Aminoglycosides modify the in vitro metachromatic reaction and murine generalized Shwartzman phenomenon induced by Salmonella minnesota R595 lipopolysaccharide.
ABSTRACT Endotoxin-neutralizing activity may be an important property for antibiotics to be used in severe sepsis. Several antibiotics, belonging to different classes, were evaluated as to their endotoxin-neutralizing ability, using the inhibition of an in vitro metachromatic assay for lipopolysaccharides and a murine generalized Shwartzman reaction model. Gentamicin, amikacin, and sisomicin have been found to share significant in vitro antiendotoxin activity at an antibiotic/endotoxin ratio as low as 1.0/5 (by weight) and to reduce the murine generalized Shwartzman reaction at an antibiotic/endotoxin ratio of 3.3/5.
- SourceAvailable from: Michael Cooperstock[show abstract] [hide abstract]
ABSTRACT: The limulus gelation assay was utilized to investigate endotoxin inactivation by a number of antibiotics in vitro. Endotoxin activity was sharply reduced by polymyxin B and sodium colistimethate. The effect of the polymyxin was not significantly inhibited by 0.001 M calcium or 90% serum. Crude endotoxins from a variety of aerobic gram-negative bacteria, including several not previously studied, could be inactivated 1 or more logs by as little as 1 mug of polymyxin B per ml, whereas Bacteroides fragilis endotoxin was poorly detoxified. A 10,000-fold range in the relative susceptibility of different endotoxins to inactivation by polymyxin B was found. The endotoxin most susceptible to polymyxin B was derived from an organism resistant to polymyxin B by disk sensitivity testing, suggesting that the bacteriocidal and endotoxin detoxifying properties of polymyxin need not be directly related.Antimicrobial Agents and Chemotherapy 11/1974; 6(4):422-5. · 4.57 Impact Factor
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ABSTRACT: The primary functions of the gut are to absorb nutrients and exclude bacteria and their products. However, under certain circumstances the gut may lose its barrier function and serve as a reservoir for systemic microbial infections. These experiments were performed to determine the mechanisms whereby endotoxin causes bacteria to escape (translocate) from the gut. Bacteria translocated from the gut to the mesenteric lymph nodes of mice challenged with nonlethal doses of Escherichia coli 026:B6 or E. coli 0111:B4 endotoxin. Physical disruption of the gut mucosal barrier appears to be the primary mechanism whereby endotoxin promotes bacterial translocation. Mucosal injury and endotoxin-induced bacterial translocation were reduced by inhibition (allopurinol) or inactivation (tung-sten diet) of xanthine oxidase activity (P less than 0.01), but were not affected by the platelet-activation factor antagonists, SRI 63-441 or BN 52021. Because the inhibition or inactivation of xanthine oxidase activity reduced both the extent of mucosal injury and endotoxin-induced bacterial translocation, the effect of endotoxin on the gut appears to be mediated, at least to some degree, by xanthine oxidase-generated, oxygen-free radicals.Journal of Clinical Investigation 08/1989; 84(1):36-42. · 12.81 Impact Factor
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ABSTRACT: The cationic polypeptide polymyxin B, has been demonstrated to form a stable molecular complex with the lipid A region of bacterial lipopolysaccharides. The interaction between polymyxin B and LPS appears to be stoichiometric, with several lines of evidence suggesting that one molecule of polymyxin B binds one monomer unit of LPS. The formation of LPS-polymyxin B complexes results in the generation of higher mol. wt aggregates with decreased isopynic density.Immunochemistry 11/1976; 13(10):813-8.
Vol. 35, No. 10
Aminoglycosides Modify the In Vitro Metachromatic Reaction and
Murine Generalized Shwartzman Phenomenon Induced by
Salmonella minnesota R595 Lipopolysaccharide
A. FOCA,l* G. MATERA,1 D. IANNELLO,2 M. C. BERLINGHIERI,1 AND M. C. LIBERTO1
Cattedra di Microbiologia, Universita' di Reggio Calabria, Policlinico "Mater Domini", Via T. Campanella,
88100Catanzaro,1and Istituto diMicrobiologia, Universita di Messina, 98100 Messina, Italy
Received 29 April 1991/Accepted 18 July 1991
Endotoxin-neutralizing activity may be an important property for antibiotics to be used in severe sepsis.
Several antibiotics, belonging to different classes, were evaluated as to their endotoxin-neutralizing ability,
using the inhibition of an in vitro metachromatic assay for lipopolysaccharides and a murine generalized
Shwartzman reaction model. Gentamicin, amikacin, and sisomicin have been found to share significant in vitro
antiendotoxin activity at an antibiotic/endotoxin ratio as low as 1.0/5 (by weight) and to reduce the murine
generalized Shwartzman reaction at an antibiotic/endotoxin ratio of 3.3/5.
It has been reported that acute vasomotor collapse could
follow clinical use of proper bactericidal antibiotics in gram-
negative sepsis (3, 21). Both in vitro and in vivo experimen-
tal evidence of antibiotic-induced massive bacteriolysis as-
sociated with release of a substantial amount of endotoxin
(lipopolysaccharide [LPS]) has been published (4, 22). In
septic patients treated with antibiotics, a sudden increase in
the level of free LPS in plasma has been detected and
correlated with the number of dead and alive bacteria in
plasma samples and with clinical outcome (21). Also, non-
lethal experimental endotoxemia may cause a self-promoted
translocation of bacteria and their endotoxins from the gut
(8). Unlike spontaneously released LPS, it has been sug-
gested that antibiotic-released LPS bears a well-exposed
toxic moiety (4), called lipid A, that is thought to be the
innermost and least-variable portion of the LPS molecule
(18). Therefore, an antibiotic with anti-lipid A activity would
be useful in the control of endotoxin-dependent sepsis se-
quelae induced by different species of gram-negative organ-
isms. The Limulus amoebocyte lysate gelation assay (LAL
assay) has been used to evaluate endotoxin neutralization by
antibiotics in vitro (1, 4, 5). However, several limitations of
the LAL assay (10) warrant a different approach to this
An endotoxin assay using the metachromatic dye 1,9-
dimethyl-methylene blue (DMB assay) has been reported to
be reproducible, specific, positively correlated to LPS tox-
icity, and less laborious and expensive than the LAL assay
and other previous endotoxin tests (11). The DMB assay has
already been used to evaluate the in vitro reactivity of
Salmonella minnesota R595 LPS (11). It has been reported
that polymyxin B binds to lipid A and reduces or abrogates
most of the biological activities and chemical properties of
LPS (15), including in vitro metachromatic reactivity in the
DMB assay (11).
The purpose of this study was to examine the LPS-
neutralizing ability of antibiotics from several classes by
comparing the effects of antibiotic-LPS mixtures with those
ofantibiotic-free LPS, both in vitro by the DMB assay and in
vivo by a model of the generalized Shwartzman reaction in
the mouse (2).
S. minnesota R595 LPS was obtained from Calbiochem
Corporation and dissolved in sterile water for injection
Chloramphenicol succinate (Carlo Erba), polymyxin sul-
fate (Burroughs-Wellcome), gentamicin sulfate (Schering-
Plough), amikacin sulfate (Bristol), tobramycin sulfate (Lilly),
sisomicin sulfate (Menarini), dibekacin (Logifarm), kanen-
ofloxacin (Sigma Tau), cefixime (Menarini), and aztreonam
(Menarini) were dissolved and diluted in sterile water for in
vitro experiments and in sterile saline (Bioindustria) for in
vivo experiments. Aminoglycoside concentrations are given
for the free base. The dye 1,9-dimethyl-methylene blue was
obtained from Serva, and the DMB reagent for the LPS
metachromatic assay was prepared as previously reported
Samples of 0.1 ml of the antibiotic to be tested were
incubated at 37°C in a sterile Falcon 96-well culture plate
(Becton Dickinson) with 0.1 ml of LPS solution to obtain
final dilutions of antibiotic of 10.0, 33.3, 100.0, 333.3 ,ug/ml
(reported in Fig. 1 as 1.0, 1.5, 2.0, and 2.5 log
respectively) against a final concentration of 500 jig/ml of
LPS. Thus, we tested the following antibiotic/LPS ratios:
0.1/5, 0.3/5, 1.0/5, and 3.3/5. Control samples with only 0.2
ml of sterile water as well as control combinations of LPS
solvent (sterile water) plus antibiotic or antibiotic solvent
(sterile water) plus LPS were incubated, in addition to other
experimental combinations. After 3 h of incubation, 0.1-ml
samples were taken from each well, immediately mixed with
2.0 ml of DMB reagent, and spectrophotometrically read
against a reference cuvette containing 2.0 ml of sterile water
(11). Experiments were done at least in triplicate.
The optical density (OD) at 535 nm of each antibiotic plus
sterile water sample was subtracted from the corresponding
OD value of antibiotic plus LPS sample, and the resulting
values were averaged and compared with averaged OD
values of antibiotic-free LPS samples. Data were shown as
means and subjected to analysis of variance. Fisher's pro-
tected least squares differences test was used to determine
significant differences between groups.
Figure 1 shows that polymyxin B, gentamicin, amikacin,
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1991, p. 2161-2164
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Effect of serial dilutions of chloramphenicol (CHL), polymyxin B (PB), gentamicin (GT), amikacin (AMK), tobramicin (TBR),
sisomicin (SSM), dibekacin (DBK), kanendomycin (KND), and netilmicin (NTL) on metachromatic reactivity (OD at 535 nm) of S. minnesota
R595 LPS (500 ,ug/ml) with 1,9-dimethyl-methylene blue. Refer to text for further details. *, P < 0.05 versus the OD of antibiotic-free LPS
control (C) by Fisher's protected least squares differences test.
tobramycin, sisomicin, dibekacin, and kanendomycin shared
the ability to reduce significantly (P < 0.05 versus antibiotic-
free LPS) the reactivity of S. minnesota R595 LPS with
1,9-dimethyl-methylene blue. In contrast, chloramphenicol
and netilmicin, as well as the remaining antibiotics tested
(data not shown), did not interfere with LPS metachromatic
assay, not even at the highest concentration used.
For in vivo experiments, the generalized Shwartzman
reaction model in the mouse (2) was used, with some
changes. Female 6-week-old NMRI mice, bred under non-
specific pathogen-free conditions (Nossan), were used.
Groups of four to five mice were injected twice, 24 h apart,
with several combinations of an antibiotic plus LPS or with
control combinations. The treatment schedule as well as
results are shown in Table 1. For both preparative injection
in the footpad and for provocative intravenous (i.v.) injec-
tion, we used an antibiotic/LPS concentration ratio of 3.3/5,
TABLE 1. Effects of antibiotics mixed with LPS on the
generalized Shwartzman reaction in mice
% of mice with
Saline + saline
LPS + saline
LPS + polymyxin B
LPS + gentamicin
LPS + amikacin
LPS + sisomicin
aLPS (5 ,ug) plus antibiotic (3.3jig)dissolved in 25 pl of saline.
bLPS (100ILg)plus antibiotic (66.7jig)dissolved in 0.5 ml of saline.
IMucocutaneous hemorrhages in one site other than the footpad.
dMucocutaneous hemorrhages in at least two sites other than the footpad.
Saline + saline
LPS + saline
LPS + polymyxin B
LPS + gentamicin
LPS + amikacin
LPS + sisomicin
which was the highest one we used during in vitro experi-
ments. Within each group the percentage of animals devel-
oping footpad swelling and/or macroscopically visible hem-
orrhagic, petechial, or echymotic lesions (5 and 18 h after
i.v. injection) was recorded for at least one of the following
mucocutaneous sites: nose tip, mouth, conjunctiva, ears, tail
end, nail beds, and anus.
Table 1 summarizes the results of the antibiotic-LPS
interaction evaluated by a model of the generalized Shwartz-
man reaction in the mouse. Animals injected twice with
sterile saline did not show any pathological signs. In con-
trast, every mouse treated with LPS plus saline in the
footpad, followed by an i.v. injection ofLPS plus saline 24 h
later, showed an obvious swollen and hemorrhagic footpad,
particularly 5 and 18 h after the second injection. At the
same time, most of the mice also showed hemorrhagic
lesions on the ears, nose tip, conjunctiva, and nail beds.
There were no dead mice; however, some animals of this
group developed transient paralysis of the hind legs.
The mice treated twice (first in the footpad and then i.v.)
with antibiotics plus LPS showed a reduced incidence of
both footpad swelling and generalized hemorrhagic symp-
toms (Table 1). No animals developed paralysis of the hind
legs in these groups.
Therefore, we demonstrate the neutralizing potential of
three aminoglycosides on LPS from S. minnesota R595 both
in vitro and in vivo. Two models are also suggested for quick
evaluation of the anti-LPS activity of antibiotics.
Our results are consistent with most of the previously
reported data on the inhibition of LPS by polymyxins and
aminoglycosides, using in vitro cell-free systems (1, 5, 11).
In addition, aminoglycosides and polymyxins are known to
interact with Pseudomonas aeruginosa (14) as well as with
Escherichia coli and Salmonella typhimurium (17) LPS.
Evaluating the inhibition by polycations of dansyl-poly-
myxin binding to LPS and lipid A, Moore et al. (14) sug-
gested the important role played by the fatty acyl tail of
VOL. 35, 1991
polymyxin B. The lack of lipidic moiety among aminoglyco-
sides might account for their reduced affinity for LPS in
comparison to polymyxin B. Furthermore, the use of ami-
noglycosides with different chemical structures allows us to
suggest that at least four primary amino groups may be
necessary to ensure an anti-LPS effect. Netilmicin, unlike
the other aminoglycosides used, has only three primary
amino groups and did not show a significant anti-LPS effect
within the range of antibiotic concentrations used for this
study. Chloramphenicol (1, 5), as well as tetracycline, ampi-
cillin, carbenicillin, and sulfisoxazole (5), has been reported
as an antimicrobial agent without demonstrable LPS-neutral-
izing potential, as shown by the lack of inhibition of the LAL
assay for LPS.
The hypothesis of the absence of anti-LPS effect for
3-lactams is further supported by our
data, which also suggest the lack of anti-LPS effect for a
monocyclic ,B-lactam such as aztreonam.
Quinolone antimicrobial agents such as ciprofloxacin have
been reported to cause a rapid and uncontrolled in vitro
release ofLPS following their prompt bacteriolytic effect (4).
In the same study, polymyxin B and gentamicin effectively
controlled the antibiotic-induced increase of free LPS in
bacterial cultures. In our study ofloxacin, a quinolone deriv-
ative, did not alter in vitro reactivity of isolated LPS,
suggesting the lack of an anti-LPS effect for quinolone.
The results of in vivo experiments were consistent with in
vitro data on the anti-LPS
aminoglycosides, thus allowing us to correlate the reduction
of an in vitro metachromatic reaction of LPS with the
decrease of LPS-induced in vivo toxicity by antibiotics, as
suggested by Keler and Nowotny (11).
A different origin and extraction procedure as well as
major structural differences between the LPS used in this
study and the LPS used by Billiau et al. (2) may account for
the lack of mortality in control mice injected with antibiotic-
free LPS. However, obvious signs of hemorrhagic lesions
appeared at many sites in mice treated with antibiotic-free
LPS, while both the number and the intensity of such lesions
were reduced in antibiotic plus LPS-injected animals and
completely absent in control mice injected with antibiotic-
LPS vehicle (sterile saline). Therefore, in this murine gener-
alized Shwartzman reaction model, polymyxin B, gentami-
cin, amikacin, and sisomicin may reduce the signs of LPS
toxicity, as reported for polymyxin B by other workers, who
used a Shwartzman reaction model in the rabbit (6, 7).
The heptoseless LPS used in the present study was
isolated from a deep rough strain and has been reported to
contain mainly 2-keto-deoxy-octonic acid plus lipid A, the
LPS toxic moiety (18). Although smooth strains are often
reported as more virulent than rough strains (19), our data
may have a significant clinical impact.
induced LPS liberation makes the toxic innermost fraction of
bacterial LPS readily available to interact adversely with
host cells (4, 9). Second, since lipid A structure shows
minimal variability among endotoxins from different organ-
isms (18), our results might extend to antibiotic-released
LPS from many gram-negative bacterial species. Third, the
innermost portion of LPS has been reported to be less
inhibited by human serum components than a smooth type
LPS (16), thus the anti-LPS effect of antibiotics could be
Moreover, the concentrations of aminoglycosides (20) and
polymyxin B (13) in plasma which may be safely achieved
during therapy are within the range of a few micrograms per
milliliter (1 to 8 pLg/ml, depending on the molecule). In
effect of polymyxin B and
antibiotic-treated humans, the highest level of free LPS in
plasma was recently found, 5 ng/ml (21). Thus, thepresent
report demonstrates that both in vitro and in vivo, an
antibiotic/LPS ratio of 3.3/5 (1,000-fold less than the ratio
achievable in patient plasma) may be able to significantly
decrease LPS activity. Also, a recent study suggested that
gentamicin, tobramicin, amikacin, and kanamycin may work
as inhibitors of LPS synthesis in gram-negative bacteria(12).
In conclusion, the present contribution may be of clinical
usefulness in the choice of an antibiotic therapy for severe
sepsis, since we demonstrate that aminoglycosides behave
as endotoxin-neutralizing molecules both in vitro and in
This work was supported by CNR, Italy (grant 890292504).
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