Novel genetic tools for studying food-borne Salmonella

https://doi.org/10.1016/j.copbio.2009.02.002Get rights and content

Nontyphoidal Salmonellae are highly prevalent food-borne pathogens. High-throughput sequencing of Salmonella genomes is expanding our knowledge of the evolution of serovars and epidemic isolates. Genome sequences have also allowed the creation of complete microarrays. Microarrays have improved the throughput of in vivo expression technology (IVET) used to uncover promoters active during infection. In another method, signature tagged mutagenesis (STM), pools of mutants are subjected to selection. Changes in the population are monitored on a microarray, revealing genes under selection. Complete genome sequences permit the construction of pools of targeted in-frame deletions that have improved STM by minimizing the number of clones and the polarity of each mutant. Together, genome sequences and the continuing development of new tools for functional genomics will drive a revolution in the understanding of Salmonellae in many different niches that are critical for food safety.

Introduction

Nontyphoidal Salmonellae are responsible for an estimated 1.4 million cases of gastrointestinal disease with 500 associated deaths in the United States, at a cost of $2 billion [1]. The number of cases worldwide probably exceeds 100 million each year. Infection generally occurs after the ingestion of contaminated food or water, and usually leads to a self-limiting enterocolitis. The disease is characterized by diarrhea, abdominal cramps, nausea, fever, vomiting, and headache lasting 7–10 days, followed by a longer period of subclinical fecal shedding. Infants, the elderly, and immunocompromised individuals are at risk for serious systemic complications and death as a result of infection.

Contaminated foods, including beef, pork, poultry, and egg products are frequent vectors responsible for the transmission of these organisms to humans. Livestock can harbor Salmonellae subclinically resulting in carcass contamination at slaughter and in the laying of contaminated eggs. In recent years, as the traditional routes of infection are better controlled, large outbreaks of nontyphoidal Salmonella infection in the United States have been attributed to fruits, vegetables, and processed foods including jalapeño peppers, cantaloupe, Malto-meal™ cereal, and peanut butter (http://www.cdc.gov/salmonella/).

Serology based on surface antigens is the standard method of classification of Salmonella. The host-range and disease can differ considerably between serovars, making such classification important. Throughout the world, the most prevalent nontyphoidal serovars isolated from human sources are serovars Typhimurium and Enteritidis and these two serovars comprise nearly 40% of isolations from human sources in the United States [2]. These serovars can be harbored subclinically in livestock for prolonged periods of time and are thus very difficult to eradicate in the absence of a detailed knowledge of the biology of the organism in this niche.

The bacterial factors necessary for Salmonellae to persist subclinically in the gastrointestinal tract of livestock and to survive and grow in other reservoirs such as crops and processed foods are only beginning to be elucidated. This knowledge will allow the development of new strategies and the identification of points in the production chain where producers can intervene to improve the safety of foods. We review the current status as well as the uses of complete genome sequence information for Salmonellae, and enhancements of genetic techniques that may rapidly increase our knowledge of the biology of this organism in these important food safety niches.

Section snippets

Complete genome sequencing of Salmonellae

Currently, the complete genome sequences of one or more representatives of six serovars have been determined, annotated, and published [3, 4, 5, 6, 7, 8••], and additional genomes in eight other serovars are complete (Table 1). Cheaper and faster sequencing technologies, such as 454 and Illumina Solexa [9], are now being applied to extensive sequence comparison of nontyphoidal Salmonellae (Table 1). The use of these tools provides a new window into genetic diversity within and between

Genetic approaches to find genes needed during colonization of foods

A better understanding of the Salmonella genes that are required for the colonization and persistence in foods will allow us to focus future safety and HACCP programs. Complete genome sequencing and annotation can suggest a likely function for some genes. However, other methods must be used to determine the function or molecular role of most genes and the particular conditions where each gene is important. Strategies currently in use for the identification of Salmonella genes involved in the

Gene expression

Determining genes that are expressed in a particular condition or environment has been used as a first step to define groups of candidate genes that are necessary for survival and growth. Microarray technology has been used to determine gene expression in particular environments, some of which are relevant to food safety [30, 31, 32, 33•], but thus far this technique has not been directly used to define Salmonella genes expressed in or on foods. Expression analysis using RNA has disadvantages,

Genes required in particular environments

Even though expression analysis and IVET identify genes expressed in a particular environment, these techniques cannot define genes that are required to colonize a given ecological niche. Furthermore, required genes may be only transiently expressed at low levels, and thus may be missed by some RNA expression and IVET-based strategies. A more direct method for finding the subset of required genes is to use forward genetic screening.

Signature tagged mutagenesis (STM) is a negative selection

Recent advances in functional genomics of Salmonellae

One limitation of using random transposon mutagenesis is that very large numbers of mutants are needed to ensure complete coverage of the genome. This factor is a significant disadvantage in circumstances where the population of bacteria experiences random loss, also termed a ‘founder effect’ or bottleneck. For example, when a population of Salmonellae passes from the intestine to systemic sites only a small fraction of the bacteria arrive in the new niche. Thus, only a few hundred or a few

The future

Complete genome sequencing of Salmonellae is allowing us to better understand their genetic diversity, to develop novel tools, and to improve existing genetic techniques to understand the complex biology of these important food-borne pathogens. Approximately half of the genes in Salmonella still have no known phenotype in the environment. Frontiers for further study of Salmonella for improved food safety using modern genetic tools are likely to include determination of the genes necessary for

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported in part by NIH grants R01 AI034829 and R01 AI052237, and R21AI057733, grant ADI-08/2006 from CONICYT, Chile and the World Bank, DOD grant BC073899, by the Texas A&M University System Health Science Center, and the generous support of Mr Sidney Kimmel. We thank Steffen Porwollik for assistance with Table 1.

References (59)

  • N. Arrach et al.

    Salmonella serovar identification using PCR-based detection of gene presence and absence

    J Clin Microbiol

    (2008)
  • E.M. Ribot et al.

    Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet

    Foodborne Pathog Dis

    (2006)
  • P.R.T. Frenzen et al.

    Salmonella cost estimate updated using FoodNet data

    Food Rev

    (1999)
  • CDC Salmonella Annual Summary 2005. Edited by Department of Health and Human Services, Centers for Disease Control and...
  • M. McClelland et al.

    Complete genome sequence of Salmonella enterica serovar Typhimurium LT2

    Nature

    (2001)
  • J. Parkhill et al.

    Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18

    Nature

    (2001)
  • M. McClelland et al.

    Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid

    Nat Genet

    (2004)
  • W. Deng et al.

    Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18

    J Bacteriol

    (2003)
  • C.H. Chiu et al.

    The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen

    Nucleic Acids Res

    (2005)
  • N.R. Thomson et al.

    Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways

    Genome Res

    (2008)
  • K.E. Holt et al.

    High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi

    Nat Genet

    (2008)
  • S. Porwollik et al.

    Evolutionary genomics of Salmonella: gene acquisitions revealed by microarray analysis

    Proc Natl Acad Sci U S A

    (2002)
  • K. Chan et al.

    Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray

    J Bacteriol

    (2003)
  • W.W. Navarre et al.

    Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella

    Science

    (2006)
  • S. Porwollik et al.

    Differences in gene content between Salmonella enterica serovar Enteritidis isolates and comparison to closely related serovars Gallinarum and Dublin

    J Bacteriol

    (2005)
  • M.S. Kang et al.

    Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica

    Appl Environ Microbiol

    (2006)
  • F.J. Reen et al.

    Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray

    Appl Environ Microbiol

    (2005)
  • H.L. Andrews-Polymenis et al.

    Host restriction of Salmonella enterica serotype Typhimurium pigeon isolates does not correlate with loss of discrete genes

    J Bacteriol

    (2004)
  • S. Porwollik et al.

    Characterization of Salmonella enterica subspecies I genovars by use of microarrays

    J Bacteriol

    (2004)
  • E.F. Boyd et al.

    Molecular genetic relationship of the Salmonellae

    Appl Environ Microbiol

    (1996)
  • B. Tankouo-Sandjong et al.

    MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars

    J Microbiol Methods

    (2007)
  • M.C. Maiden et al.

    Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms

    Proc Natl Acad Sci U S A

    (1998)
  • S. Kim et al.

    Multiplex PCR-based method for identification of common clinical serotypes of Salmonella enterica subsp. enterica

    J Clin Microbiol

    (2006)
  • C.R. Jackson et al.

    Prevalence of streptogramin resistance in enterococci from animals: identification of vatD from animal sources in the USA

    Int J Antimicrob Agents

    (2007)
  • B. Swaminathan et al.

    Surveillance for human Salmonella infections in the United States

    J AOAC Int

    (2006)
  • S. Cho et al.

    Allele distribution and genetic diversity of VNTR loci in Salmonella enterica serotype Enteritidis isolates from different sources

    BMC Microbiol

    (2008)
  • D. Boxrud et al.

    Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage typing for subtype analysis of Salmonella enterica serotype Enteritidis

    J Clin Microbiol

    (2007)
  • S. Octavia et al.

    Single-nucleotide-polymorphism typhing and genetic relationships of Salmonella enterica serovar Typhi isolates

    J Clin Microbiol

    (2007)
  • S. Baker et al.

    High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and pathotypes within an urban district of Jakarta, Indonesia

    J Clin Microbiol

    (2008)
  • Cited by (0)

    View full text