Journal of Microbiological Methods 77 (2009) 178–183

Contents lists available at ScienceDirect

Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

Laser interferometric and cultivation methods for measurement of colistin/ampicilin

and saponin interactions with smooth and rough of Proteus mirabilis
lipopolysaccharides and cells
Michał Arabski a,⁎, Sławomir Wąsik b, Kazimierz Dworecki b, Wiesław Kaca a
a
Department of Microbiology, Institute of Biology, The Jan Kochanowski University in Kielce, ul. Świętokrzyska 15, 25-406, Kielce, Poland
b
Department of Biophysic, Institute of Physic, The Jan Kochanowski University in Kielce, ul. Świętokrzyska 15, 25-406, Kielce, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Laser interferometry is commonly used in permeability studies of soluble substances. In this study a
Received 3 November 2008 modification that allowed testing partially insoluble mixtures is presented. The modification relies on the
Received in revised form 23 January 2009 measurement of diffusion from 1% agarose gel. As a model for this study, two Proteus mirabilis strains were
Accepted 27 January 2009
used that differ in polysaccharide content: smooth P. mirabilis S1959 strain and its Re-type mutant, strain
Available online 1 February 2009
R45. By laser interferometry and precipitation it is shown that R45 lipopolysaccharide is more effective in
Keywords:
binding colistin. It has been shown with the laser interferometric method that saponins, which are
Proteus mirabilis detergent-like substances of plant origin, partially enhance the interaction of colistin with the S and Re types
LPS of P. mirabilis. These results were confirmed with whole cell Proteus studies. The saponin partially inhibited
Colistin the growth of the S and Re P. mirabilis strains at doses of 31–500 μg/ml. A sub-inhibitory dose — 15 μg/ml of
Saponin saponins alone do not reduced the numbers of P. mirabilis S1959 and R45 cells. However, the presence of
Laser interferometry colistin or amipicillin and 15 μg/ml of saponins reduced the amount of P. mirabilis S1959 and R45 cells. The
saponins enhanced sensitivities of S and R P. mirabilis cells towards colistin and amipicillin. One may
proposed that saponins binds to lipid A part of LPS may resulted on an increase in bacterial cell wall outer-
membrane permeabilities and by that facilitated antibiotics penetration into the bacterial cells. In conclusion,
the laser interferometric method is a useful tool for studies of lipopolysaccharide–antibiotic interactions even
if the tested substances are not fully soluble in water.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction Colistin (polymyxin E) is a polycationic antibiotic member of the

Proteus mirabilis is an opportunistic uropathogen that can cause
complicated urinary tract infections (UTIs). P. mirabilis is a clinically
important species, accounts for up to 10% of uncomplicated urinary
tract infections, is the fifth most common cause of nosocomial urinary
tract infections, and may also cause wound infections and sepsis in
hospitalised individuals (Rozalski et al., 1997). Infections of the upper
urinary tract can lead to acute pyelonephritis, bladder/renal stones,
fever, and bacteriaemia (Senior, 1979; Burall et al., 2004; Melekos and
Naber, 2000; Mobley and Hausinger, 1989; Mobley and Belas, 1995). It
has also been suggested that P. mirabilis strains and its antigens are
causative agents in the pathogenesis of rheumatoid arthritis (Rozalski
et al., 1997). Nosocomial strains of P. mirabilis may manifest resistance
to several antimicrobial agents, including extended-spectrum cepha-
losporins, fluoroquinolones, and aminoglycosides (Daza et al., 2001;
Gupta et al., 2001). Proteus strains are also inherently resistant
towards polymyxins (Shimizu et al., 1977).
⁎ Corresponding author. Tel.: +48 41 3496307; fax: +48 41 3496292.
E-mail address: arabski@ujk.kielce.pl (M. Arabski).
polymyxin family. This drug shows amphipathic character and might
interact with both the polar heads and the alkyl chains of phospholipids.
Therefore, the mechanism of colistin's action is associated with the
disruption of phospholipid bilayers through electrostatic interactions
with its components. The resistance of P. mirabilis to colistin is associated
with the interaction of its lipopolysaccharides (endotoxins, LPSs) with
the antibiotic. The complete substitution of phosphate groups of lipid A
of the LPS with 4-amino-arabinose reduced the electrostatic interactions
of colistin with outer membrane of Proteus spp. (McCoy et al., 2001;
Vinogradov et al., 2000). In the presented study we proposed to use
saponin to abolish the inherent resistance of P. mirabilis S1959 and its Re
mutant (R45) to colistin measured by modified laser interferometric
system. The saponins are a group of glycosides consisting of a polycyclic
aglycone and an ether bond to a sugar side chain. These natural, non-
ionic detergents have cytotoxic, haemolytic, molluscicidal, anti-inflam-
matory, antifungal, anti-yeast, antibacterial, and antiviral activities.
Saponins have also been sought after in the pharmaceutical industry
because some of them form the starting point for the semi-synthesis of
steroidal drugs (Spark et al., 2004; Sen et al., 1998). Saponins have
mainly surface-active properties and we suggest that these might

0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2009.01.020
 M. Arabski et al. / Journal of Microbiological Methods 77 (2009) 178–183 179

disturb the permeability of the bacterial outer membrane. In the OH, USA). The reaction mixtures contained saponin at concentra-
presented studies we proposed new, modified laser interferometric tions of 2–4000 μg/ml and a bacterial suspension of S1959 or R45
methods for testing the interaction of positively charged colistin with P. mirabilis at 103 cells/ml in each probe in final volumes of 300 μl of
smooth and rough Proteus lipopolysaccharides. The interaction was LB medium. Additionally we analyzed the effect of saponin on the
tested in the presence of saponins. resistance of P. mirabilis S1959 and its mutant R45 to colistin. The
Laser interferometric methods are widely used for studying diffusion reaction mixtures contained colistin at concentrations of 8, 16, 32,
through membranes or in polymeric media (Dworecki, 1995; Dworecki 64, 125, 250, and 500 μg/ml, saponin at 15 μg/ml, and the bacterial
et al., 2003, 2006). In previous studies we showed that laser inter- suspension at 103 cells/ml in each probe in final volumes of 300 μl
ferometry may be used to study the interactions of antibiotics with of LB medium. We used ampicillin (Sigma Chemical Co., St. Louis,
Proteus LPSs (Arabski et al., 2007). In this study we used a new, modified MO, USA) at appropriate concentrations as controls. The incuba-
laser interferometric system and microbiological methods to evaluate tions proceeded for 18 h at 37 °C. The bacterial suspensions were
whether the saponin influenced the colistin and ampicillin resistance of measured by spectroscopy at 630 nm using EL 340 Microplate
smooth and rough P. mirabilis strains. As microbiological methods we Readers (MTX Lab Systems, USA). Experiment was made in three
used LPS/colistin precipitation assay and analysis of P. mirabilis growth independent experiments. Bacterial viability was expressed in terms
in the presence of saponin and colistin to verify results of investigation of colony-forming units (CFU/ml).
using modified laser interferometric method.
2.4. Data analysis
2. Materials and methods
The data were analysed using the Statistica (StatSoft, Tulsa, OK,
2.1. Culture conditions and lipopolysaccharide extraction USA) software package. All the values in this study are expressed as
the mean± SD from three independent experiments. If no significant
P. mirabilis O10, S1959 (OXK, O3), and its two R mutants Re (R45) differences between variations were found as assessed by the Snedecor–
originated from the Institute of Microbiology and Immunology, Fisher test, the differences were compared by the ANOVA test.
University of Lodz, Poland. The bacteria were cultivated in nutrient
broth (Sigma Chemical Co., St. Louis, MO, USA). P. mirabilis strains 3. Results
were cultivated under aerobic conditions in a fermenter (Chemap AG,
Switzerland) in nutrient broth (BTL, Poland) under the controlled 3.1. The formation of LPS and colistin aggregates in the presence of
conditions (37 °C, pH 7.4–7.6, pO2 75–85%). Cells were harvested at saponin
the end of the logarithmic growth phase, centrifuged (5000 ×g,
30 min), washed with distilled water and lyophilized. The complete Fig. 1 shows the aggregate formation by the LPSs of P. mirabilis
structures of the lipid A portion, core oligosaccharides, and the O- S1959 or R45 at 15–500 μg/ml concentrations with constant amounts
polysaccharides of the S1959 and R45 LPSs were presented previously of colistin (4.00 mg/ml). The mixtures of LPSs and colistin were
(Rozalski et al., 1997; Vinogradov et al., 1994, 2000; Ziółkowski et al., incubated for 18 h at 37 °C. We observed significant differences
1997; Amano et al., 1996). Lipopolysaccharides were isolated from the
smooth type Proteus strain (S1959) by the hot phenol-water method
(Westphal and Jann 1965) and from the R form (R45) by the phenol-
chloroform-petroleum ether (PCP) method (Galanos et al., 1969). The
LPSs were purified by DNase and RNase (Sigma Chemical Co., St. Louis,
MO, USA) treatment and ultracentrifugation, as described (Ziółkowski
et al., 1997; Amano et al., 1996). All LPSs were essentially free of
nucleic acids and contained less than 2% protein.
Saponin from Quillaja saponaria bark was obtained from Sigma
Chemical Co., St. Louis, MO, USA. The main aglycone (sapogenin, 20–
35%) moiety is quillaic acid, a triterpene of predominantly 30-carbon
atoms of the Δ12-oleanane type. The aglycone is bound to various
sugars including glucose, glucuronic acid, galactose, xylose, apiose,
rhamnose, fucose and arabinose.

2.2. Colistin/LPS/saponin precipitation assay

Stock solutions of P. mirabilis O3 and R45 LPSs were diluted with

appropriate amounts of water prior to addition of colistin. The re-
action mixtures contained the LPSs at concentrations of 15, 31, 62, 125,
250, or 500 μg/ml and 4 mg/ml colistin (Sigma Chemical Co., St. Louis,
MO, USA) (in each probe in final volumes of 200 μl. Saponin was
derived from a stock (500 μg/ml) solution and added to the sus-
pensions to give a final concentration of 75 μg/ml prior to the incu-
bation with colistin (3 h, 37 °C). Precipitation proceeded for 18 h at
37 °C. Colistin/LPS or colistin/LPS/saponin aggregates were detected
by spectroscopic measurement at 630 nm using EL 340 Microplate
Readers (MTX Lab Systems, USA).

2.3. Bacteria growth in the presence of saponin and colistin/ampicillin

Fig. 1. Precipitation of colistin (4 mg/ml) with P. mirabilis R45 or S1959 proceeded for
18 h at 37 °C in the presence of saponin at 15, 75, or 150 μg/ml. Colistin/LPS aggregates
The stock solution of saponin (Sigma Chemical Co., St. Louis, MO, were detected by spectroscopy at 630 nm. Statistical analysis was done by ANOVA. The
USA) was diluted in Luria-Bertani medium (LB) (MP Biomedicals, results are the means of three independent experiments.
180 M. Arabski et al. / Journal of Microbiological Methods 77 (2009) 178–183
Fig. 2. Sketch of the laser interferometric system used in measuring antibiotic release from agarose gel to pure water. A description of the scheme is presented in Materials and methods.
between the turbidity of colistin with R45 and S1959 LPS. The ag-
gregates began to form at concentrations of 250 μg/ml for R45 LPS and
500 μg/ml for S1959 LPS, respectively. In the presence of different
doses of saponins the turbidity of the LPS–colistin mixtures increased
in a saponin dose-dependent manner. On the basis of this experiment
we used endotoxins at a concentration of 250 μg/ml and saponin at
75 μg/ml to measure interferometrically the antibiotic release from 1%
agarose. This simply method is not sensitive enough to detect minimal
LPS and colistin concentrations for aggregates formation.
3.2. Laser interferometric analysis of colistin diffusion
The laser interferometric method is commonly used to determine
the diffusion of molecules fully dissolved in water solutions. Turbid
solutions cannot be tested by this method. We therefore modified this
method to evaluate the concentration boundary layers of antibiotic
release from the agarose gel to pure water (Fig. 2). Here we proposed a
modification of the laser interferometric method by immobilising the
tested molecules in agarose gel and measuring the amount of released
substances. The concentrations of S1959 or R45 P. mirabilis LPS, colis-
tin, and saponin used in laser interferometry were established on the
basis of a precipitation assay. We filled the lower part of the cuvette
with an aqueous 1% agarose solution with the LPS/colistin or LPS/
colistin/saponin probes while in the upper part of the cuvette there
was pure water. With such a configuration of the measurement system
the solution in the upper part of the cuvette remains undisturbed and
stable concentration boundary layers are created. The aggregate-free
colistin concentration is uniform in the planes parallel to the gel-
solution interface and gradients occur only in the vertical direction. All
experiments were performed at a temperature of 22 °C.
The amount of aggregate-free colistin, N(t), which diffuses in time
t from a gel solution to water was calculated by integrating the con-
centration profile according to:
Z δ
Nðt Þ = S C1 ðx; t Þdx;
0
where C1(x,t) denotes the concentration of colistin at a point situated
at a distance x from the gel–water interface, S the surface area of
the gel–water interface (S = 70 × 10− 6 m2), and δ the concentration
boundary layer thickness.
The values C1(x,t) and δ were determined experimentally by
means of laser interferometry. The measurement set-up for the inter-
ferometric investigations of the substance transport was presented
previously (Arabski et al., 2007; Dworecki, 1995; Dworecki et al., 2003,
2006). It consists of a Mach–Zehnder interferometer with an He–Ne
laser, a system of two measurement cuvettes, a TV-CCD camera,
and a computer with a system for the acquisition and processing of
interference images. The computer program used to analyze these
images allows, among other things, ascertaining the concentration
profiles and concentration boundary layer thicknesses. The interfer-
ograms, which appear due to the interference of two laser beams, are
determined by the refraction coefficient of the solute, which in turn
depends on the concentration of the substance. When the solute is
uniform, the interference fringes are straight and they bend when a
concentration gradient appears. The concentration profile C(x,t) is
determined by the deviation of the fringes from a straight course.
Since the concentration C and the refraction coefficient are assumed
to be linear, we have:
λdðx; t Þ
C ðx; t Þ = C0 + a ;
hf
where C0 is the initial substance concentration, a the proportion-
ality constant between the concentration and the refraction index
(a = 2.92 × 105 mol/m3 for the colistin aqueous solution), λ the
wavelength of the laser light, h the distance between the fringes in
the field where they are straight lines, and f the thickness of the
solution layer in the measurement cuvette. The concentration
boundary layer thickness δ was defined as the distance from the
gel–water interface to the point at which the deviation d of the
interference fringe from its straight line run is 10% of the fringe
thickness. By recording the interferograms at a given time interval
one can reconstruct the concentration profiles at different times.
Such profiles were used to calculate the membrane permeability
coefficient.
The system under study consists of two glass cuvettes (internal
dimensions: 70 mm high, 10 mm wide, 7 mm optical path length). The
measurement cuvettes are made of optical glass of high uniformity.
The data presented in Figs. 3 and 4 show that the modification of
the laser interferometric method (by using a gel instead of solutions)
is suitable for qualitative and quantitative diffusion measurements.
Fig. 3 shows the amount of free colistin released from 1% agarose gel in
the presence of both P. mirabilis LPSs (R45, S1959) and saponins. The
concentration profiles depicted in Fig. 4 are the result of mathematical
analysis of the interferograms presented as inserts in Fig. 4.
Mathematical analysis and comparison of the time-dependent
concentration profiles indicate that free colistin from the LPS/colistin
Fig. 3. The amount of aggregate-free colistin released from 1% agarose gel in the
presence of P. mirabilis LPS (R45 or S1959) at 250 μg/ml and saponin at 75 μg/ml at
room temperature measured by the laser interferometric system. Representative results
are presented.
 M. Arabski et al. / Journal of Microbiological Methods 77 (2009) 178–183 181

Fig. 5. The effect of the saponin on the growth of P. mirabilis S1959 or R45 after 18 h at
37 °C. Error bars denote SD Statistic analysis was made by ANOVA. The results displayed
Fig. 4. The concentration profiles of aggregate-free colistin released from 1% agarose gel are the mean of three independent experiments.
in the presence of P. mirabilis LPS (R45 or S1959) at 250 μg/ml and saponin at 75 μg/ml
after 40 min of culture at room temperature measured by the laser interferometric
system. Inside the figure are the interferograms for 40 min of the experiment.
Representative results are presented. Saponin alone does not influence the release of
Based on the data presented in Fig. 5, we used a constant amount
colistin (results not shown). of saponin at a concentration of 15 μg/ml to evaluate its influence on
the growth of the Proteus strains in the presence of antibiotics (Fig. 6).
The 15 μg/ml of saponin was the highest amount that did not sig-
aggregates immobilised in 1% agarose was more efficiently released nificantly affect bacterial growth. In addition to colistin, we chose
from smooth P. mirabilis S1959 LPS (35.03 × 109 mol/m2) than from the beta-lactam antibiotic ampicillin, which does not aggregate with
rough R45 LPS (12.79 × 109 mol/m2). After 40 min the amount of free LPSs. As expected, both strains were sensitive to ampicillin and re-
colistin released from the S1959 LPS/colistin aggregates was 2.74 sistant to colistin (Fig. 6). After 18 h at 37 °C we observed sharp
times higher than from the rough R45 LPS/colistin mixtures. decreases in CFU/ml of the rough P. mirabilis R45 and smooth S1959
The amount of free colistin not bound to LPSs and released from the strains in the presence of ampicillin at a dose of 8 μg/ml. The presence
agarose gel was lower in the presence of saponin (75 μg/ml). After of 15 μg/ml saponin retarded the effectiveness of ampicillin towards
40 min of incubation the amount of released colistin was 1.22 times the P. mirabilis R45 strain. This effect was much less visible in the
lower for S1959 LPS/colistin than for S1959 LPS/colistin/saponin ag-
gregates, i.e. 35.03 × 109 mol/m2 versus 28.63 × 109 mol/m2, respec-
tively. The difference was even greater for the rough (Re type) form of P.
mirabilis LPS. It was 2.27 less for R45 LPS/colistin than for the R45 LPS/
colistin/saponin mixtures (12.79 × 109 mol/m2 and 5.62 × 109 mol/m2,
respectively).
The concentration boundary layers occurrence reflected the amount
of the substance released from agarose gel. The substance diffusion
was non-linear from time point to time point in each experiment. It
increases rapidly for initial times and slower for later times. Such a
course of changes in substance flow is predetermined by significant
concentration changes on the boundary of the agarose gel solution. The
concentration boundary layers formation process leads to a significant
reduction in the substance concentration difference on the agarose gel.

3.3. The effect of saponin and colistin/ampicillin on bacterial growth

The above experiments were done with highly purified LPS, co-
listin, and saponin mixtures. The question was if the observed dif-
ferences in the interactions of smooth and rough LPSs with colistin
would be detected with whole bacteria as well. The influence of
saponin on cultures of smooth P. mirabilis S1959 and its deep rough
R45 strain of this control experiment are presented in Fig. 5. Fig. 5
shows the effects of saponin at concentration ranging from 1.12–
500 μg/ml on the growth of the P. mirabilis S1959 and R45 strains
incubated for 18 h at 37 °C. We observed statistically significant dose-
dependent decreases in CFU/ml of both Proteus strains in the presence
of saponin. The number of cells of P. mirabilis S1959 was reduced by
31 μg/ml of saponin. In the case of the deep rough strain R45, 62 μg/ml
Fig. 6. The effect of 15 μg/ml saponin on the growth of P. mirabilis R45 or S1959 after
saponin was needed for significant bacterial cell reduction. This in- 18 h at 37 °C in the presence of colistin or ampicillin in the concentration range of 0–
dicated a higher sensitivity of the smooth cells P. mirabilis S1959 to 500 μg/ml. Error bars denote SD. Statistic analysis was made by ANOVA. The results are
the saponin than its rough Re type mutant. the means of three independent experiments.
182 M. Arabski et al. / Journal of Microbiological Methods 77 (2009) 178–183
case of the smooth S1959 strain. In contrast to ampicillin, the growth
of the P. mirabilis R45 or S1959 strains in the presence of a constant
amount of saponin was statistically reduced in the presence of rising
concentration of colistin. In the R45 and S1959 strains this was ob-
served at doses of 62 and 250 μg/ml of colistin, respectively.
The results with whole bacterial Proteus cells confirmed the data
on colistin/LPS/saponin interaction determined by the laser inter-
ferometric method. Saponin was more effective in influencing
colistin's action against the rough R45 than the smooth S1959 strain.
This was also the case with the isolated S1959 and R45 LPSs tested by
aggregation and by the diffusion method.
4. Discussion
In this study we used two P. mirabilis LPSs that differ in their
polysaccharide content. The smooth (S) P. mirabilis S1959 strain pro-
duces complete LPS with the O-specific polysaccharide containing
lysine residue. The deep-rough mutant of S1959 (R45 strain, Re type)
synthesizes LPS depleted of the O-specific part and the core oligo-
saccharide (Kaca and Ujazda, 1996). It is well known that the LPSs
of Gram-negative bacteria, in addition to other factors, are respon-
sible for resistance against polymyxin types of antibiotics as a result
of 4-amino-arabinose-substituted phosphate group in the lipid A part
of the LPS with colistin interaction (Boll et al., 1994, Brandenburg
et al., 2002a,b; Helander et al., 1996; Kaca et al., 1990). The in vitro
interaction of the LPSs and colistin resulted in aggregate formation
and the precipitation of LPS/colistin mixtures from water solution.
Also, LPSs isolated from naturally colistin-resistant Proteus strains
precipitate antibiotic. The laser interferometric method used in this
study showed that 2.4 times less antibiotic diffused from S1959LPS/
colistin than from R45LPS/colistin aggregates (see Fig. 3). The dif-
ferences in colistin binding by the two P. mirabilis LPSs might be due
to differences in their polysaccharide content. The long O-specific
polysaccharide and core oligosaccharide of S1959 LPS may prevent
access of colistin to the lipid A part of the LPS. In addition, the poly-
saccharide parts of S1959 LPS make these molecules more hydrophilic
than R45 LPS.
The goal of the presented study was to discover the role of
detergent-like substances, i.e. saponins, on LPS–colistin interactions.
We showed (Fig. 1) that pre-incubation of both LPSs with saponins
increased, in a saponin dose-dependent manner, LPS/colistin aggre-
gate formation. This observation was also confirmed by analysing the
concentration boundary layers by interferometry, which showed that
significantly much less colistin diffused from the S1959 and R45 LPSs
aggregates pre-incubated with saponins (Fig. 3). The similar action of
the saponins on S and Re LPSs may indicate that lipid A is targeted by
saponins. The disaggregation of LPS aggregates might by responsible
for enhancing the immunogenicity and immune response on LPSs
(Takahashi et al., 1990).
The mechanism of saponins influence on the eucaryotic biological
membranes is based on their affinity for membrane bound sterols. The
specific interactions with cholesterol within membranes result in
formation of aggregates, which build pores, thus allowing penetration
even of large molecules (Karabaliev and Kochev, 2003). About 90% of
the surfaces of naturally cholesterol-free Gram-negative bacterial cell
wall outer-membranes are covered by LPS. We suggest that saponin
might interact with lipid A part of Proteus LPSs and by that increasing
the permeability of bacterial cell wall. The saponin partially inhibited
the growth of the S and Re P. mirabilis strains at doses of 31–500 μg/
ml. A sub-inhibitory dose — 15 μg/ml of saponins alone do not
reduced the numbers of P. mirabilis S1959 and R45 cells. However, the
presence of colistin or amipicillin and 15 μg/ml of saponins reduced
the amount of P. mirabilis S1959 and R45 cells. Ampicillin is a beta-
lactam antibiotic which inhibits transpeptidation during bacterial
cell-wall peptidoglycan synthesis and both Proteus strains used were
sensitive to it, contrary to colistin. The saponins enhanced sensitivities
of S and R P. mirabilis cells towards colistin and amipicillin. One may
proposed that saponins binds to lipid A part of LPS may resulted on an
increase in bacterial cell wall outer-membrane permeabilities and by
that facilitated antibiotics penetration into the bacterial cells.
In conclusions, the presented study showed that the modified laser
interferometric method might be used for antibiotic/LPS diffusion
studies. The results of the model studies with purified LPSs were
confirmed in a whole bacterial cell experiment. Further studies are
needed to discover the exact mechanism of interaction of LPSs, antibiotic
and saponins.
Acknowledgements
The work was supported by grant no. NN304411433 from the
Ministry of Science and Higher Education, Poland.
References
Amano, K.I., Cedzyński, M., Swierzko, A.S., Kyohno, K., Kaca, W., 1996. Comparison of
serological reactions of Rickettsiae-infected patients and rabbit anti-Proteus OX
antibodies with Proteus OX2, OX19 and OXK lipopolysaccharides. Arch. Immun.
Therap. Exp. 44, 235–240.
Arabski, M., Wąsik, S., Dworecki, K., Kaca, W., 2007. Laser interferometric determination
of ampicillin and colistin transfer through cellulose biomembrane in the presence
of Proteus vulgaris O25 lipopolysaccharide. J. Memb. Sci. 299, 268–275.
Boll, M., Radziejewska-Lebrecht, J., Warth, C., Krajewska-Pietrasik, D., Mayer, H.,
1994. 4-amino-4-deoxy-L-arabinose in LPS of enterobacterial R-mutants and
its possible role for their polymyxin reactivity. FEMS Immunol. Med. Microbiol.
8, 329–341.
Brandenburg, K., Arraiza, M.D., Lehwark-Ivetot, G., Moriyon, I., Zähringer, U., 2002a. The
interaction of rough and smooth form lipopolysaccharides with polymyxins as
studied by titration calorimetry. Thermochim. Acta 394, 53–61.
Brandenburg, K., Moriyon, I., Arraiza, M.D., Lewark-Yvetot, G., Koch, M.H.J., Seydel, U.,
2002b. Biophysical investigations into the interaction of lipopolysaccharide with
polymyxins. Thermochim. Acta 382, 189–198.
Burall, L.S., Harro, J.M., Li, X., Lockatell, C.V., Himpsl, S.D., Hebel, J.R., Johnson, D.E.,
Mobley, H.L., 2004. Proteus mirabilis genes that contribute to pathogenesis of
urinary tract infection: identification of 25 signature-tagged mutants attenuated at
least 100-fold. Infect. Immun. 72, 2922–2938.
Daza, R., Gutierrez, J., Piedrola, G., 2001. Antibiotic susceptibility of bacterial strains
isolated from patients with community-acquired urinary tract infections. Int. J.
Antimicrob. Agents 18, 211–215.
Dworecki, K., 1995. Interferometric investigations of near-membrane layers. J. Biol. Phys.
21, 37–49.
Dworecki, K., Ślęzak, A., Drabik, M., Ornal-Wąsik, B., Wąsik, S., 2006. Determination of
the membrane permeability coefficient under concentration polarisation condi-
tions. Desalination 198, 326–334.
Dworecki, K., Wąsik, S., Ślęzak, A., 2003. Temporal and spatial structure of the
concentration boundary layers in a membrane system. Physica A 326, 360–369.
Galanos, C., Luderitz, O., Westphal, O., 1969. A new method for the extraction of R
lipopolysaccharides. Eur. J. Biochem. 9, 245–249.
Gupta, K., Sahm, D.F., Mayfield, D., Stamm, W.E., 2001. Antimicrobial resistance among
uropathogens that cause community-acquired urinary tract infections in women:
a nationwide analysis. Clin. Infect. Dis. 33, 89–94.
Helander, I.M., Kato, Y., Kilpeläinen, I., Kostiainen, R., Lindner, B., Nummila, K., Sugiyama, T.,
Yokochi, T., 1996. Characterization of lipopolysaccharides of polymyxin-resistant and
polymyxin-sensitive Klebsiella pneumoniae O3. Eur. J. Biochem. 237, 272–278.
Kaca, W., Radziejewska-Lebrecht, J., Bhat, U.R., 1990. Effect of polymyxins on the
lipopolysaccharide-defective mutants of Proteus mirabilis. Microbios 61, 23–32.
Kaca, W., Ujazda, E., 1996. Studies of antibiotic resistance of rough and smooth Proteus
mirabilis strains and influence of polymyxin E on their lipopolysaccharide composi-
tion. Acta Microbiol. Pol. 45, 161–168.
Karabaliev, M., Kochev, V., 2003. Interaction of solid supported thin lipid films with
saponin. Sensors and Actuators B 88, 101–105.
McCoy, A.J., Liu, H., Falla, T.J., Gunn, J.S., 2001. Identification of Proteus mirabilis mutants
with increased sensitivity to antimicrobial peptides. Antimicrob. Agents Che-
mother. 45, 2030–2037.
Melekos, M.D., Naber, K.G., 2000. Complicated urinary tract infections. Int. J. Antimicrob.
Agents 15, 247–256.
Mobley, H.L., Belas, R., 1995. Swarming and pathogenicity of Proteus mirabilis in the
urinary tract. Trends Microbiol. 3, 280–284.
Mobley, H.L., Hausinger, R.P., 1989. Microbial ureases: significance, regulation, and
molecular characterization. Microbiol. Rev. 53, 85–108.
Rozalski, A., Sidorczyk, Z., Kotelko, K., 1997. Potential virulence factors of Proteus bacilli.
Microbiol. Mol. Biol. Rev. 61, 65–89.
Sen, S., Makkar, H.P.S., Muetzel, S., Becker, K., 1998. Effect of Quillaja saponaria saponins
and Yucca schidigera plant extract on growth of Escherichia coli. Lett. Appl. Microbiol.
27, 35–38.
Senior, B.W., 1979. The special affinity of particular types of Proteus mirabilis for the
urinary tract. J. Med. Microbiol. 12, 1–8.
 M. Arabski et al. / Journal of Microbiological Methods 77 (2009) 178–183
Shimizu, S., Iyobe, S., Mitsuhashi, S., 1977. Inducible high resistance to colistin in Proteus
strains. Antimicrob. Agents Chemother. 12, 1–3.
Spark, S.G., Light, M.E., van Staden, J., 2004. Biological activities and distribution of plant
saponins. J. Ethnopharmacol. 94, 219–243.
Takahashi, H., Takahashi, T., Morein, B., Putney, S., Germain, R.N., Berzofsky, J.A., 1990.
Induction of CD8+ cytotoxic T-cells by immunization with purified HIV-1 envelope
protein in ISCOMs. Nature 344, 873–875.
Vinogradov, E., Radziejewska-Lebrecht, J., Kaca, W., 2000. The structure of the
carbohydrate backbone of core-lipid A region of the lipopolysaccharides from Pro-
teus mirabilis wild-type strain S1959 (serotype O3) and its Ra mutant R110/1959.
Eur. J. Biochem. 267, 262–269.
183
Vinogradov, E., Thomas-Oates, J., Brade, H., Holst, O., 1994. Structural investigation of
the lipopolysaccharide from Proteus mirabilis R45 (Re-chemotype). J. Endotoxin.
Res. 1, 99–206.
Westphal, O., Jann, K., 1965. Bacterial lipopolysaccharides: extraction with phenol-
water and further application of the procedure. In: Whistler, R.C. (Ed.), Methods in
Carbohydrate Chemistry. Academic Press, Inc., New York, pp. 5–83.
Ziółkowski, A., Shashkov, A.S., Swierzko, A.S., Senchenkova, S.N., Toukach, F.V.,
Cedzyński, M., Amano, K.I., Kaca, W., Knirel, Y.A., 1997. Structures of the O-antigen
of Proteus bacilli belonging to OX group (serogroup O1–O3) used in Weil–Felix test.
FEBS Lett. 411, 221–224.