Arvind A. Bhagwat

Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR

A protocol enabling simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains was devised and evaluated using artificially contaminated fresh produce. Association of Official Analytical Chemists (AOAC)-approved polymerase chain reaction (PCR) detection methods for three human pathogens were modified to enable simultaneous and real-time detection with high throughput capability. The method includes a melting-curve analysis of PCR products, which serves as confirmatory test. The modified protocol successfully detected all three pathogens when fresh produce was washed with artificially contaminated water containing E. coli O157:H7 and S. typhimurium down to the predicted level of 1 to 10 cells/ml and L. monocytogenes at 1000 cells/ml. The ability to monitor several pathogens simultaneously will save time and increase our ability to assure food safety.

Characterization of Enterohemorrhagic Escherichia coli Strains Based on Acid Resistance Phenotypes

ABSTRACT 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.

Suppression of fungal beta-glucan-induced plant defence in soybean (Glycine max L) by cyclic 1,3-1,6-beta-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6-β-glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6-β-glucans. Here we demonstrate that both the fungal and the bacterial β-glucans are ligands of β-glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal β-glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6-β-glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6-β-glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6-β-glucans. Another type of cyclic β-glucan, a 1,2-β-glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the β-glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plants defence response.

Rapid detection of Salmonella from vegetable rinse-water using real-time PCR

Abstract A PCR-based method for the detection of Salmonella spp. from fresh vegetable rinse-water was developed. The method is a modification of an existing Association of Official Analytical Chemist (AOAC)-approved protocol and is compatible for high throughput analysis. The protocol is sensitive enough to detect contamination in mixed-salad (made up of approximately 80% leaf lettuce, 10% red cabbage and 10% carrots by weight) that was washed with water, which was artificially contaminated with Salmonella spp. at the estimated level of 1–10xa0cellsxa0ml −1 . The modified protocol, which includes a confirmatory melt–curve analysis of PCR products, requires 8–10xa0h. The method should help implementation of the HACCAP program for fresh produce.

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 organisms 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.

Comparative analysis of transcriptional regulatory elements of glutamate‐dependent acid‐resistance systems of Shigella flexneri and Escherichia coli O157:H7

The ability to withstand an acid-challenge of pH 2.5 or less by Shigella flexneri is a necessary trait for virulence and is generally believed to be restricted to the stationary-phase of growth. Earlier reports indicated the glutamate-dependent acid-resistance (GDAR) system of S. flexneri is under the regulation of rpoS, the gene encoding alternative sigma factor that is induced in the stationary-growth phase. The present study reports that unlike Escherichia coli O157:H7, S. flexneri cells when grown in minimal medium, require acid-induction in the stationary-growth phase for a functional GDAR. When grown on complex medium at pH 5.5, GDAR of S. flexneri was vigorous compared to the cells grown at pH 7.5. No acid-induction was required for the stationary phase E. coli cells grown on either minimal or complex growth media. Distinct differences in the gadA, gadBC, gadE, and hdeA (but not in rpoS) transcript levels were observed in the stationary-growth phase cells between the two pathogens grown on minimal medium. Additionally, rpoS-independent acid-induction of GDAR in the logarithmic growth phase that has been recently observed in E. coli strains [FEMS Microbiol. Lett. 227 (2003) 39-45] was not detected in the S. flexneri rpoS mutant. Although some differences in the DNA sequence at the upstream regulatory elements of gadBC were noticed, they do not appear to be significant and involvement of additional regulators in S. flexneri is anticipated, which also may explain the observed differences in the GDAR of two enteric pathogens.

Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies.

Gene expression studies require high quality messenger RNA (mRNA) in addition to other factors such as efficient primers and labeling reagents. To prevent RNA degradation and to improve the quality of gene array expression data, several commercial reagents have become available. We examined a conventional hot-phenol lysis method and RNA stabilization reagents, and generated comparative gene expression profiles from Escherichia coli cells grown on minimal medium. Our data indicate that certain RNA stabilization reagents induce stress responses and proper caution must be exercised during their use. We observed that the laboratory reagent (phenol/EtOH, 5:95, v/v) worked efficiently in isolating high quality mRNA and reproducibility was such that reliable gene expression profiles were generated. To assist in the analysis of gene expression data, we wrote a number of macros that use the most recent gene annotation and process data in accordance with gene function. Scripts were also written to examine the occurrence of artifacts, based on GC content, length of the individual open reading frame (ORF), its distribution on plus and minus DNA strands, and the distance from the replication origin.

Isolation and characterization of an ndvB locus from Rhizobium fredii.

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in β‐1, 2‐glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of β‐1, 2‐glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment‐specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for β‐1, 2‐glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).

Fresh-Cut Lettuce in Modified Atmosphere Packages Stored at Improper Temperatures Supports Enterohemorrhagic E. coli Isolates to Survive Gastric Acid Challenge

Incidences of foodborne outbreaks involving enterohemorrhagic Escherichia coli strains with mutations in a key regulatory gene, rpoS, have been reported. Incentives, if any, for losing this regulatory function are not clear since the RpoS regulator is required for the expression of several environmental stress tolerance genes. RpoS also positively regulates 2 of the 3 acid-resistance systems of E. coli under aerobic growth conditions and enables the pathogen to survive gastric acid challenge. We selected 7 enterohemorrhagic E. coli isolates, 6 of which are known to carry defective rpoS gene, and then analyzed resistance to synthetic gastric juice after the strains were inoculated on fresh-cut lettuce and stored under modified atmosphere packaging (MAP) conditions. Subatmospheric oxygen partial pressures in MAP enabled all 6 rpoS-defective isolates to induce acid resistance over the 8-d storage period if the temperature was >or= 15 degrees C. No acid resistance was induced for MAP-stored lettuce left at temperatures <or= 10 degrees C or for lettuce packed and stored under aerobic conditions. The data underscore the impending danger of abusive storage temperatures especially with regard to the application of MAP to extend the shelf life of fresh produce. The results also highlight the biological significance of having multiple acid-resistance pathways and the complex regulatory network of enterohemorrhagic E. coli strains.

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.