Journal of Antimicrobial Chemotherapy (2002) 49, 863-866
© 2002 The British Society for Antimicrobial Chemotherapy
Antibiotic tolerance of peritoneal bacterial isolates in dialysis fluids
Faculties of 1Pharmacy and 2Medicine, University of Manitoba; Departments of 3Pharmacy, 4Infectious Diseases and 5Nephrology, St Boniface General Hospital, Winnipeg, Manitoba, Canada
Received 6 August 2001; returned 19 November 2001; revised 17 December 2001; accepted 23 January 2002.
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Abstract |
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The objective was to test antibiotic activity against Staphylococcus epidermidis and Pseudomonas aeruginosa in fresh dianeal fluid and spent dialysate from patients undergoing chronic ambulatory peritoneal dialysis. MICs and MBCs were measured and compared with those in standard broth. For S. epidermidis, there was some reduction in cefazolin and vancomycin activity in dialysis fluids. For P. aeruginosa, ciprofloxacin and tobramycin MICs increased significantly, and all isolates were tolerant to ceftazidime and piperacillin in dialysis fluids. Dialysis fluids can significantly impair antibiotic activity. The clinical implications warrant further study of antibiotic pharmacodynamics in the treatment of peritonitis.
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Introduction |
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Peritonitis is a significant cause of morbidity and mortality in patients on chronic ambulatory peritoneal dialysis (CAPD). Normal skin flora, including Staphylococcus epidermidis, are the most common aetiological agents in CAPD peritonitis.1 Standard treatments involve intraperitoneal administration of cefazolin for methicillin-susceptible strains or vancomycin for methicillin-resistant infections. Pseudomonas aeruginosa peritonitis can be a difficult infection to eradicate, even with prolonged treatments with multiple anti-pseudomonal antibiotics.
In the treatment of infectious diseases, the MIC is the standard microbiological basis of antibiotic susceptibility testing.2 The interpretation of MIC data, however, does not consider peritonitis-specific factors, such as the relatively high intraperitoneal antibiotic concentrations and the effect of dialysis fluids on antibacterial activity.
Previous studies have shown that in vitro antibiotic activity against pathogens like S. epidermidis can be significantly impaired by fresh and spent dialysis fluids collected from patients without infection.3,4 Relatively few investigations have been conducted with the more virulent P. aeruginosa.5,6 The goal of the present study was to compare antibiotic MICs and minimal bactericidal concentrations (MBCs) for S. epidermidis and P. aeruginosa in standard Mueller–Hinton broth, fresh dextrose-based dianeal fluid (Dianeal), a new icodextrin-based dianeal fluid (Extraneal) and spent Dianeal dialysate from patients with confirmed culture-negative peritonitis.
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Materials and methods |
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Two clinical strains of S. epidermidis (methicillin susceptible, isolate no. 754; methicillin resistant, isolate no. 036) and P. aeruginosa (isolate nos 427 and 310) isolated from the peritoneal dialysate of patients with peritonitis were studied. Staphylococcus aureus (ATCC 29213) and P. aeruginosa (ATCC 27853) were used as controls.
Antibiotics including cefazolin (Sigma–Aldrich Canada Ltd, Oakville, Ontario, Canada), ciprofloxacin (Bayer Inc., Etobicoke, Ontario, Canada), gentamicin and vancomycin (Sigma–Aldrich) were tested against S. epidermidis. Ciprofloxacin, tobramycin, ceftazidime (Eli Lilly & Company, Greenfield, IN, USA), imipenem (Merck Frost Canada & Co., Kirkland, Quebec, Canada) and piperacillin (Wyeth-Ayerst Research, Pearl River, NY, USA) were studied against P. aeruginosa.
The standard broth medium was Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 25 mg/L calcium and 12.5 mg/L magnesium. Fresh Dianeal with 2.5% dextrose (Baxter, Mississauga, Ontario, Canada) and Extraneal with 7.5% icodextrin (Baxter) were used. Fresh dianeal fluids were supplemented with cation-supplemented Mueller–Hinton broth (CSMHB) (90:10), because preliminary experiments demonstrated that fresh dianeal fluids did not support bacterial growth over 24 h. Spent Dianeal dialysate from three patients with confirmed culture-negative peritonitis was collected, filter sterilized (0.22 µm membrane filter) and pooled.
Macrodilution MICs and MBCs were measured in CSMHB and all dialysis fluids in triplicate on separate occasions, using the methods described by the NCCLS.2,7 A significant change in MIC was defined as a difference of at least two doubling dilutions. Impaired bactericidal activity or tolerance was defined as an MBC:MIC ratio > 32.7
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Results |
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As demonstrated in Table 1, dialysis fluids had some effect on the antibiotic activity against S. epidermidis but not S. aureus. Cefazolin and ciprofloxacin MICs for isolates 036 and 754, respectively, increased four-fold in all dialysis fluids. For both isolates, cefazolin and vancomycin MBC:MIC ratios increased significantly from 2–4 in standard broth to 16–32 in spent dialysate.
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MICs and MBCs for P. aeruginosa are shown in Table 2. Ciprofloxacin and tobramycin MICs for two isolates increased four- and eight-fold, respectively. Ceftazidime and piperacillin MICs were unchanged, but MBC:MIC ratios were significantly elevated and tolerant in all dialysis fluids. Imipenem activity was unchanged except for ATCC 27853, which was tolerant in dialysis fluids.
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Discussion |
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Other studies have investigated the effects of fresh and spent glucose-based dianeal fluids on antibiotic activity. To our knowledge, this is the first to expand these investigations to Extraneal, an iso-osmolar, glucose-free solution that may produce fewer haemodynamic effects and less damage to peritoneal membranes. Whereas previous studies used spent dialysate from patients without infection, our experiments were conducted with fluid from those with confirmed culture-negative peritonitis.
Our study demonstrated two significant limitations in applying MIC data to the treatment of CAPD peritonitis. First, MIC results in standard broth differed in some cases from those in dialysis fluid. In general, MICs for S. epidermidis were not affected, whereas ciprofloxacin and tobramycin MICs for P. aeruginosa were significantly increased. In clinical practice, standard MICs would not be indicative of the antibiotic–pathogen interaction in vivo.
MICs do not measure bactericidal activity in dialysis fluids. All P. aeruginosa isolates were tolerant to ceftazidime and piperacillin. Consequently, routine MIC testing would not detect tolerant pathogens that might evade the lethal effects of antibiotics resulting in treatment failure.
Different growth characteristics of S. epidermidis in dialysis fluids could explain the variable effects on antibiotic activity. In particular, growth condition is an important factor in the acquisition of bacterial tolerance, since slower growth can result in reduced kill by antibiotics such as b-lactams. Others have demonstrated that fresh dianeal fluids are bacteriostatic, whereas spent dialysate with a higher pH and the presence of amino acids and proteins supports bacterial growth.8 Changes in growth patterns of coagulase-negative staphylococci have also been associated with cation concentrations that vary during dwell times.9 In addition, Wilcox et al.10 showed the effect of 5% CO2 on bacterial growth rates and MICs.
Some difference in antibiotic activity against P. aeruginosa could be explained by environmental conditions such as pH. For example, ciprofloxacin MICs and MBCs were relatively high in the fresh dianeal fluids (pH 5.5) compared with spent dialysate (pH 7.5) and CSMHB (pH 7). Shalit et al.5 observed increases in ciprofloxacin and tobramycin MBCs in fresh dianeal fluid but not in fluids buffered to pH 7.4. The effects of cations, specifically Ca2+ and Mg2+, on the cellular uptake and antibacterial effects of aminoglycosides are also well documented.2 Finally, different growth characteristics of P. aeruginosa in dialysis fluids could have contributed to the tolerance to ceftazidime and piperacillin.
Considerable debate surrounds the clinical significance of impaired in vitro antimicrobial activity in dialysis fluids. The relevance of the application of routine MIC data to CAPD peritonitis is questionable; however, would susceptibility testing in dialysis fluid be more predictive of treatment outcome? Craddock et al.6 concluded that diminished ceftazidime and gentamicin activity, and bacterial adherence to catheters were important in the clinical failure of five episodes of pseudomonal peritonitis. Another study, how-ever, found no association between reduced ciprofloxacin activity in peritoneal dialysate and treatment response in 34 episodes of Gram-positive peritonitis.4 Further studies of antimicrobial pharmacodynamics in the treatment and clinical outcome of CAPD peritonitis are required.
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Acknowledgements |
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Ciprofloxacin was provided by Bayer Inc.; tobramycin and ceftazidime by Eli Lilly & Company; imipenem by Merck Frost Canada & Co.; and piperacillin by Wyeth-Ayerst Research. Fresh dianeal fluids were supplied by Baxter. Research support was provided by the Kidney Foundation Corporation of Canada, Manitoba Branch.
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Footnotes |
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* Corresponding author. Tel: +1-204-474-8414; Fax: +1-204-474-7617; E-mail: Zelenits{at}ms.umanitoba.ca
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References |
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1 . Zelenitsky, S., Barns, L., Findlay, I., Alfa, M., Ariano, R., Fine, A. et al. (2000). Analysis of microbiological trends in peritoneal dialysis-related peritonitis from 1991 to 1998. American Journal of Kidney Diseases 36, 1009–13.[Web of Science][Medline]
2 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Wayne, PA.
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Wilcox, M. H., Geary, I. & Spencer, R. C. (1992). In-vitro activity of imipenem, in comparison with cefuroxime and ciprofloxacin, against coagulase-negative staphylococci in broth and peritoneal dialysis fluid. Journal of Antimicrobial Chemotherapy 29, 49–55.
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Ludlam, H., Johnston, L. & Hopkins, P. (1992). Susceptibility testing of bacteria recovered from patients with peritonitis complicating continuous ambulatory peritoneal dialysis. Antimicrobial Agents and Chemotherapy 36, 1097–101.
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Shalit, I., Welch, D. F., San Joaquin, V. H. & Marks, M. I. (1985). In vitro antibacterial activities of antibiotics against Pseudomonas aeruginosa in peritoneal dialysis fluid. Antimicrobial Agents and Chemotherapy 27, 908–11.
6 . Craddock, C. F., Edwards, R. & Finch, R. G. (1987). Pseudomonas peritonitis in continuous ambulatory peritoneal dialysis: laboratory predictors of treatment failure. Journal of Hospital Infectection 10, 179–86.
7 . National Committee for Clinical Laboratory Standards. (1999). Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Guideline M26-A. NCCLS, Wayne, PA, USA.
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McDonald, W. A., Watts, J. & Bowmer, M. I. (1986). Factors affecting Staphylococcus epidermidis growth in peritoneal dialysis solutions. Journal of Clinical Microbiology 24, 104–7.
9 . Morton, A. R., Evans, G., Shannon, A. & Zoutman, D. (1994). Growth of coagulase negative staphylococci in peritoneal dialysate fluid. Effect of calcium concentration. ASAIO Journal 40, M431–4.[Medline]
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Wilcox, M. H., Smith, D. G., Evans, J. A., Denyer, S. P., Finch, R. G. & Williams, P. (1990). Influence of carbon dioxide on growth and antibiotic susceptibility of coagulase-negative staphylococci cultured in human peritoneal dialysate. Journal of Clinical Microbiology 28, 2183–6.
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