Department of Pharmacology, School of Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy
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Abstract |
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Introduction |
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The effect of antibiotic MICs can be investigated and visualized easily in vitro; the basic concept of antibiotic therapy is to try to obtain similar results in vivo.
The most commonly adopted method of antibiotic administration in the treatment of infectious diseases is intermittent administration and in this situation the pharmacokinetic curves show that antibiotic concentrations have a sinusoidal behaviour. They exceed MICs only for a certain period of time, after which they drop to below the MIC until the next administration starts the cycle again.
Although these sub-MIC concentrations do not kill bacteria, they are still capable of modifying their physico-chemical characteristics and the architecture of their outermost surface, and of interfering with some important bacterial cell functions such as adhesiveness, surface hydrophobicity, fimbriation, motility and hostbacteria inter-actions, such as phagocytosis, killing and the production and release of reactive oxygen species (ROS) from phagocytes.28 The final result is a reduction in bacterial virulence factors.
On the basis of these effects, and in accordance with the National Committee for Clinical Laboratory Standards,9 a sub-MIC concentration can be defined as the concentration of an antimicrobial agent that is not active on bacterial growth but is still active in altering bacterial biochemistry and shape in vitro and in vivo, and thus reducing bacterial virulence.
Knowledge of the effects on bacteria exposed to sub-MIC concentrations, and the correlation of these pharmaco-dynamic findings with pharmacokinetic curves, is considered to be useful for optimizing therapy.2,10,11
Cefodizime is a recently developed broad-spectrum, third-generation cephalosporin with a 2-aminothiazolyl group. Its molecule is characterized by the presence of a 3-methyl-5-carboxymethyl-1,3-thiazole-2-thio chain at position 3' of the dihydrothiazine rings. This side chain confers a longer elimination half-life and some new immunological properties.12 Although some scattered information exists in the literature,13 the effects of sub-MIC concentrations of cefodizime have not yet been fully investigated.
The aim of the present study was to investigate the pharmacodynamic activity of such concentrations on various bacterial cell functions in order to evaluate its ability to interfere with bacterial virulence factors.
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Materials and methods |
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Bacterial strains and culture conditions.
Two Escherichia coli strains taken from clinical isolates (urinary infections), and lyophilized E. coli ATCC 25922 (D.I.D., Milan, Italy), were used.
Cell suspensions of each organism were prepared from overnight cultures in tryptic soy broth (Sigma, Milan, Italy) at 37°C under static conditions. The organisms were harvested, washed three times in phosphate buffered saline (PBS) (0.02 M phosphate and 0.15 M NaCl pH 7.3) and adjusted to 13 x 108 organisms/mL, as determined by direct microscopic counts, using interference contrast microscopy, in a PetroffHouser bacterial counting chamber (Thomas Scientific, Swedesboro, NJ, USA).
The bacterial strains (inoculum 106 bacteria/mL) were tested to establish the MIC of cefodizime by using serial two-fold dilutions in MuellerHinton broth (Oxoid, Milan, Italy). The MIC was defined as that concentration of antibiotic that led to no visible bacterial growth after overnight incubation at 37°C.
Collection of epithelial cells.
Human buccal epithelial cells were collected by scraping the mucosa of each cheek of apparently healthy non-smoking donors with a sterile plastic spatula, which was then twirled in 2 mL PBS. The suspensions obtained from three to five subjects were pooled. The epithelial cells were passed through a needle (150 µm diameter) to disrupt cell aggregates, and washed by centrifuging three or four times to free them from debris and non-adherent bacteria (200g, 10 min, 21°C).
PBS was added to the washed cell suspensions to give 3 x 105 cells/mL as determined by a direct microscopic count (interference contrast microscopy) in a Bürker chamber (Passoni, Milan, Italy).
In vitro adhesion assay.
The ability of the bacteria to adhere to epithelial cells was investigated by mixing 1:1 volumes of adjusted suspensions of bacteria and epithelial cells in polystyrene tubes. The tubes were rotated end-over-end at 10 rpm for 60 min at 37°C. The epithelial cells were separated from the non-adherent bacteria by means of centrifugation at 100g for 7 min. The final epithelial cell pellet was resuspended in a small quantity of PBS, placed on a round microscope coverslip and dried.
Scanning electron microscopy.
The coverslip with the cells was fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.1 for 60 min. After dehydration in graded alcohols, the coverslips underwent critical-point drying, were coated with 200 Å gold, and then bacteria counted with a scanning electron microscope (SEM).
The adhesiveness of the bacteria to the epithelial cells was determined by counting the number of epithelial cells with 40 adhering bacteria per 100 cells.14 Each test was performed twice. Control epithelial cell suspensions were always included to provide data on the number of bacteria that were already attached (natural acquisition) when the cells were collected.
Inhibition test.
The bacteria grown in the presence of sub-inhibitory concentrations of cefodizime (37°C for 18 h), ranging from 1/2 to 1/128 x MIC, or with the same amount of medium but without any antibiotic, were harvested and resuspended in PBS at a final concentration of 3 x 108 bacteria/mL. They were challenged with epithelial cells as in the adherence assay, and the samples were prepared for SEM analyses.
Haemagglutination assay
Anti-coagulated guinea pig and human group A erythrocytes were collected, washed three times in saline and finally suspended 1:1 in Alsever solution (home-made) (2.05% glucose, 0.8% sodium citrate, 0.42% NaCl, 0.055% citric acid pH 6.1). The washed erythrocytes were then stored at 4°C and used within 5 days.
The haemagglutination tests15,16 were performed by pipetting 20 µL of a 4 x 108/mL suspension of erythrocytes in saline and 20 µL of the bacterial suspension (1 x 109 cells/mL) on to microscope slides. The slides were rotated gently at ice temperature for 5 min, and then read again after 10 min. To test for mannose sensitivity, 20 µL of 1% mannose solution was added to a duplicate slide containing undiluted bacteria. The haemagglutination test was applied to samples of each bacterial strain grown with the different sub-MIC concentrations of cefodizime.
The results were recorded as grade 4 when coarse clumping was complete in a very short period of time (a few seconds); 3 when moderate clumping commenced within 30 s; 2 when fine clumping appeared only after 1 min; 1 when very fine granules appeared after 35 min; and 0 when no clumping was visible.15
Hydrophobicity assay
The hydrophobicity of the bacterial cell surfaces was investigated using the salt aggregation test with slight modifications.17 Briefly, a bacterial suspension of 25 µL (1 x 108 bacterial cells/mL in 0.001 M sodium phosphate buffer pH 6.3) was mixed with an equal volume of ammonium sulphate [(NH4)2SO4)] at a concentration of 1.8 M, pH 6.3. The bacteria/salt solution mixture was rocked gently for 2 min at 20°C, and aggregation was read visually against a black background using a stereomicroscope.
This method was originally used to distinguish between fimbriated and non-fimbriated E. coli, but it is clear from the physico-chemical principle of the interaction18 that it can be usefully adopted to investigate cell surface hydrophobicity19 and the effect of drugs. To quantify bacterial hydrophobicity better, the number of aggregates with 20 bacteria was counted in randomly chosen microscopic fields. The test was repeated for each strain and for each sub-MIC concentration of cefodizime.
Motility
The bacteria were grown overnight in tryptic soy broth at 37°C, and a 5 µL portion of the cell suspension (3 x 108 cells) was placed on the agar surface of the semi-solid swarming medium (agar motility) (home-made) (1% tryptone, 0.5% NaCl, 0.25% agar dissolved in distilled water pH 7.1).5
After inoculation, the assay plates (Petri dishes diameter 9 cm, containing 10 mL of medium) were placed in a water-saturated plexiglass incubator (37°C) (home-made). The plates were illuminated obliquely and viewed against a dark background, and the diameter of the swarming zones was measured with a ruler at regular time intervals.
The assays were performed on each strain grown overnight with the different sub-MIC concentrations of cefodizime and inoculated on to plates with the swarming medium containing the same sub-MIC concentrations. The control plates had no antibiotic.
Filamentation
The morphology of bacilli does not change at the same time or in the same ratios for all of the organisms in a given population of bacteria exposed to a given concentration of an antibiotic20 and so, in order to verify the exact kinetics of morphological changes at different times, a 700 µL sample was withdrawn from the cultures at 1, 2, 4 and 8 h and processed for scanning electron microscopy.
This procedure21 was adopted after control samples had shown that the removal of 700 µL did not interfere with growth, and so it was possible to compare the evolution of the changes in the same culture.
For each determination, the 700 µL sample was added to 2 mL of broth and centrifuged at 450g; the final pellet was resuspended in 100 µL of PBS, placed on a round microscope coverslip and dried. The coverslip was then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.1, for 2 h. After dehydration in graded alcohols, the coverslip underwent critical-point drying, was coated with 200 Å gold and observed in an SEM. The microscopic fields to be counted were selected by means of random scanning.
The morphological characteristics before and after incubation with the different sub-MIC concentrations of cefodizime were classified and quantified without the observer knowing the concentration of antibiotic or the duration of incubation. The lengths of the filaments, and their proportions in the total number of microorganisms per 100 randomly observed bacteria, were recorded. The normal length of E. coli can vary from 1.2 to 2.3 µm: at the middle of the division cycle it is about 5 µm. Organisms of up to 15 µm were classified as short filaments and those longer than 15 µm as long filaments.20
Microelectrophoretic mobility measurement
The microorganisms were harvested before and after incubation with sub-MIC concentrations of cefodizime, washed and finally resuspended in standard phosphate buffer (1.194 g/L Na2HPO412 H2O, 0.454 g/L KH2PO4). Velocity measurements were made at the determined stationary levels in a flat Choucroun cell22,23 (home-made) maintained at 25°C. This cell is particularly well suited for pathogenic organisms since the complete apparatus can be easily cleaned and autoclaved.22
A minimum of 30 individual timings over a distance of 20 µm, with the current passing first in one and then in the other direction, were made for the determination of the mean mobility value. The electrophoretic mobility was expressed as µm/s/V/cm and calculated from the mean velocity of the bacteria, the specific resistance of the suspension and the current.
Phagocytosis and killing
Collection of human polymorphonuclear leucocytes (PMNs).
Peripheral venous blood was drawn from healthy adult donors into heparinized (5 U/mL) syringes. The blood (5 mL) was stratified on 3 mL Polymorphoprep (Nycomed Pharma, Oslo, Norway), and the PMNs separ-ated by means of density gradient centrifugation. When necessary, any residual erythrocytes in the granulocyte preparation were lysed with NH4Cl solution pH 7.4, 0.15 mol/L.
The PMNs were collected and washed in glutaminecontaining RPMI 1640 (Sigma) after being passed through a 150 µm internal diameter needle in order to disrupt cell aggregates. They were then tested for viability by means of trypan blue exclusion. The final cell suspension was adjusted to the cell numbers needed for each test by counting in a Bürker chamber with interference contrast microscopy.
Phagocytosis and bacterial killing.
The phagocytic capacity of the PMNs, as well as their ability to kill bacteria, were determined using a fluorochrome assay (acridine orange stain), which distinguishes viable from dead microorganisms intracellularly. The procedure of Bellinati-Pires et al.24 was used with slight modifications.
Equal volumes of PMNs (2 x 106 cell/mL) and preopsonized bacteria (2 x 107 bacteria/mL) were mixed in tubes at a ratio of 1:10 (PMN/bacteria) in a final volume of 0.5 mL. The tubes were incubated at 37°C and rotated end-over-end (6 rpm) for 30 min. Phagocytosis was stopped by placing each tube in an ice bath and adding 0.5 mL of ice-cold medium to the suspension of bacteria and PMN. The non-ingested bacteria were removed by centrifugation (100g for 7 min) and two washes. The pellet was stained with 200 µL of 14.4 mg/L acridine orange pH 7.2 (Sigma) in medium for 1 min. Immediately after staining, 1 mL of ice-cold Hanks' buffered salt solution (HBSS) was added to the PMN suspension, which was then centrifuged at 160g for 7 min at 4°C. The cells were washed twice with ice-cold HBSS and kept in an ice bath until microscopic examination.
Just before the observation of each cell sample, a drop of the cell suspension was wet-mounted on a microscope slide and sealed with nail varnish. It was then examined immediately under oil immersion by means of a UV epifluorescence microscope equipped with an excitation filter at 450490 nm, a beam split mirror at 510 nm and a cut-off filter at 520 nm. A short time interval of no longer than 10 min was established for each slide reading: if this was not sufficient, another slide was prepared from the ice-cold suspension. A total of 100 PMNs was observed for each slide.
The number of cells phagocytosing at least three bacteria/100 PMNs gave the percentage phagocytosis, whereas the average number of bacteria in phagocytosing cells gave the phagocytic index. The percentage of killed bacteria was obtained using the formula: [number of dead bacteria/ (number of dead + live bacteria)] x 100. The killing index was the average number of dead bacteria in phagocytosing PMNs.
After staining with acridine orange to avoid the overestimate of phagocytosis that may occur if E. coli are attached to the surface but not yet internalized in the PMNs, the technique of Hed25 and Goldner et al.26 was used to quench the extracellular membrane-adherent microorganism fluorescence by crystal violet (500 mg/L for 20 s). Since crystal violet does not penetrate PMNs intracellularly, it does not alter the fluorescence of ingested microorganisms.
The same procedure was followed in order to investigate the effect of the exposure of PMNs to sub-MIC concentrations of cefodizime.
Measurement of oxidative burst response by chemiluminescence (luminol-amplified chemiluminescence)
Luminol-amplified chemiluminescence (LACL) was investigated using a slightly modified version of the procedure of Robinson et al.27 for pathogenic organisms. In brief, 0.1 mL of a suspension of PMNs (1 x 106 cells/mL) and 0.30 mL HBSS with Ca2+ and Mg2+ plus 0.05 mL 104 M luminol [Sigma; diluted from a first stock solution in dimethylsulphoxide (DMSO)] was introduced into a 3 mL flatbottomed polystyrene vial. This vial was placed in a lightproof chamber of the Luminometer 1250 (Bio Orbit, Turku, Finland), and the carousel rotated to bring the sample in line with the photomultiplier tube in order to record background activity. A suspension of pre-opsonized killed Candida albicans cells (2 x 107 cells/mL) in a final volume of 0.05 mL was added, and the resulting light output in mV was recorded continuously on a chart recorder and, simultaneously, by means of a digital printout set for 1 or 10 s recording integrals.
All of the constituents of the mixture were kept at 37°C during the reaction by means of water passing from a thermostatically controlled circulator through a polished hollow metal sample holder. No mixing was done during the recording. The gain control was set to give a reading of 10 mV for a built-in standard. A background subtraction control zeroed the instrument before the addition of the opsonized cells. The patterns of LACL responses were determined by calculating the initial slope, peak (mV), time to peak, slope of declining response and area under the curve (AUC). The peaks correlate well with the other parameters, and so a first analysis can be obtained simply in terms of peak counts. However, since oxidant radical production is a phenomenon with its own time course, a simple peak (which freezes measurement at a single time) does not completely characterize the phenomenon over time, and so the data are also expressed as a curve. The analysis was completed by investigating the effects of sub-MIC concentrations of cefodizime on PMN oxidative bursts.
Data analysis
The statistical significance of the differences was calculated using the t test and when necessary, the analysis of variance between treatments, followed by multiple pair comparisons according to Dunnett's test when the differences were statistically significant. Each test was repeated six to nine times for each concentration; the mean values ± S.D. are reported. The differences were considered statistically significant when P 0.05.
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Results |
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During the control test performed before the antibiotic challenge, all of the bacterial strains adhered well to human epithelial cells, with a certain degree of variability in the number of bacteria per cell.
The mean of the MICs of cefodizime, observed for the strains investigated, was 1 mg/L and, after incubation with sub-MIC concentrations, bacterial adhesiveness significantly decreased. The data are summarized in Table I and the normalized percentages of inhibition versus control are shown in Figure 1
. Instead of a peak of inhibition at 1/2 x MIC (as frequently occurs in these studies), the inhibition of adhesiveness was high at the sub-MIC concentrations from 1/2 to 1/8 x MIC; the figures then progressively returned to control values. The inhibition curve was still significant at 1/32 x MIC.
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The haemagglutination and hydrophobicity assays were not tested between 1/2 and 1/8 x MIC because the presence of a large number of filamentous forms interfered with the analysis of these two parameters. The assays were thus performed between 1/16 and 1/128 x MIC, and significant inhibition was still present at 1/16 x MIC (Tables II and III).
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The means of the different radii of the swarming halo read at different times and sub-MIC concentrations are shown in Table IV. On average, the inhibition of swarming was significant down to 1/32 x MIC.
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The morphological changes induced by the sub-MIC concentrations of cefodizime consisted mainly in filament-ation. Table V shows the frequency of normal length, short/long filaments and ghosts observed for every 100 microorganisms at the different times and concentrations. Interference with the normal shape of bacilli is conditioned by both the time of exposure and the concentration of antibiotic. The maximum interference was reached at 1/8 x MIC.
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Microelectrophoretic mobility
The microelectrophoretic mobility findings are shown in Table VI; for the same reasons as above (filamentous forms), the measurements started from 1/16 x MIC. In one strain, a significant reduction in electrophoresis was present down to 1/32 x MIC; in a second strain, this occurred at 1/16 x MIC; in the last strain no significant differences from control values were observed.
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When E. coli is challenged with sub-MIC concentrations of cefodizime a high incidence of filamentation is induced mainly from 1/2 x MIC to 1/8 x MIC. The presence of long and very long filaments interferes with the analysis of phagocytosis and killing because a PMN can be filled by only one long filament while normally activated PMNs generally phagocytose more than one normal length bac-illi. We found that the extension of the study from 1/16 to 1/128 x MIC (sub-MIC concentrations with progressive disappearance of filamentation) made it possible to evaluate phagocytosis and killing more precisely.
The number of bacteria associated with PMNs (% phagocytosis and phagocytic index) after exposure to 1/161/128 x MIC of cefodizime was not significantly different from that of the unexposed bacteria (Table VII). The interesting finding was that the overall killing rate at 1/16 x MIC was still significantly higher than in the control (Table VII
). The cefodizime sub-MIC concentrations did not change the percentage of phagocytosis or the phagocytic index, but significantly increased the killing of bacteria down to 1/32 and 1/16 x MIC (Table VII
).
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Possible interferences with oxidative bursts were investigated by exposing the neutrophils to different sub-MIC concentrations of cefodizime. The findings shown in Figure 2, in which the data are expressed as percentages versus control, indicate that cefodizime did not interfere with this important aspect.
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Discussion |
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Sub-MIC concentrations of cefodizime have been shown to be able to reduce E. coli adherence to human epithelial cells at concentrations down to 1/32 x MIC and, given that the mean MIC observed was 1 mg/L, this means that the interference with bacterial adhesiveness is still significant at 0.03 mg/L. From 1/2 to 1/8 x MIC an unusually strong inhibition of adhesiveness has been observed which correlates with the very high degree of filamentation observed at those concentrations.13
The induction of long filaments caused by the binding of cefodizime to penicillin-binding protein 3 (PBP3) peaked at 1/8 x MIC, which was the same sub-MIC at which the maximum inhibition of adhesiveness occurred. This is in agreement with the findings of Ofek et al.,30 who found that the elongated forms are less adhesive than normal forms. The presence of a high degree of filamentation also influenced the readability of the other parameters investigated, and forced us to extend the study from 1/8 to 1/128 x MIC; in this type of study the investigation is generally stopped after the first two or three sub-MIC concentrations.
The phenomenon of surface hydrophobicity is often cited when interpreting bacterial adhesiveness because it has been observed that adhesiveness increases with increasing and decreases with decreasing hydrophobicity.28,29,31 The reduction in this parameter induced by cefodizime was still significant at 1/16 x MIC. The presence of surface appendages, such as fimbriae (pili) or fibrils, increases hydrophobicity and haemagglutination and renders microbial cells more adhesive than non-fimbriate cells32,33 and so to complete the information concerning this aspect both hydrophobicity and haemagglutination were also investigated; the result was a significant reduction down to 1/16 x MIC for both parameters.
The electrophoretic mobility of bacteria is also a measurement of their physico-chemical properties and the results obtained with sub-MIC concentrations of cefodizime (which are in agreement with those of Nomura et al. for Klebsiella pneumoniae34) reflect the ability of the drug to interfere with the normal behaviour of this parameter. Comparative analysis of the effects of these virulence factors shows that hydrophobicity, fimbriation and electrophoretic mobility provide useful information about the alterations induced by cefodizime on the physico-chemical characteristics of the molecular architecture of the outermost surface of bacteria; but the interference of cefodizime on adhesiveness gives better evidence of the biological effect of very low concentrations because the interaction with human cells mimics the mechanisms occurring in disease in vivo, which are not present in the case of other parameters. The fact that sub-MIC concentrations of cefodizime are capable of weakening some important virulence factors is also confirmed by the significant reduction in motility (swarming), which reduces the possibility of forming new colonies and spreading infection away from the first point of contact.
In the complex interaction between bacteria expressing virulence factors and a biological environment containing defensive eukaryotic cells such as phagocytes, but in the presence of an antimicrobial agent whose potency decreases over time, it is necessary to consider the possible interference of sub-MIC concentrations of cefodizime with the defensive functions of PMNs. Cefodizime concentrations below the MIC did not interfere with phagocytosis (% phagocytosis and phagocytic index) or with the oxidative bursts of neutrophils, but killing increased.
Investigations of antibioticbacteria interactions have concentrated upon killing [minimal bactericidal concentrations (MBC)] or growth inhibition [minimal inhibitory concentrations (MIC)], and less attention has been paid to interference with the expression of bacterial virulence factors (sub-minimal inhibitory concentrations). The knowledge that, even if sub-MIC concentrations of antibiotics do not kill or inhibit bacteria, they are still able to interfere with the parameters of bacterial virulence is important in relation to their therapeutic presence between administrations or in peripheral tissue for a certain period of time, after MBC or MIC has been reached.
Looking at the pharmacokinetic curve (Figure 3) of a common single daily dose of cefodizime, 1 g im35 and given that the average overall effects of cefodizime concentrations below the MIC on the expression of virulence factors can still be considered significant down to about 1/16 x MIC (which corresponds to 0.06 mg/L), the interpolation of this value with the pharmacokinetic curve shows that the effects of sub-MIC concentrations may last for as long as 12 h after the MIC value has been reached.
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Acknowledgments |
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Notes |
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References |
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Received 9 March 1999; returned 3 August 1999; revised 7 September 1999; accepted 3 October 1999