Why is streptomycin more effective against gram negatives?
A Bradbury
give me any additional information on how it works and why gram
negatives are more sensitive than gram positives?
Ivan Kobrinsky
> why gram negatives are more sensitive than gram positives?
G- are more sensitive than G+ because of their different type of cell
walls. This difference in cell walls between gram positives and gram
negatives affect its resistance or its sensivity to to antibiotics and
the host defense component complement. Both types of cell walls are
pretty impermeable, but G- have as some of its major virulence factors
a component of the cell wall called lipopolysaccharide (LPS) coat or
endotoxin. LPS coat is the outer covering and basis for gram negative
bacterial endotoxin, which causes anaphylaxis. Brought on the point you
can say the thick peptidoglycan of the gram positives preventslysis by
complement porin proteins of the gram negatives (thin peptidoglycan)
outer membrane prevent entry of larger antibiotics, but the outer
membrane called LPS coat is simply sensitive to lysis by complement.
I.e. gram negative organisms are salmonella, shigella, escherichia
coli and pseudomonas while thoose of staphylococcus, streptococcus,
clostridium and anthrax are gram positive organisms.
lynx
"A Bradbury" <abra...@taptonschool.co.uk> wrote in message
news:1113303588.2...@z14g2000cwz.googlegroups.com...
>I know that streptomycin inhibits protein synthesis, but can anybody
> give me any additional information on how it works and why gram
> negatives are more sensitive than gram positives?
>
against most Gram positive organisms (as well as Gram negatives). The MICs
of sensitive organisms [both G+ and G-) are similar.
Streptomycin was discovered, IIRC, in screens for anti-Mycobacterial
activity. However, it's clinical indications, besides TB, were mostly for
Gram negatives (as is true for most aminoglycosides). Streptomycin is hardly
used now due to toxicity. I would say that the perception that it is not
effectrive in gram positives is due to their acquired resistance (either
through mutation of ribosomal protein S12 or via plasmid borne streptomycin
degrading enzymes) [although this occurs in Gram negatives too, of course] -
rather than to intrinsic insensitivity to the drug. Probably the MIC90s for
gram positives are higher than those for Gram negatives (reflecting the
overall frequency of resistance).
--
lynx
lynx
"Ivan Kobrinsky" <kob...@gmx.net> wrote in message
news:1113363208.2...@g14g2000cwa.googlegroups.com...
wasn't
thinking about the in vivo question you address. On the other hand, Gram
negatives are much less sensitive to many antibiotics because of their LPS
which prevents the entry of many types of antibiotics. The small, cationic
aminoglycosides can get through the porins of the LPS, and in fact, can
themselves lead to disruption of LPS.
A. Bradbury, where did you get your information that streptomycin was more
effective on G- than G+ bacteria?
--
lynx
Ivan Kobrinsky
>Hmmm, interesting Ivan. I guess I'm such an in vitro microbiologist,
>I wasn't thinking about the in vivo question you address. On the
>other hand, Gram negatives are much less sensitive to many antibiotics
>because of their LPS which prevents the entry of many types of
>antibiotics. The small, cationic aminoglycosides can get through the
>porins of the LPS, and in fact, can themselves lead to disruption of
>LPS.
Of course, interesting points of view. The aminoglycoside streptomycin
is primary used for G-, in vivo it can't be effective used to G+ (only
given the case of an endogenous resistance disharmony).
A Bradbury
the actual mechanism of action from the newsgroup "ribosomes" which
might be of interest.
My impression of the relative sensitivity of G+ versus G- came from
fairly general information on various websites of a non-academic
nature. I'm now starting to think that it might have been misleading.
My initial question arises from an A level (17-18 year old) project
done by one of my students using antibiotic discs on bacterial lawns of
E. coli and Staph. albus. E.coli does indeed apear to be more
sensitive to streptomycin. Does anyone know what the MIC would be for
these two organisms.
Incidentally, the same project showed greater sensitivity of Staph.
albus to penicillin G. A more easily explained result.
John Gentile
at abra...@taptonschool.co.uk wrote on 4/13/05 10:01 AM:
>
> My initial question arises from an A level (17-18 year old) project
> done by one of my students using antibiotic discs on bacterial lawns of
> E. coli and Staph. albus. E.coli does indeed apear to be more
> sensitive to streptomycin. Does anyone know what the MIC would be for
> these two organisms.
If I read this right, are you saying that you think the E. coli is more
sensitive to the drug because the zone size is bigger (as in "appear to be
more sensitive")? You can't compare the plain zone size around a disk from
one organism to another of different groups. There is a very complex process
involved in deciding how large the zone has to be for each organism as
compared to MICs and broth dilutions. A smaller zone for one bug/drug combo
can be sensitive while a larger zone can be resistant for another bug. NCCLS
sets zone size limits for Sensitive, Intermediate and Resistant
interpretations for each organism group and drug.
--
John Gentile Editor, Rhode Island Apple Group
yjg...@cox.net RIAG Web page: www.wbwip.com/riag/
"I never make mistakes, I only have unexpected learning opportunities!"
JEDilworth
Mueller Hinton agar (thickness of the agar determines rate of diffusion,
plus pH and all that other good stuff), and the inoculum must be
standardized to 0.5 McFarland turbidity. The plate must be incubated in
non-CO2 enriched conditions at 35 degrees C for 16-18 hours.
Sensitivity depends on the drug and the bug. The size of the zone size,
as John has said, is irrelevant, i.e. a large zone size doesn't
necessarily equate to being sensitive. The reverse is also true; a small
zone size can indicate sensitivity depending on the drug and bug being
tested.
Judy Dilworth, M.T. (ASCP)
Microbiology
"John Gentile" <yjg...@cox.net> wrote in message
news:BE833B70.18B5E%yjg...@cox.net...
Jenny Gibbs
giving this result were flawed. Could anyone specify some of the
uncontrolled variables, assuming that conditions were roughly equally
favourable for both strains tested (though incubated at room temperature
rather than optimal temperature- possibly this didn't help); the volume/
thickness of agar was constant; concentration of Streptomycin was a constant
(using pre-manufactured discs); they were incubated on a multi-medium agar
jelly.
Sorry, being only at A-Level standard I wasn't sure exactly what some of the
variables mentioned by JEDilworth meant, e.g. McFarland turbidity.
Thanks very much for the help.
"JEDilworth" <bact...@nospamhortonsbay.com> wrote in message
news:x9OdnVx7obF...@buckeye-express.com...
GS
of school before going to college or university. You would not encounter
many of these terms at that point.
Try thinking more closely about how you have stated your results - eg "in
this experiment, the zone size obtained for organism A was X mm and the zone
size for organism B was Y mm and X is greater than Y." That's data. "X mm is
greater that Ymm therefore organism A is inhibited by a lower concentration
of antibiotic than organism B" is an interpretation of the data. "Organism A
is more sensitive to the antibiotic than organism B" is a separate
interpretation, and its meaning depends on what you mean by "sensitive" and
"resistant". By all means look critically at your experimental method; but
if you've done the experiment several times and you've got the same result
each time, then the data are probably correct.
It may help to clarify what "sensitive" and "resistant" mean here - most of
the comments you have had are from clinical microbioligists who are involved
in helping diagnose and treat illnesses. To us, "sensitive" means that we
expect that a patient infected with the organism can be successfully treated
with the antibiotic. "Resistant" means that we don't expect this. It's a
predicition. In a laboratory, all you can do is measure the effect of the
antibiotic IN THE LABORATORY - and to do that effectively, you need known
points of reference. A zone size means only that the organism is inhibited
to some degree. You need a standard to be able to decide if the inhibition
is enough to call the organism "sensitive". If you are making that
judgement on the basis of a measurement of the zone, then you must have
pre-existing knowledge of what zone sizes mean. That information comes from
testing antibiotics against bacteria in standardised conditions. In a disk
diffusion test you will find that the zone size you get from the same
organism and antibiotic disks is different when you use different types or
thicknesses of agar or different densities of bacterial suspension to
inoculate the plates. In practice, it is impossible to have everything
exactly the same every time, so you would normally include a known reference
or control strain in your test - ie an organism similar to the test organism
and known to be sensitive to the antibiotic under test. If your test zone
size is at least as big as the reference zone size, you can class your test
organism as "sensitive". Note that you still have an uncontrolled
variable - the density of the inocula of your test and reference strains.
Finally - remember that you will probably learn more from the experiments
that don't work the way you expect that the ones that do - and you're in
good company - Pasteur and Fleming have been here too.
GS
"Jenny Gibbs" <Jenny...@Sheffield23.fsnet.co.uk> wrote in message
news:d3t7rg$jmv$1...@news6.svr.pol.co.uk...
Bob
<Jenny...@Sheffield23.fsnet.co.uk> wrote:
>I'm increasingly getting the impression that the experimental techniques
>giving this result were flawed. Could anyone specify some of the
>uncontrolled variables, assuming that conditions were roughly equally
>favourable for both strains tested (though incubated at room temperature
>rather than optimal temperature- possibly this didn't help); the volume/
>thickness of agar was constant; concentration of Streptomycin was a constant
>(using pre-manufactured discs); they were incubated on a multi-medium agar
>jelly.
>
The point is that the method cannot be used for what you are trying to
use it for, period. Both Judy and John have said this. If you want to
know a MIC, measure a MIC.
Zone tests are useful within the context of a particular organism and
particular drug under particular conditions, because someone has
worked out, empirically, a relationship between MIC and zone size.
bob
JEDilworth
fairly ignorant of other countries - it's a flaw that is fairly
universal, unfortunately.
It sounds as if you're dabbling with things that are difficult to apply
quality control to. Coming to a conclusion about zone sizes without the
appropriate background in what produces sensitive/resistant zone sizes
means that your experiment will be uninterpretable.
In clinical labs, discs are tested weekly with known strains of
antibiotics with predictable zone sizes. If the zone size for that
antibiotic falls outside of these ranges, then the disc is rendered "out
of control" and steps need to be taken to get it back into control.
Usually, that means the disc is either too old or hasn't been stored in
the correct conditions and have deteriorated.
A McFarland standard is just someone's name tacked on to a known
turbidity standard. You can buy these commercially or make your own.
Certain procedures require a 0.5 McFarland, some a 1, some a 2, and so
on up to about 6 or so. The higher the number the greater the turbidity.
Unless you have some way of QC'ing your discs, then your method is
flawed. You can't test for unknowns unless your "knowns" are in range.
That being said, certain drugs will only work on gram negatives and
others on gram positives. Since streptomycin isn't routinely used in
clinical labs, except for treating tuberculosis, I can't say off-hand
which one it is good for. Some are for both, and this may be one. I'd
have to do some research. I believe streptomycin used to be used back in
the 50's, but, at least for regular bacteria, it is not used now. I've
never reported it out on routine cultures, and I've worked in micro labs
since 1974.
Again, I may be way off base, as I'm not sure what the original purpose
of this experiment was.....:-(
GS, your posting makes a lot of sense!
Judy Dilworth, M.T. (ASCP)
Microbiology
"GS" <n...@home.com> wrote in message
news:d3u1gf$nsp$1...@hercules.btinternet.com...
Tex Bandrew
According to it, streptomycin only affects (p 711) gram-negative and
mycobacteria (a gram-positive with a high GC content) (p 414).
Staphylococcus is a gram-positive with a low GC content (p 399).
The mode of action is interfeering with polypeptide chain initiation.
I just started university, so if i got anything wrong, pls correct me, as i
will learn from it.
Anyways the only way i could see that streptomycin should affect other
gram-positive is if there is some kind of semisynthetich drug, but i dont
know is such a drug exsist.
Tex
"Bob" <bbx...@excite.com> wrote in message
news:gl3561hvtd2gbhd62...@4ax.com...
Mike McWilliams
> I chekked my textbook "Brock Biology of Microorganisms"
>
> According to it, streptomycin only affects (p 711) gram-negative and
> mycobacteria (a gram-positive with a high GC content) (p 414).
> Staphylococcus is a gram-positive with a low GC content (p 399).
>
> The mode of action is interfeering with polypeptide chain initiation.
>
> I just started university, so if i got anything wrong, pls correct me, as i
> will learn from it.
>
> Anyways the only way i could see that streptomycin should affect other
> gram-positive is if there is some kind of semisynthetich drug, but i dont
> know is such a drug exsist.
>
> Tex
Well tex, there may be other things to consider here. If only a select
few gram positive organisms are being affected (like mycobacteria which
don't really thrive in environments away from their hosts), perhaps it
may have something to do with the peptidoglycan layer acting as a bit of
a sheild. It is also worth considering that if streptomycin is
inhibiting protein synthesis, it may be acting in a way that is more
effective against gram - translation initiation as opposed to gram +
translation initiation for whatever reason.
I did a little looking
"Streptomycin is effective against gram-negative bacteria, although it
is also used in the treatment of tuberculosis. Streptomycin binds to the
30S ribosome and changes its shape so that it and inhibits protein
synthesis by causing a misreading of messenger RNA information."
http://www.elmhurst.edu/~chm/vchembook/654antibiotic.html
so the misreading of mRNA prevents translation. Chances are if you were
to do a strict comparison of the 30S ribosome of gram - and gram +, the
site where streptomycin is binding in gram - would be different enough
in gram + to result in total death for gram -, and probably significant
inhibition of growth in gram +, but not total death.
however, in mycobacteria (these are metabolically weak organisms ie.
they rely on their host for a lot of biochemical processing), the effect
of streptomycin may well downregulate protein synthesis to the point
where they do not survive. Another possibility is that the 30s subunit
in mycobacteria is more closely related to gram - bacteria than to gram
+ bacteria, resulting in greater inhibition. Bacteria have many examples
of horizontal gene transfer.
Something useful to do would be to do some sequence comparisons (learn
how to use expasy.org) between the 30s subunits of gm +, gm -, and
mycobact. using blast or something. There is also likely a research
paper which goes into detail about the mechanism of inhibition of
streptomycin, as scientists generally don't just make things up, or take
random guesses as to the activity of antibiotics.
Also if you quote a textbook include the edition. Brock has had many
editions.
Heres a mycobacterial paper:
Microbial Drug Resistance
Investigation of the In Vitro Activity of Streptomycin Against
Mycobacterium tuberculosis
Jun 2002, Vol. 8, No. 2: 147-149
heres a bit of it
"The mechanism of action of streptomycin is inhibition of protein
synthesis of mycobacteria in the ribosome. Resistance emerges when
mutations appear in genes encoding 16S rRNA and protein S12. The
activity of streptomycin against 1,496 M. tuberculosis strains was
investigated; 1,186 and 196 strains corresponded to pulmonary and
extrapulmonary specimens, respectively. For 114 strains, the source was
not indicated. Initially, the BACTEC 460 TB system was used for
antibiotic susceptibility testing. Since 1996, the ESP II system was
used. The strains ATCC27294 (sensitive to streptomycin, rifampin,
ethambutol, and isoniazid) and ATCC35820 (resistant to streptomycin)
were used as controls. An overall resistance rate of 2.2% was obtained.
In all cases secondary resistance was observed. Multiresistance was
observed in 23 strains."
See, now I have an idea that the mechanism of inhibition in mycobacteria
is different than in gm - org. the 16s rRNA and protein S12 are implicated.
The problem with asking a simple question is that the answers are
invariably far too complex to pursue to completion (there is no
completion).
To look back at yours, it was worded awkwardly, but you wanted to know
about semisynthetic analogs of streptomycin that should affect gm +?
The answer is it probably couldnt be an analog of streptomycin, because
streptomycin doesn't inhibit gm + org. for the most part, due to
differences in the 30s ribosomal subunit.
A good way to kill gm + org. are to use beta-lactamase oriented
antibiotics like penicillin or most other -illins.
Anyhow.. the Brock text is excellent, I still have mine and refer to it
every now and then.
Cheers, Mike
lynx
"Mike McWilliams" <michael.m...@drdc-rddc.gc.ca> wrote in message
news:11153035...@coyote.suffield.drdc-rddc.gc.ca...
Actually, the original query was from early April and a number of
possibilities were discussed at that time. In fact, streptomycin is pretty
broad spectrum, active against many/most "wild type" gram positives in vitro
(in the test tube) and their ribosomes are sensitive to it as well. It is
not indicated for use against gram positives (except TB) clinically - and
there were a number of speculations as to why [perhaps related to resistance
in clinical populations or to host factors]. I can't find any simple
explanation for this. But the textbook statement that most gram positives
are not sensitive to it is wrong (at least for "wild type" organisms).
As to 16s RNA and protein s12 - M. TB has a single copy of rRNA - and so
single mutations can give rise to resistance in the rRNA. In gram
negatives - and actually most other bacteria, there are many copies of rRNA
genes and resistance due to rRNA mutations doesn't arise with detectable
frequency [except upon repeated rounds of selection]. The mutations in S12
arise in Gram negatives [and Gram positives, such as B. subtilis, for
example] as well as TB. So, simply working back from the resistance pattern
is a bit misleading.
The aminoglycosides, like streptomycin, cause translational misreading [via
ribosome binding] at concentrations much lower than that at which they
inhibit protein synthesis - and it is likely that this function is the one
that leads to "cidality" (and MIC).
Gentamicin, another aminoglycoside (and there are many natural product
aminoglycosides as well as semisynthetic ones) is used clinically against
certain Gram positive infections - and is certainly active against gram
positives. It doesn't bind to precisely the same part of the 30s ribosome
as does streptomycin, and is not subject to resistance by mutation of a
ribosomal protein gene. There are, however, many plasmid borne
aminoglycoside degrading enzymes which cause resistance - and they are
generally specific to subclasses of aminoglycosides.
--
lynx
Tex Bandrew
Thanks for the things you wrote, they was enligthening.
Just want to clear up, that i dident post the initial question, im just
chekking these newsgroups to see if i can learn anything.
My Brock Biology of Microorganisms is the 10th international edition.
well...have a nice day :)
Tex
"Mike McWilliams" <michael.m...@drdc-rddc.gc.ca> wrote in message
news:11153035...@coyote.suffield.drdc-rddc.gc.ca...