In the beginning…

Young_d'Herelle“From a modern microbiological viewpoint the motivation behind the experiments that led to the discovery of bacteriophage is hard to understand. What was the purpose of filtering a bacterial culture to remove the bacteria, then remixing the filtrate with a fresh bacterial culture? We all know the outcome of this classic experiment, but why was it done in the first place? To fathom the intent of the original investigator, Félix d’Herelle, one has to put aside twenty-first century ideas and recover the context of microbiology and disease in the early twentieth century.

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Phage-Mediated Biocontrol of Plant Pathogens (2001 to “current”)

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

I gave the opening talk at the 2nd International Symposium, “New Stages of Phage Biocontrol of Plant Diseases”, held September 18, 2014, at Hiroshima University, Japan. Though my talk was at best peripheral to the emphasis of the symposium, i.e., watch here, I did strive to get into the spirit of things by tracking down references to phage-mediated biocontrol of plant pathogens. Clearly I did not succeed in finding every last one of these references, but nevertheless I probably IDed the ones that “everybody” in the field knows about, and maybe perhaps then some. I’ve sorted these by year plus have indicated the target pathogen as well as the disease that is caused by that pathogen. Where possible I’ve provided a link to the article, though note that I’m providing no promises regarding your potential to find all of these articles online for free! Shown only are experimental articles, and note that I have not confirmed the validity of many of these. So if you know better, or can otherwise help by adding to this list, please let me know!

Here are those papers published in the Twenty-First Century (2001 and newer) up to at least the date of my talk:

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Going Bottomless in the Plaque World

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

Bottom agar = Hard agar = Solid media

Top agar = Soft agar = Sloppy agar = Semi-Solid media.

I’m embarrassed to say that I’ve had this article in my reference database since August of 2007: Rizvi, S., Mora, P.T. (1963). Bacteriophage plaque-count assay and confluent lysis on plates without bottom agar layer. Nature 200:1324-1325. It is only today, however, and apparently as a form of avoidance behavior (there’s a talk I’m supposed to be working on), that I’ve obtained the reprint and set out to read it.

Their second sentence reads, “Having spent considerable time on preparation of ‘bottom’ agar plates for the agar layer assay by the plaque count method and for high-titre bacteriophage stock preparation on a large number of plates by the confluent lysis method…” And thus they are off striving to do something about this by investigating “…the possibility of saving time by using plates without ‘bottom’ agar for assay and stock preparation.”

The media they used for their ‘bottomless’ agar consisted of the following:

  • 10 g Difco bacto-casamino-acids (acid hydrolysed casein)
  • 15 g Difco bacto-nutrient broth
  • 10 g Sucrose
  • 1g Dextrose
  • 5g Crystalline magnesium sulphate
  • 5 g Sodium chloride
  • 8g Agar

Efficiencies of plating in testing phages T1 through T7 they found to be essentially 100% for T1, T3, and T7, and basically 50% for the rest. To the extent that my interpretation of the ‘smudges’ provided in Nature’s PDF can be trusted, the per plate yields for confluent lysis phage preps were more or less the same with versus without bottom agar. Consistently for the latter they note: “The yields obtained on plates without ‘bottom’ agar were slightly better than the yields obtained on plates with ‘bottom’ agar.”

They also note that, “Confluent lysis can be adapted for large-scale bacteriophage production by carrying it out on large stainless steel trays.”

Historical Referencing:

These authors also cite four, mostly Mark Adams-dominated publications for plaque count method (first three) and confluent lysis stock preparation (last). These are:

(1) Gratia, A. (1936). Des relations numeriques entre bactéries lysogenes et particules de bacteriophage. Ann. Inst. Pasteur, 57:652-676.

(2) Adams, M. H. (1950). Methods of Study of Bacterial Viruses. (Methods in Medical Research, 2:1) The Year Book Publishers, Inc., Chicago.

(3) Adams, M. H. (1959). Bacteriophages. Interscience Publishers, Inc., New York.

(4) Swanstrom, M., and Adams, M. H. (1951). Agar layer method for production of high titer stocks. Proc. Soc. Exp. Biol. and Med. 78:372-375.

GOT (phages in your breast) MILK?

Whether it be the suckling newborn, trend-following athlete, or odd lactophile, human breast milk consumers are ingesting more than just calcium and protein in their morning bottle (or shake). Aside from the complete panel of nutritional components and immune boosters that breast milk offers, –proteins, sugars, fats, vitamins, fatty acids, enzymes, antibodies, and leukocytes – it also creates a gulf stream for the vertical transmission of bacteria from the producer to the intestinal tract of the consumer. And where there are bacteria, phage is sure to follow.

Jeremy Barr and the Rowher group are investigating the mother-to-infant transmission of phages in breast milk, and more specifically, what role phages may play in intestinal mucosal immunity.

At the Viruses of Microbes conference last July in Zurich, Barr presented data on the detection Virus-Like-Particles (VLPs) in breast milk samples from five different women. VLPs selected for cesium-chloride density, chloroform resistance, particle size, and dsDNA fluorescence were found from 103 – 104 VLP/mL in each samples, showing that, indeed, phages are present in breast milk.

They are currently investigating what types of phages are actually present by sequencing of the human breast milk virome of mothers, and corresponding stool samples from their breast milk-drinking babies.

While waiting for the answer as to “who” is present in breast milk, they have already starting probing the question of the potential role of phages at their final destination: the infant intestinal tract. To test the ability of phage to adhere and remain in the intestinal tract, T4 phage was mixed with human breast milk, infant formula, or buffer at 107 PFU/mL and layered onto mucus-producing gut epithelial cells. Significantly higher amounts of phage mixed with real milk were recovered after washing and scraping gut cells than for formula- or buffer- phage solutions. This increase, however, was accrued in a in a lactation stage-dependent fashion, with early stage milk supporting greater phage adherence.

So, phages are present in human breast milk and, when coupled with milk, they can persist at the intestinal mucosal surface, but do they have an effect on infection? The first step in the establishment of bacterial infections is bacterial adherence to host epithelial receptors, and breast milk alone contains oligosaccharides that have been shown to prevent mucosal adhesion of such unwanted, disease-causing bacteria or viruses (Bode, 2012). When epithelial cells were cells pre-incubated with phage in breast milk prior to infection with Escherichia coli, significantly fewer bacteria were able to adhere to the host cell surface as compared to only breast milk, or phage-treated formula or buffer.

Clearly, mother’s milk has something that formula has yet to fit into a can.

 

…Keep an eye out for an upcoming publication that will detail the specific components of human breast milk that are necessary for this phage-mucosal interaction, as well as for human breast milk virome!

VoM, Zurich, 2014 “Bacteriophage in human breast milk provide infant mucosal immunity.” Information presented by Jeremy Barr, PhD

Phages & Flamingos

For my first post on this blog, I wanted to share the following paper with you, The virus’s tooth: cyanophages affect an African flamingo population in a bottom-up cascade (see below). It captured my attention after Dr. Brian Jones visited my lab earlier in 2014 where he gave a lecture on flamingos in Kenya (Lesser Flamingo, Phoeniconaias minor). As an extremophile specialist he had been invited to a workshop organized by the Kenyan government to find out why the flamingos have disappeared from Lake Nakuru, a local alkaline lake. According to Brian, many theories were offered up during the workshop, but none of them with sufficient evidence, mainly because of a lack of long-term monitoring of the lakes’ ecosystem.

The following paper presents a possible explanation: Phages caused it! The researchers hypothesize that cyanophages are at the root of a bottom-up cascade causing the flamingo’s main food source, the cyanobacterium Arthrospira fusiformis, to be broken down causing massive drops in flamingo numbers. 

A question of where did the flamingos go, was partly answered accidently at a sampling expedition of my lab, the Centre for Microbial Ecology and Genomics (CMEG, University of Pretoria). Each year a bunch of researchers of CMEG and collaborators make a trip to the Namib Desert to investigate the local arid ecosystems. When driving to the closest town, Walvis Bay, about a 90 minute away located at the Atlantic Ocean, many people stop at the actual bay to watch huge gatherings of the Lesser Flamingo. Sadly, we have no records of how many years the flamingos have been gathering there and if there stay there year-round or not.


The virus’s tooth: cyanophages affect an African flamingo population in a bottom-up cascade

Link to the article

ABSTRACT

Trophic cascade effects occur when a food web is disrupted by loss or significant reduction of one or more of its members. In East African Rift Valley lakes, the Lesser Flamingo is on top of a short food chain. At irregular intervals, the dominance of their most important food source, the cyanobacterium Arthrospira fusiformis, is interrupted. Bacteriophages are known as potentially controlling photoautotrophic bacterioplankton. In Lake Nakuru (Kenya), we found the highest abundance of suspended viruses ever recorded in a natural aquatic system. We document that cyanophage infection and the related breakdown of A. fusiformis biomass led to a dramatic reduction in flamingo abundance. This documents that virus infection at the very base of a food chain can affect, in a bottom-up cascade, the distribution of end consumers. We anticipate this as an important example for virus-mediated cascading effects, potentially occurring also in various other aquatic food webs.