Staph And Psueo Burn

International Journal of Antimicrobial Agents 10 (1998) 223 – 228

Original article

Susceptibility of adherent organisms from Pseudomonas aeruginosa and Staphylococcus aureus strains isolated from burn wounds to antimicrobial agents Elz; bieta Anna Trafny Department of Microbiology and Epidemiology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland Received 20 May 1998; accepted 1 June 1998

Abstract To assess the bactericidal effects of ciprofloxacin, netilymicin, and polymyxin B on adherent Pseudomonas aeruginosa organisms and also the bactericidal effects of ciprofloxacin, vancomycin and teicoplanin on adherent Staphylococcus aureus cells, a simple end-point microplate assay, based on the method described by Miyake et al. was used in the present study. As results of the assay, the minimal inhibitory concentration (MICADH) values are taken, which express the susceptibility of the bacterial cells spontaneously released from the surface of adherent microcolonies to antimicrobial agents. Also, a minimal bactericidal concentration (MBCADH) value was read, which is defined as the lowest antibiotic concentration required to kill the sessile bacterial cells. For twenty P. aeruginosa strains and nineteen S. aureus strains isolated from burn wounds, an enhanced resistance against bactericidal action of the applied antibiotics was observed when bacterial cells were attached to polystyrene surface. The MICADH values were comparable with the conventional MIC values only for ciprofloxacin and netilmicin for P. aeruginosa strains. The MBCADH values exceeded many times the conventional MBC values for the majority of strains. The validity of the assay was estimated in the experiment designed to determine the concentration of ciprofloxacin that should be released topically from the collagen dressing to prevent the biomaterial from microbial colonization and allow the decontamination of the wound. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Antimicrobial resistance; Sessile bacteria; Collagen dressing

1. Introduction Biofilm is a characteristic mode of growth for microorganisms invading host tissues in chronic infections and also for those colonizing the surface of medical devices or implantable materials [1,2]. The common feature of bacteria in sessile phase of growth is their increased resistance to antimicrobial agents compared to their planktonic counterparts [3,4]. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) are two parameters relative to antibiotic susceptibility of planktonic cells but their appropriateness to serve as sole 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0924-8579(98)00042-9

guide to antibiotic treatment of foreign body and chronic tissue infections has been already argued by others [5,6]. The reports exist on the tissue and biomedical-centered infections which failed to respond to treatment with antibiotics to which organisms were fully susceptible on conventional susceptibility testing [7,8]. Hence, there is a need for an alternative method to test the antibiotic concentration required to eradicate the adherent bacteria which is of great interest for clinicians seeking an advice on the bactericidal activity of the drug, especially in the patients with chronic infection or with impaired immune system e.g. in burn patients. The wounds of burn patients are often covered with bio-

224

E.A. Trafny / International Journal of Antimicrobial Agents 10 (1998) 223–228

compatible dressings or composite grafts of cultured skin cells that possess high susceptibility to burn wound microorganisms, therefore the concentration of antimicrobial agents sufficient to prevent the biomaterials from microbial colonization or to eradicate sessile organisms is also of great importance [9,10]. The goal of the work reported here was, (i) to compare the MICs and the MBCs of five antibiotics either for planktonic organisms from P. aeruginosa and S. aureus strains isolated from burn wounds by conventional testing or for sessile bacteria from these pathogens using the simple end-point microplate method [11]; (ii) to determine whether MBC values obtained for the adherent P. aeruginosa organisms by the microplate method would have any predictive value for the prevention of collagen dressings [12] containing ciprofloxacin from microbial colonization.

2. Materials and methods

2.1. Bacteria Twenty P. aeruginosa and nineteen S. aureus strains were isolated from burn wounds in the Burn Unit of the IInd Municipal Hospital in Siemianowice, Poland and kindly provided by Dr S. Sakiel. All P. aeruginosa strains were nonmucoid. Among S. aureus isolates eight were classified as methicillin resistant (MRSA). Bacterial strains were grown at 37°C for 24 h on Bacto-Agar plates (Difco Laboratories, USA). The bacteria were harvested and suspended in PBS (phosphate buffered saline, pH 7.4) to a final density of 1 – 5 × 105 CFU/ml, unless otherwise stated. Bacterial concentrations were calculated by densitometry and confirmed by plate counting.

2.2. Antimicrobial agents The antimicrobials used in this study were purchased as follows, ciprofloxacin (Ciprobay 100) from Bayer, Germany; polymyxin B sulfate from Pfizer GmbH, Germany; netilmicin sulfate (Netromycine) from Schering-Plough, USA; vancomycin hydrochloride (Vancocin) from Lilly Deutschland GmbH, Germany and teicoplanin (Targocid) from Marion Merrell Dow, France. Stock solutions of antibiotics were prepared in PBS and then were diluted in the nutrient broth to achieve the desired concentrations.

2.3. Determination of MIC and MBC for planktonic bacteria The MICs of antibiotics for bacterial strains tested were determined by macrodilution broth method using doubling dilutions prepared in 1 ml of Oxoid Nutrient

Broth (Unipath, England) [13]. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth after 20 h at 37°C. The MBCs of antibiotics for particular strains were determined as follows, 100 ml of solution from all dilution test tubes without obvious turbidity was subcultured to agar plates without antimicrobial. The concentration of antibiotic corresponding to its concentration in the tube from which the solution produced no colonies on the agar plates (reduction in number below 10 CFU per plate) was treated as the MBC [14].

2.4. Determination of MIC and MBC for sessile bacteria Determination of these parameters for sessile bacteria was performed using a simple end-point microplate method of Miyake et al. [11] with modifications as follows, the bacterial suspensions (100 ml) of P. aeruginosa and S. aureus individual strains at a density of 0.5− 1× 106 CFU/ml were incubated in previously sterilized round-bottomed microplates for 20 h at 37°C to allow the bacteria to attach to the polystyrene. Then, the plates were washed with PBS-T (PBS containing 0.1% of Tween 80) using Microplate Washer (Dynatech, USA) to remove the loosely bound bacteria. The 2-fold dilutions of antibiotics in nutrient broth were added to the twenty consecutive microplate wells (in at least two repetitions) and incubated for 20 h at 37°C. The lowest concentration of antibiotic at which there was no bacterial growth observed in the well was read as the MIC value for the spontaneously released bacteria from adherent microcolonies (MICADH). After emptying the plates, the fresh nutrient broth was added to the microplate wells, and the plates were incubated at 37°C. After 24 h, the MBC value for adherent bacteria (MBCADH) was taken as the lowest concentration of antibiotic at which there was no growth of bacteria in the microplate wells. All operations were performed in the laminar flow cabinet.

2.5. Colonization of collagen dressing containing ciprofloxacin by P. aeruginosa strains Collagen dressing was composed of two collagen sponges (manufactured from bovine tendons by Tissue Bank, Morawica, Poland) and intermediate active layer with incorporated ciprofloxacin (0.04 mg/cm2), prepared as described previously [12]. After 24 h of incubation of the dressing in the 5 ml of PBS, the concentration of ciprofloxacin released from the dressing was 12.891.9 mg/ml (n= 8) as determined spectrophotometrically (A275). The 1× 3 cm rectangular sections of the dressing were placed inside the tubes containing 5 ml on nutrient broth that were inoculated with 50 ml of bacterial suspension of individual P.

E.A. Trafny / International Journal of Antimicrobial Agents 10 (1998) 223–228

aeruginosa strains at the density of 107 CFU/ml and incubated for 24 h at 37°C. Enumeration of viable P. aeruginosa cells still attached to the dressing surface after that treatment was performed by sonication of the dressing and plating of the released bacterial cells as described previously [15].

3. Results Among twenty P. aeruginosa strains isolated from burn wounds, for six strains the MIC values of ciprofloxacin were evaluated as equal or higher than 2 mg/ml; these strains were considered as resistant to ciprofloxacin. For four strains the MICs for netilmicin were higher than 8 mg/ml and all the MICs of polymyxin B for all strains tested but one were below 0.5 mg/ml. Among the nineteen S. aureus strains examined in this study, seven strains were resistant to ciprofloxacin (the MICs equal or higher then 8 mg/ml); none of them was resistant either to teicoplanin or vancomycin. All above values were established for the planktonic bacteria. When susceptibility was tested in the microplate assay for the sessile bacteria from P. aeruginosa strains, the MICADH for ciprofloxacin and netilmicin did not differ significantly from the MICs evaluated for the planktonic bacteria except for polymyxin B where the differences were observed. The correlation coefficients between the MIC and MICADH were respectively: r= 0.97 for ciprofloxacin, r = 0.95 for netilmicin, and r= −0.17 for polymyxin B. For S. aureus strains the correlation coefficients between the MlCs evaluated for planktonic and sessile bacteria were as follows, r = 0.02 for teicoplanin, r = 0.03 for vancomycin and r =0.5 for ciprofloxacin. However, for all these antimicrobial agents except ciprofloxacin to which susceptibility was tested on S. aureus strains, the MICADH was maximally eight times higher from the MIC evaluated for floating bacteria from all P. aeruginosa and S. aureus strains tested. Surprisingly, for S. aureus strains which were susceptible to ciprofloxacin (MIC50.125) the highest ratio (MICADH/MIC =64) was observed and as a consequence all S. aureus strains but three displayed high resistance to ciprofloxacin in the MICADH assay. The high ratio MICADH/MIC and lack of correlation between the results of MIC and MICADH reflects the increased resistance to antimicrobial killing of the newly formed daughter cells shed from the microcolonies attached to the plastic surface. In Figs. 1 and 2 the MICs and the MBCs for planktonic and sessile bacteria were compared for both groups of microorganisms. The MBCADH values for five antimicrobial agents examined in this study were markedly elevated and even the ratio MBCADH/ MBC= 500 might be observed for the antibiotics tested

225

on the both groups of pathogens. For P. aeruginosa cells, the highest MBCADH/MBC ratio was observed for netilmicin, followed by ciprofloxacin and polymyxin B (for the latter antibiotic the mean ratio for twenty strains tested was equal to 6.89 9.4). The comparable low MBCADH/MBC ratio was observed for teicoplanin (the mean= 6.898.1) among the organisms from S. aureus strains. For vancomycin the mean ratio was equal 14.29 19.7, and for ciprofloxacin and S. aureus strains the mean ratio was the highest among all antibiotics tested. The validity of the MBCADH assays to determine the antibiotic concentration sufficient to kill adherent bacteria was examined in the following experiment: microcolonies of P. aeruginosa strains grown on the surface of the collagen dressings were challenged with ciprofloxacin released from the active layer of the dressing in a concentration exceeding the conventional MBC value for all twenty strains tested. However, it was

Fig. 1. Comparison of P. aeruginosa clinical strains susceptibility to ciprofloxacin, netilmicin and polymyxin B expressed as the MIC (A) and the MBC (B) for planktonic bacteria, and also as the MICADH (C) and the MBCADH (D) for sessile bacteria. The data are presented as box plots, where left and right boundaries of the box mark respectively the 25th and the 75th percentiles. A line within the box indicates the median and the whiskers on the left and right side of the box display the 10th and the 90th percentiles. The points represent the outlying values. Plots were generated using Sigma Plot Software (Jandel Corporation, USA).

226

E.A. Trafny / International Journal of Antimicrobial Agents 10 (1998) 223–228

Fig. 2. Comparison of S. aureus clinical strains susceptibility to ciprofloxacin, teicoplanin and vancomycin expressed as the MIC (A) and the MBC (B) for planktonic bacteria, and also as the MICADH (C) and MBCADH (D) for sessile bacteria. The data are presented as described in Fig. 1.

observed that for seven strains the bacteria were not completely eradicated from the dressing (reduction in number below 20 CFU per 1 cm2 of the dressing). For all these seven strains the MBCADH was at least two times higher than the concentration of released ciprofloxacin. As it is seen in Fig. 3, the number of viable sessile bacterial cells which remained still attached to the surface of dressing was not dependent of the MBC values displayed for the strains tested, but was quantitatively dependent on the MBCADH values determined for P. aeruginosa strains when sessile bacteria were assayed for their susceptibility to ciprofloxacin.

4. Discussion Biofilm is a biomass of bacteria and slime. This mode of bacterial growth allows the embedded bacteria to be less accessible to the bactericidal action of antibiotics and human immune system defense mechanisms. The bacterial biofilm is a dynamic structure that is influenced by substrate accessibility on the biofilm surface and the hydrodynamic conditions including shear stress

[16]. The method that incorporates the shear forces affecting the biofilm preformed on membrane filters under laminar flow conditions in a modified Robbins Device (MRD) has been recently described for testing the antimicrobial susceptibilities of bacteria [17]. However, one could meet some difficulties adopting this method in the routine laboratory because it requires specialistic equipment as MRDs and apparatus for continuous flow of bacteria and fresh broth. Simple and cost-saving methods of determination the susceptibility of biofilm bacteria to antimicrobial agents have been described in the literature but these methods do not fulfill the above conditions for true biofilm development. The steady-state bacteria attached to the solid surfaces are usually challenged with antibiotics. However, despite this incomplete methodology used to produce the biofilm in vitro, it had been possible to predict in vivo the outcome of device-related infections using these methods [5–7]. The procedure that was applied in our study is based on the method developed to investigate the antibiotic resistance of the freshly attached S. aureus cells, which have not yet formed a biofilm. The attachment of the organisms to plastic surface was performed by centrifugation of bacterial suspension at 450× g for 10 min., followed by 1-h incubation at 37°C. In the method described here, the incubation time necessary for the bacteria to settle down, attach and develop the microcolonies on the plastic surface was prolonged to 20 h at 37°C. After elimination of non-adherent organisms by several rinses, the sessile organisms were exposed to antimicrobial agents. At the same time, the microcolonies grown on the plastic start to release spontaneously newly formed daughter cells. Their release and viability in the microplate wells despite the presence of the increased concentrations of antimicrobial agent reflects the resistance of the daughter cells to the drug tested. This increased antibiotic resistance of the spontaneously released cells was shown when P. aeruginosa microcolonies were treated with polymyxin B or S. aureus microcolonies were challenged with ciprofloxacin, vancomycin or teicoplanin. The similar phenomenon of the increased resistance to antimicrobial killing of the released bacteria from adherent biofilms has been observed for S. aureus growing on fibronectin-coated surfaces treated with oxacillin, vancomycin and fleroxacin [4]. Evans et al. [18] have reported that the cells dislodged from a chemostatgrown P. aeruginosa biofilm demonstrate no increase in resistance towards ciprofloxacin as could be observed also in our experiments. Determination of the MBCADH in the microplate assay demonstrated that the susceptibility of both pathogens to antimicrobial killing decreased up to 500 fold when cells were grown adherent to the surface of microtiter plate. Some of the adherent P. aeruginosa

E.A. Trafny / International Journal of Antimicrobial Agents 10 (1998) 223–228

227

Fig. 3. Killing of P. aeruginosa cells adherent to the collagen dressing surface by ciprofloxacin released from the dressing at a concentration of 12.8 mg/ml. (A) the number of adherent bacterial cells after the treatment with drug versus the MBC values for planktonic bacteria; (B) the number of adherent bacterial cells after the treatment with drug versus the MBCADH values for sessile bacteria. Twenty P. aeruginosa clinical S strains were tested in the experiments performed twice. Strains for which the MBCADH values were below 16 mg/ml data are not presented because these strains the bacterial cells were completely eradicated from the surface of collagen dressing.

strains would be completely killed by ciprofloxacin at a concentration many times higher than the mean maximum plasma concentration of ciprofloxacin (4.29 1.1 mg/l) in burn patients [19]. Similarly, high resistance to bactericidal action of ciprofloxacin was demonstrated for adherent S. aureus organisms. In burn patients, the median serum teicoplanin concentration was reported to equal 12.8 mg/l at 12 h after a single intravenous dose of the drug (12 mg/kg). The median concentration of teicoplanin in fluid from the burn wound was 60% of the serum antibiotic concentration [20]. Vancomycin mean trough serum concentration (Cmin) above the values of 13–32 mg/l may be associated with its ototoxicity and Cmin values above 20 mg/l with its nephrotoxicity [21]. The discrepancy of the above data with MBCADH values obtained in the microplate assay for the strains isolated from burn wounds may explain clinical failures in the treatment of the serious infections in immunocompromised patients. The one exception that could be observed in our studies is polymyxin B, the antibiotic that is often applied topically. Topical application of antibiotic is more effective and allows higher local concentration of drug than after systemic administration. The adherent organisms from the most of the P. aeruginosa strains (90%) were susceptible to concentration of polymyxin B below 10 mg/ml. This concentration can be easily achieved with its topical application [22,23]. The usefulness of the microplate assay for evaluation of the bactericidal activity of antibiotics (MBCADH) was confirmed in the experiments designed to determine the concentration of ciprofloxacin, which should be re-

leased from collagen dressing to prevent collagen biomaterial from microbial colonization and to decontaminate the wounds on which the dressing is to be applied. The ciprofloxacin released at a concentration exceeding the MBC values on conventional testing of all P. aeruginosa strains did not eradicate the bacteria attached to the surface of collagen dressing from all these strains for which the MBCADH values were determined to be higher than the concentration of drug released from dressing. Based on the MBCADH values for the strains usually contaminating the burn wounds, it is possible to predict the maximal local concentration of the drug that would protect the biomaterials from microbial colonization and control of microbial contamination in the wound bed.

References [1] Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annu Rev Microbiol 1995;49:711 – 45. [2] Lewis WJ, Sherertz RJ. Microbial interactions with catheter material. Nutrition 1997;13:5S – 9S. [3] Arizono T, Oga M, Sugioka Y. Increased resistance of bacteria after adherence to polymethyl methacrylate. Acta Orthop Scand 1992;63:661 – 4. [4] Chuard C, Vadaux P, Waldvogel FA, Lew DP. Susceptibility of Staphylococcus aureus growing on fibronectin-coated surfaces to bactericidal antibiotics. Antimicrob Agents Chemother 1993;37:625 – 32. [5] Widmer AF, Frei R, Rajacic Z, Zimmerli W. Correlation between in vivo and in vitro efficacy of antimicrobial agents against foreign body infections. J Infect Dis 1990;162:96–102.

228

E.A. Trafny / International Journal of Antimicrobial Agents 10 (1998) 223–228

[6] Jones JW, Paull SN. Effect of bigfilm culture on antibiotic susceptibility of lactobacilli causing endocarditis. I Infect 1995;31:80 – 1. [7] Widmer AF, Colombo VE, Gachter A, Thiel G, Zimmerli W. Salmonella infection in total hip replacement: test to predict the outcome of antimicrobial therapy. Scand J Infect Dis 1990;22:611 – 8. [8] Kobayashi H. Quinolones for respiratory tract infections. Antibiotics Chemother 1997;1:6 (letter) [9] Boyce ST, Supp AP, Warden GD, Holder IA. Attachment of an aminoglycoside, amikacin, to implantable collagen for local delivery in wounds. Antimicrob Agents Chemother 1993;37:1890 – 5. [10] Boyce ST, Warden GD, Holder IA. Noncytotoxic combinations of topical antimicrobial agents for use with cultured skin substitutes. Antimicrob Agents Chemother 1995;39:1324 – 8. [11] Miyake Y, Fijiwara S, Usui T, Suginaka H. Simple method for measuring the antibiotic concentration required to kill adherent bacteria. Chemotherapy 1992;38:286–90. [12] Grzybowski J, Kolodziej W, Trafny EA, Struzyna J. A new anti-infective collagen dressing containing antibiotics. J Biomed Mater Res 1997;33:163–6. [13] Trafny EM, Stepinska M, Antos M, Grzybowski J. Effect of free and liposomeencapsulated antibiotics on adherence of Pseudomonas aeruginosa to collagen type I. Antimicrob Agents Chemother 1995;39:2645–9. [14] Isenberg HD. Antimicrobial susceptibility testing: a critical evaluation. J Antimicrob Chemother 1998;22:73–86. [15] Trafny EA, Kowalska K, Grzybowski J. Adhesion of Pseudomonas aeruginosa to collagen biomaterials: effect of amikacin

.

[16] [17]

[18]

[19]

[20]

[21]

[22]

[23]

and ciprofloxacin on the colonization and survival of the adherent organisms. J Biomed Mater Res 1988 (in press) Van Loosdrecht MCM. Biofilm structures. Wat Sci Tech 1995; 8:35 – 43. Dominque G, Ellis B, Dasgupta M, Costerton JW. Testing antimicrobial susceptibilities of adherent bacteria by a method that incorporates guidelines of the National Committee for Clinical Laboratory Standards. J Clin Microbiol 1994;32:2564– 8. Evans DJ, Allison DG, Brown MR, Gilbert P. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother 1991;27:177 – 84. Garrelts JC, Jost G, Kowalsky SF, Krol GJ, Lettieri JT. Ciprofloxacin pharmacokinetics in burn patients. Antimicrob Agents Chemother 1996;40:1153 – 6. Steer JA, Papini RP, Wilson AP, Dhillon S, Hichens MF, McGrouther DA, Frame JD, Parkhpuse N. Pharmacokinetics of a single dose of teicoplanin in burn patients. J Antimicrob Chemother 1996;37:545 – 53. Leader WG, Chandler MHH, Castiglia M. Pharmacokinetics optimization of vancomycin therapy. Clin Pharmacokinet 1995;28:327 – 42. Walton MA, Carino E, Herndon DN, Heggers JP. The efficacy of polysporin first aid antibiotic spray (polymyxin B sulfate and bacitracin zinc) against clinical burn wound isolates. J Burn Care Rehabil 1991;12:116 – 9. Boyce ST, Holder IA, Supp AP, Warden GD, Greenhalgh DG. Delivery and activity of antimicrobial drugs released from human fibrin sealant. J Burn Care Rehabil 1994;15(25):1–255.