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1.  Methods and tools for comparative genomics of food-borne pathogens 
Foodborne pathogens and disease  2008;5(4):487-497.
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 food-borne pathogen surveillance and subsequent risk assessment to enhance the safety of the food supply.
doi:10.1089/fpd.2008.0117
PMCID: PMC2614369  PMID: 18713064
2.  Methods and Tools for Comparative Genomics of Foodborne Pathogens 
Foodborne Pathogens and Disease  2008;5(4):487-497.
Abstract
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.
doi:10.1089/fpd.2008.0117
PMCID: PMC2614369  PMID: 18713064
3.  Sensitivity of Escherichia albertii, a Potential Food-Borne Pathogen, to Food Preservation Treatments▿  
Applied and Environmental Microbiology  2007;73(13):4351-4353.
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°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.
doi:10.1128/AEM.03001-06
PMCID: PMC1932773  PMID: 17468283
4.  Isolation and Characterization of Listeria monocytogenes Isolates from Ready-To-Eat Foods in Florida†  
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.
doi:10.1128/AEM.00435-06
PMCID: PMC1489337  PMID: 16820508
5.  Functional Heterogeneity of RpoS in Stress Tolerance of Enterohemorrhagic Escherichia coli Strains 
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 β-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.
doi:10.1128/AEM.02842-05
PMCID: PMC1489321  PMID: 16820496
6.  Characterization of Enterohemorrhagic Escherichia coli Strains Based on Acid Resistance Phenotypes†  
Infection and Immunity  2005;73(8):4993-5003.
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.
doi:10.1128/IAI.73.8.4993-5003.2005
PMCID: PMC1201262  PMID: 16041014
7.  Inoculation onto Solid Surfaces Protects Salmonella spp. during Acid Challenge: a Model Study Using Polyethersulfone Membranes 
Salmonellae are the most frequently reported cause of outbreaks of food-borne gastroenteritis in the United States. In clinical trials, the oral infective dose (ID) for healthy volunteers was estimated to be approximately 1 million cells. However, in reports from various outbreaks, the ID of Salmonella species associated with solid foods was estimated to be as few as 100 cells. We found that fresh-cut produce surfaces not only provided suitable solid support for pathogen attachment but also played a critical role in increasing the acid tolerance of the pathogen. However the acidic nature of certain produce played no role in making salmonellae resistant to stomach acidity. Inoculation onto fresh-cut produce surfaces, as well as onto inert surfaces, such as polyethersulfone membranes and tissue paper, increased the survival of salmonellae during acid challenge (50 mM Na-citrate, pH 3.0; 37°C; 2 h) by 4 to 5 log units. Acid challenge experiments using cells inoculated onto polyethersulfone membranes provided a model system suitable for studying the underlying fundamentals of the protection that occurs when Salmonella strains are associated with solid foods. The surface-associated acid protection, which was observed in several Salmonella strains, required de novo protein synthesis and was independent of stationary-phase sigma transcription factor.
doi:10.1128/AEM.68.1.86-92.2002
PMCID: PMC126570  PMID: 11772613
8.  Availability of Glutamate and Arginine during Acid Challenge Determines Cell Density-Dependent Survival Phenotype of Escherichia coli Strains 
Applied and Environmental Microbiology  2001;67(10):4914-4918.
The cell density-dependent acid sensitivity phenotypes of Escherichia coli strains K-12 and O157:H7 were examined with reference to three possible mechanisms of acid resistance. There was no evidence of any diffusible substance released from dead cells which could influence the cell density-dependent acid survival phenotype. Instead, cell density-dependent acid survival phenotype was associated with induction of glutamate- and arginine-decarboxylase acid survival pathways and concomitant availability of glutamate and arginine during acid challenge.
doi:10.1128/AEM.67.10.4914-4918.2001
PMCID: PMC93249  PMID: 11571202
9.  Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant 
Applied and Environmental Microbiology  1991;57(12):3496-3501.
Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.
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PMCID: PMC184002  PMID: 16348601
10.  Legume-Rhizobium Interactions: Cowpea Root Exudate Elicits Faster Nodulation Response by Rhizobium Species 
Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH4+ in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH4+, indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.
PMCID: PMC241921  PMID: 16345989

Results 1-10 (10)