Where the Wild Microbes Are: A New Theory on How Pathogens Survive Food Processing

Produce borne diseases have recently been gracing the front pages of our media. Our spinach has E. coli, our onions have Hepatitis A virus and E. coli, our strawberries have Listeria, and our tomatoes and peanut butter have Salmonella. Not to mention the countless tons of ground beef tainted with pathogenic E. coli.

Common sense says that washing and proper handling of our food should simply be enough to prevent illness outbreaks. It has now been hypothesized that many bacteria were able to "hide" within and among the plant cells, protected by their sturdy cell wall. Or even that some pathogenic bacteria were able to enter the cells and remain protected from traditional washing methods.

An article in this month's Applied and Environmental Microbiology looks at a much different method of bacterial survival on produce. They hypothesize that these bacteria are taking refuge in various protozoa, and subsequently are protected from washing and other sanitation methods due to being held either within the cell or an exogenous cell-derived vesicle.

The authors used protozoal isolates from store-bought spinach and romaine lettuce. They measured the concentrations of various microbes from the surface, and cultured a set of naturally occurring protists from these samples.

They show that not only are certain pathogenic bacteria (like E. coli 0157:H7) able to be taken up by protozoa, but that in many cases, they survive to be ejected into the world around, surrounded by a vesicle. The flourescent image at above shows this event happening with E. coli 0157:H7 and a strain of Tetrahymena.
Previous studies have shown that the bacteria in these extracellular vesicles are able to survive for the long-term, even in harsh conditions.

This paper shows us that microbial ecology is much more complex than we perceive, and that simple and obvious solutions may not always be the best. We now know that wild protozoa living on our produce can sequester pathogenic bacteria, and furthermore wet produce allows vesicles to be created containing viable bacteria.

This makes me wonder about the current trend in grocery stores to spray there fresh produce with water at specific time intervals. If protists are doing what this study shows they are doing, and vesicles form on wet leaf surfaces, this practice may be something we must reconsider for our food safety.

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Gourabathini, P., Brandl, M.T., Redding, K.S., Gunderson, J.H., Berk, S.G. (2008). Interactions between Food-Borne Pathogens and Protozoa Isolated from Lettuce and Spinach. Applied and Environmental Microbiology, 74(8), 2518-2525. DOI: 10.1128/AEM.02709-07

A Fatty Acid Synthetase is Necessary for Active TB Infection

Mycobacterium tuberculosis is the most common infectious agent in the world. Nearly 1/3 of the world population is infected, and a drastic number of these are increasingly antibiotic resistant. MDR-TB and XDR-TB are becoming more commonplace everyday, especially in regions that are combating HIV infections.

As such, it is incredibly important to study how this bug works. It is one of the most difficult organisms to work with due to a wide variety of factors. These include: slow growth rate, pathogenicity, very waxy cell walls, high levels of illegitimate recombination, and a bunch of other nuances that make the mycobacteria unique among its relatives.

One unique feature of the mycobacteria, is their unique acid-fast staining pattern. It has been shown that when treated with isoniazid (the most prescribed anti-TB drug), mycobacteria lose this pattern of staining. Therefore, one can extrapolate that their outer cell wall (filled with extremely long-chain lipids, called mycolic acids) plays an important role in both the lifestyle of M. tb. and its pathogenesis.

Two genes have been identified as having roles in mycolic acid synthesis, kasA and kasB. KasA has previously been shown to be essential (J Bacteriol. 2005 Nov;187(22):7596-606.), with kasB serving a non-essential accessory function. This paper, from Bill Jacobs’ lab at AECOM, shows that although kasB is nonessential, it is necessary for acid-fastness AND it has a large role in pathogenesis.

They first knocked out kasB using a specialized transducing phage. Knockouts of kasB had a severe deficiency in colony growth and a large change in colony morphology (Fig. below).

Also, kasB mutants lacked acid fastness and were exceptionally more sensitive to rifampicin (Fig. below).

Further biochemical analysis showed that kasB is likely involved in the production of specific mycolic acids.

However, the story gets more exciting. Immunocompetant mice that were infected with the kasB mutant, showed no signs of infection—there was no formation of granulomas and no mortality. In contrast, mice infected with wild-type died within 356 days. What is interesting to note is that although mice infected with kasB mutants did not show pathology, they still had measurable amounts of bacterial growth—indicating a persistent infection that was not able to become active (Fig. below).

The same study shows that kasB mutants act nearly identically to wild-type in mice that are immunocompromised. This means that mycolic acid production by kasB plays a role in the immune systems ability to hold M. tb. infections in check.

The authors suggest a variety of implications that their very exciting findings bring to light. For one, due to the phenotype, kasB mutants could be used specifically to study persistant infection. Secondly, kasB is clearly a candidate for deletion in an attenuated vaccine strain. Finally, and I think most importantly, this mutant shows a potential drug target for secondary drug therapy. Drugs that act on kasB or kasA could be developed in conjunction with rifampicin and isoniazid treatment.

XDR-TB cases are present in 41 countries, including the US. It is vital that we develop new lines of drugs and continue identifying drug targets. TB presents an event where scientists, public health departments, and drug industry must come together and work as a single entity. The amount of TB cases is ever increasing and I fear that the days of the sanitarium may once return to our country.

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Bhatt, A., Fujiwara, N., Bhatt, K., Gurcha, S.S., Kremer, L., Chen, B., Chan, J., Porcelli, S.A., Kobayashi, K., Besra, G.S., Jacobs, W.R. (2007). Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proceedings of the National Academy of Sciences, 104(12), 5157-5162. DOI: 10.1073/pnas.0608654104

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Suggested Readings
Molecular Genetics Mycobacteria
Mycobacteria Protocols (Methods in Molecular Biology) (Methods in Molecular Biology)
The White Plague: Tuberculosis, Man, and Society
Timebomb : The Global Epidemic of Multi-Drug Resistant Tuberculosis

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I'll Have My Bacteria Extra-CRISPR

The microbial cold war between species has been widely studied and is, in my opinion, one of the most interesting topics in current biology.

Fungi make compounds to destroy bacteria, bacteria make compounds to destroy fungi. But it doesn't stop there. Phages mutate constantly to evade bacterial defenses. We've known about a handful of bacterial defenses against phage-- repressor systems, restriction enzymes, receptor modification, DNA modification, etc.

We have utlized these systems to our advantage--our first effective antibiotic (Penicillin G) came from the fungus Penicllium, a vital antifungal (cyclohexamide) comes from the bacteria Streptomyces, our ability to clone and subclone is only useful because we can specifically cut DNA with restriction enzymes, etc.....

But now, there is another form of bacterial defense. We have evidence for, what I will call, the "special forces" of bacterial defense systems. Heck, I'd liken this to a form of adaptive immunity.

CRISPR (clustered, regularly interspaced, short, palindromic repeats) sequences have been found in 40% of all sequenced Bacteria and 90% or sequenced Archaea. Essentially, you have an array of nearly identical repeats which form RNA stem-loops. Between the repeats, you find unique DNA that has sequence similarity to phages. There are also a handful of unique genes that are closely associated with the arrays.

The idea is that the bacteria are using an RNA interference mechanism. The CRISPR array gets transcribed and then spliced at the loop structures--forming small RNAs. These sRNAs associate with phage nucleic acids, recruit degradation genes, and subsequently denature the phage nucleic acid. See the figure at right taken from the review article.

You find a distinct correlation between the number of CRISPR sequences and resistance. You also can add specific phage sequence to the array and grant immunity. If you then take that sequence away, immunity is lost.

What is interesting (but not necessarily surprising)is that a small population of phage remained resistant to the system. It was found that these phage had a slightly different nucleic acid sequence at the site. BUT, given time, the bacteria can acquire CRISPR sequence against the phage.

It's an evolutionary arms race!

That we should be exploiting!There are a variety of ramifications that this system can and will have on the future of molecular biology.

For one, a system of spoligotyping could be developed based on the unique spacer regions in the CRISPR array.

Apparently a large problem in dairy industry (specifically yogurt and cheese) is that bacterial cultures get lost to phage. This system could help prevent that by allowing the development of ultra-resistant strains.

A more unique (and as far as I know, untested) use would be the development of a system to knock out genes in vivo. You could imagine setting up a CRISPR system on a plasmid with specificity for a specific gene (or multiple genes) you want to knock out. The CRISPR system would do what it does naturally and degrade the gene's transcript. Since these regions are only 26-72bp, it would certainly be an awesome tool to have--especially for organisms notoriously recalcitrant to genetic manipulation (like my favorite bugs: the Mycobacteria). Genes could be effectively silenced using only a plasmid, without the need for messy recombination systems.

Sorek, Kunin, and Hugenholtz. "CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews Microbiology. 6, 181-186 (March 2008)

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Sorek, R., Kunin, V., Hugenholtz, P. (2008). CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Reviews Microbiology, 6(3), 181-186. DOI: 10.1038/nrmicro1793