Microbial sequencing for food applications is gaining momentum, but challenges remain

Microbes bring us a wide variety of foods, transforming texture and intensifying flavors. Jake Lahne, posted a great overview of some of the good microorganisms in cheese - like Penicillium molds in Cabrales cheese shown on the right - that adds to other ingredients such as milk, salt and coagulants.

While modern cheeses are made with preselected cultures, traditional cheeses carry dozens of types of microbes, some highly unusual and uncharacterized.


Lactic acid bacteria, including lactococci and lactobacilli, not only convert the basic milk sugar, lactose, into lactic acid but also make the cheese inhospitable to many spoilage organisms and is the first step towards deliciousness. Streptococci are also important in cheese and yogurt-making, adding flavor to alpine (Emmental, Gruyere, etc) and Italian hard (Grana Padana, Pecorino Romano, etc) cheeses. Lactococcus lactis, Staphylococcus, Trichococcus, and Monascus are strongly associated with the 36 key aroma compounds of Monascus-fermented cheese. Lactococcus lactis was found to be the dominant bacterium while Monascus was confirmed to be the dominant fungus.

Propionobacter shermanii, are able to digest acetic acid and convert it to sharp, sweaty-smelling propionic acid and carbon dioxide. Several species of propionibacteria also inhabit human skin, producing less wanted odors.

Most of the molds that grow on cheese are species of Penicillium, but some cheeses, like St. Nectaire, develop others such as blue mold, P. roqueforti and P. glaucum in blue cheese. Blues include Roquefort, Stilton, Gorgonzola, and Cabrales, and goat cheese Monte Enebro.
White molds, which are found on the outside of all types of soft-ripened cheeses, are subspecies of P. camembertii (also called P. candidum). These white molds produce enzymes that break down the milk proteins and producing garlicky or earthy, also ammonia smells.

Room-clearing ability of Epoisses, Münster, and Limburger owe to the smear bacteria officially known as Brevibacter linens. They need salty (up to 15%), moist environments to grow,and create stinky odor compounds, producing oniony or garlicky, fishy, and sweaty aromas. The aroma of the washed-rind cheeses is often compared to smelly feet - and, yes, brevibacter grow well on human skin.

But it is not only cheese that carries myriads of microbes. There are many other foods. And not all of the bacteria we consume with the foods is good for you.

Genome sequencing was predicted to bring practical benefits to the field of microbial food safety, identifying and controlling emerging microbial pathogens. It is still not as readily available and inexpensive as needed for practical applications, but a few pilot projects have showed a promise.

GenomeWeb's Andrea Anderson recently published this article about academic researchers and public health agencies exploring the use of genomics-based approaches to complement existing food safety and surveillance methods.

Common foodborne pathogens include E. coli 0157:H7, Salmonella, Listeria, and Campylobacter, but there are many more in need of identification. Having effective ways to distinguish between dangerous and neutral microbes is crucial for food safety.

Many identification methods exist, but whole-genome sequencing could give unprecedented wealth of information, allowing predictions about the nature of organisms, their potential sources and associated risk,

In a paper published in the Journal of Food Protection in May, USDA's Ward and his colleagues reported on their findings from a multi-locus genotyping study of more than 500 Listeria monocytogenes isolates collected by the USDA-FSIS from a variety of ready-to-eat foods.
"Integration of PFGE and DNA-sequence-based sub-typing provides an improved framework for prediction of relative risk associated with L. monocytogenes strains from [ready-to-eat] foods," they wrote.

In another recent paper, Ward and collaborators from Colorado State University used genotyping to show that a virulence-decreasing inlA mutation in L. monocytogenes was more common in isolates from ready-to-eat than from isolates from actual human listeriosis cases.
Honisch presented a poster outlining work done with collaborators from London's Health Protection Agency at the American Society for Microbiology annual meeting in San Diego this May, describing how the team used the Sequenom MassArray platform to do multi-locus sequence typing, or MLST, on hundreds of Salmonella isolates. Honisch told GWDN that the approach is promising, in part, because mass spec is high-throughput and generates very reproducible data.
During a session at the recent ASM meeting, Eric Brown, a microbiologist with the US Food and Drug Administration, explained that the FDA has been exploring the use of Roche 454 sequencing to characterize Salmonella isolates and to find markers for tracing outbreak strains back to their source.
And in Canada, the NML's Gilmour was lead author on a paper appearing in BMC Genomics this February in which researchers used the Roche 454 GS FLX platform to sequence the genomes of two L. monocytogenes strains isolated during a 2008 outbreak of listeriosis in Canada that killed 22 people and caused serious illness in dozens more.
"This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events," Gilmour and his co-authors wrote, "and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole-genome sequences in the context of epidemiology."
Even so, Gilmour said it will take time for whole-genome sequencing to become a standard traceback method — largely due to remaining bioinformatics challenges.
"It's our job to learn how to use those [sequencing] technologies and glean the interesting information or the informative information," Gilmour said. "That's kind of the bottleneck we're at right now, is developing those bioinformatics tools to take that raw data and quickly parse through it and find relevant information."

There is still a long way before genome-sequencing or methods developed based on sequencing results will be standardized and incorporated into practice, but the results look promising and are opening new horizons for health applications.

References

Ward TJ, Evans P, Wiedmann M, Usgaard T, Roof SE, Stroika SG, & Hise K (2010). Molecular and phenotypic characterization of Listeria monocytogenes from U.S. Department of Agriculture Food Safety and Inspection Service surveillance of ready-to-eat foods and processing facilities. Journal of food protection, 73 (5), 861-9 PMID: 20501037

Van Stelten A, Simpson JM, Ward TJ, & Nightingale KK (2010). Revelation by single-nucleotide polymorphism genotyping that mutations leading to a premature stop codon in inlA are common among Listeria monocytogenes isolates from ready-to-eat foods but not human listeriosis cases. Applied and environmental microbiology, 76 (9), 2783-90 PMID: 20208021

St-Gelais D, Lessard J, Champagne CP, & Vuillemard JC (2009). Production of fresh Cheddar cheese curds with controlled postacidification and enhanced flavor. Journal of dairy science, 92 (5), 1856-63 PMID: 19389943

Rossetti L, Fornasari ME, Gatti M, Lazzi C, Neviani E, Giraffa G. (2008). Grana Padano cheese whey starters: microbial composition and strain distribution. Int J Food Microbiol. 2008 Sep 30;127(1-2):168-71. Epub 2008 Jun 12.PMID: 18620769

Flórez AB, Mayo B. (2006) Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE. Int J Food Microbiol. 2006 Jul 15;110(2):165-71. Epub 2006 Jun 27.PMID: 16806553

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