In Vitro Activity and Durability of a Combination of an Antibiofilmand an Antibiotic against Vascular Catheter Colonization

Mohammad D. Mansouri,a,b Richard A. Hull,a Charles E. Stager,a,b Richard M. Cadle,a,b Rabih O. Darouichea,b

Baylor College of Medicine, Houston, Texas, USAa; Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, USAb

Catheter-associated infections can cause severe complications and even death. Effective antimicrobial modification of cathetersthat can prevent device colonization has the potential of preventing clinical infection. We studied in vitro the antimicrobial ac-tivities of central venous catheters impregnated with N-acetylcysteine (NAC), an antibiofilm agent, and a broad-spectrum antibi-otic against a range of important clinical pathogens. NAC-levofloxacin-impregnated (NACLEV) catheters were also evaluated fortheir antiadherence activity. NACLEV catheters produced the most active and durable antimicrobial effect against both Gram-positive and Gram-negative isolates and significantly reduced colonization (P < 0.0001) by all tested pathogens compared tocontrol catheters. These in vitro results suggest that this antimicrobial combination can potentially be used to combat cathetercolonization and catheter-associated infection.

Intravascular catheters are essential in managing critically ill pa-tients. However, vascular catheter-associated infections could

result in dire consequences leading to excessive morbidity andmortality, longer hospital stays, and higher health care costs, withan average treatment cost of $25,000 per episode (1–5).

Catheter-associated infections are generally initiated by micro-bial colonization of the catheter surface and formation of a super-ficial biofilm layer (6). Cell surface proteins and polysaccharideproduction by bacterial cells reportedly contribute to the forma-tion of biofilm that can then protect pathogens by impeding boththe penetration of antibiotics and the function of phagocytic im-mune cells, thus hindering the ability to combat colonizing patho-gens (7–9).

Impregnation or coating of catheters with antimicrobial agentshas commonly been used to prevent bacterial colonization of vas-cular catheters (8). However, some existing antimicrobial-treatedcatheters designed to prevent catheter colonization may have par-tial clinical efficacy, particularly against drug-resistant pathogens,and limited durability of antimicrobial activity (8, 10–13) partlydue to their inability to control biofilm formation and combatbiofilm-nested microorganisms, which can have MICs of up to1,000 times higher than their MICs against their free-floatingplanktonic counterparts (8, 14–16).

N-acetylcysteine (NAC), a commonly used inhalation muco-lytic therapy for chronic bronchitis and an FDA-approved intra-venous injection for the treatment of acetaminophen toxicity(17), has favorable pharmacokinetics when used in hemodialysispatients (18). NAC also adversely affects bacterial growth andpolysaccharide production and disrupts disulfide bonds in mucus,reducing the viscosity of secretions. These properties may contrib-ute to the prevention and disruption of biofilm around differentpolymeric and metallic surfaces (9, 19–21). Not only does NACdiminish the formation of biofilm by common pathogens (19, 22,23), it also possesses some in vitro intrinsic antimicrobial activityagainst both Gram-positive and Gram-negative bacteria (24).Taking into consideration the therapeutic safety record and theantibiofilm ability of NAC combined with antimicrobial activityof a broad-spectrum antibiotic, impregnation of intravascularcatheters with this unique combination can be a promising ap-

proach for reducing catheter colonization and potential subse-quent catheter-associated infection.

Seven-French, triple-lumen, polyurethane central venouscatheter (CVC) segments (Cook Inc., Bloomington, IN) were im-pregnated with a combination of NAC and levofloxacin, neomy-cin, or gentamicin by immersion in an agitated solution that com-prises 100 mg/ml of NAC and 100 mg/ml of the antibiotic,followed by drying overnight and rinsing to remove any unboundcompound (25). Since the size of the zone of inhibition (ZI) gen-erated by antimicrobial-coated devices is reportedly correlatedwith their in vivo antimicrobial efficacy to prevent colonization(26), we assessed the antimicrobial activity of impregnated cathe-ter segments at baseline and after exposure to human serum overdifferent periods of time against a panel of important clinical iso-lates. One-centimeter segments of NAC-antibiotic-impregnatedand nonimpregnated control catheters were individually placed inhuman serum at 37°C for 3, 7, 10, 14, and 30 days, with a weeklychange of serum. Catheter segments removed from sera and base-line catheter segments (no serum incubation) were assessed for ZI,using a modified Kirby-Bauer diffusion assay (27–29), against me-thicillin-resistant Staphylococcus epidermidis (MRSE), methicil-lin-resistant S. aureus (MRSA), vancomycin-resistant Enterococ-cus (VRE), Pseudomonas aeruginosa, Klebsiella pneumoniae, andEscherichia coli. Briefly, bacterial suspensions at 108 CFU/ml werestreaked uniformly onto Mueller-Hinton agars using cottonswabs, and 1-cm catheter segments were then half embedded inthe center of agar plates, which were then incubated at 37°C over-night. These in vitro experiments were designed to select for fur-ther evaluation the most active and durable NAC-antibiotic com-bination with the broadest spectrum of antimicrobial activity.

Since NAC-levofloxacin-impregnated (NACLEV) catheter

Received 9 August 2012 Returned for modification 27 August 2012Accepted 20 October 2012

Published ahead of print 31 October 2012

Address correspondence to Mohammad D. Mansouri, mdmansouri@aol.com.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.01646-12

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segments displayed the most potent (ZI � 15 mm), widest spec-trum, and longest durability of antimicrobial activity againsttested pathogens, we further evaluated their antiadherence effi-cacy by culture methods and microscopic observations.

We assessed the anticolonization activity of NACLEV cathetersby using a modified Robbins device (MRD) (24) that consists of anacrylic apparatus with an enclosed internal groove that can se-quentially accommodate up to 25 1-cm catheter segments. Varia-tions of this device have been described in other studies (30–32).Twenty 1-cm segments of NACLEV or control catheters were ster-ilely placed in the MRD in sequence. The MRD was then closed,sealed, and connected to a circulating stream of bacterial suspen-sion of MRSE, MRSA (levofloxacin sensitive), MRSA (levofloxa-cin resistant), VRE, P. aeruginosa, K. pneumoniae, or E. coli at 2 �105 CFU/ml using a peristaltic pump with a flow rate of 1 ml/minaround and through the lumens of each catheter segment for 24 hat 37°C. The system was then flushed with one MRD volume ofnormal saline. Segments were then removed, individually placedin 1 ml of normal saline, and cultured quantitatively using thestandard sonication technique (24, 28). Since the MRD devicecould only accommodate one bacterial suspension and either theimpregnated or nonimpregnated catheter segments, this processwas repeated for each organism and for each type of catheter. Wespecifically tested the activity of NACLEV catheters against levo-floxacin-resistant MRSA to evaluate the possible synergetic anti-microbial effect of this combination.

To visualize the antibiofilm and antibacterial effects of the NAC-levofloxacin combination, two 0.5-cm segments of NACLEV orcontrol catheters were individually incubated in a 2 � 105-CFU/mlsuspension of a Gram-positive (MRSA) or Gram-negative (K. pneu-moniae) representative pathogen at 37°C overnight. Catheter seg-ments were then briefly rinsed with normal saline, fixed in a glutar-aldehyde buffer solution, dehydrated, and observed under a JEOL(Peabody, MA) NeoScope JCM-5000 scanning electron microscope(SEM) (33). Samples were coded, and the SEM operator was blindedin regard to the catheter impregnation and treatment. The degree ofcatheter colonization was assessed comparatively between the im-pregnated and control segments based on the clusters of organismsand the amount of superficial biofilm observed.

We used Stata software (Stata Corp., College Station, TX) forour statistical analyses. P values of �0.05 indicated a statisticalsignificance.

In general, NACLEV catheters displayed larger zones of inhi-bition against all tested pathogens compared to NAC-neomycin-

or NAC-gentamicin-impregnated catheters, both at baseline andafter incubation in human serum for different time periods. Rep-resentative images of these zones are shown in Fig. 1a to c.NACLEV catheter segments generally produced the largest ZIsagainst MRSA, a very important pathogen associated with cathe-ter-related infections, compared to NAC-gentamicin- or NAC-neomycin-impregnated catheters at each time period. NAC-gen-tamicin-impregnated catheters had larger ZIs than NAC/neomycin-impregnated catheters at baseline and at different timeperiods after exposure to human serum. Nonimpregnated controlcatheter segments did not produce any zone of inhibition againstany organism. The graphical antimicrobial durability of thesecatheters is depicted in Fig. 2a to f.

Since NACLEV catheters displayed the most potent and durableantimicrobial activity against all test pathogens, we selected this com-bination for further evaluation and showed that this combinationsignificantly (P � 0.0001) reduced all bacterial colonization com-pared to control catheter segments. NACLEV catheter segments ex-posed to P. aeruginosa were completely sterile, whereas the controlsegments averaged 1.3 � 106 CFU/segment. More importantly,NACLEV catheters significantly (P � 0.0001) reduced catheter colo-nization by levofloxacin-resistant MRSA. The mean colony countsextracted from catheter segments are displayed in Fig. 3.

SEM images revealed that considerably fewer bacterial cells ofMRSA and K. pneumoniae were attached to NACLEV cathetersthan control catheter segments (Fig. 4a to d). In addition, therewere less visible biofilms observed on NACLEV catheter segmentsversus nonimpregnated control segments when exposed to eitherorganism.

Despite many infection control measures and implementationof guidelines, the prevention of infections associated with medicaldevices, particularly vascular catheters, remains challenging. Theundue morbidity, excessive mortality, and rising cost associatedwith managing catheter-associated bloodstream infections arestaggering (34, 35). Patients often spend 10 to 40 additional daysin hospitals as a result of acquiring these infections (36–38). How-ever, recently established U.S. Medicare policy changes prohibitreimbursing hospitals and health care facilities for the cost ofmanaging certain health care-acquired complications that are rea-sonably preventable, including vascular catheter-associatedbloodstream infections (39). Furthermore, the Guidelines for thePrevention of Intravascular Catheter-Related Infections issued bythe Centers for Disease Control and Prevention (CDC) and theInfectious Diseases Society of America (IDSA) recommend the

FIG 1 (a to c) Zones of inhibition produced by NAC-antibiotic-impregnated catheter segments after incubation in serum.

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use of antimicrobial-impregnated CVCs in patients whose cathe-ters are expected to remain indwelling for more than 5 days if,after successful implementation of a comprehensive strategy todecrease rates of central line-associated bloodstream infection, therate remains above the goal set by the individual institution (5). Ameta-analysis showed that currently available anti-infective CVCsstill have an overall colonization rate of about 14% (345/2,491)compared to a rate of 24% (607/2,524) for standard untreatedcatheters (13). Furthermore, the duration of antimicrobial activity

sharply decreases with a catheter’s indwelling time. The overallcolonization rates of indwelling anti-infective versus standardcatheters were 12% versus 24%, 23% versus 28%, and 17% versus16% for catheters placed for 5 to 12 days, 13 to 20 days, and �20days, respectively. After 20 days of catheter placement, there isvirtually no difference in the colonization rates between antimi-crobial-treated and untreated standard catheters (13).

Since pathogenic colonization of a catheter can occur days afterits implantation, it would be optimal for antimicrobial-treated

FIG 2 (a to f) Antibacterial activity of NAC-antibiotic-impregnated central venous catheters against common clinical pathogens based on zone of inhibition.

FIG 3 Anticolonization activity of NAC-levofloxacin-impregnated catheter segments exposed to different bacterial suspensions in MRD. Error bars indicatestandard errors of the means (SEM) for bacterial colony counts per centimeter of catheter. An asterisk indicates a significant difference in mean bacterial colonycounts (P � 0.0001) between control and NACLEV devices.

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catheters to have a sustained activity for as long as it is safe andnecessary. However, the activities of many antimicrobial-treatedcatheters usually diminish rapidly during the first few days ofplacement in patients due to the rapid and uncontrolled release ofthe antimicrobial agents into the bloodstream. A measured releasecan ensure a sustained antimicrobial activity against microorgan-isms as reflected by the durable zones of inhibition produced byNACLEV catheters after incubation in human serum in vitro. SEMcolonization images corroborate the adherence assay findings, in-dicating that NACLEV catheters substantially reduce bacterialcolonization and biofilm formation by both Gram-positive andGram-negative bacteria.

NACLEV catheter segments exposed to bacterial suspensionsdisplayed 53-fold (with VRE) to more than 1.2 � 106-fold (with E.coli) fewer average numbers of adherent bacterial cells than con-trol catheter segments and completely impeded adherence of P.aeruginosa bacterial cells. Although levofloxacin alone is clinicallyeffective against a small percentage of clinical cases of MRSA in-fections, the incorporation of sufficient and safe concentrations oflevofloxacin and NAC into catheters may synergistically alleviatethis limitation based on the strong in vitro anticolonization activ-ity of NACLEV catheters against levofloxacin-resistant MRSA inthe MRD. Since our MRD could accommodate all segments fromeach comparing group (impregnated or nonimpregnated) at eachrun, we eliminated the need to perform multiple independentobservations, promoting consistency in our results.

These promising in vitro results suggest that NACLEV cathe-ters may protect against catheter colonization, without whichcatheter-associated infection would not evolve. Although statisti-cally significant, these data may not translate into clinical signifi-cance, and therefore, it is important to evaluate these catheters invivo as well. In light of these favorable results, we have initiated ananimal study to evaluate the efficacy of NACLEV cathetersagainst catheter colonization and catheter-associated infec-tion. Future research will also assess the safety of this novelapproach.

(This work was presented in part at the 49th Annual Meeting ofthe Infectious Diseases Society of America [IDSA], Boston, MA,October 2011.)

ACKNOWLEDGMENTS

This study was supported by grants from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID; grantsAI074010 and AI096702).

We thank Don Shult and Tom Loesch for their technical support withthe SEM.

Baylor College of Medicine (Houston, TX) is the assignee for patentsthat describe the method of incorporating agents onto catheters and theuse of the combination of NAC and antibiotics. M.D.M. and R.O.D.,employees of the mentioned institution, are inventors of one or more ofthese patents. However, there is no licensing activity regarding these pat-ents at this time.

FIG 4 (a and b) Catheter segment incubated in MRSA. (c and d) Catheter segment incubated in Klebsiella pneumoniae.

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