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JAC Advance Access published online on August 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm295
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vitro effectiveness of the antibiotic lock technique (ALT) for the treatment of catheter-related infections by Pseudomonas aeruginosa and Klebsiella pneumoniae

Mi Young Lee1,{dagger}, Kwan Soo Ko1,2,{dagger}, Jae-Hoon Song1,3 and Kyong Ran Peck3,*

1 Asian-Pacific Research Foundation for Infectious Diseases (ARFID), Seoul 135-710, Korea 2 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea 3 Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea

* Corresponding author. Tel: +82-2-3410-0322; Fax: +82-2-3410-0041; E-mail: krpeck{at}smc.samsung.co.kr

Received 18 June 2007; returned 29 June 2007; revised 10 July 2007; accepted 11 July 2007


   Abstract
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Objectives: To determine the adequate antibiotic treatment, the concentration of antibiotics and the duration of treatment with the antibiotic lock technique (ALT) for treatment of central venous catheter-related infections caused by Klebsiella pneumoniae and Pseudomonas aeruginosa.

Methods: To investigate the in vitro effectiveness of four candidate antibiotics, amikacin, ceftazidime, cefepime and ciprofloxacin, two isolates of both K. pneumoniae and P. aeruginosa forming biofilms were selected. The polyurethane (PU) films were incubated for 5 days to allow for bacterial colonization or biofilm production. After 5 days, the biofilm-formed PU films were exposed to each of the antibiotics (1, 5 and 10 mg/mL) for 1, 3, 5, 7, 10 and 14 days. The presence of the remaining bacteria in the biofilm was evaluated by the determination of viable cell counts.

Results: All of the antibiotic treatments effectively removed P. aeruginosa biofilm within 3–5 days. Among the four antimicrobial agents tested in this study, ciprofloxacin showed superior bactericidal activity. The biofilms of both species were eliminated by 5 mg/mL ciprofloxacin within 3 days. In all cases, P. aeruginosa strains were removed more rapidly than K. pneumoniae strains. All antibiotics eradicated the susceptible K. pneumoniae strain, K144, within 5 days. One strain of K. pneumoniae, K139, which was resistant to all tested antibiotics, was not eradicated by amikacin (1, 5 and 10 mg/mL) or 1 mg/mL ceftazidime.

Conclusions: These results show that ciprofloxacin, cefepime, ceftazidime and amikacin might be used as an effective ALT for treatment of catheter-related infections caused by antibiotic-susceptible K. pneumoniae and P. aeruginosa. This study suggests that the duration of treatment against catheter-related infection by Gram-negative bacilli can be reduced to 3–5 days when using antibiotics to which the organisms are susceptible in vitro, even at a concentration of 1 mg/mL.

Key Words: biofilms , ciprofloxacin , cefepime


   Introduction
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Concerns about catheter-related infections are increasing in modern medicine. Catheter-related infections occur with the establishment of microbial biofilms on the catheter surface.1–3 Owing to frequent failure of treatment of these infections based on conventional antimicrobial susceptibility testing, it has become more complex and difficult to treat such infections. In general, more than 100 times the antimicrobial concentrations are needed to kill biofilm-forming bacteria than to kill bacteria in solution.4 It is known that the most effective treatment is the removal and replacement of the infected device. However, this process is accompanied by significant technical problems and costs.5

Instead of the removal of the device, the antibiotic lock technique (ALT) is recommended by the guidelines of the Infectious Diseases Society of America (IDSA) and the Centers for Disease Control and Prevention (CDC) as an effective therapeutic option for catheter-related infections.6,7 In the ALT, a concentrated antibiotic solution is instilled into the central venous catheter (CVC) lumen and allowed to dwell for several hours or days. Antimicrobial choices for ALT depend on biofilm-forming bacterial pathogens, characteristics of infected bacteria and the pharmacodynamics of antimicrobial agents used in the ALT. More than half of all catheter-related infections are caused by Gram-positive bacteria such as Staphylococcus aureus and coagulase-negative staphylococci (CoNS).8 Thus, most studies on ALT have focused on staphylococcal infections.1,9,10 However, Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella pneumoniae also cause catheter-related infections and should not be ignored.6 Experimental evidence for selection of antimicrobial agents and antimicrobial concentrations against Gram-negative bacteria has rarely been shown.

We examined the in vitro activity of ALT using a variety of antimicrobial agents to determine the optimal concentration of antibiotics and adequate duration of locking therapy for treatment of P. aeruginosa and K. pneumoniae biofilms.


   Materials and methods
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Bacterial strains used in this study

Two strains of P. aeruginosa and two strains of K. pneumoniae were used in this study: P. aeruginosa (ATCC 9027 and P66) and K. pneumoniae (K139 and K144). P. aeruginosa ATCC 9207 is known to form biofilms and has been used in a previous study. P. aeruginosa P66 and K. pneumoniae K139 were isolated from catheterized urine from patients with a brain tumour as underlying disease. K. pneumoniae K144 was isolated from the vascular catheter of a patient with diabetes mellitus, chronic renal failure and kidney transplantation. All strains were confirmed as biofilm-forming. None of the strains produced extended-spectrum ß-lactamase.

Determination of MICs and MBCs

The MIC was determined by the broth microdilution method according to the CLSI.11 S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922 were used as controls. The MBC was also determined from subcultures on 5% sheep blood agar plates with 0.1 mL of each, from the control, the first tube with visible growth and tubes without visible growth.10 The MBC was defined as the lowest concentration of antibiotic that reduced the bacterial population by ≥99.9% of the original inoculum.12

Biofilm formation

The polyurethane (PU) sheet, which was prepared by the method of Golomb and Shpigelman,13 was cut into 1 x 1 cm square pieces (thickness, 0.3 mm) and sterilized with ethylene oxide gas. Bacterial strains were grown on tryptic soy agar (TSA) plates at 37°C overnight. A 0.5 McFarland bacterial suspension (~108 cfu/mL) was prepared and diluted with tryptic soy broth (TSB) supplemented with 0.25% sucrose, to yield a final inoculum of ~1.5 x 107 cfu/mL. The sheet was placed in 10 mL of a bacterial suspension of TSB. The PU sheet was shaken at 100 rpm during incubation at 37°C for 5 days to allow colonization or biofilm production.

In vitro antibiotic lock model

After 5 days of incubation, the PU pieces were rinsed with PBS solution to remove planktonic bacteria. They were transferred to TSB supplemented with 0.25% sucrose containing each of the following antimicrobial agents: ciprofloxacin, amikacin, ceftazidime and cefepime. Antimicrobial agents were used at concentrations of 1, 5 and 10 mg/mL. Ciprofloxacin could not be evaluated at a concentration of 10 mg/mL because of precipitation. TSB without antimicrobials was used as a control for each experiment. The bacterial killing activities of the antibiotic solutions were assayed after lock periods of 1, 3, 5, 7, 10 or 14 days. In each set of lock period and antibiotic solution, different PU sheets were used. Antibiotic lock solutions were replaced every 2 days. Biofilm eradication was evaluated by the determination of viable counts. The PU pieces were removed from the bacterial culture and washed twice with 10 mL of sterile PBS in order to remove planktonic bacteria and antimicrobial residue. Then, the washed PU pieces were subjected to high-speed vortexing in 3 mL of sterile PBS, followed by sonication using a VCX-400 sonicator (Sonics & Materials, Inc., Danbury, CT, USA) (120 s, 30% cycle, pulse 3.5 s). The number of viable biofilm bacteria was counted by 10-fold serial dilution and plating on blood agar. The experiments were repeated three times, and the mean values for the biofilm bacteria were compared between groups for each antibiotic.


   Results
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MICs and MBCs

The MICs and MBCs of the antimicrobial agents for each of the two strains of P. aeruginosa and K. pneumoniae are shown in Table 1. P. aeruginosa ATCC 9027 was susceptible to all antibiotics tested in this study, whereas P66 was resistant to ceftazidime and cefepime. The two K. pneumoniae strains also showed different antimicrobial resistance profiles. K139 was resistant to all antibiotics used in this study, whereas K144 was susceptible to all of the antibiotics. No strains except P. aeruginosa P66 against ceftazidime showed tolerance to any of the antimicrobial agents tested in this study, that is MBC/MIC > 8.


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  		Table 1. MICs and MBCs of antimicrobial agents for P. aeruginosa and K. pneumoniae strains

 
Effect of ALT

For P. aeruginosa, four antimicrobial agents completely eradicated the biofilm bacteria within 3 days (Tables 2 and 3). In particular, the PU films were sterilized after 1 day of exposure to 5 mg/mL ciprofloxacin and 3 days to 5 mg/mL amikacin. Ciprofloxacin, amikacin or ceftazidime at 1 mg/mL eradicated all P. aeruginosa within 3 days. Cefepime at 1 mg/mL completely killed the ATCC 9027 and P66 strains within 3 or 5 days, respectively.


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  		Table 2. Effect of ALT for treatment of P. aeruginosa ATCC 9027 (no. of cfu/sheet, mean ± SD)

 


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  		Table 3. Effect of ALT for treatment of P. aeruginosa P66 (no. of cfu/sheet, mean ± SD)

 
For K. pneumoniae, the PU films were sterile within 5 days of exposure to any of the concentrations of ciprofloxacin (Tables 4 and 5). While K. pneumoniae K144 was eradicated by 1 mg/mL amikacin (Table 5), the K139 strain was not sterilized by any of the concentrations of amikacin (Table 4). Although 1 mg/mL ceftazidime did not eradicate K. pneumoniae K139, it killed all K144 bacteria within 3 days of the lock period. K. pneumoniae biofilms were killed within 7 days of exposure to cefepime (Tables 4 and 5).


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  		Table 4. Effect of ALT for treatment of K. pneumoniae K139 (no. of cfu/sheet, mean ± SD)

 


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  		Table 5. Effect of ALT for treatment of K. pneumoniae K144 (no. of cfu/sheet, mean ± SD)

 

   Discussion
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It has been estimated that 75 000 cases of catheter-related infection occur every year in the United States, which results in additional expenditure of $300 million to $2.3 billion.6 Despite some controversy, it is known that catheter-related infections cause excess mortality and prolonged hospitalization.14,15 At this point, removal of the catheter is the most effective treatment for catheter-related infections, especially in patients with severe sepsis or septic shock. However, the catheter may be left in, and it is generally recommended that infections be treated with appropriate antimicrobial drugs when the patient is in a stable condition.16 In particular, the ALT is recommended as an alternative therapeutic option for catheter-related infections.6,7

Reliable experimental data on the selection of effective antimicrobial agents and antimicrobial concentrations for ALT have not been sufficient. Most investigations have focused on staphylococcal infections.8 Actually, CoNS and S. aureus are the most frequently found pathogens in catheter-related infections. However, Gram-negative bacteria are also important pathogens in catheter-related infections.16 In particular, P. aeruginosa is one of the common organisms that causes catheter-related infections and severe, often difficult-to-eradicate bloodstream infections.17 P. aeruginosa is one of the best-studied models for biofilm formation.8,18 Although K. pneumoniae is a less frequently found pathogen in catheter-related infections, it should not be neglected.4,19

In this study, all four antibiotics were found to be effective in eradicating P. aeruginosa biofilms. Both P. aeruginosa strains were killed by 1 mg/mL of any antibiotics within 3–5 days. It is noteworthy that although strain P66 showed resistance to ceftazidime (MIC, 16 mg/L) and cefepime (MIC, 16 mg/L), it was still successfully eradicated by the antibiotics. In spite of tolerance of strain P66 against ceftazidime, the efficacy of ALT was not reduced.

K. pneumoniae strains did not show consistent results (Tables 4 and 5). The effectiveness of ALT against K. pneumoniae seems to be partially associated with antibiotic resistance. Ceftazidime at 1 mg/mL was enough to kill a ceftazidime-susceptible strain, K144, within 3 days. Conversely, for the ceftazidime-resistant strain, K139, higher concentrations (5–10 mg/mL) were needed to eradicate biofilm bacteria. Even though the efficacy of ALT against resistant strains of P. aeruginosa and K. pneumoniae was inconsistent with antibiotic resistance, most of the antibiotics eradicated susceptible strains within 3 days at a concentration of 1 mg/mL. Higher concentrations of 5–10 mg/mL sterilized biofilm bacteria more rapidly.

The present study showed that ciprofloxacin was the most effective agent in eradicating both of the P. aeruginosa and K. pneumoniae strains. Even 1 mg/mL ciprofloxacin killed both of the P. aeruginosa and K. pneumoniae biofilms within 5 days, and 5 mg/mL could eradicate all strains within 3 days. In a previous study, we suggested that ciprofloxacin was superior to vancomycin in ALT for S. epidermidis and S. aureus biofilms.10 Thus, ciprofloxacin could be considered as a therapeutic option for treatment of mixed catheter-related infections with staphylococci and Gram-negative bacilli, including antimicrobial-resistant pathogens with relatively low MICs.

Our study concentrated on the effectiveness of antimicrobials against catheter-related infections, but more consideration is needed in the ALT. Besides common antibiotics, minocycline-edetate calcium disodium (MEDTA), taurolidine-polyvinylpyrolidine, ethanol and citrate-taurolidine have been recently suggested as potential catheter lock solutions.17,20 Particularly, taurolidine has broad-spectrum activity and does not carry the risk for side effects and resistance. Although their effectiveness could not be investigated in this study, it is worth considering them as other options for the treatment of catheter-related infections. In addition, the interaction between the antibiotics and anticoagulants in catheter lock solutions needs to be evaluated in future studies, because antibiotics are commonly used with anticoagulants to prevent thrombosis or obstruction of the catheter.

Catheter-related infections by Gram-negative bacilli currently must be treated according to antimicrobial susceptibility testing. However, there have been no reports on the optimal duration of therapy. Instead, treatment periods of 10–14 days have been suggested empirically in most cases.6,16 This study suggests that the duration of treatment against catheter-related infections by Gram-negative bacilli can be reduced to 3–5 days when we use antibiotics to which the organisms are susceptible in vitro, even at a concentration of 1 mg/mL. However, this suggestion must not be decisive because it is based entirely on in vitro studies. Thus, prospective clinical trials for further evaluation of the clinical efficacy of ALT are required for <5 days with antibiotics at 1–5 mg/mL.


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This study was partly supported by the SBRI (Samsung Biomedical Research Institute) (grant C-A4-316-1), the Basic Research Program of the Korea Science and Engineering Foundation (R01-2002-000-00467-0) and the ARFID (Asian-Pacific Research Foundation for Infectious Diseases).


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None to declare.


   Footnotes
 
{dagger} These authors contributed equally to this paper. Back


   Acknowledgements
 
We thank Dr Jin Ho Lee (Hannam University, Korea) for providing polyurethane films.


   References
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1 . Curtin J, Cormican M, Fleming G, et al. Linezolid compared with eperezolid, vancomycin, and gentamicin in an in vitro model of antimicrobial lock therapy for Staphylococcus epidermidis central venous catheter-related biofilm infections. Antimicrob Agents Chemother (2003) 47:3145–8.[Abstract/Free Full Text]

2 . Raad II, Hann HA. Intravascular catheter-related infections: new horizons and recent advances. Arch Intern Med (2002) 162:619–24.

3 . Eggimann P. Prevention of intravascular catheter infection. Curr Opin Infect Dis (2007) 20:360–9.[CrossRef][Medline]

4 . Carratalà J. The antibiotic-lock technique for therapy of ‘highly needed’ infected catheters. Clin Microbiol Infect (2002) 8:282–9.[CrossRef][Web of Science][Medline]

5 . Toltzis P. Antibiotic lock technique to reduce central venous catheter-related bacteremia. Pediatr Infect Dis J (2006) 25:449–50.[CrossRef][Web of Science][Medline]

6 . Mermel LA, Farr BM, Sherertz RJ, et al. Infectious Diseases Society of America; American College of Critical Care Medicine; Society for Healthcare Epidemiology of America. Guidelines for the management of intravascular catheter-related infections. Clin Infect Dis (2001) 32:1249–72.[CrossRef][Medline]

7 . O'Grady NP, Alexander M, Dellinger EP, et al. Guidelines for the prevention of intravascular catheter-related infections. Centers for Disease Control and Prevention. MMWR Recomm Rep (2002) 51:1–29.[Medline]

8 . Bestul MB, VandenBussch HL. Antibiotic lock technique: review of the literature. Pharmacotherapy (2005) 25:211–27.[CrossRef][Web of Science][Medline]

9 . De Sio L, Jenkner A, Milano GM, et al. Antibiotic lock with vancomycin and urokinase successfully treat colonized central venous catheters in pediatric cancer patients. Pediatr Infect Dis J (2004) 23:963–5.[CrossRef][Web of Science][Medline]

10 . Lee JY, Ko KS, Peck KR, et al. In vitro evaluation of the antibiotic lock technique (ALT) for the treatment of catheter-related infections cased by staphylococci. J Antimicrob Chemother (2006) 57:1110–5.[Abstract/Free Full Text]

11 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S16 (2006) Wayne, PA, USA: CLSI.

12 . Anhalt JP, Washington JA II. Bactericidal tests. In: Laboratory Procedures in Clinical Microbiology—Washington JA II, ed. (1985) Second Edition. New York: Springer-Verlag.

13 . Golomb G, Shpigelman A. Prevention of bacterial colonization on polyurethane in vitro by incorporated antibacterial agent. J Biomed Mater Res (1991) 25:937–52.[CrossRef][Web of Science][Medline]

14 . Pittet D, Tarara D, Wenzel RP. Nosocomial bloodstream infection in critically ill patients. Excess length of stay, extra costs, and attributable mortality. JAMA (1994) 271:1598–601.[Abstract/Free Full Text]

15 . Wenzel RP, Edmond MB. The evolving technology of venous access. N Engl J Med (1999) 340:48–50.[Free Full Text]

16 . Fätkenheuer G, Cornely O, Seifert H. Clinical management of catheter-related infections. Clin Microbiol Infect (2002) 8:545–50.[CrossRef][Web of Science][Medline]

17 . Sherertz RJ, Boger MS, Collins CA, et al. Comparative in vitro efficacies of various catheter lock solutions. Antimicrob Agents Chemother (2006) 50:1865–8.[Abstract/Free Full Text]

18 . Banin E, Brady KM, Greenberg EP. Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm. Appl Environ Microbiol (2006) 72:2064–9.[Abstract/Free Full Text]

19 . Poole CV, Carlton D, Bimbo L, et al. Treatment of catheter-related bacteremia with an antibiotic lock protocol: effect of bacterial pathogen. Nephrol Dial Transplant (2004) 19:1237–44.[Abstract/Free Full Text]

20 . Betjes MGH, van Agteren M. Prevention of dialysis catheter-related sepsis with a citrate-taurolidine-containing lock solution. Nephrol Dial Transplant (2004) 19:1546–51.[Abstract/Free Full Text]


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This Article
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