Wednesday, September 3, 2008

PHOTORHABDUS: A LUMINESCENT HUMAN PATHOGEN


1. INTRODUCTION: A BIT OF SC
IENCE FICTION," PHOTL++", A BACTERIA MAKING EYES GLOW AND THEN ROT...!

DID YOU SEE THE GOOGLE ADSENSE ADS ON THE RIGHT?



In "We Share", the novel on bacteria I am writing, the cave-living colony of bacteria which I call a bactorg will feel threatened by changes in its environment. To defend itself, it will launch a series of attacks on humans and animals outside the cave.

This will be seen initially as a bioterrorist attack. Soon, considering the variety of viruses and bacteria used and their complete strangeness, the scientists in the novel will understand that there is more at work than just "plain", human-invented bioterrorism... Indeed, they will be confronted with attacks devised by an organism, the bactorg, which has acquired, over its millions of years, a total mastery of cellular bioengineering at the evolutionary time scale.

It has built all sorts of strange bacterial and viral mutants and has enslaved many insects and higher animals. It is a frightful enemy. However, it is a bit naïve too, It does not really know the world outside the cave and does not really understand what we are or what are the constraints of massive terrestrial and airborne bioterrorism agents dissemination.

To build my directory of potential bactorg weapons, I have thus, at this stage, to find in the literature on "bacterial emerging pathogens", "viruses" and "bacterial curiosa", a set of dramatic infections using insects and animal vectors, bacterial and viral pathogens. I need my pathogens to induce nightmarish (yet plausible) illnesses. It is also mandatory that they use advanced and original signalling and logical processing methods, they need to be intelligent weapons.


I have thus to select carefully my pathogens for scientific interest and drama potential.

In this post, I'll present one of my favourites "Photarhabdus luminescent". In the novel, I modify it of course to amplify its effects and I call it PhotL++.

The real PhotL it is a bacteria living in nematodes (small worms) which infect the guts of many insects. The nematodes release the bacteria into the insect blood circulation and then the bacteria kill the insect. From my point of view, the real PhotL it has three interesting characteristics:
  • It is the only terrestrial bacteria which is luminescent... the question is why ??? This will be a big question in "WE SHARE"
  • Until recently, it was known to infect only insects but it is now emerging as a human pathogen.
  • It secretes many powerful toxins and also antibiotics
Just extrapolating a little bit, I will thus invent "PhotL++", a nicely dramatic mutant.

PhotL++ causes awful abscesses in humans and make them glow in the dark. It also attacks the eyes creating strong conjunctivitis and keratitis (inflammations of the conjunctiva -the outermost layer of the eye and the inner surface of the eyelids- and the cornea). The inflammated eyes present first an abundant, opaque purulent discharge and tears. Then they become red and glowing in the dark (very bright green light). Finally, they rot and the brain is attacked; the infected animal or person dies. Is that sufficiently awful? I guess so. As you will see, temporal lobe epilepsy is high drama stuff.

I will add one more twist: the emission of photoluminescence by PhotL++ will be influenced by electromagnetic radiations which can make its intensity pulsate

(I have a literature reference proposing this, see later).

Moreover, a PhotL colony will use its light signal to coordinate its actions and those of its symbiont, the nematode.

But enough on science fiction. When I had my initial nightmare about luminous bacteria, I was quite excited, here was an ideal weapon... Imagine, your eyes glowing in the dark, pulsating and then rotting...

Then I went hunting for it in the real world (I mean on the WEB) and as usual, I was flabbergasted... Reality is almost always better than fiction... My brainchild was almost existing. The real PhotL is known as Photorhabdus luminescent. In the following sections, I will just present the facts about it... just plain facts.


2. PHOTORHABDUS LUMINESCENT, THE FACTS

Meet Nick Waterfield, from Bath University, a microbiologist studying phosphorhabdus and many other microbes (sorry for the distorsion of the photo, for some reason, I can't get it right).

One of its many ideas is that insects are a neglected field of study when it comes to microbiology. They are an enormous reservoir, harboring many potentially harmful species. Their immune systems is, strangely enough (at least to me), quite close to ours. So the bacteria which have adapted to them have just small evolutionary steps to take to get their metaphorical teeth into us. So Nick's idea is that we should study more closely the bacteria infecting insects and that's what he does.
What follows is closely inspired from a text on his website on one of his pet subject, Photorhabdus (PhotL). It is the only known terrestrial bioluminescent bacterium. It is a pathogen of insects. It lives in the gut of a nematode. An ideal candidate for the "WE SHARE" casting.

Click here to have a link to Nick's site.

2.1 THE LIFE OF PHOTL:
Infective young nematodes carrying PhotL search in the soil for their insect prey until they encounter one (often a larva). Then, they scratch their way into the insect's blood circulation and "vomit" up Photorhabdus into the blood where it secretes toxins and virulence factors that rapidly kill the insect.

Studies on the insecticidal-complex produced by PhotL have revealed that several extracellular macromolecules such as proteases, lipases and broad-spectrum antibiotics confer its insecticidal ability which is wide ranged (PhotL is proposed as a pesticide but that raises important security concerns. Its killing ability is wide ranging and it might very well kill useful insects). Imagine a few mutations and we have a fearful human pathogen.

The bacteria replicate rapidly and convert the insect tissues into more bacteria that serve as a food source for the nematodes which may then reproduce. It is around the time of insect death (when the food source will soon be exhausted... and signalling of that fact needed) that the bioluminescence of the insect corpse due to the bacteria can be seen. This will be important. Hereafter a couple of photo from Nick's site: on the left, two insect larvas glowing a little bit before being destroyed by the bacteria. On the left, a larva exploding and ejecting its full load of nematodes. (courtesy of Dr. Nick Waterfield, Bath University)


A load of bacteria in an insect, just below the collagen outside sheat.








2.2 RELATION OF PHOTL AND YERSINIA PESTIS
(the black plague microbe): More stuff for nightmares, lateral transfer of genetic material between Photorhabdus and Yersinia has been demonstrated and is probably a result from their common association with insects as bacterial pathogens .

The association with yersinia pestis opens up interesting avenues for my novel. A few centuries ago (in 1720), there was a famous black plague epidemics in Provence, just where I need it. Yersinia and PhotL were there and were associated. Could they have been incorporated as part of the multispecies, underground Provencal bactorg community at that time? Could they then have been kept dormant until now for two centuries and then suddenly reactivated and released outside the cave as an answer to some quorum sensing set of signals from the outside eenvironment having suddenly reached the bactorg after the earthquake?

Remark: However note that the black plague has recently been the subject of alternative theories attributing its origin to other causes like anthrax or a virus like ebola…?

2.3 BIOLUMINESCENCE IN PHOTL:Bioluminescence is the production of visible light by a chemical reaction in a living organism (see the photo above). Bioluminescence is rarely reported in clinical bacteriology laboratories because bacterial bioluminescence is seen primarily in marine species. Some fishes indeed have an organ in which they grow a large colony of resident luminescent bacteria which provide them with a powerful light useful to attract preys or mates. But what could be the usefulness of light in a bacterium living in the gut of a worm inside an insect? Photorhabdus are the only terrestrial bacteria known to exhibit this property.A useful signalling system?

A culture of PhotL growing and swarming on an agar plate. It emits a faint light visible in the dark. Do you remember the fractal shapes of bacterial growth we saw when we discussed Eshel Ben Jacob's work? I think that this might be another example of it. Do these cultures grow in difficult conditions? Can we experiment on them by modulating their growth conditions like Eshel did? Will we observe nice fractals?

WHY DO PHOTL EMIT LIGHT? Energetically speaking, bioluminescence is a costly process, difficult to justify on an evolutionary basis if it has no clear role. Current theories include some unknown biochemical role or even that it is a lure to tempt fresh insect victims into range. I do not very much believe in them.

When the insect resources have been exhausted, the bacteria provide the nematode with an unknown "food signal" which switches them into a developmental state known as an infective juvenile. At this point they re-package the bacteria before bursting from the insect corpse in search of fresh victims. Could light play a role? A remark: Worms with enough food cease to emit light.


2.4 PHOTORHABDUS, AN EMERGING HUMAN PATHOGEN

Photorhabdus has never been isolated as free living in the environment. However, recent cases of human infections due quite plausibly to Photorhabdus have been reported in the US, Australia and Nepal. Many other cases might be misdiagnosed due to the failure of laboratories to recognise this unexpected pathogen.

Let us describe a clinical case (reported by Dr. Gerrard, Gold Coast Hospital, Queensland, Australia).

a 29-year-old woman with an intensely painful and swollen right foot (3). Two days before presentation, she had cleared debris and weeds from her country property while barefoot. She was started on oral amoxicillin-clavulanic acid in the emergency department, but by the next day her foot had become even more swollen, erythematous, and painful. She was admitted to the hospital and started on intravenous antistaphylococcal (flucloxacillin) antibiotics. Abscess before and after treatment with antibiotics... (courtesy of Dr. Gerrard)

Despite treatment, a local abscess formed. This was incised, and pus was sent to the laboratory for culture. Three days later, a gram-negative rod was isolated in pure culture. The Vitek GNI card identified the organism as Flavobacterium sp. It took them a lot of ingenuity to identify Phot L as the culprit, Thanks to them...

Another case (Dr Alice Weisfeld, Microbiology Specialists Incorporated, Houston, Texas )
A 54-year-old male presented to the emergency department of a local Houston hospital during July 2003. He was a ranch hand who believed that he was bitten by a spider on his left
breast. He presented with multiple carbuncles on his left chest wall and multiple pustular nodular lesions over his extremities. The patient, who has a family history of diabetes, had a blood sugar level of 400 on admission. His temperature was 101°F, his blood pressure was 135/70, his respiratory rate was 20, and his pulse was 60. Culture of the left-breast abscess showed moderate numbers of methicillin-resistant Staphylococcus aureus and an unremarkable gram-negative rod identified by a MicroScan Neg Urine Combo Panel Type 34 on the MicroScan WalkAway (Dade Behring, Inc., MicroScan Division, West Sacramento, CA) as Pseudomonas oryzihabitans. An identical gram-negative rod was isolated from four of four blood culture bottles from two separate venipunctures. However, it was identified on the same system as Providencia rustigianii.
Both isolates were sent to a local reference laboratory (Microbiology Specialists Incorporated). Each isolate produced two colony types, which exhibited annular hemolysis and swarming on blood agar (Fig. 1 and 2). Annular hemolysis is unusual in that there is no hemolysis immediately around the colony but there is a thin line (about 2 mm wide) of hemolysis about 12 mm from the edge of the colony. Each isolate was oxidase negative, catalase positive, and motile, with a nondiffusible yellow to dirty-brown pigment. Neither isolate reduced nitrate to nitrite, but both fermented glucose (Table 1). The isolate was finally identified as Photorhabdus asymbiotica (formerly Xenorhabdus luminescens) on the basis of weak bioluminescence when tryptic soy agar slants grown at either 25°C or 35°C were observed in the dark.

The organism identifications were subsequently confirmed by the Centers for Disease Control and Prevention (Atlanta, US) using conventional biochemicals (1) and a number of other rapid identification systems.

Source of infection: The source of human infection is not yet known, although an invertebrate vector(why not a spider bite, which would be nice for we SHARE in which spiders play a big role) is suspected. Indeed, cases occur indeed in warm wet months, usually after rain storms, and the victims are often people working in the outdoors. Moreover, the abscesses appear on feet and legs.

REmark that with th eclimate change in Some parts of Provence, the climate there is becoming dryer in summer but winters are warmer and wetter, ideal for PhotL...?

CONCLUSION: PHotL associated with severe soft tissue and systemic infections, and is now considered as “emerging human pathogen”.

ANGEL GLOW:
Notice that some people believe that PhotL was the cause of a phenomenon called "angel glow", soldiers wounds glowing in the dark which were observed in people lying on the ground for days during the war of independence in the US. The soldiers with glowing wounds were recovering better, hence the name.
The hypothesis is that PhotL was infecting them by contact with the soil (either due to nematodes or free living bacteria). They were killing other bacteria with their antibiotics, hence the better survival probability. At that time, they were not pathogenic for humans... things change.

All together, I have an ideal scheme for my glowing wounds, I have just to suppose that PhotL is now pathogenic for humans, that it attacks not only the legs but also the eyes and that in its final stage, it infects the brain and kill people... a piece of cake.

A last twist I promised you: Why does the light emission from PHOTL++ pulsate?

Glowing rotting eyes are nice but in addition, in "WE SHARE", the light intensity is always changing.

Admittedly, PhotL does not do that. Why does PHOTL++ do it? The real reason is because it is nice. The second reason is that it allows PhotL to modulate its signal and make it more complex... Advanced signalling... more sophisticated language!

How does it do it? Consider the following reference:

Electromagnetic field effect on luminescent bacteria Berzhanskaya and al. IEEE transactions on Magnetics Vol 31, Issue 6, Nov. 1996 - pp. 4274-4275
Abstract: The effects of electromagnetic fields with frequencies varying from 36 to 55GHZ on the bioluminescent activity of bacteria were investigated. EMFs resulted in a decrease of bioluminescence which depended on frequency. The time of adaptation of cells to the EMF was longer than the intrinsic temporal constants of the bioluminescent signals. The effect was non thermal. Magnetic storms resulted in an increase of bioluminescence.

That's a good start. I'll leave you on this.

A NOTE ON THE USE OF PhotL as a biopesticide (see before), cf the following paper:

Biosafety concerns on the use of Photorhabdus luminescens as biopesticide: experimental evidence of mortality in egg parasitoid Trichogramma spp. Sharad Mohan1,* and Naved Sabir - CURRENT SCIENCE, VOL. 89, NO. 7, 10 OCTOBER 2005

Photorhabdus luminescens exhibit biopesticidal potential against important pests, independent of its host nematode. Indeed, it secretes powerful toxins and large spectrum antibiotics. The question is: can it also attack useful non-targeted organisms? The authors have tested this. They tested the bio-ecological compatibility of PhotL in vitro, against the common biocontrol (and thus useful) agent Trichogramma living as parasites inside the eggs of the rice grain moth, Corcyra cephalonica. Most Corcyra egg-shells became flaccid and there was significant reduction of up to 84% in the emergence of Trichogramma adults. The nematode carrying the bacterium within its gut had no effect on the emergence. This result points to the bio-ecological hazards of indiscriminate use of P. luminescens as a biopesticide. Due to its wide host range, the use of P. luminescens in a pest management programme must be questionned until it is proven safe for non-target organisms

EXTRAPOLATION FOR "WE SHARE": I might decide that PhotL has been used as a pesticide in Provence. Unknown to us, PhotL has been transformed by the insects they invaded which were containing other bacteria transmitted to them by the bactorg (remember what I call enslaving).
They've become PhotL++ and they carry death with them.They are thus there in the soil in large quantities but do not attack humans. Then some signal (probably transmitted by a phage) from the bactorg reaches them and they become active...
PhotL++ uses light as a modulated signal to control it sexpansion. But its light generation mechanism is receptive to EMF influence. In the cave, this was making no harm, EM fields were too weak. But outside, they receive EM radiations from the NSA-like site in the legion camp. It disorganizes them completely and they can't control themselves anymore... a recipe for disaster.

That's it folks?. Have a good night.