Identification of an Antagonistic Probiotic Combination Protecting Ornate Spiny Lobster (Panulirus ornatus) Larvae against Vibrio owensii Infection

Evan F. Goulden, Michael R. Hall, Lily L. Pereg, and Lone Høj PLoS One. 2012; 7(7): e39667.

Diseases of marine animals, particularly farmed shrimp, shellfish and fish, are an increasing priority from a somewhat off-beat veterinary health perspective. From my perspective, this is a nice system for probiotic development because it is so obvious that yogurt isn't going to be the probiotic of choice. This opens up the choice of organisms more broadly; and in this case, marine isolate collections are the obvious source of probiotic strains to prevent infection with a pathogen.

In the formulation of a probiotic, resource competition and active antagonism are two major mechanisms by which the probiotic may prevent the pathogen from infecting a sensitive host (immunostimulation, anti-virulence, and direct predation are other strategies which have been investigated; in some sense, immunostimulation is actually a live vaccine so I wouldn't put it in the same category). The advantage of resource competition as a strategy is relative robustness. If the probiotic (single strain or multi-strain) fills a niche required for pre-infection colonization, it is likely difficult for the pathogen to evolve around that requirement or outcompete the probiotic. Antagonism, on the other hand, tends to be brittle; evolution to resistance is straight-forward in a number of well-characterized cases (including several bacteriocins). The selection to resistance is strong and the path typically involves loss or truncation of a receptor.

The attempt to formulate antagonistic Escherichia coli probiotics in enteric diseases has hit dead ends historically as a result of resistance. However, attempts in Streptococcus mutans (for caries prevention) has been successful enough to lead to a marketed product (Oragenics Inc). In this current paper, a large number (~500) of marine isolates were screened for antagonism against a type strain of the pathogen. 500 isolates may not seem like much to high-throughput screeners for small molecules; but the handling and management of that many environmental isolates and the execution of the assay on that scale could be significant. These isolates were screened against a single strain of the pathogen.

This is a point of criticism. The routine underestimate of microbial, particularly pathogen, diversity, is a very serious blind spot. Many researchers falsely presume that a single model strain can reasonably represent the pathogen population for many assays. Even worse, researchers may presume that some set of traits makes a model 'relevant' or creates a 'bad bug' - a priority organism - and that diversity away from the model is irrelevant. In this case, strain V. owensii DY05 (http://www.ncbi.nlm.nih.gov/pubmed/22307306, http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2009.01850.x/full) is a recognized lobster pathogen which induces high mortality. However, despite there being some other known strains which are pathogenic in prawns, the apparent known diversity of lobster pathogens appears minimal. The prawn pathogens which are related to the lobster pathogen are not particularly pathogenic in the lobsters and mention of a second lobster pathogen strain is not made in papers addressing the phylogeny of these Vibrio (http://eprints.jcu.edu.au/23845/; http://www.ncbi.nlm.nih.gov/pubmed/22055753). As a result, a therapy which addresses this single pathogenic strain, DY05, may do so for reasons which are unrelated to the general model for lobster pathogenesis. This may create a brittle therapy. The appropriate solution is to find a range of lobster pathogenic strains and do a screen against all of them.

The antagonism/co-culture assays were conducted both in isolation (agar well method) and in biofilms; a sound experimental practice; the subsequent screen eliminated potential pathogens of the lobsters. The biofilm assay was crude - crystal violet microwell assay - but traditional. Given that there is only one pathogen strain included, it seems an appropriate assay.

Members of 7 genera antagonized DY05; drawn from new marine isolates as well as an established marine strain collection. From the 91 isolates of these 7 genera, ease of culture and relationship to putative human pathogens restricted the total list to 16. It seems that this screen may be overly aggressive at this stage of probiotic development; but understandably convenient. From the remaining 16, the multispecies biofilm study eliminated 6 strains as potentially enhancing pathogen biofilm formation, through unknown mechanisms. Exclusion, competition and displacement studies were each done; antagonistic strains performed these tasks differently.

Four strains were advanced into live protection/treatment trials. One thing that was skipped: testing whether the strains antagonized each other... so it isn't clear exactly why the four together are less efficacious either of the two best, or a pair comprised of those two, but it may be that one of the four antagonizes the others. Regardless, they found a pair which reduced the mortality impact of pathogen exposure to statistically undetectable. This is an unquestionably impressive result; but it also depended on a relatively dense inoculum of the protective organisms. The authors suggest that this observation is normal in the literature; the claim requires more investigation (that is, more reading to do...).

This paper is part of a growing field for multi-species probiotics, exemplified by the Clostridium difficile work at the Sanger Institute (http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002995). This paper did not engage with the microbiome/metagenomic sequencing field directly, as the C. difficile research did, but could probably benefit from engagement with those methods. Perhaps that is yet to come, given the development in this area, led by the Høj group.

So, since the time that I started this blog to now, the field I titled the blog to address has virtually exploded. The human microbiome, particularly in its relationship to other microbiomes is still more than a little uncharacterized - but now, people are trying. Process is still a giant mess but pattern is seeing rapid advances.

This is very encouraging in one respect; I am able to give talks about the microbiome and say things that are 'factual.' We are introgressing into the areas where ignorance reigned supreme.

Some of my greatest joy, though, with a bit of schadenfreude, I suppose, is the ability to finally convince the old guard - with their reliance on culture techniques and classic in vitro biochemistry, their vast taxonomies and their stodgy assurance that they knew which pathogens were important, what was merely a clinical 'contaminant' and their oversimplified Koch's postulates, that they not only knew very little - but that most of what they thought/think they know is actually error. Erroneous. False.

Yes, that's the stuff of which revolutions are made. Every microbiologist knew 'how little we know.' How few organisms were named and characterized, how complex biochemistry and genetics was, etc. What they didn't know was how deeply flawed their generalizations were. The assumption was largely that 'refinements' were necessary. That new pathogens would be discovered, for instance, not that the entire mode of pathogenesis would be, for many diseases, overturned; that Koch's postulates and the germ theory would have to be thrown out for 'dybiosis,' that the host-microbiome function would be so sophisticated and genetically interwoven.

We are in a brave new world and much unlearning needs to happen. The next generation of researchers will have little need for the hopelessness that comes from wondering if anything big is yet to be discovered.

Anyway, there are plenty of interesting papers to discuss.

Let's start anew with one of the recent microbiome papers:

Phylogenetic Characterization of Fecal Microbial

Communities of Dogs Fed Diets with or without

Supplemental Dietary Fiber Using 454 Pyrosequencing

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842427/pdf/pone.0009768.pdf

Inflammatory bowel diseases were one of the great unknowns a decade or so ago. Still, the debates about the microbial, genetic, immunologic components rage. There is probably some truth, it turns out, to all three views. Root causes may not, frankly, exist in this case; that is, the disease state may be a result of a spiral, as the immunity and microbiomes, assisted by predispositions, behaviors, environmental exposures, migration events, lead from one state to another until the gut is irreversibly (or near irreversibly) broken. At least now, we can say that the bowel flora is abnormal, that the immune response is abnormal, and that normalizing these things as best we can is helpful. The place of worms in the health-compatible flora is unknown - both for 'primitive' living conditions and 'modern living conditions' and may have something to do with it, but we can't say that for certain.

The results of this paper were generally unsurprising. Diet and flora interact in a repeatable fashion, at least at the phyla level. There was no obvious priority effects/order effects with the diets used in conventional dogs (useful result). The OTUs per dog were ~300; pretty normal. Using V3 gave them enough data to be dangerous but there really isn't much to say about function or anything like that. It's a good first data set and will certainly be foundational for later work on dog GI disease; which appears to be their next step.

Vibrio harveyi Associated with Aglaophenia octodonta (Hydrozoa, Cnidaria)

Microbial Ecology, November, 2006
Volume 52(4) 603-608

Classically, Vibrio harveyi is thought of as a symbiote of squid. It's light production is a clear benefit to the squid as a form of camouflage and the symbiosis is very specialized. However, the luciferase light production system is much more broadly distributed in nature, and further more, V. harveyi live in the water column and other place, away from the squid.

This paper highlights an alternative symbiosis - or is it simply an association? Are the V. harveyi on this hydrozoa the same way they would be on a rock? Is it just a surface for colonization? Or is there a real colonization specific to the hydrozoa? Is it beneficial to the hydrozoa as well as squid, or for the hydrozoa, are the vibrio just parasites, conveniently tamed by the squid?

First, let me express my extreme jealousy at the guys who had the job of scuba diving off the coast of Apulia, Italy, home of much wonderful food...

The choice of buffered peptone, a waring blender, and 2216 Marine Agar are all fairly standard. This isolation strategy was paired with microscopic observation, but no direct molecular techniques. In so much as it was a simple recovery operation, the methods used appear completely sufficient to achieve their end. However, we can't suggest that the complete diversity was recovered.

What seems odd is the choice to move from a partial 16S sequence to a series of 'Bergey's' style phenotypic tests for strain identification. After all, with isolates in hand, it might have been trivial to sequence the RecA (see Phylogeny of Vibrio cholerae Based on recA Sequence; Infection and Immunity, Stine et al 2000, 68 (12) 7180...). Hsp60 could have been used equally well. Or maybe a nice MLSA strategy could have been pursued (Journal of Clinical Microbiology, May 2003, p. 2191-2196, Vol. 41, No. 5; Journal of Clinical Microbiology, March 2004, p. 1280-1282, Vol. 42, No. 3). Given the right 3 or so genes, they might have great confidence in the outcome.

Regardless, they found a fluorescing vibrio colonizing the cnidarians. Other cnidarians produce their own light, but this one does not, and perhaps the bacteria provide this function. Did colonization relax the selection on light production and obviate the native luminescence? Or did the loss come first? How useful is the light, how much is produced by the bacteria and how does it compare?

Also, there is speculation in the article about the role of chitinases... but no real follow up. It is certainly a pain to produce flakes of squid pen and test for growth on chitin in minimal media, but it could clarify whether these bacteria actually can eat the chitin.

Still, we need experiments with antitbiotics and such to test whether the cnidarians are helped or harmed by these colonists. It would also be nice to see a sampling of diverse cnidarians at a few places, with several vibrio from each, to see whether there are founder populations of vibrio from the water, or perhaps whether the bacteria spread with the cnidarians (co-evolution - parasite, pathogen, epiphyte...), etc.

So, all in all, an intriguing paper but frustratingly light on conclusions, and perhaps missing some important/helpful molecular phylogenetic data. Hopefully this will be filled in in future publications.

Identification of Escherichia coli O157:H7 Genomic Regions Conserved in Strains with a Genotype Associated with Human Infection

Marina Steele, Kim Ziebell, Yongxiang Zhang, Andrew Benson, Paulina Konczy, Roger Johnson, and Victor Gannon

Applied and Environmental Microbiology 73(1), Jan. 2007, 22-31.


I'll presume we all know that E. coli O157:H7 is code for the most common seriously pathogenic form of E. coli currently present in the food supply. The numbers and letters represent the surface 'antigens' that are used to do a preliminary identification of strains of bacteria - the antigens are related to the structures on a bacterial cell surface that are accessible, presumably, to the immune system - but also accessible to assays for testing cell type. Because they are accessible to the immune system, antibodies (particularly monoclonals) can be generated to them and then used in immunoassays, which are very rapid and sensitive. The now classic 'ELISA' (enzyme-linked immunosorbent assay) allows single cells to be identified by their antigens almost immediately. The O antigen is part of the "LPS" of the gram negative cell wall, it is made of sugars. The K antigen is part of the 'capsule' layer - a protective layer of polymerized sugars more loosely connected with the cell. H antigens are derived from the flagella, which are also important to the cell in colonizing a host and are exposed on the cell surface.

Anyway, these antigens can also be used as markers for strongly selected genetic markers - certain antigens are better under certain conditions; particularly, some are more useful for evading predation (diversifying selection acts in this case) and some are more useful for evading the immune system.
Whatever the case, there are hundreds of antigens (~200 O, ~100 K, ~55 H). This allows for tens of thousands of combinations, helping sort the E. coli into clonal monophyletic populations. Of course, it is not necessary that this be true for any given combination, that it is clonal or monophyletic.

Everyone cares about O157:H7 because it keeps showing up in the ill and recently deceased - and in their spinach, tacos, etc. It also shows up in the rectum of ruminants (cattle, sheep) where it doesn't seem to cause the animals any trouble. Certainly, these E. coli have learned some tricks, and modern agriculture encourages their spread. One argument goes that animals being fed grain, not hay, has changed the stomach conditions to permit these E. coli to spread more readily (Diez-Gonzalez et al, 1998; Russell et al, 2000; Russell et al, 2000). The idea is that the E. coli are becoming acid tolerant. Stomach acid is one of the main barriers for humans to prevent infection by bacteria; it protects the established symbionts against temporary invaders. Without going into too much discussion of this point, there is reason to doubt the conclusion that the E. coli are really being selected for acid resistance by cattle diets (Carolyn J. Hovde, Paula R. Austin, Karen A. Cloud, Christopher J. Williams, and Carl W. Hunt, Applied and Environmental Microbiology, 65(7), July 1999, 3233-3235; Grauke LJ, Wynia SA,Sheng HQ, Yoon JW, Williams CJ, Hunt CW, Hovde CJ. Vet Microbiol. 2003 Sep 1;95(3):211-25; Science, April 1999, Vol. 284. no. 5411, p. 49; reviewed, "E. coli O157:H7 in hay- or grain-fed cattle," Dale Hancock and Tom Besser, October 12, 2006, citation not obvious). The evidence for this appears to be more 'suggestive' than 'conclusive.' Anyway, simply lowering the infectious dose may make it easier to become ill, but there must be other things going on to cause O157:H7 to be a problem.

One may be that these E. coli have learned tricks to get into plant roots. There are lots of studies about this issue as well:
JV. Gagliardi and JS. Karns. Environmental Microbiology 2002, Volume 4(2) 89.
Warriner K, Ibrahim F, Dickinson M, Wright C, Waites WM. J Food Prot. 2003 Oct;66(10):1790-7.
The second is interesting in that it presages the spinach problem by showing that E. coli can get internal to spinach cells, so the bacteria could never be washed off. Thus, eating raw spinach fertilized 'organically' with pathogen contaminated manure might create a hazard that cannot be dealt with by surface sterilization.

Anyway, back to the topic of the paper at hand: the relationships among these 0157:H7 bacteria that are causing all this mess.
There are apparently two major families of these bacteria. While even the dominant human pathogens don't seem to cause disease in animals, it seems from most experiments I've seen, that they also don't colonize the animals particularly stably. This doesn't really surprise me - stable intestinal colonization of a conventional (previously naturally colonized by bacteria) host by a laboratory isolate is pretty difficult, in the literature and in my experience. This is true even with large doses of the inocula.

As an aside, infectious doses vary greatly, and it may depend on everything short of the phase of the moon - and even that, in women. One of the great stories of early microbiology was when Koch was trying to demonstrate the germ theory of disease, and he had isolated the cholera causative agent (Vibrio cholera). He had a huge flask of this nasty bug, and another science professor, doubting Koch, postulates and all, decided to demonstrate his scorn. He drank the entire flask of the supposed cause of misery and massive suffering, still one of the most serious diseases - and he didn't get sick. I guess he had taken his vitamins.

Anyway, there are the human-derived Lineage I strains and the bovine Lineage II strains of E. coli O157:H7. they both appear around the world, but one tends to be in the cattle, the other, in disease. They appear to have diverged prior to global dispersal. So, perhaps, all those O157:H7 in cattle that people are all stressed about aren't a big deal... The authors in this paper decided to figure out what genes were different among the lineages. They found a bunch of regions that were different, and then characterized them to look for virulence factors which may make Lineage I a problem and Lineage II a bunch of bullcrap, literally.

They used 30 strains for the secondary screen - 10 Lineage I and 20 Lineage II, to confirm the differences found in a smaller primary set of 4 strains. Then they did a tertiary screen with 119 additional strains.

They found a bunch of regions that were relevant. The big finding is that most of these were associated with potential viruses, plasmids, and transposons - mobile genetic elements that can thereby change their background and associations. This begins to explain the apparent recent origin of the pathogens. Some of the genes look like obvious problems - colonization elements, hemolysin genes, outer membrane molecules. Others are not obvious.

One thing that this says is that these factors might reassemble in different combinations in other backgrounds. O157:H7 might have just assembled all the cool bad genes, a sort of Ocean's 11 of E. coli, all at once.

Another interesting thing is that it looks like 'lineage II' is just lineage I that has lost elements - perhaps there is selection against virulence elements in the cattle. Perhaps there is general pressure to be less virulent, not more. this would be very good for us and our food supply.

This is a very cool article on molecular ecology and evolution in the context of pathogenesis and symbiosis. It makes for some cool findings and begins to suggest some interesting approaches for prevention and therapy. For instance, curing bacteria of phage would likely be enough to eliminate many virulence factors without killing the cells. Another interesting point - if we can keep bacteria from being infected with phage, they might not emerge to virulence, if that is a major source of virulence factors which are otherwise being purified by selection.

Identification of Escherichia coli O157:H7 Genomic Regions Conserved in Strains with a Genotype Associated with Human Infection

Marina Steele, Kim Ziebell, Yongxiang Zhang, Andrew Benson, Paulina Konczy, Roger Johnson, and Victor Gannon

Applied and Environmental Microbiology 73(1), Jan. 2007, 22-31.


I'll presume we all know that E. coli O157:H7 is code for the most common seriously pathogenic form of E. coli currently present in the food supply. The numbers and letters represent the surface 'antigens' that are used to do a preliminary identification of strains of bacteria - the antigens are related to the structures on a bacterial cell surface that are accessible, presumably, to the immune system - but also accessible to assays for testing cell type. Because they are accessible to the immune system, antibodies (particularly monoclonals) can be generated to them and then used in immunoassays, which are very rapid and sensitive. The now classic 'ELISA' (enzyme-linked immunosorbent assay) allows single cells to be identified by their antigens almost immediately. The O antigen is part of the "LPS" of the gram negative cell wall, it is made of sugars. The K antigen is part of the 'capsule' layer - a protective layer of polymerized sugars more loosely connected with the cell. H antigens are derived from the flagella, which are also important to the cell in colonizing a host and are exposed on the cell surface.

Anyway, these antigens can also be used as markers for strongly selected genetic markers - certain antigens are better under certain conditions; particularly, some are more useful for evading predation (diversifying selection acts in this case) and some are more useful for evading the immune system.
Whatever the case, there are hundreds of antigens (~200 O, ~100 K, ~55 H). This allows for tens of thousands of combinations, helping sort the E. coli into clonal monophyletic populations. Of course, it is not necessary that this be true for any given combination, that it is clonal or monophyletic.

Everyone cares about O157:H7 because it keeps showing up in the ill and recently deceased - and in their spinach, tacos, etc. It also shows up in the rectum of ruminants (cattle, sheep) where it doesn't seem to cause the animals any trouble. Certainly, these E. coli have learned some tricks, and modern agriculture encourages their spread. One argument goes that animals being fed grain, not hay, has changed the stomach conditions to permit these E. coli to spread more readily (Diez-Gonzalez et al, 1998; Russell et al, 2000; Russell et al, 2000). The idea is that the E. coli are becoming acid tolerant. Stomach acid is one of the main barriers for humans to prevent infection by bacteria; it protects the established symbionts against temporary invaders. Without going into too much discussion of this point, there is reason to doubt the conclusion that the E. coli are really being selected for acid resistance by cattle diets (Carolyn J. Hovde, Paula R. Austin, Karen A. Cloud, Christopher J. Williams, and Carl W. Hunt, Applied and Environmental Microbiology, 65(7), July 1999, 3233-3235; Grauke LJ, Wynia SA,Sheng HQ, Yoon JW, Williams CJ, Hunt CW, Hovde CJ. Vet Microbiol. 2003 Sep 1;95(3):211-25; Science, April 1999, Vol. 284. no. 5411, p. 49; reviewed, "E. coli O157:H7 in hay- or grain-fed cattle," Dale Hancock and Tom Besser, October 12, 2006, citation not obvious). The evidence for this appears to be more 'suggestive' than 'conclusive.' Anyway, simply lowering the infectious dose may make it easier to become ill, but there must be other things going on to cause O157:H7 to be a problem.

One may be that these E. coli have learned tricks to get into plant roots. There are lots of studies about this issue as well:
JV. Gagliardi and JS. Karns. Environmental Microbiology 2002, Volume 4(2) 89.
Warriner K, Ibrahim F, Dickinson M, Wright C, Waites WM. J Food Prot. 2003 Oct;66(10):1790-7.
The second is interesting in that it presages the spinach problem by showing that E. coli can get internal to spinach cells, so the bacteria could never be washed off. Thus, eating raw spinach fertilized 'organically' with pathogen contaminated manure might create a hazard that cannot be dealt with by surface sterilization.

Anyway, back to the topic of the paper at hand: the relationships among these 0157:H7 bacteria that are causing all this mess.
There are apparently two major families of these bacteria. While even the dominant human pathogens don't seem to cause disease in animals, it seems from most experiments I've seen, that they also don't colonize the animals particularly stably. This doesn't really surprise me - stable intestinal colonization of a conventional (previously naturally colonized by bacteria) host by a laboratory isolate is pretty difficult, in the literature and in my experience. This is true even with large doses of the inocula.

As an aside, infectious doses vary greatly, and it may depend on everything short of the phase of the moon - and even that, in women. One of the great stories of early microbiology was when Koch was trying to demonstrate the germ theory of disease, and he had isolated the cholera causative agent (Vibrio cholera). He had a huge flask of this nasty bug, and another science professor, doubting Koch, postulates and all, decided to demonstrate his scorn. He drank the entire flask of the supposed cause of misery and massive suffering, still one of the most serious diseases - and he didn't get sick. I guess he had taken his vitamins.

Anyway, there are the human-derived Lineage I strains and the bovine Lineage II strains of E. coli O157:H7. they both appear around the world, but one tends to be in the cattle, the other, in disease. They appear to have diverged prior to global dispersal. So, perhaps, all those O157:H7 in cattle that people are all stressed about aren't a big deal... The authors in this paper decided to figure out what genes were different among the lineages. They found a bunch of regions that were different, and then characterized them to look for virulence factors which may make Lineage I a problem and Lineage II a bunch of bullcrap, literally.

They used 30 strains for the secondary screen - 10 Lineage I and 20 Lineage II, to confirm the differences found in a smaller primary set of 4 strains. Then they did a tertiary screen with 119 additional strains.

They found a bunch of regions that were relevant. The big finding is that most of these were associated with potential viruses, plasmids, and transposons - mobile genetic elements that can thereby change their background and associations. This begins to explain the apparent recent origin of the pathogens. Some of the genes look like obvious problems - colonization elements, hemolysin genes, outer membrane molecules. Others are not obvious.

One thing that this says is that these factors might reassemble in different combinations in other backgrounds. O157:H7 might have just assembled all the cool bad genes, a sort of Ocean's 11 of E. coli, all at once.

Another interesting thing is that it looks like 'lineage II' is just lineage I that has lost elements - perhaps there is selection against virulence elements in the cattle. Perhaps there is general pressure to be less virulent, not more. this would be very good for us and our food supply.

This is a very cool article on molecular ecology and evolution in the context of pathogenesis and symbiosis. It makes for some cool findings and begins to suggest some interesting approaches for prevention and therapy. For instance, curing bacteria of phage would likely be enough to eliminate many virulence factors without killing the cells. Another interesting point - if we can keep bacteria from being infected with phage, they might not emerge to virulence, if that is a major source of virulence factors which are otherwise being purified by selection.