An in vitro evaluation of the antibiotic/heparin lock to sterilize central venous haemodialysis catheters

Lavern M. Vercaignea,b,*, Sheryl A. Zelenitskya,c, Ian Findlaya,d, Keevin Bernsteinb,e and S. Brian Pennerb,e,f

a Division of Clinical Sciences and Practice, Faculty of Pharmacy, University of Manitoba, Winnipeg; b Manitoba Provincial Dialysis Program, Winnipeg; c St Boniface Research Centre, Winnipeg; d Victoria General Hospital, Winnipeg; e Department of Internal Medicine (Section of Nephrology), Faculty of Medicine, University of Manitoba, Winnipeg; f Departments of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
This in vitro study investigated the ability of antibiotic/heparin locks to sterilize central venous haemodialysis catheters (CVCs) inoculated with methicillin-resistant Staphylococcus epidermidis (MRSE). Isolates of MRSE were incubated in broth inside CVCs. The catheters were then drained and filled with either vancomycin/gentamicin/heparin (VGH), cefazolin/gentamicin/ heparin (CGH) or control locks for 48 h. The catheters were drained, filled with fresh broth and again incubated. The final catheter solutions were sampled and the remaining volumes filtered. The samples, filters and catheter segments were examined for growth. For two isolates, both the VGH and CGH locks sterilized the catheters. Bacterial counts of the remaining two isolates were significantly reduced by >99%, but the catheters were not sterilized after the instillation of a single-antibiotic/heparin lock.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Central venous catheters (CVCs) are commonly used for vascular access to carry out haemodialysis. Bacteraemia may occur and result in morbidity and mortality through complications of septicaemia and potential development of osteomyelitis, infective endocarditis or septic arthritis.1 Since the reported efficacy of systemic antibiotics alone is only 25–32%,1,2 catheter removal and replacement at an alternative site has been suggested as standard therapy.3

To further investigate and develop the use of intraluminal antibiotics in the treatment of catheter-related bacteraemia in the haemodialysis population, we initially verified the in vitro stability of high concentrations of antibiotics (vancomycin, cefazolin and ceftazidime 10 000 mg/L and gentamicin 5000 mg/L) and heparin (5000 IU/mL) in CVCs at 37°C.4 As a follow-up, this study investigated the use of the antibiotic/heparin lock procedure to sterilize CVCs infected with methicillin-resistant Staphylococcus epidermidis (MRSE) in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Antibiotic/heparin locks

Vancomycin 10 000 mg/L was combined with gentamicin 5000 mg/L and heparin sodium 5000 IU/mL (VGH lock). Similarly, cefazolin 10 000 mg/L was combined with gentamicin 5000 mg/L and heparin sodium 5000 IU/mL (CGH lock). Dual-lumen, 200 mm, polyurethane catheters were obtained from Gamcath (Hospal Gambro, Hechingen, Germany; code no. GDC-1120).

Bacterial strains

Four isolates of MRSE, causing single-pathogen bacteraemia, were used (Table 1Go). Methicillin resistance was defined as a MIC of >=0.5 mg/L of oxacillin according to current National Committee for Clinical Laboratory Standards guidelines.5 Two isolates of MRSE (H523 and H440) were also resistant to gentamicin.


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Table 1. Minimum inhibitory and bactericidal concentrations (mg/L) for S. epidermidis
 
Experiments

Bacterial isolates were grown on tryptic soy agar (TSA) plates at 35°C for 18–24 h. A 0.5 McFarland (c. 108 cfu/mL) bacterial suspension was prepared and diluted with cation-supplemented Mueller–Hinton broth (CSMHB) to yield a final inoculum of c. 106 cfu/mL. Using sterile procedures in a laminar flow hood, 2.5 mL of the suspension were injected into the lumens of the catheters. The catheters were capped and incubated at 35°C for 24 h. The catheters were then drained and filled with either (a) VGH lock, (b) CGH lock or (c) heparin 5000 IU/mL solution as the control and then incubated for 48 h. The catheters were drained, filled with fresh CSMHB and incubated for a final 24 h.

Samples (0.1 mL) of the final solution were collected and serially diluted in normal saline at 4°C, and 10 and 100 µL aliquots were plated in duplicate on blood agar. After 24 h of incubation, viable colonies between 10 and 100 per plate were counted. Using this method, our limit of detection was 102 cfu/mL. In addition, the final solution drained from the catheters was filtered through a 0.45 µm cellulose nitrate membrane filter and rinsed three times with 10 mL of normal saline to remove residual antibiotic. The filter was then incubated on TSA plates at 35°C for 48 h. The tip and a 2 cm proximal section were cut from each catheter, placed on blood agar and incubated for 48 h. Experiments were conducted in triplicate on separate occasions.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
For all four isolates, bacterial counts >108 were maintained after administration of the heparin control lock for 48 h, confirming the viability of S. epidermidis inside the CVCs.

Results after administration of the VGH and CGH locks are summarized in Table 2Go. Two of the MRSE isolates (H900 and H863) were sterilized by both the VGH and CGH locks. We subsequently investigated whether the combination of antibiotics was necessary or whether the two isolates could be killed by a single-antibiotic/heparin lock. Isolate H863 was not sterilized by vancomycin/ heparin or cefazolin/heparin, but there was >97% kill in both cases. In addition, the gentamicin/heparin lock eradicated the organism from the final solution; however, 23 colonies grew from the filter and three colonies grew from the catheter segments. Isolate H900 was not sterilized by the vancomycin/heparin or cefazolin/heparin locks, but growth was reduced by >96% in both cases. Catheters growing isolate H900 were sterilized by the gentamicin/ heparin-lock combination.


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Table 2. Bacterial growth in catheter solutions, filters and catheter segments
 
For the remaining two MRSE isolates (H523 and H440), the VGH and CGH locks did not completely sterilize the catheters, but bacterial counts in the final solution were significantly reduced by >99% compared with the control (Table 2Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Our results show that a single 48 h instillation of the VGH or CGH lock sterilizes or significantly reduces MRSE growth inside CVCs in vitro. When the single-antibiotic/ heparin locks were separately tested against these isolates, only gentamicin/heparin administration produced undetectable bacterial counts from the final catheter solutions; however, one isolate (H863) continued to grow on filter and catheter segments. Vancomycin/heparin and cefazolin/ heparin administration did not sterilize the catheters, but they significantly reduced bacterial growth by 96–98% compared with the control. For these isolates, the combination of gentamicin with either vancomycin (VGH lock) or cefazolin (CGH lock) appeared to eradicate the bacteria more effectively.

No significant differences in growth reduction were observed between the vancomycin- and cefazolin-containing antibiotic/heparin locks. The relative effectiveness of cefazolin compared with vancomycin in our study may be explained by the relatively low cefazolin MICs of 2–8 mg/L compared with the high cefazolin concentrations in the lock (10 000 mg/L).

Small, observational clinical trials using intraluminal antibiotics have been conducted previously.6 A small trial in haemodialysis patients using a 4 h continuous antibiotic infusion followed by an antibiotic lock successfully eradicated 100% (13/13) of catheter-related sepsis cases, without catheter removal.6 Although promising, a small sample size and a non-comparative, observational design limit the applicability of the results. The use of the 4 h infusion is also less practical for treating outpatients between dialysis treatments. Capdevila et al.6 successfully utilized a vancomycin/heparin lock procedure in haemodialysis patients, but the use of vancomycin may not be necessary if high concentrations of other antibiotics like cefazolin/gentamicin are effective against bacteria within the slime layers on catheter luminal surfaces.7–10 Notably, the combination of cefazolin (10 000 mg/L), gentamicin (5000 mg/L) and heparin (5000 IU/mL) used in this study remains in solution at 37°C for over 72 h; however, when preparing the solution at room temperature, a slight haze begins to develop on standing (c. 30 min). Other combinations (cefazolin 5000 mg/L, gentamicin 2500 mg/L and heparin 5000 IU/mL) should also be sufficient.

Because of the similar efficacy of vancomycin or cefazolin lock combinations in this study, the use of the CGH lock and systemic antibiotics may be sufficient for empirical treatment until blood culture and susceptibility results are obtained. In our study, two isolates were sterilized and the other two had significant reductions in bacterial counts compared with controls after one administration of the CGH lock. Thus, S. epidermidis reported as being methicillin resistant responded well to the CGH lock. This is most likely due to the large concentration of antibiotic combination dwelling inside the catheter.

Although this in vitro catheter study was limited to four different isolates causing bacteraemia in dialysis patients, the results warrant further investigation in prospective clinical trials. The duration of use of the antibiotic/heparin lock must also be investigated. Clinical investigations should target a longer antibiotic/heparin lock duration.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
These results indicate that the VGH or CGH locks sterilize or significantly reduce MRSE growth (>99%) in CVCs in vitro and therefore warrant further investigation in prospective, randomized, blinded clinical trials.


    Acknowledgements
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
The authors would like to thank the Kidney Foundation of Canada (Manitoba Branch) for their research support.


    Notes
 
* Correspondence address. 407 B Pharmacy Building, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba, Canada R3T 2N2. Tel: +1-204-474-6043; Fax: +1-204-474-7617; E-mail: lavern_vercaigne{at}umanitoba.ca Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
1 . Marr, K. A., Sexton, D. J., Conlon, P. J., Corey, G. R., Schwab, S. J. & Kirkland, K. B. (1997). Catheter-related bacteremia and outcome of attempted catheter salvage in patients undergoing hemodialysis. Annals of Internal Medicine 127, 275–80.[Abstract/Free Full Text]

2 . Moss, A. H., Vasilakis, C., Holley, J. L., Foulks, C. J., Pillai, K. & McDowell, D. E. (1990). Use of a silicone dual-lumen catheter with a Dacron cuff as a long-term vascular access for hemodialysis patients. American Journal of Kidney Diseases 16, 211–5.[ISI][Medline]

3 . Pearson, M. L. (1996). Guideline for prevention of intravascular device-related infections. Part I. Intravascular device-related infections: an overview. The Hospital Infection Control Practices Advisory Committee. American Journal of Infection Control 24, 262–77.[ISI][Medline]

4 . Vercaigne, L. M., Sitar, D. S., Penner, S. B., Bernstein, K., Wang, G. Q. & Burczynski, F. J. (2000). Antibiotic–heparin lock: in vitro antibiotic stability combined with heparin in a central venous catheter. Pharmacotherapy 20, 394–9.[ISI][Medline]

5 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Villanova, PA.

6 . Capdevila, J. A., Segarra, A., Planes A. M., Ramirez-Arellano, M., Pahissa, A., Piera, L. et al. (1993). Successful treatment of haemodialysis catheter-related sepsis without catheter removal. Nephrology, Dialysis, Transplantation 8, 231–4.[Abstract]

7 . Farber, B. F., Kaplan, M. H. & Clogston, A. G. (1990). Staphylococcus epidermidis extracted slime inhibits the antimicrobial action of glycopeptide antibiotics. Journal of Infectious Diseases 161, 37–40.[ISI][Medline]

8 . Darouiche, R. O., Dhir, A., Miller, A. J., Landon, G. C., Raad, I. I. & Musher, D. M. (1994). Vancomycin penetration into biofilm covering infected prostheses and effect on bacteria. Journal of Infectious Diseases 170, 720–3.[ISI][Medline]

9 . Slack, M. P. & Nichols, W. W. (1982). Antibiotic penetration through bacterial capsules and exopolysaccharides. Journal of Antimicrobial Chemotherapy 10, 368–72.[ISI][Medline]

10 . Sheth, N. K., Franson, T. R. & Sohnle, P. G. (1985). Influence of bacterial adherence to intravascular catheters on in-vitro antibiotic susceptibility. Lancet ii, 1266–8.

Received 6 August 2001; returned 9 October 2001; revised 14 December 2001; accepted 21 December 2001