USA

Benzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during the experimental evolution of K-12. Transcriptomes of 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 , 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 or of the acid fitness island from to the end of the gene encoding Gad regulators and the multidrug pump genes Benzoate tolerance was also increased by deletion of multidrug component gene , RpoS posttranscriptional regulator gene , adenosine deaminase gene , hydrogenase gene (Hyd-3), and the RNA chaperone/DNA-binding regulator gene Chloramphenicol resistance was decreased by mutations in genes for global regulators, such as RNA polymerase alpha subunit gene , the Mar activator gene , and Deletion of lipopolysaccharide biosynthetic kinase gene decreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism, such as (encoding tyrosine biosynthesis) and (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. 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.
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http://dx.doi.org/10.1128/AEM.00966-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677852PMC
August 2019

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

Curr Microbiol 2017 Sep 8;74(9):1005-1014. Epub 2017 Jun 8.

National Institutes of Health Library, Division of Library Services, Office of Research Services, National Institute of Health, Building 10, Bethesda, MD, 20892, USA.

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.
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http://dx.doi.org/10.1007/s00284-017-1267-1DOI Listing
September 2017

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

Food Chem 2017 Nov 25;235:145-153. Epub 2017 Apr 25.

Food Composition Methods Development Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, USDA-ARS, Beltsville, MD 20705, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.foodchem.2017.04.143DOI Listing
November 2017

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

Appl Environ Microbiol 2017 06 31;83(12). Epub 2017 May 31.

Department of Biology, Kenyon College, Gambier, Ohio, USA

Acid-adapted strains of 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 IS insertion or IS-mediated deletion in , while population B11 had a point mutation affecting the arginine activator The and mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an (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 , , and Gad). Other strains showed downregulation of H consumption mediated by hydrogenases ( and ) which release acid. Strains F9-2 and F9-3 had a deletion of and showed downregulation of FNR-dependent genes (, , , , and ). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism. 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.
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http://dx.doi.org/10.1128/AEM.00442-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452808PMC
June 2017

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

PLoS One 2016;11(9):e0163511. Epub 2016 Sep 27.

Laboratory of Mucosal Pathogens and Cellular Immunology, Food and Drug Administration-Center for Biologics Evaluation and Research, New Hampshire Avenue, Silver Spring, Maryland, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163511PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5046385PMC
September 2016

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

Arch Microbiol 2016 May 2;198(4):353-62. Epub 2016 Feb 2.

Environmental, Microbial, and Food Safety Laboratory, Beltsville Agriculture Research Center, USDA-ARS, Beltsville, MD, USA.

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.
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http://dx.doi.org/10.1007/s00203-016-1191-yDOI Listing
May 2016

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

Curr Microbiol 2013 May 18;66(5):522-6. Epub 2013 Jan 18.

Diet Genomics and Immunology and Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, 10300 Baltimore Avenue, BARC-E, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1007/s00284-012-0298-xDOI Listing
May 2013

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

Foodborne Pathog Dis 2012 Jul 25;9(7):632-7. Epub 2012 Jun 25.

Environmental Microbial and Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, Beltsville, Maryland 20705-2350, USA.

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.
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http://dx.doi.org/10.1089/fpd.2011.1090DOI Listing
July 2012

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

Arch Microbiol 2012 Jun 26;194(6):541-8. Epub 2012 Jan 26.

Environmental Microbial and Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Bldg. 173, BARC-E, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1007/s00203-012-0791-4DOI Listing
June 2012

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

J Proteome Res 2012 Mar 8;11(3):1720-7. Epub 2012 Feb 8.

Soybean Genomics and Improvement Laboratory, USDA-ARS , Beltsville, Maryland 20705, United States.

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.
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http://dx.doi.org/10.1021/pr200933dDOI Listing
March 2012

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

Methods Mol Biol 2011 ;765:297-307

NIH Library, Office of Research Services, National Institutes of Health, Bethesda, MD, USA.

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.
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http://dx.doi.org/10.1007/978-1-61779-197-0_17DOI Listing
January 2012

Arginine-dependent acid-resistance pathway in Shigella boydii.

Arch Microbiol 2011 Mar 7;193(3):179-85. Epub 2010 Dec 7.

Environmental Microbial & Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Bldg. 002, Room 117, BARC-W, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1007/s00203-010-0656-7DOI Listing
March 2011

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

Foodborne Pathog Dis 2010 Dec 31;7(12):1551-8. Epub 2010 Aug 31.

Environmental Microbial and Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, Maryland 20705-2350, USA.

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.
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http://dx.doi.org/10.1089/fpd.2010.0634DOI Listing
December 2010

Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri.

Arch Microbiol 2010 Mar;192(3):167-74

College of Food Science and Technology, Northwest A&F University, 712100 Yangling, China.

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.
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http://dx.doi.org/10.1007/s00203-009-0538-zDOI Listing
March 2010

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

Curr Microbiol 2009 Dec 29;59(6):641-5. Epub 2009 Aug 29.

Environmental Microbial and Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1007/s00284-009-9486-8DOI Listing
December 2009

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.

FEMS Microbiol Lett 2009 Mar;292(1):13-20

Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1111/j.1574-6968.2008.01462.xDOI Listing
March 2009

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats.

Acta Vet Hung 2008 Dec;56(4):451-8

Food Safety Laboratory, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350, USA.

A real-time PCR assay was evaluated for the rapid detection (10 h) of Salmonella in meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated with Salmonella enterica serovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.
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http://dx.doi.org/10.1556/AVet.56.2008.4.3DOI Listing
December 2008

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

Microbiology (Reading) 2009 Jan;155(Pt 1):229-237

Diet Genomics and Immunology Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Bldg. 002, BARC-W, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1099/mic.0.023747-0DOI Listing
January 2009

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

Int J Food Microbiol 2009 Jan 14;129(1):93-8. Epub 2008 Nov 14.

Produce Quality and Safety Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705-2350, USA.

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.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2008.11.007DOI Listing
January 2009

Characterization of Shiga toxin-producing Escherichia coli strains isolated from swine feces.

Foodborne Pathog Dis 2008 Dec;5(6):827-38

Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038, USA.

The virulence gene and antibiotic resistance profiles of Shiga toxin-producing Escherichia coli (STEC) strains belonging to 58 different O:H serotypes (219 strains) isolated from swine feces were determined. Of the 219 isolates, 29 (13%) carried the stx(1) gene, 14 (6%) stx(2), 176 (80%) stx(2e), 46 (21%) estIa, 14 (6.4%) estIb, 10 (4.6%) fedA, 94 (42.9%) astA, 25 (11.4%) hly(933), and one (0.46%) cdt-III. None of the strains possessed the elt, bfp, faeG, fanA, fasA, fimF(41a), cnf-1, cnf-2, eae, cdt-I, or cdt-IV genes. The strains were also tested for antibiotic susceptibility using 16 antibiotics. The STEC isolates displayed resistance most often to tetracycline (95.4%), sulfamethoxazole (53.4%), kanamycin (38.4%), streptomycin (34.7%), and chloramphenicol (22.4%). An E. coli serotype O20:H42 strain, which was positive for stx(2e) and astA, was resistant to all of the antibiotics tested except for amikacin. In addition, 52 of the swine isolates, representing 16 serogroups and 30 different serotypes, were examined for their ability to withstand acid challenge by three types of acid resistance (AR) pathways, AR1 (rpoS dependent), AR2 (glutamate dependent), and AR3 (arginine dependent). None of the strains was defective in the AR1 resistance pathway, while one strain was defective in the AR2 pathway under aerobic growth conditions but fully functional under anaerobic growth conditions. Among the three AR pathways, the AR3 pathway offered the least protection, and 8 out of 52 strains were defective in this pathway. The strain that was defective in AR2 was fully functional in the AR3 pathway. Since AR plays a vital role in the survival and virulence of these strains, differences among the isolates to induce AR pathways may play a significant role in determining their infective dose. This study demonstrates that swine STEC comprise a heterogeneous group of organisms, and the possession of different combinations of E. coli virulence genes indicate that some swine STEC can potentially cause human illness.
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http://dx.doi.org/10.1089/fpd.2008.0147DOI Listing
December 2008

Methods and tools for comparative genomics of foodborne pathogens.

Foodborne Pathog Dis 2008 Aug;5(4):487-97

Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, Maryland 20705-2350, USA.

A comparison of genome sequences and of encoded proteins with the database of existing annotated sequences is a useful approach to understand the information at the genome level. Here we demonstrate the utility of several DNA and protein sequence comparison tools to interpret the information obtained from several genome projects. Comparisons are presented between closely related strains of Escherichia coli commensal isolates, different isolates of O157:H7, and Shigella spp. It is expected that comparative genome analysis will generate a wealth of data to compare pathogenic isolates with varying levels of pathogenicity, which in turn may reveal mechanisms by which the pathogen may adapt to a particular nutrient supply in certain foods. These genome sequence analysis tools will strengthen foodborne pathogen surveillance and subsequent risk assessment to enhance the safety of the food supply.
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http://dx.doi.org/10.1089/fpd.2008.0117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2614369PMC
August 2008

Detection of Salmonella species in foodstuffs.

Methods Mol Biol 2008 ;429:33-43

Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, USDA, Beltsville, MD, USA.

Conventional methods to detect Salmonella spp. in foodstuffs may take up to 1 wk. Methods for pathogen detection are required. Real-time detection of Salmonella spp. will broaden our ability to screen large number of samples in a short time. This chapter describes a step-by-step procedure using an oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon; MB) in a real-time polymerase chain reaction (PCR) assay. The capability of the assay to detect Salmonella species from artificially inoculated fresh- and fresh-cut produce as well as ready-to-eat meats is demonstrated. The method uses internal positive and negative controls which enable researchers to detect false-negative PCR results. The procedure uses the buffered peptone water for the enrichment of Salmonella spp. and successfully detects the pathogen at low level of contamination (2-4 cells/25 g) in <24 h.
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http://dx.doi.org/10.1007/978-1-60327-040-3_3DOI Listing
September 2008

Role of RNA polymerase sigma-factor (RpoS) in induction of glutamate-dependent acid-resistance of Escherichia albertii under anaerobic conditions.

FEMS Microbiol Lett 2008 Jun 14;283(1):75-82. Epub 2008 Apr 14.

Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD, USA.

Escherichia albertii is a potential enteric food-borne pathogen with poorly defined genetic and biochemical properties. Acid resistance is perceived to be an important property of enteric pathogens, enabling them to survive passage through stomach acidity so that they may colonize the mammalian gastrointestinal tract. We analyzed glutamate-dependent acid-resistance pathway (GDAR) in five E. albertii strains that have been identified so far. We observed that the strains were unable to induce GDAR under aerobic growth conditions. Mobilization of the rpoS gene restored aerobic induction of this acid-resistance pathway, indicating that all five strains may have a dysfunctional sigma-factor. On the other hand, under anaerobic growth conditions where GDAR is induced in an RpoS-independent manner (i.e. in Shigella spp. and Escherichia coli O157:H7 strains), only three out of five E. albertii strains successfully induced GDAR. The remainder of the two strains exhibited dependence on functional RpoS even under anaerobic conditions to express GDAR, a regulatory function previously considered to be redundant. The data indicate that certain E. albertii strains may have an alternate RpoS-dependent pathway for acid-resistance under anaerobic growth conditions.
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http://dx.doi.org/10.1111/j.1574-6968.2008.01153.xDOI Listing
June 2008

Sensitivity of Escherichia albertii, a potential food-borne pathogen, to food preservation treatments.

Appl Environ Microbiol 2007 Jul 27;73(13):4351-3. Epub 2007 Apr 27.

Food Technology and Safety Laboratory, USDA-ARS, ANRI, BARC-EAST, Beltsville, MD 20705, USA.

Escherichia albertii is a potential food-borne pathogen because of its documented ability to cause diarrheal disease by producing attachment and effacement lesions. Its tolerances to heat (56 degrees C), acid (pH 3.0), and pressure (500 MPa [5 min]) were evaluated and found to be significantly less than those of wild-type E. coli O157:H7.
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http://dx.doi.org/10.1128/AEM.03001-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932773PMC
July 2007

Characterization of Listeria monocytogenes isolated from retail foods.

Int J Food Microbiol 2007 Jan 22;113(1):47-53. Epub 2006 Sep 22.

Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA.

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2006.07.010DOI Listing
January 2007

Isolation and characterization of Listeria monocytogenes isolates from ready-to-eat foods in Florida.

Appl Environ Microbiol 2006 Jul;72(7):5073-6

Florida State Department of Agriculture and Consumer Services, Tallahassee, Florida, USA.

Of 3,063 ready-to-eat food samples tested, 91 (2.97%) were positive for Listeria monocytogenes, and lineage 1 strains outnumbered lineage 2 strains 57 to 34. Seventy-one isolates (78%) exhibited multiple antibiotic resistance, and an L. monocytogenes-specific bacteriophage cocktail lysed 65 of 91 (71%) isolates. Determining phage, acid, and antibiotic susceptibility phenotypes enabled us to identify differences among strains which were otherwise indistinguishable by conventional methods.
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http://dx.doi.org/10.1128/AEM.00435-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489337PMC
July 2006

Functional heterogeneity of RpoS in stress tolerance of enterohemorrhagic Escherichia coli strains.

Appl Environ Microbiol 2006 Jul;72(7):4978-86

Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Bldg. 002, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA.

The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher beta-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.
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http://dx.doi.org/10.1128/AEM.02842-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489321PMC
July 2006

Characterization of enterohemorrhagic Escherichia coli strains based on acid resistance phenotypes.

Infect Immun 2005 Aug;73(8):4993-5003

Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Bldg. 002, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA.

Acid resistance is perceived to be an important property of enterohemorrhagic Escherichia coli strains, enabling the organisms to survive passage through the acidic environment of the stomach so that they may colonize the mammalian gastrointestinal tract and cause disease. Accordingly, the organism has developed at least three genetically and physiologically distinct acid resistance systems which provide different levels of protection. The glutamate-dependent acid resistance (GDAR) system utilizes extracellular glutamate to protect cells during extreme acid challenges and is believed to provide the highest protection from stomach acidity. In this study, the GDAR system of 82 pathogenic E. coli isolates from 34 countries and 23 states within the United States was examined. Twenty-nine isolates were found to be defective in inducing GDAR under aerobic growth conditions, while five other isolates were defective in GDAR under aerobic, as well as fermentative, growth conditions. We introduced rpoS on a low-copy-number plasmid into 26 isolates and were able to restore GDAR in 20 acid-sensitive isolates under aerobic growth conditions. Four isolates were found to be defective in the newly discovered LuxR-like regulator GadE (formerly YhiE). Defects in other isolates could be due to a mutation(s) in a gene(s) with an as yet undefined role in acid resistance since GadE and/or RpoS could not restore acid resistance. These results show that in addition to mutant alleles of rpoS, mutations in gadE exist in natural populations of pathogenic E. coli. Such mutations most likely alter the infectivity of individual isolates and may play a significant role in determining the infective dose of enterohemorrhagic E. coli.
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http://dx.doi.org/10.1128/IAI.73.8.4993-5003.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201262PMC
August 2005