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Posted: Sun Sep 20th, 2009 17:10 |
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Listeria L-forms: Discovery Of An Unusual Form Of Bacterial Life
ScienceDaily (Sep. 19, 2009) — ETH Zurich researchers have discovered a new life form of Listeria monocytogenes, an opportunistic pathogen responsible for serious food poisoning. These bacteria can reproduce and proliferate as so-called L-forms. The methods to detect these bacteria should now be adapted.
For over 100 years, it was known that bacteria may lose their cell wall and can still survive. However, it was believed that this phenomenon was merely an artefact and that bacteria without cell walls do not remain viable. Recent research of a group headed by ETH Zurich Professor Martin J. Loessner, which has just been published in Molecular Microbiology, shows that bacteria without a cell wall can be a stable form of bacterial life. Astonishingly, not only can Listeria survive without a cell wall, they are even able to reproduce and proliferate.
From cheese to the brain
Listeria (Listeria monocytogenes) are pathogens causing dangerous and often fatal cases of food-borne infections, and are frequently found in milk products such as vacherin soft cheese. The bacteria invade the human body through the epithelial cells of the intestine and spread from cell to cell., which renders them invisible to the immune system. Listeria can cross both the blood-brain barrier and the placenta barrier. Having reached the brain, they cause severe inflammation of the brain, which can be fatal. Listeria can also endanger fetuses and pregnant women.
Membrane instead of a cell wall
Listeria cells normally appear as small rods. If they shed their cell wall, e.g. through contact with certain antibiotics such as penicillin, they become spherical and enlarge greatly. These cell wall deficient cells are surrounded by a single membrane only. As an intermediate stage between this L-form and the rod-shaped parental cells, there is an intermediate stage from which the bacteria can rebuild their cell wall. However, once Listeria has reached the complete L-form status, there may be no way back.
The change from the normal form to the L-form is accompanied by many changes in cell metabolism and gene activity. Almost 280 of the genes of normal and L-form Listeria showed differing activity. While genes responsible for stress regulation were activated in the L-forms, genes for metabolism and energy balance were strongly repressed. The researchers interpret this as the bacterial response and active adaptation to its new lifestyle. Loessner says “L-form Listeria really have a very stressful life.”
“Culturing” the L-forms of bacteria is not easy. They need to be “bred” in a liquid medium and do not normally form colonies, so plating on a petri dish is not possible. Although L-form Listeria cells are capable of reproducing themselves, this can take time: formation of a visible colony within tubes containing a soft medium takes at least six days, compared to 16 to 20 hours for normal cells.
A new mechanism of division
The researchers were amazed by their observations on how mother cells produce L-form daughter cells. First, new vesicles form inside the large L-form cells. When these are large enough, the mother cell bursts and releases the daughter cells. At this point, these have the full genetic make-up of the mother cell, but it is still unclear how the genetic material is transferred. Interestingly, their metabolism does not start up until they have been released from the mother cell.
L-forms can grow in milk
The researchers had a reason for investigating this strange form of bacterial life: a large epidemic of Listeria with many fatalities in the US about 20 years ago. Although it is clear that this was the result of the consumption of contaminated milk, and the pathogens could be detected both on the farm from which the milk originated and in the patients who had consumed the milk, Laboratories were unable to find Listeria in the milk itself. One possible explanation is that the bacteria had been present in the milk in their reversible L-form and had thus been undetectable. Loessner says, “This is because the L-form can reproduce in milk just as well as under laboratory conditions.”
L-form Listeria can also outwit the immune system. Although macrophages, i.e. phagocytes, ingest the spherules, they seem unable to kill them in a timely fashion. While normal Listeria cells are killed after about 30 minutes, the L-forms can survive for much longer inside a macrophage. The ETH Zurich professor feels that “the immune system may have a problem if macrophages cannot recognise the L-forms as a pathogen.”
Pathologists sometimes reported small bubble-shaped objects in brain sections from animals that had died of listeriosis, but it has hitherto been impossible to classify these properly. Loessner hypothesizes that these could also have involved L-form Listeria.
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rosana
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Posted: Tue Sep 22nd, 2009 09:53 |
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I was going to add this article to the forum, and I am happy to see that somebody has referred to it. Here is the abstract:
Cell wall-deficient bacteria referred to as L-forms have
lost the ability to maintain or build a rigid peptidoglycan
envelope. We have generated stable, nonreverting
L-form variants of the Gram-positive
pathogen Listeria monocytogenes, and studied the
cellular and molecular changes associated with this
transition. Stable L-form cells can occur as small
protoplast-like vesicles and as multinucleated, large
bodies. They have lost the thick, multilayered murein
sacculus and are surrounded by a cytoplasmic membrane
only, although peptidoglycan precursors are
still produced. While they lack murein-associated
molecules including Internalin A, membraneanchored
proteins such as Internalin B are retained.
Surprisingly, L-forms were found to be able to divide
and propagate indefinitely without a wall. Time-lapse
microscopy of fluorescently labelled L-forms indicated
a switch to a novel form of cell division, where
genome-containing membrane vesicles are first
formed within enlarged L-forms, and subsequently
released by collapse of the mother cell. Array-based
transcriptomics of parent and L-form cells revealed
manifold differences in expression of genes associated
with morphological and physiological functions.
The L-forms feature downregulated metabolic functions
correlating with the dramatic shift in surface to
volume ratio, whereas upregulation of stress genes
reflects the difficulties in adapting to this unusual,
cell wall-deficient lifestyle.
"Listeria monocytogenes L-forms respond to cell wall
deficiency by modifying gene expression and the mode
of division"
Molecular Microbiology (2009) 73(2), 306–322
Simone Dell’Era,1 Carmen Buchrieser,2
Elisabeth Couvé,3 Barbara Schnell,1 Yves Briers,1
Markus Schuppler1* and Martin J. Loessner
It is quite an interesting article. Worth reading in a very difficult field.
Regards,
Francisco Calvo, MD
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Deedee
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Posted: Tue Sep 22nd, 2009 17:24 |
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"Surprisingly, L-forms were found to be able to divide
and propagate indefinitely without a wall."
Sounds like a sci-fi monster. 
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Phillyguy
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Posted: Tue Sep 22nd, 2009 18:55 |
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To me this answers one of the significant questions that I had regarding the use of long-term, IV antibiotics such as rocephin for lyme. If the L-forms are able to replicate without reverting back to a cell wall form, then it is impossible to cure this type of infection with beta lactam antibiotics regardless of the length of treatment or dose. As such, it would appear that only palliation is provided by this regimen as has been pointed out by Dr. Marshall on several occasions.
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NorCalJim
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Posted: Mon Sep 28th, 2009 22:53 |
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Looks to fit in well with this and the bacteriality sites information...
NorCalJim
Last edited on Mon Sep 28th, 2009 23:07 by NorCalJim
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tussilago
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Posted: Wed May 23rd, 2012 19:09 |
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Here is some more litterature on l-forms.
On the website Bacteriality, Amy Proal mentions Ernst
Almquist, a Swedish professor who also studied bacteria
and l-forms. Around 1920 he published "booklets", papers on l-forms.
http://bacteriality.com/2007/08/18/history/
After a trip to the library, here is what it is written in his booklets from around 1920:
Titel: "Pathogenic bacterias' redmodelling/transformation during saprophytic growth"
Translation of Saprophytic- which lives on dead or decaying tissue
"My interest in the subject was brought about partly
because the bacterias' biology was so one-sidedly treated
in litterature. The culture was restricted to certain,
conventional medium and the description of the species
has been kept down to a few caracters, and the meaning of the
species chemical reactions has been overexaggerated.
The result of this has become an artificial attempt
to simplify nature. A rod should always be a rod and
a coccus coccus. When an other form was noticed by the eye,
then the observer right away named it involution form,
a Naegele-itic term [Wikipedia: from Naegel's rule].
The Koch'ish law concerning the spread of thyfoid of
the gut, cholera,.. and so many other epidimical diseases
states that the infected person practically produce
all viruses... This law is supported by the perception
of bacterias unchangeability... It has lead to beautiful
discoveries and forceful measures, but many
has missunderstood the state of things/facts and believed
that the law was prooven in all.
It is necessary to expand Koch's work hypothesis...
The discovery of remodeling under saprophytic condition
with accompanying changes and variations, changed
virulence, the rise of indefinite small forms- bring on
new work hypothesis.
During the last year, I have carried out some of the experiments with doctor G. Koraen.
1. bacterium typhi
In dried nutrition, on pieces of potatoe or on dry agar,
the tyfoid rod quickly transforms and develop forms,
that are not seen during conventional culture. The phenomenon
starts after a few days, after 2-3 week it reaches its
peak and after a month it is all over. Picture one,
shows shows how a bunch of rods changes after two weeks.
Spheres lie free or are attached to the rods as exogene formations.
One of the spheres is growing. Often we
find protoplasmatic substances in every possible formation.
In the previous thesis, I have described how the spheres
grow to rods, to spheres or delicate/the finest small formations,
which do not allow to be coloured. In picture two, it
is seen a bunch of small formations that developed out of a big sphere.
Before this phenomenon is all over, the same rods form
spheres straight away, when they are well nutritioned at
body temperature. There are many medium where
the described transformation takes place at a low
temperature, without any direct formation of spheres.
However, the spheres appears immediately when the rods are
brought to good nutrition at 35 degrees. So react the
tyfoid bacteria to algea mud, fecals and dirth and so on.
... In 1908, I and Wange have studied how these form
directly under the microscope; we saw how they leaved the
big exogenely formed spheres or directly out of the rods.
3. spirillum cholerae
as the thyfoid rod, this one form exogene spheres, that
develop along the sides or at the tip of the spirillum; in
quite abundance also thick threads and other protoplasmatic
forms. The spheres grow to new spheres, not through
division but sort of like yest svamps though budding. Not
selldom, one sees a small comma bacteria (shaped like a
comma) emerge.
The ablility to form growing spheres seems only to occur
during culture at lower temperatures, not at body
temperature. P.G Olsson has grown the spirillum... they
could be brought back to ordinary (wild type) cholerae spirillum.
6. bacterium acidi propionici c.
This ..bacteria out of Swedish cheese is not pathogenic,
but is reviewed though because it has many similarities to
diptheria... picute 6 shows protoplasmatic forms along rods..."
bacteriu urinarium n. sp.
During 20 years, I have had the opportunity to follow a
case of bakteriuri (urinary infection?) that was aquired
during sectio alta [a Cystolithotomy is the surgical
removal of bladder stones via a lower abdominal incision].
The bacteria is always present in large abundance in the
urine. It gives the urine a strong smell,... A the clinics,
the bacteria is referred to as B. coli but it does not
belong to the specie and might not even be related to it.
Because it is almost always immobile, does not coagulate
milk, but some individuals can make the colony at the blue
agar plate red. It grows pretty good at 10 degrees. At low
temperatures this specie form a tangle of threads with
exogene spheres and smaller protoplasmatic forms. It is not so different to b. dysenteria."
The remodelling, studied by me, is important to the
speices' parasitism. The indefinate number of small forms
facilitate the spread and render easier infringement in
the tissues. The virulence can also increase.
There are advantages even with the saprophytical existance.
Many bacteria adpat to an environment, where it not directly
(from the first moment) can spread. Other species win
through a remodelling capacity to grow of one nuitrition,
in which it can not proliferate before the remodeling.
I think that each and everyone, that know a lot about micro
organisms' big capabillity to adapt and remodelling, should
admire Linné's sharp look when he express, that the nature
is never so admirable when it comes to the smallest organisms. "
------------------------------------------------------
I am not too sure about the science behind this next piece
of litterature is good enough to post on the forum, but here it goes...
In 1971, Swedish physician etc Håkan Gnarpe published the
article "L-phase bacteria in urinary tract infections"
http://www.ncbi.nlm.nih.gov/pubmed?term=(%22swedish%22%5BLanguage%5D)%20AND%20l-forms
... certain factors in the urinary tract contributes to
increase the frequency of l-phase isolation from this organ
system. In the kidney, foremost in the papilla, and in
the urine one has normally unusual high electrolyte
concentrations (300-1200mosm/L) compared to other organs
and body liquids. L-phase organisms that are very sensitive
to hypotonic (solution) environment, thus have great oppotunities to
escape osmosis lysis which probably occurs in other
organs.
To protect from osmotic lysis, electrolysis
concentrations over 400mosm/L are neeeded, even higher
concetrations are needed if bacteria in l-phase are induced
from gram posisitive bacteria. This has been shown
experimentally by for example Alderman and Freedman (1963)
who could get survival of l-phase organisms in the
kidney's marrow but not subkutis(?). The presence of UREA in
high concentrations in kidney tissue and urine (200mg-pro
cent) might probably also stabilize l-phase organisms at
otherwise low electrolyte concentrations (Braude et al 1968).
An other important factor is the PH-value. Normally urine
has a somewhat soure reaction due to what we eat. Edebo
(1961) could show that certain bacteria in l-phase can be
protected from lysis even at very low electrolysis
concentrations provided that one kept a low enough PH-value
(around 5). My own investigations have confirmed this but
has also showned that bacteria in l-phase can grow in
hypotonic solutions if the PH is kept between 5, 0 and 5,5
(Gnarpe and Edebo 1970).
In several investigations one has demonstrated the
importance of keeping the electrolyte at a certain
concentration. One found that bacteria in l-phase can no
longer be detected in urine when osmosis-ialisation went
below 220 mosm/L (Braude et al 1968). In my own experiments
we have similar results...
The testing can take its course, same procedure but it is important
that the urine is as concentrated as possible so that the
electrolyte concentration can protect the l-phase from
osmosis lysis. Morning urine is preferable. ... a suitable
transport medium is found to be 0,6 M sackaros-0,015 M
magnesium solution. The presence of sackralose stabilize
the urine osmoticly and the magnesium ion has an allover
concentration effect on the cellmembranes (Weibull 1953). Last edited on Wed May 23rd, 2012 23:32 by tussilago
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tussilago
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Posted: Fri May 25th, 2012 21:10 |
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Here are some more early papers on what the authors believe are l-forms.
Published in 1916, they describe how many different
bacteria, assumably all bacteria, has the ability to form
l-forms.
Titel: "Life cycles of the bacteria"
Journal of Agricultural Research
By F (Felix) Löhnis and N.R. Smith
The papers can be read/view and printed out as PDF:s on this webpage:
http://naldc.nal.usda.gov/catalog?f%5Bname_facet%5D%5B%5D=Lohnis%2C+F.
They also discuss if what they see is contamination or
something completely else than l-forms. They seem to name them:
Vegetative cells, gonidia, symplasm and regenerative bodies
In 1916, they write:
"A comparative study of 24 Azotobacter cultures and 18
strains of other bacteria now revealed the fact that those
wide morphological differences first observed with
Azotobacter are by no means restricted to this one group of
bacteria. Similar variations occured with all bacteria
generally...
Therefore, not only for diagnostic and
systematic purposes are these facts of fundamental
importance but also for all lines of research in
agricultural and medical bacteriology....At present we
merely wish to inform agricultural and also medical
bacteriologists about these newly discovered facts and to
ask for their cooperation...
Indeed, numerous items in the
bacteriological literature, for instance, show that the
formation of gonidia and the budding of bacteria have been
observed quite frequently. Yet again the authoritatative
statement that bacteria multiply exclusivelty by fission
apparently had been sufficient to prevent thorough research
in this direction, and the credous adherence to "standard
methods" unfortunately explains only too well why the
turning point in the life cycles of the bacteria has been
completely overlooked.
... Those "granules" which precede the formation of the
normal endosphores inside the bacterial cells are actually
nothing else than gonigia. When budding, the gonidia
frequently develop into thick-walled regenerative bodies,
which do not only germinate in the same manner as
endospores but may also acuire quite a considerable
resistance to heat... Normal heat-resistant endospores
showing polar germination have bee found in 13 of our
Azotobacter culturres... During our first investigations
upon this subject only 4 of 11 strains possessed this
faculty, which they had acuired between 1908 and 1912.
culture 7 gave some bodies looking like endospores in 1914,
but they were not resistant to heat. This faculty has now been fulle developed.
Practically all of the many artificial substrates
recommended in the bacteriological literature are much too
concentrated, especially for soil organisms. For many of
our experiments we used the following mineral solution to good advantage...0,02 procent...
In 1887 Sorokin... His illustrations, reproduced in several
textbooks, show clearly that he also found a budding
bacterium without becoming aware of this fact... Many type
of irregular "involution" forms, so frequently observed
with other spirilla, belong also to this type of growth.
DISCUSSION OF RESULTS
It is true that several authors before us have already
spoke of the "life cycles" of bacteria. In most cases,
however, they meant only the straight forward (not
"cyclic") development, consisting in stretching and
dividing of the cells, sometimes combined with formation
and germination of the endospores. Fuhrman, who also wrote
upon the "Entwicklungskresise" of bacteria, made some
correct observations concerning the formation and further
development of gonidia. He was wrong howeverer, in... In
this direction Fokker came much closer to the truth.
It is not impossible, of course, that by a thorough sifting
of the litterature we shall discover some entirely
forgotten author who was already on the right track. So
far as we know now, only one bactreiologist has previously
seen all the different stages of the growth typicalk of the
full life cycle of the bacteria. We refer to De Negri's
important "Untersuchungen zur Kenninis der
Corynebacterien", which appeared this spring, when we had
just began to prepare this paper for publication.
For instance, those curious but heretofore entirely
unexplainable regular seasonal variations in the activity
of the bacteria in soils, quite frequently observed in
europe as well as in America during the last years, now
seem to become explainable as a result of the seasonal
effect upon the different modes of multiplication and
propagation of the bacteria. A similar dependency on this
factor then would exist as with pother organismsAt least we
can hardly consider it being merely an accidental
coincidence that essentially the same annual curve, showing
a maximum in spring and another one in autumn, is also
followed by lower fresh-water algea, where, as Transeau's
careful investigations have shown, the temporary prevalence
of spore formation and of vegetative processes apparently
represents the principal causes of these variations.
Concerning pathological problems, we readily admit that we
are entirely laymen. However, we feel sure that this branch
of bacteriology also would win considerably by making use
of our observations.
They show that Henri's very interesting results obtained
with B. anthracis could easily be duplicated with this or
other pathogenic species simply by studying the relation of
virulence and type of growth.
That the discovery of the complete life cycles of the
bacteria solves also some problems in general biology has
been indicated earlier in this paper, when Fokker's theory
concerning the development of bacteria from granules in
milk or blood was discussed...
It goes without saying that we will readily furnish
subcultures of these strains used in our studies to everone
who asks for them. But it probably would be more
interesting and surprising to our fellow bacteriologist if
they would make some investigations with their own well-
stock cultures along the lines discussed in the foregoing
pages. Even a renewed microscopical study of the old
stained preparates may become very instructive. For
example, the senoir author also did not know that for more
than 11 years he had in his collection, patiently waiting
to the photographed, that fine preparate now shown in
figure 6 until, as stated before, he decided to take down
his "theoretical blinders." We have already mentioned that
a careful study of the illustrations contained in our daily
used textbooks will now reveal several things which we were
so trained not to see".
Löhnis and Smith reference Ernst Almquist in their work above.
Around 1918, Ernst Almquist in turn discuss their publications in his booklet.
They, Löhnis and Smith, published an other paper in 1914.
Here is a more recent paper on bacillus subtilis l-form:
http://www.ncbi.nlm.nih.gov/pubmed/22122227
Some internet sources on Felix Löhnis:
http://www.nature.com/nature/.../127099a0.html
In German: http://www.uni-leipzig.de/unigeschichte/professorenkatalog/leipzig/Loehnis_97/
http://de.wikipedia.org/wiki/Felix_L%C3%B6hnis
Here are some more quotes from Lohnis and Smith's paper in 1916,
but I am not sure their conclusions are good enough to post on the forum:
"So far as we are aware, only one other author has spoken
of similar observations. In 1892 Förster found occationally
that Chromatium Okenii sometimes entered into some
"primitive copulation". Among the drawing accompanying his
paper, a sketch made from a photomicrograph seems to us
most trustworthy. Its conformity with our Azotobacter
illustrations is practically complete. Observations in the
hanging-drop clearly showed that there is some interference
between the plasmatic substances in the conjunt cells or
even some direct mixing of them.... The determination of
the actual psyciological signification of this conjunction
must be left, of course, to a more thorough investigation."Last edited on Sat May 26th, 2012 02:33 by tussilago
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Ron
Foundation Staff
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Posted: Sat May 26th, 2012 06:44 |
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Wow, 1916!!! 
In 1892 Pasteur was still alive.
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tussilago
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Posted: Sat May 26th, 2012 13:14 |
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they probably draw a lot of wrong conclusion in the paper (mentioned on Wikipedia)
But, yes, it seems they have theories about pleomorphism and perhaps l-forms, early on.
Do they somewhere correctly describe the phenomenon of cell wall deficient bacteria just like other researchers have described how listeria and E coli forms cell wall deficient (eg researchers mentioned in above post and researchers at Collin College, Texas?
Do pleomorphism better describe their work than the word "l-forms"?Last edited on Sat May 26th, 2012 13:29 by tussilago
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Dr Trevor Marshall
Foundation Staff
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Posted: Sat May 26th, 2012 13:23 |
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During the late 1940's there were a number of papers (from Harvard, I recall) talking about how Penicillin was capable of causing microbes to turn to the L-form and enter cells. But it was all swept under the carpet.
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Carricol
Support Team
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Posted: Sun May 27th, 2012 01:11 |
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Here is a 1957 paper concerning L-Forms and Penicillin. It references some earlier papers some of which go back into the 40s.
http://mic.sgmjournals.org/content/17/1/64.full.pdf
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