Department of Surgery, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, BB 1734, New York, NY 10032, USA
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Abstract |
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Introduction |
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In this study, we have evaluated the risk of developing resistance by Staphylococcus epidermidis to the antibiotics minocycline and rifampicinused in the Cook Spectrum catheter and the antisepticschlorhexidine and silver sulphadiazineused in the Arrowgard catheter. Some other commonly used antiseptics (p-chloro-m-xylenol, triclosan and polyhexamethylene bis-biguanide) were also evaluated. The efficacy of both antibiotic and antiseptic catheters against S. epidermidis passaged through different antimicrobials was also studied. S. epidermidis was chosen for this study since it is one of the major pathogens associated with catheter-related infections. This study provides some insight into the relative risk of emergence of resistant mutants and the efficacy of antimicrobial catheters against antibiotic-resistant bacteria.
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Materials and methods |
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A coagulase-negative strain of S. epidermidis (ATCC 35983) and two clinical strains, one from Harlem Hospital (H) and the othera rifampicin-resistant strainfrom Columbia Presbyterian Hospital (RIF-r1), both in New York, were used.
Antimicrobials
Minocycline (MIN), rifampicin (RIF), chlorhexidine acetate (CHA) and p-chloro-m-xylenol (PCMX) were purchased from Sigma Chemical Co. (St Louis, MO, USA), triclosan (TC) was obtained from Ciba-Geigy (High Point, NC, USA) and silver sulphadiazine (AgSD) and poly-hexamethylene bis-biguanide (PHMB) from Marrion Merrell Dow (Kansas City, MO, USA) and Zeneca Biocides (Wilmington, DE, USA), respectively.
Culture media
Trypticase soy broth (TSB), trypticase soy agar (TSA) and drug neutralizing agar (DE)12 were purchased from Fisher Scientific Co. (Atlanta, GA, USA). The composition of drug neutralizing broth (LTSB)8 was: 3% TSB, 0.5% proteose peptone, 0.1% tryptone, 0.5% sodium thiosulphate, 0.6% sodium oleate, 2% lecithin and 5% Tween 80.
Soaking medium
A proteinaceous medium containing 5% bovine adult serum (BAS) and 5% total parenteral nutrition fluid (TPN) in phosphate-buffered saline (PBS) was constituted in the laboratory. BAS and PBS were purchased from Sigma. TPN was obtained from ColumbiaPresbyterian Medical Center Research Pharmacy (New York, NY, USA).
Catheters
Antibiotic and antiseptic catheters used in this study were triple lumen 7 French catheters. The antibiotic catheter (Cook Spectrum), impregnated with MIN and RIF, was obtained from Cook Critical Care (Bloomington, IN, USA). Two types of antiseptic catheters, I and II, were used in the study and were obtained from Arrow International, Inc. (Reading, PA, USA). Antiseptic catheter I is impregnated with CHA and AgSD on the external surface only (Arrowgard Blue). Antiseptic catheter II is impregnated with AgSD and higher levels of CHA on the external surface and chlorhexidine alone on the luminal surface (Arrowgard Plus). Arrowgard Plus is a new catheter approved recently by the Food and Drug Administration (FDA) and is also used clinically.
MIC and MBC determinations
MIC was determined by a standardized tube dilution method in TSB, as described previously.13 Briefly, two-fold serial dilutions of the drugs were prepared in 5 mL TSB. Stock solutions (10 000 and 2000 mg/L) of all the drugs, made in 50% reagent alcohol, were diluted and added to TSB-containing tubes keeping alcohol at a concentration of 2.5% v/v in all tubes, including the control. An overnight culture grown in TSB was diluted and added to all the tubes for a cell density of 104 cfu/mL. The tubes were incubated for 2448 h at 37°C and checked for turbidity. In addition to the individual agents, the drug combinations studied were CHA and AgSD in 3:1 ratio (CHA+AgSD) based on the levels present in the antiseptic catheter II, CHA and TC in 3:1 ratio (CHA+TC) and MIN and RIF in 1:1 ratio (MIN+RIF) as used in the antibiotic catheter.14 For MBC determination, 0.1 mL aliquots from all tubes showing no visible growth were plated out on DE agar and incubated for 2448 h. MBC was defined as the lowest antimicrobial concentration that killed 99.9% of the inoculum.
Development of resistant bacteria
Culture tubes containing 5 mL of TSB and the drugs were inoculated with diluted overnight culture to obtain 1 x 104 cfu/mL in each tube. Drug concentrations ranged from three doubling dilutions above to three doubling dilutions below the MIC for each agent. After incubation at 37°C for 24 h the culture below the MIC tube was used to prepare the inoculum for the next transfer (104 cfu/mL). After 10 and 20 passages through subinhibitory concentrations of the antimicrobials, cultures below the MIC tube were subcultured and used for susceptibility testing. The clinical isolate (H) was also evaluated for resistance development against RIF, MIN+RIF and CHA+AgSD. Stability of developed resistance was checked by passaging the cultures 20 times through drug-free medium and testing the MIC.
Susceptibility testing
This was carried out by determining the changes in MIC and measuring zones of inhibition produced using catheter segments.
(i) MICs against passaged strains were determined by tube dilution method as described earlier.
(ii) Zones of inhibition produced by the antimicrobial catheters against the original and passaged isolates were determined using the modified KirbyBauer method.8 Briefly, 0.5 cm segments of the test catheters were embedded in TSA plates seeded with 0.3 mL of overnight culture (diluted to 1 x 108 cfu/mL). After incubation at 37°C the zones of inhibition, including the diameter of the catheters, were read. The catheters were tested before and after shaking for 7 and 14 days in proteinaceous medium (1 mL/cm) on a rotary shaker at 100 rpm and at 37°C. The medium was changed daily. The clinical isolate, RIF-r1, was also tested for susceptibility by zones of inhibition.
Rates of kill of antiseptics
Antiseptics (CHA, TC and AgSD) were added in the required concentration (530 mg/L), singly and in combination, to 1 mL of TSB containing 20% BAS and inoculated with 0.1 mL of a suspension of S. epidermidis ATCC 35983 (1 x 107cfu/mL). Samples (0.2 mL) were withdrawn 2, 5 and 10 min after addition of the culture and serial dilutions were made in LTSB. Aliquots (0.5 mL) of the appropriate dilution were plated out on DE agar and incubated for 2448 h. The drug combinations used were CHA+TC (3:1) and CHA+AgSD (3:1) at a total concentration of 530 mg/L.
Correlation between MIC and zone size of antibiotic and antiseptic catheters
Strains of S. epidermidis with different MICs (high and low) of RIF, isolated at various stages of serial passage, were used for this study. The zones of inhibition of catheters pre-soaking, and 7 and 14 days post-soaking were determined using these strains as described earlier.
Bacterial adherence studies
Control (uncoated), antiseptic and antibiotic catheter segments (4 cm long and sealed at both ends) were soaked for 7 and 14 days in proteinaceous medium (1 mL/cm) as stated earlier and tested for in vitro adherence of S. epidermidis ATCC 35983. The culture was grown overnight, at 37°C, in TSB supplemented with 0.25% glucose15 and was diluted 1:5 with fresh medium. This was incubated further for 46 h, at which time the absorbance (at 600 nm) was 0.250.3. The culture was then centrifuged and the cells washed twice in PBS. These were then resuspended in PBS supplemented with 0.5% glucose15 (glucose was added as a substrate for viability and slime production) and sonicated for 1 min to obtain a uniform suspension. After dilution to a density of 12 x 105 cfu/mL, four catheter segments were suspended in 6 mL of this culture in a culture tube, and incubated at 37°C. After 24 h, the catheters were removed, rinsed twice in saline and 1 cm was cut off at both ends. The remaining catheter pieces (2 cm in length) were then suspended in 4 mL LTSB and sonicated in an ultrasonic bath at 40 kHz for 20 min. Aliquots of the LTSB extract (0.5 mL) were then plated out on DE agar and incubated at 37°C for 2448 h.
Evaluation of the neutralizing efficacy of drug inactivating media
To avoid false-negative results due to carry-over of drugs to the subculture media, drug-inactivating media were used. The efficacy of the neutralizing media was tested by suspending 2 cm segments of control (uncoated), antiseptic and antibiotic catheters in 4 mL LTSB inoculated with an overnight culture of S. epidermidis so as to give a density of 1 x 103 cfu/mL. These were sonicated at 40 kHz for 20 min. Aliquots of this suspension (0.5 mL) were plated out on TSA and DE plates and incubated at 37°C for 2448 h to determine the colony count (0.5 mL aliquot of LTSB, similarly inoculated but not sonicated, was used as control).
Estimation of drug levels in catheters
Antibiotic levels were determined using a modification of the method described by Darouiche & Hamill.16 Briefly, 1 cm catheter segments (ends were open to ensure diffusion of drugs from the luminal surface) were extracted with 5 mL of methylene chloride by sonication for 15 min and by intermittent vortexing for 45 min. The extracts were dried in air and the residue was suspended in 1 mL of buffer consisting of 60% KH2PO4 and 40% acetonitrile, adjusted to pH 3.25 using 80% ortho-phosphoric acid. Two-hundred microlitres of the sample were loaded on to a SEP-PAK C18 cartridge (Waters Corporation, Milford, MA, USA) pre-washed with buffer. Elution was carried out using the same buffer at a flow rate of 1 mL/min and 1 mL fractions were collected. The absorbance of the eluting fractions was read at 339 nm. MIN was eluted in the first two fractions and RIF was eluted between the fifth and seventh fractions. Standard amounts of the two antibiotics similarly processed showed 8590% recovery from the column. The initial levels of antibiotics in the catheter were also estimated by this method. The amounts of antiseptics in the catheters were estimated using methods described previously.7 Briefly, chlorhexidine levels were determined by extracting 1 cm catheter segments with 4 mL of reagent alcohol for 20 min. Aliquots (1 mL) of the extract were diluted with an equal volume of water and measured spectrophotometrically at 259 nm for chlorhexidine levels. After alcohol extraction the catheter segments were re-extracted with 2 mL methylene chloride and 4 mL of 1% nitric acid and left overnight. These were then centrifuged and the aqueous layer was read at 254.5 nm for sulphadiazine levels. Silver and sulphadiazine have been shown to be released in similar proportions at various times of soaking7 and hence AgSD levels were calculated based on the sulphadiazine levels. The initial levels of CHA and AgSD in the catheters were also determined using these methods.
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Results |
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A 25 000-fold increase in the MIC of RIF was detected in S. epidermidis (ATCC strain and clinical isolate H) after 10 passages while no significant increase (two-fold) was detected in the MIC of MIN after 20 passages (Table I). The increase in the MIC of the antibiotic combination (MIN+RIF) was 10-fold for the ATCC strain and 16-fold for the clinical isolate H. In the case of the antiseptics (CHA, PHMB and PCMX) and antiseptic combinations (CHA+AgSD and CHA+TC), there was no substantial change in MIC, even after 20 passages (one- to two-fold). However, an eight-fold increase in the MIC of triclosan was seen, when used alone (Table I
). The MICs of all the passaged isolates were stable and did not change after 20 transfers through drug-free medium. There were no significant changes in the MBC of any of the drugs except RIF and MIN+RIF. In the case of RIF there was a >10-fold increase in the MBC for the ATCC strain and 10 000-fold for the clinical isolate H (Table I
), while for MIN+RIF the increase was >2000-fold for both the strains.
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The sensitivity of the different cultures, obtained after passage through RIF (RIF-r2) and MIN+RIF (MINRIF-r) to MIN, CHA, PCMX, PHMB, TC, CHA+TC and CHA+AgSD was not significantly different from that of the susceptible strain (Table II). However, the MINRIF-r strain had an elevated MIC of RIF (80-fold), while the RIF-r2 strain had a higher MIC of MIN+RIF (16-fold), both relative to that of the susceptible strain.
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The zones of inhibition of the antiseptic catheters against the susceptible strain and the cultures obtained after 20 passages through CHA+AgSD, MIN alone and RIF alone were similar (1314 mm). After 7 and 14 days soaking (this was done to simulate the release of drug from the intravenous portion of the catheters or in the case of where there is serous fluid in the subcutaneous space), the antiseptic catheter I had zone sizes of 4.3 and 3.5 mm, respectively, against the susceptible strain while for the antiseptic catheter II these were 7.2 and 5.6 mm, respectively. Since the antiseptic catheter I had very low activity at day 7 due to diffusion of most of the drugs, only the antiseptic catheter II, which has higher quantities of drugs, was used for further evaluation There was no difference between the zones of the antiseptic catheter II for the susceptible strain and RIF-r2 strain at any of the time intervals tested. Antiseptic catheters produced larger zones (89 mm) against the MINRIF-r strain and the RIF-resistant clinical isolate (RIF-r1) compared with the susceptible strain (7 mm) at all periods of testing (Figure 1).
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To evaluate whether the combinations described in this study exhibit enhanced or synergic antimicrobial efficacy as compared with the respective drugs when used singly, the rates of kill of individual and combination drugs were determined. The CHA+TC combination was the most effective in achieving a total kill within 5 min followed by the CHA+AgSD combination, which produced total kill within 10 min. CHA alone gave a 2.5 log10 reduction in 10 min (Figure 3).
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This experiment was carried out to determine changes in zone sizes of the antiseptic and antibiotic catheters when tested against RIF-r2 isolates with varying degrees of resistance. The isolates (with different MICs) were obtained at different stages of serial passage through RIF. Unlike RIF (MIC increased >25 000-fold after passage), the MIC of MIN and CHA+AgSD did not change significantly and that of MIN+RIF changed only 16-fold (MINRIF-r isolate).
Zone sizes of the antibiotic catheters against the parent (MIC 0.02 mg/L) and MINRIF-r strains (MIC 0.2 mg/L) were c. 22 and 10 mm respectively after 14 days of soaking. Table III shows that zone sizes of antibiotic catheters decreased with the increase in MIC of RIF-r2 strains. However, for the antiseptic catheter group no such decrease was seen.
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There was no difference in the number of bacteria recovered from the control and the antiseptic catheter groups when their sonicated extracts were plated either on TSA or DE plates (103 cfu/mL in all the groups). On the other hand, with the antibiotic catheter, no colonies were detected on TSA plates while the colony counts on DE plates were similar to those obtained with the control and antiseptic catheter. These results show that the use of LTSB as a suspending fluid during sonication and DE agar plates for subculturing is effective in neutralizing the drugs present in both the antiseptic and antibiotic catheters. Both the sonicated control group and non-sonicated LTSB culture yielded 103 cfu/mL.
Drug levels, zone sizes and bacterial adherence
Since the antiseptic catheter exhibits relatively lower zones of inhibition than that of antibiotic catheters, and antibiotic catheters with zones below 15 mm have been reported to be ineffective in preventing catheter colonization,9 we evaluated the correlation between the zone size, drug levels and bacterial adherence of the antiseptic and antibiotic catheter pre- and post-soaking in media.
Drug levels.
It was observed that for both the antiseptic catheter II and the antibiotic catheter, the rates of release of the two impregnated drugs were different. MIN was released more quickly than RIF, altering the ratio of MIN to RIF from approximately 1:1 at day 0 to 1:6.5 at day 7 and 1:5.8 at day 14. In the case of the antiseptic catheter II CHA was released more quickly than AgSD, changing the ratio from 3.2:1 at day 0 to 1:1 and 2:1 at day 7 and day 14, respectively (Table IV).
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Discussion |
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It has been suggested that the combination of MIN and RIF used in the antibiotic catheter may have a lower risk of developing resistance.18 This was based on the observations that (i) there is good tissue penetration of both agents into infected tissue and (ii) emergence of RIF resistance can occur only in patients infected with high concentrations of bacteria (mutations conferring RIF resistance can occur with a frequency of 106108)19,20 and the level of colonization of catheter rarely exceeds 104 cfu/cm. However, in a clinical study using 1681 catheters, Sherertz et al.21 found that out of the 12.3% of colonized catheters, 31% had 105107 cfu/cm. Resistance to RIF has been found to occur in staphylococci2226 and in one study, these were isolated from patients with central venous catheters.27 We were also able to isolate mutants resistant to RIF (RIF-r2 strain) after 10 passages with two S. epidermidis strains. In contrast, development of resistance to MIN did not occur even after 20 passages. Similar in vitro results have been reported by other investigators.28 However, MIN-resistant strains of S. epidermidis and Staphylococcus aureus have been isolated from patients.29,30 Our studies indicate that MIN has a protective effect on RIF resistance development, since when RIF was used in combination with MIN during passage, resistance of this isolate (MINRIF-r) to RIF increased only 80-fold in contrast to 25 000-fold when RIF alone was used (Table II). The MINRIF-r strain also had an elevated MIC of MIN+RIF (10-fold) because of the higher MIC of RIF, since the MIC of MIN for this strain was unaltered. The protective effect of MIN on RIF has been shown previously by Yourassowsky et al.31 in methicillin- and gentamicin-resistant S. aureus.
The development of resistance to antiseptics was negligible, except to TC, which showed an eight-fold increase in MIC when used singly (Table I). TC-resistant mutants of Escherichia coli, but not Gram-positive bacteria have been isolated.3234 When TC was combined with CHA, the MIC did not change significantly, probably due to the synergic effect of CHA and TC in rapidly killing the organism (Figure 3
). CHA and AgSD also produced a rapid kill. Although low-level, unstable resistance to chlorhexidine in antibiotic-resistant S. aureus35,36 and stable resistance to chlorhexidine in Pseudomonas spp. have been reported,5 emergence of chlorhexidine-resistant strains of either Gram-positive or Gram-negative species has not been reported for the past 8 years with the use of CHA+AgSD catheters. We were also unable to obtain strains resistant to CHA+AgSD after 20 passages through subinhibitory concentrations. Lack of resistance development to these antiseptic combinations may be due to their action at multiple target sites in the bacteria1 resulting in synergic efficacy of these agents (Figure 3
). Based on our results the small increase in the MIC of antiseptics (two-fold after 20 passages) may not have clinical significance.
We evaluated the correlation between zone size and antibacterial efficacy of antibiotic and antiseptic catheters. Sherertz et al.9 have suggested that a zone size of at least 15 mm is necessary to prevent catheter colonization. The antiseptic catheter that produced small zones of inhibition after 7 and 14 days soaking (7.2 and 5.6 mm, respectively) prevented bacterial adherence. On the other hand, the antibiotic catheter showed adherence in spite of a larger zone size (14 mm; Table IV). These results indicate that unlike in the case of antibiotic catheters, the relatively smaller zones of inhibition with antiseptic catheters do not appear to be predictive of lack of efficacy.
Zone size may be related to solubility and diffusion characteristics of the agents involved, while ability to resist colonization may depend on the type of antimicrobial action such as static or cidal and slow or rapid kill. Evaluation of activity of the antimicrobial catheters against passaged strains shows that the antibiotic catheter has substantially smaller zones against the MINRIF-r (10 mm) and RIF-r2 strains (4.1 mm) compared with the susceptible strain (14.1 mm) after 14 days (Figure 2). This may be due to lack of adequate MIN levels (20.3 µg/cm) in the catheter and the elevated MIC of RIF for these strains. This catheter showed reduced activity after 7 and 14 days soaking, even against strains with relatively low-level resistance to RIF (Table III
). The antiseptic catheter showed similar activity against original and passaged strains (Figure 1
).
Since resistance to RIF is known to occur clinically, the question arises as to whether indwelling antibiotic catheters will be effective against late onset infection by RIF-r organisms when substantial amounts of antibiotics, especially MIN, have presumably diffused out of the catheters. On the other hand, the zones of inhibition of the antiseptic catheters against the susceptible and RIF-r2 strains were similar at all time intervals, suggesting that there is no cross-resistance to the antiseptics used in the catheter. In fact, larger zones were observed against the MINRIF-r strain and RIF-r1 strain (clinical isolate).
In a clinical study of comparison of antibiotic and antiseptic catheter I, the antibiotic resistance profile of bacteria isolated from colonized catheters in both groups was similar.14 Our study suggests that should an antibiotic catheter be infected with RIF-resistant organisms, the risk of catheter colonization may be higher compared with the antiseptic catheter. The possible emergence of organisms resistant to the antimicrobials used in these catheters needs to be monitored constantly.
In summary, resistance can develop more easily for the combination of antibiotics than for the antiseptics and more readily for RIF than for MIN. While antibiotic catheters have been found to be effective in clinical studies and the emergence of resistant bacteria has not been reported with the use of these catheters to date, their efficacy when challenged with RIF-resistant bacteria remains to be seen. Antiseptic catheters may have a low risk of colonization by antibiotic-resistant bacteria. Both antibiotic and antiseptic catheters should be continually monitored and the resistance profile of bacteria recovered from colonized catheters determined in order to detect the possible emergence of resistant bacteria.
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Acknowledgments |
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Notes |
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References |
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Received 28 September 2000; returned 13 December 2000; revised 8 January 2001; accepted 19 January 2001