Effect of Antibiotics on Cell Surface Hydrophobicity of Bacteria ...

Microbiology Chemotherapy 2003;49:237–242 DOI: 10.1159/000072447

Received: September 13, 2002 Accepted after revision: March 6, 2003

Effect of Antibiotics on Cell Surface Hydrophobicity of Bacteria Causing Orthopedic Wound Infections Tama´s Kustos a Ildiko´ Kustos b Ferenc Kila´r c Ga´bor Rappai d Béla Kocsis b Departments of a Orthopedics, and b Medical Microbiology and Immunology, c Central Research Laboratory, Faculty of Medicine, and d Department of Statistics and Demography, Faculty of Economics, University of Pécs, Pécs, Hungary

Abstract Background: Despite antibiotic prophylaxis and treatment, the incidence of wound infections in orthopedic surgery is significant. Postoperative wound infection is a multifactorial process, which can be modified by several bacterial factors. Cell surface hydrophobicity of bacteria is a very important physicochemical feature, which has a great influence on the ability of bacteria to adhere to the surface of host cells or medical implants. Methods: In this study, the hydrophobic properties of thirteen bacterial strains (coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa) isolated from patients with postoperative deep wound infections following orthopedic procedures were determined by the salt aggregation test. Results were compared to the hydrophobicity of three Hungarian standard bacterial strains. The modifying effect of four antibiotics (cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin) – applied most often in our Department for prophylaxis and treatment of patients – were analyzed. Results: The cell surface hydrophobicity of cer-

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tain strains showed considerable changes after antibiotic treatment. These alterations indicated the decrease in hydrophobicity. Supra-inhibitory concentrations (2! minimum inhibitory concentrations, MIC) of the antibiotics were able to induce more frequent alterations in hydrophobicity than sub-inhibitory (0.5! MIC) levels. Conclusions: Alterations in cell surface hydrophobicity caused by antibiotics can modify the adhesion process and thus the pathogenicity of bacterial strains. These changes should be taken into consideration in the management of proper antibiotic prophylaxis and in the treatment of orthopedic patients. Copyright © 2003 S. Karger AG, Basel

Introduction

Studies on bacterial cell surface characteristics such as charge and hydrophobic properties provided valuable insights into the interaction of bacteria with host cell membranes [1]. Several molecules contribute to the hydrophobic effect of bacterial cells. Some of them are covalently bound to the cell wall (staphylococcal proteins [2]), or to the cytoplasmic membrane (lipoteichoic acid in Streptococcus pyogenes [3]), while others are secreted biomolecules (polysaccharides of Acinetobacter calcoaceticus [4]).

Ildiko´ Kustos Department of Medical Microbiology and Immunology Faculty of Medicine, University of Pécs, Szigeti u´t 12 HU–7643 Pécs (Hungary) Tel. +36 72 536 001 1905, Fax +36 72 536 253, E-Mail [email protected]

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Key Words Antibiotic treatment W Hydrophobicity W Orthopedic surgery W Wound infections

Materials and Methods Bacterial Strains Three Hungarian standard bacterial strains were used in the experimental series: Staphylococcus aureus NIH Hungary 118003, Staphylococcus saprophyticus NIH Hungary 120008 and Pseudomonas aeruginosa NIH Hungary 170000. In addition, 13 bacterial strains isolated from the wounds of patients were analyzed: three coagulase-negative staphylococci (KT24, KT26, KT278); five S. aureus (KT1, KT3, KT23, KT25, KT27), and five P. aeruginosa (KT2, KT7, KT25Ps., KT28 and KT39). The strains were isolated and identified by standard bacteriological methods [8, 9]. Bacteria were cultivated with shaking (100 rpm) at 37 ° C in 100 ml of a medium, which contained bacteriological peptone (1.667 w/v %), Na2HPO4 (0.11 w/v %), glucose (0.389 w/v %), beef extract (0.195 w/v %), and MgCl2 (0.023 w/v %), at pH = 7.2, till they reached the logarithmic phase of their multiplication. Bacteria were collected by centrifugation (4,500 g, 15 min, 4 ° C), and washed with physiological saline. Antibiotic Treatment of Bacteria Four antibiotics were used: Zinacef (Eli Lilly Italia, Sesto Fiorentino, Italy) containing cefuroxime; Claforan (Hoechst Marion Roussel, France) containing cefotaxime; Aktil (Richter, Hungary) containing amoxicillin and clavulanic acid with a ratio 2:1; and Amikin (Bristol-Myers Squibb, USA) containing amikacin. The minimum inhibitory concentrations (MICs) were determined by tube dilution method. The lowest concentrations of the antibiotics inhibiting the growth of the bacterial strains after a 24-hour incubation were regarded as MIC. To examine the effect of antibiotic treatment, bacteria were cultivated in the medium described above. In the logarithmic phase of multiplication, 0.5! or 2! MIC of the antibiotics was administered to the culture medium and incubated for 90 min.

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Determination of Bacterial Cell Surface Hydrophobicity – Salt Aggregation Test One drop (10 Ìl) of control or treated bacterial suspension (5 ! 109 bacteria/ml) in 0.04 M sodium phosphate buffer (pH = 6.8) was mixed with 10 Ìl of increasing concentrations (0.05–4.00 M ) of ammonium sulfate solution. The mixture was gently agitated for 1 min in a glass. The lowest concentration of ammonium sulfate at which aggregation of bacteria occurred was regarded to be characteristic of the cell surface hydrophobicity of the bacterial strain [10]. Statistical Analysis The one-sample one-sided Z test was applied under the following conditions: (a) sample size is greater than 30; (b) distribution is normal, and (c) standard deviation is not higher than the control value (i.e. the sampling error is maximum 100%).

Results

MICs of Antibiotics The MICs of the four selected antibiotics for the three standard strains and the bacteria isolated from orthopedic wounds are summarized in table 1. Bacterial Cell Surface Hydrophobicity Cell surface hydrophobicities of the control and antibiotic-treated coagulase-negative staphylococci, S. aureus and P. aeruginosa strains are shown in figure 1. In each figure, we presented the (NH4)2SO4 concentration characteristic for the untreated strain, and the changes after 0.5! and 2! MIC of (a) cefuroxime, (b) cefotaxime, (c) amoxicillin + clavulanic acid and (d) amikacin. Hydrophobicity of the bacterial cell surface changed in several cases after exposure to the different antibiotics. These alterations imply that visible cell aggregation occurred at higher (NH4)2SO4 concentration, thus decreasing hydrophobicity. Changes in surface hydrophobicity were more often observed after treatment with supra-inhibitory concentration of the antibiotics. Statistical Analysis The one-sample one-sided Z test was applied to determine significant antibiotic-induced changes in the cell surface hydrophobicity. Table 2 lists the changes which reach statistical significance at different control values and at different significance levels (size of type I error). In medical research, the generally accepted significance level is 1%; these data are shown in italics (table 2). At a significance level of 1%, significant changes in cell surface hydrophobicity were noted in 39 cases following antibiotic treatment.

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Adhesion of bacteria to the cell surface is the first, significant step of infection. The rapid phase of adhesion depends on a variety of nonspecific, relatively weak physicochemical forces. Hydrophobic interaction is assumed to be the most important factor among them [5]. Cell surface hydrophobicity might be influenced by several factors, from the medical aspect antibiotics are the most important [6]. Antibiotics can modify several bacterial activities: protein and DNA synthesis, and production of some enzymes, virulence factors and surface components [7]. The aim of our work was to determine the cell surface hydrophobicity of 13 bacterial strains isolated from wounds of orthopedic patients, and to compare their surface properties with three Hungarian standard strains. Furthermore, we analyzed the modifying effect of four antibiotics on the hydrophobic effect of bacteria. These drugs were selected since they are the most frequently applied antibiotics in our Department for prophylaxis and treatment of patients.

Table 1. MIC of cefuroxime, cefotaxime, amoxicillin + clavulanic acid and amikacin in the bacterial strains exam-

ined MIC, Ìg/ml cefuroxime

Strains S. saprophyticus NIH Hungary 120008 S. aureus NIH Hungary 118003 P. aeruginosa NIH Hungary 170000 Clinical isolates Coagulase-negative Staphylococcus S. aureus P. aeruginosa

Table 2. Changes in ammonium sulfate

concentrations that reach statistical significance at different control values and significance levels

1.56 0.8 1100 0.4–0.8 0.4–0.8 100–1 100

cefotaxime

amoxicillin + amikacin clavulanic acid

3.125 0.8 50

0.2 0.2 400

0.8–6.25 0.8–3.125 50–1 100

0.2 0.2–0.4 200–800

0.4 0.4 1.56 0.4–1.56 0.8–6.25 1.56–12.5

Control value m/l

Significance level (size of type I error) 0.10%

0.50%

1%

2%

2.5%

5%

10%

1 1.5 2 2.5 3 3.5 4

0.56 0.85 1.13 1.41 1.69 1.97 2.26

0.47 0.71 0.94 1.18 1.41 1.65 1.88

0.42 0.64 0.85 1.06 1.27 1.49 1.70

0.37 0.56 0.75 0.94 1.12 1.31 1.50

0.36 0.54 0.72 0.89 1.07 1.25 1.43

0.30 0.45 0.60 0.75 0.90 1.05 1.20

0.23 0.35 0.47 0.58 0.70 0.82 0.94

1% significance level is marked in italics.

Hydrophobicity of bacteria has been recognized as an important determinant of bacterial adherence to host tissues or implantable medical biomaterials [5]. In our experimental series, we determined the cell surface hydrophobicity of 13 bacterial strains isolated following orthopedic interventions. Visible cell aggregation of untreated coagulase-negative staphylococci, S. aureus and P. aeruginosa strains occurred between 1.0 and 3.0 M of ammonium sulfate. We could not detect any significant difference between the hydrophobicity of clinical isolates and the Hungarian standard coagulase-negative staphylococci (2.5 M ), S. aureus (2.0 M) and P. aeruginosa (2.0 M) strains. Both the standard strains and the orthopedic isolates were subjected to sub-inhibitory and supra-inhibitory an-

Antibiotic Effect on Bacterial Hydrophobicity

tibiotic treatment with cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin. Hydrophobicity of the cell surface was decreased in several cases after exposure to different types of antibiotics, so the bacterial surface became more hydrophilic. These changes could be observed in the hydrophobicity of both the standard strains and the clinical isolates. According to the statistical analysis, more than 39% of the changes were significant (at the 1% significance level). We would like to emphasize that not all of the changes were significant, but the tendency was the same in all cases: agglutination occurred at higher (NH4)2SO4 concentrations under antibiotic effect. Similar results were published by Hostacka and Karelova [6] when Shigella dysenteriae type 1 strain was treated with aminoglycosides. Braga and Reggio [11] found that brodimoprim decreased the surface hydrophobicity of S. aureus strains. These results indicate that bac-


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Discussion

Fig. 1. Concentrations of (NH4)2SO4 solutions to characterize cell surface hydrophobicity of bacteria. Hydrophobici-

ties of coagulase-negative staphylococci, S. aureus and P. aeruginosa strains of the untreated strains and the changes after sub-inhibitory (0.5! MIC) and supra-inhibitory (2! MIC) treatment of cefuroxime (a); cefotaxime (b); amoxicillin + clavulanic acid (c) and amikacin (d) are presented.



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terial strains having more hydrophilic surface show decreased adhesion to living tissues and to medical implants as well. Hydrophobicity of bacteria is associated with their adhesive ability and virulence [12]. Supra-MIC values of antibiotic treatment were able to induce alterations in the cell surface hydrophobicity more

frequently than sub-inhibitory concentrations. Consequently, proper perioperative prophylaxis and treatment is able to diminish the possibility of postoperative wound infections in orthopedic patients. Quantitative correlations between cell surface hydrophobicity, adhesive ability and virulence require further investigations.

Antibiotic Effect on Bacterial Hydrophobicity


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