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High-Throughput SNP Analysis of Food and Waterborne Pathogens

Investigators
Whittam, Thomas
Institutions
Michigan State University
Start date
2008
End date
2013
Objective
The goal of this project is to develop and use a high-throughput instrument specifically designed for rapid SNP analysis. The primary tool is the Beckman Coulters GenomeLab SNPstreamTM Genotyping System 2 which will be used to investigate the relationship between O157 genotype and virulence.

The specific objectives are to devise, develop, and perform high throughput SNP analyses of bacterial genomes.

  1. Develop and test an extensive collection of greater than 500 O157 strains from clinical, animals and contamination sources.
  2. Optimize SNP genotyping system for O157 strains using the SNPstream to classify strains into genotypes and assess relationships between disease severity and host and clinical factors.
  3. Develop high throughput SNP genotyping systems for other food and waterborne pathogens, including food borne Salmonella serovars and Clostridium difficile.
More information
NON-TECHNICAL SUMMARY: Shiga toxin-producing Escherichia coli cause large outbreaks of food and water borne disease and contribute to thousands of sporadic cases of illness each year in the U.S. Analysis of single nucleotide polymorphisms (SNPs) in clinical isolates of E. coli O157:H7 has recently proved to be useful for investigating sources of human disease. The proposed research is to use SNP analysis to study sources of continuation and infections of E. coli O157 and then expands this to a variety of pathogens and causes of disease. The power of the SNP approach is that it can been set to be rapid, accurate, and projected to large numbers of strains so food, water, and contamination can be quantified in population sizes and frequencies.

APPROACH: Initially, we will develop a minimal set of 32-36 SNP loci for rapid genotyping of O157:H7 strains using the SNPstream system. The first step will be to design the primers for 12 SNP loci which can be multiplexed for the PCR and primer extension reactions. We will test the new assays against known O157 SNP genotypes from the preliminary work by RT-PCR. Once the 12-plex is optimized, we will construct a second and third 12-plex which will, in total provide a 36 SNP genotyping system. With the 3 or 4 sets of 12-plex reactions, we will than apply high- throughput screening for all 36 loci in at least 500 clinical O157 strains from the STEC Center collection. We will extend this analysis with the SNPstream by examining strains collected from many US states in the past 25 years. If the system works easily and accurately, then we can further extend it to O157 from bovine, food and environmental sources. In the following years, we will extend the SNP system development to other pathovars and pathogens.

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Escherichia coli O157:H7 have been shown to be unusually acid resistant, which could account for their low infectious dose and their ability to contaminate acidic foods. These E. coli produce two glutamate decarboxylase (GAD), which are encoded by the gadA and gadB genes and are involved in acid resistance. We compared the nucleotide sequences of the gadA and gadB in 16 strains of pathogenic E. coli revealed 3.8% and 5.0% polymorphism in the two genes, respectively. Phylogenetic analysis reveals two cases in which specific gadA and gadB alleles were more closely related to one another than to other alleles at the corresponding locus. Furthermore the results indicate that O157:H7 strains appear to not have undergone a gene conversion event in gadA and gadB. This suggests that regulation of GAD expression is distinct in O157:H7 compared to other E. coli strains which may have played a role in the evolution and spread of this acid resistant pathogen. To compare survival of E. coli (EHEC) O157:H7 to EHEC strains of other serotypes we used a simulated gastric environment to quantify the effect of storage in an acidic food, apple juice, on subsequent bacterial survival in the simulated gastric environment. Survival was measured in a model stomach system (MSS) for two clonal groups of EHEC, serotype O157:H7 and strains of serotypes O26:H11 and O111:H8. We found significant differences between the two EHEC groups, with the average survival rate of O157 strains in the MSS twice as great as the O26/O111 strains. Strains of the two groups also differed in the quantity of injured cells in MSS and in the transcript levels of the GAD genes (measured by QPCR) in stationary phase before cultures entered the MSS. One hypothesis to account for the predominance of E. coli O157:H7 in foodborne illness is that this pathogen represents an emergent clone with enhanced durability. We compared survival of E. coli O157:H7 strains to EHEC O26:H11 and O111:H8 strains in conditions simulating the gastric environment. Survival was measured in a model stomach system (MSS) pH 2.5. There were significant differences between the two groups, with the average survival rate of O157 strains in the MSS twice as great as the O26/O111 strains. The enhanced survival was also seen after incubation in apple juice. To understand the genes effected by apple juice, we tested transcriptomes of mid-exponential and stationary phase cells after 10 minutes in apple juice (pH 3.5) using microarrays probing (5,100 ORFs). A total of 522 genes of O157 were significantly induced upon exposure of exponential and stationary phase cells to apple juice. Genes involved in response to low nitrogen conditions, nac and glnK, had high (10x) induction rates. Fewer genes were induced in stationary phase cells by exposure to apple juice. Overall the results indicate that E. coli O157:H7 strains have superior ability to survive simulated gastric acidity compared to the non-O157 EHEC, and become acid resistant rapidly upon entry into stationary phase.
PARTICIPANTS: Teresa M. Bergholz (Large), Graduate Research Assistant, Department of Food Science, Michigan State University, East Lansing, Mich. Dr. Bergholz finished her in Ph.D. and left MSU in 2007 to join Cornall as a postdoc. James T. Riordan, Postdoctoral researcher, National Food Safety and Toxicology, , Michigan State University, East Lansing, Mich. TARGET
AUDIENCES: Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens that have been linked to serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. EHEC comprises E. coli O157:H7 and several other serotypes of Shiga toxin-producing E. coli. EHEC are transmitted via a variety of food vehicles, such as ground beef, apple juice and cider, salami, lettuce, and sprouts. An important characteristic of EHEC is the ability to survive in acidic environments. This project used conditions designed to simulate the human stomach to evaluate the acid resistance ability of EHEC and other pathogenic E. coli strains. The results indicate that E. coli O157:H7 can survive better than other E. coli in these acidic conditions. Recent outbreaks in transmitted by vegetable foods (spinach and lettuce) have caused more severe diseases caused by a subtype of O157:H7 strains. Our project is working to understand which genes are involved in the acid resistance and enhanced survival that vary among subtypes of E. coli O157:H7. These findings may explain how these pathogens survives in nature and spreads in the human food chain to cause outbreak of disease.

IMPACT: 2007/01 TO 2007/12
Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens that have been linked to serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. EHEC comprises E. coli O157:H7 and several other serotypes of Shiga toxin-producing E. coli. EHEC are transmitted via a variety of food vehicles, such as ground beef, apple juice and cider, salami, lettuce, and sprouts. An important characteristic of EHEC is the ability to survive in acidic environments. This project used conditions designed to simulate the human stomach to evaluate the acid resistance ability of EHEC and other pathogenic E. coli strains. The results indicate that E. coli O157:H7 can survive better than other E. coli in these acidic conditions. Recent outbreaks in transmitted by vegetable foods (spinach and lettuce) have caused more severe diseases caused by a subtype of O157:H7 strains. Our project is working to understand which genes are involved in the acid resistance and enhanced survival that vary among subtypes of E. coli O157:H7. These findings may explain how these pathogens survives in nature and spreads in the human food chain to cause outbreak of disease.

PROGRESS: 2006/01/01 TO 2006/12/31
Escherichia coli O157:H7 have been shown to be unusually acid resistant, which could account for their low infectious dose and their ability to contaminate acidic foods. These E. coli produce two biochemically identical isoforms of glutamate decarboxylase (GAD), which are encoded by the gadA and gadB genes and are involved in acid resistance. These genes are unusual among the Enterobacteriaceae in that they occur only in E. coli and Shigella. We compared the nucleotide sequences of the gadA and gadB in 16 strains of pathogenic E. coli revealed 3.8% and 5.0% polymorphism in the two genes, respectively. Alignment of the homologous genes identified a total of 103 variable sites including 21 fixed nucleotide differences between the loci, which are located in the first 82 codons of the genes. Phylogenetic analysis based on synonymous substitutions reveals two cases in which specific gadA and gadB alleles were more closely related to one another than to other alleles at the corresponding locus. Furthermore the results indicate that O157:H7 strains appear to not have undergone a gene conversion event in gadA and gadB. This suggests that regulation of GAD expression is distinct in O157:H7 compared to other E. coli strains which may have played a role in the evolution and spread of this acid resistant pathogen. To compare survival of enterohaemorrhagic E. coli (EHEC) O157:H7 to EHEC strains of other serotypes we used a simulated gastric environment to quantify the effect of storage in an acidic food, apple juice, on subsequent bacterial survival in the simulated gastric environment. Survival was measured in a model stomach system (MSS) for two clonal groups of EHEC: 14 EHEC 1 strains of serotype O157:H7 and 12 EHEC 2 strains of serotypes O26:H11 and O111:H8. We found significant differences between the two EHEC groups, with the average survival rate of O157 strains in the MSS twice as great as the O26/O111 strains. Strains of the two groups also differed in the quantity of injured cells in MSS and in the transcript levels of the GAD genes (measured by QPCR) in stationary phase before cultures entered the MSS. The results indicate that E. coli O157:H7 strains have superior ability to survive simulated gastric acidity compared to the non-O157 EHEC, and become acid resistant rapidly upon entry into stationary phase, which may underlie the low infectious dose of this pathogen.

IMPACT: 2006/01/01 TO 2006/12/31
In the past period, we discovered that pathogenic E.coli O157:H7 has a measurably superior ability to survive in growth conditions simulating the gastric acidity and nutrient complexity encountered in the stomach. The mechanisms enhancing survival are not well understood but may involve differential regulation of the glutamate decarboxylase genes. These experiments are providing insights into mechanisms for reducing bacterial hazards in food and reducing the burden of infectious foodborne illness.

PROGRESS: 2005/01/01 TO 2005/12/31
Escherichia coli O157:H7 have been shown to be unusually acid resistant, which could account for their low infectious dose and their ability to contaminate acidic foods. These bacteria have evolved at least 3 distinct mechanisms of acid resistance (AR) including two amino acid decarboxylase dependent systems (arginine and glutamate) and a glucose-catabolite repressed system. We quantified the survival rates for each AR mechanism separately in commensal strains and clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones. Bacteria of these clonal groups have serotypes O157:H7, O26:H11, O111:H8, and O121:H19. Members of the STEC clones were not significantly more acid resistant than the commensal strains using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/mL per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on magnesium concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems, and significantly less than the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant using these specific mechanisms. To characterize acid resistance of E. coli O157:H7 and other STEC under more natural conditions, survival rates were measured in a model stomach system (MSS) for two clonal groups of STEC: 14 strains of E. coli O157:H7 and 12 strains of E. coli O26:H11 and O111:H8. The MSS simulates conditions in the human stomach as it contains synthetic gastric fluid and baby food, and is maintained at body temperature. Under these conditions, there were significant differences between the two groups, with the average survival rate of O157 strains in the MSS twice as high as the O26/O111 strains. The two STEC groups also differed in the percentage of injured cells in MSS, in the expression levels of glutamate decarboxylase genes (measured by quantitative PCR), and in the ability to survive storage in apple juice (pH 3.5) at warm (22 C) and cold (4 C) temperatures. The results indicate that E. coli O157:H7 has superior ability to survive simulated gastric acidity compared to the non-O157 STEC, and becomes acid resistant rapidly in stationary phase, which may underlie the low infectious dose of this pathogen.

IMPACT: 2005/01/01 TO 2005/12/31
In the past period, we discovered that pathogenic E.coli O157:H7 has a measurably superior ability to survive in growth conditions simulating the gastric acidity and nutrient complexity encountered in the stomach. The mechanisms enhancing survival are not well understood. By elucidating these mechanisms at the molecular level, we hope to understand the reasons why this pathogen is so readily transmitted in certain food matrices. These insights may provide clues for reducing bacterial hazards in food and reducing the burden of infectious foodborne illness.

PROGRESS: 2004/01/01 TO 2004/12/31
In the past year, we have developed a more realistic set of conditions for measuring the acid resistance (AR) of foodborne pathogens, including Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC). The model stomach system (MSS) is a culturing environment designed to mimic conditions in the stomach and consists of synthetic gastric fluid mixed with baby food at pH 2.5. The MSS is incubated at 37C prior to inoculation, and held at 37C for 3 h on a rocking platform once inoculated. The inoculated MSS is sampled every hour for 3 h after a 15s homogenization in the Stomacher 400C lab blender. All plating is done with a spiral plater (Spiral Biotech) and viable cell numbers (CFU/ml) are estimated the next day with the Q count plate scanner. To test the reproducibility of this model system, we compared survival of the STEC strains in the MSS, in the MSS after storage in apple juice, and in apple juice alone. Apple juice without preservatives or added sweetener was purchased at a local grocery store, adjusted to pH 3.5 with malic acid, filter sterilized, and stored frozen in 1 L aliquots. Strains were inoculated at 106 CFU/mL into 100mL of apple juice stored at 4C and 22C. Inoculated apple juice was sampled every day for 4 days and plated on LB agar to enumerate survivors. After 1 day in apple juice, 15 mL of juice was transferred to the MSS to determine if the survival of a strain was influenced by storage in apple juice at either temperature. Survival in the MSS was markedly different between the E. coli O157:H7 and O26:H11 strains, and this difference increases dramatically after 24h storage in apple juice. After storage in apple juice at 4C, there was no significant difference in the average survival rate of the O157:H7 strains compared to the initial average survival rate of this group in the MSS. Survival of both groups of strains in the MSS after storage in apple juice at 22C is greatly reduced compared to initial survival rates, possibly due to a larger number of cells being injured during 22C storage. The results support the hypothesis that E. coli O157:H7 strains have a survival advantage compared to E. coli O26:H11 strains in complex acidic foods.

IMPACT: 2004/01/01 TO 2004/12/31
The results suggest that the low infective dose for pathogenic E.coli O157:H7 depends on an unknown mechanism of survival in acidic environments. By understanding the interactions between food matrices and acid resistance, we expect ultimately to find new ways to reduce the hazards of foodborne illness through transmission of bacterial pathogens in food vehicles.

PROGRESS: 2003/01/01 TO 2003/12/31
We have completed experiments that quantified survival rates for clinical isolates in acidic growth conditions. The growth conditions are designed to restrict the bacteria to using one of three acid resistance mechanisms. Results for three groups of pathogenic E. coli (serotypes O157:H7, O121:H19, and O26:H11) and six commensal strains show that the pathogenic serogroups were not significantly more acid resistant than the commensal strains. The average survival rate for the O157:H7 clone (28 strains) was significantly less (p<0.001) than that of the other pathogens in the glutamate and oxidative systems, suggesting that strains have not evolved superior acid resistance in these mechanisms.

IMPACT: 2003/01/01 TO 2003/12/31
The results suggest that the low infective dose for pathogenic E.coli O157:H7 depends on an unknown mechanism of survival in acidic environments. Discovery of the molecular basis of this mechanism may provide clues for reducing bacterial contamination on foods or reduction of the levels in the bovine reservoir.

PROGRESS: 2002/01/01 TO 2002/12/31
Pathogenic strains of Escherichia coli O157:H7 are unusually acid resistant, have a low infectious dose, and are able to contaminate acidic foods. Foster and colleagues have defined three acid resistance mechanisms in E. coli, two amino acid decarboxylase dependent systems (arginine and glutamate) and a glucose-catabolite repressed system that confer enhanced survival in low pH environments for extended periods of time. Over the past year, we have quantified the survival rates for clinical isolates representing three common clonal groups of Shiga-toxin producing E. coli (O157:H7, O121:H19, and O26:H11) for each acid resistance mechanism. The results of the acid resistance test indicate that pathogenic strains are not significantly more acid resistant than the commensal strains using these mechanisms. The glutamate system provided the best protection in an acidic environment for all groups of isolates, with 0.1 log reduction or less per hour. We also examined the gene sequences for the glutamate decarboxylation-antiporter system, which plays a role in maintaining neutral internal pH in acidic environments. The glutamate system consists of the gadA and gadB genes that encode two isoforms of glutamate decarboxylase. Comparison of the nucleotide sequences of the gadA and gadB in 12 strains of pathogenic E. coli revealed 1.3% and 1.6% polymorphism in the two genes, respectively. Thirty-six variable sites were polymorphic for the same nucleotides in both genes suggesting past gene conversions or intergenic recombination. The results indicate that at least two gene conversion events have occurred after the gad gene duplication in the evolution of this acid resistant mechanism in pathogenic E. coli. We have also made progress in comparing the evolved RpoS+ strain and the ancestor by 2-D protein electrophoresis. The analysis resolved 600-800 spots and identified about 20 differences under acid challenge. We have submitted 3 spots for MALDI mass spectrophotometric analysis to see if our procedure results in protein spots that can be identified by this method. The next step will be to compare mutant constructs to the original ancestor and evolved strains to separate out the effect of RpoS -reactivation for other changes.

IMPACT: 2002/01/01 TO 2002/12/31
The experiments are directed toward shedding light on the basis of acid resistance in E.coli O157:H7 that promotes survival of bacteria during food processing and preparation. An understanding of the molecular mechanisms of acid resistance will help define the characteristics that allow some foodborne pathogens to cause large outbreaks of illness.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
MICL02019
Accession number
192045
Categories
Natural Toxins
Escherichia coli
Bacterial Pathogens
Commodities
Meat, Poultry, Game
Produce