Journal of Infection and Public Health (2008) 1, 105—112 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Unexpected induction of resistant Pseudomonas aeruginosa biofilm to fluoroquinolones by diltiazem: A new perspective of microbiological drug—drug interactionଝ Walid F. ElKhatib, Virginia L. Haynes, Ayman M. Noreddin ∗ University of Minnesota, Duluth, College of Pharmacy, Pharmacy Practice and Pharmaceutical Sciences, 1110 Kirby Dr. Life Sciences 232, Duluth, MN 55812, USA Received 15 August 2008; received in revised form 21 October 2008; accepted 22 October 2008 KEYWORDS Summary The increase of multi-drug resistant Pseudomonas aeruginosa infec- Diltiazem; tions is a worldwide dilemma. At the heart of the problem is the inability to treat Pseudomonas established P. aeruginosa biofilms with standard antibiotic therapy, including flu- aeruginosa; oroquinolones. We address a previously unstudied question as to the effect of a Biofilm; commonly prescribed calcium channel blocker (CCB) diltiazem on the biofilm growth. Resistance; Real-time monitoring of the overall growth and killing of P.aeruginosa biofilm during fluoroquinolones therapy in the presence and absence of diltiazem was performed. Fluoroquinolone In this study, we demonstrate that for P. aeruginosa biofilms, resistance to the first- line fluoroquinolones may be induced by the commonly prescribed calcium channel blocker diltiazem. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. All rights reserved. Introduction approved for the treatment of angina, hyper- tension and cardiac arrhythmia [1]. It acts as Diltiazem is a class III calcium channel blocker a calcium antagonist that controls calcium ion (CCB) belonging to benzothiazepines and has been influx in cardiac and vascular smooth muscle cells through slow voltage-gated L-type channels in the ଝ This manuscript was presented in part at the Design of Med- cell membrane. The stereoselective interaction of ical Devices conference April 2008, University of Minnesota, diltiazem affects the binding domain of the ␣1 Minneapolis, MN. ‘‘A High-Throughput Biofilm Model of Treat- subunit of L-type calcium channel. Diltiazem is a ment Efficacy for Pseudomonas aeruginosa Device Associated potent vasodilator that is generally well-tolerated Infections’’. Acceptance No. 027535. [1,2]. As such, it is a commonly used treat- ∗ Corresponding author. Tel.: +1 218 726 6028; fax: +1 218 726 6500. ment for elderly patients including those who are E-mail address: [email protected] (A.M. Noreddin). diabetic [3]. 1876-0341/$ — see front matter Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. All rights reserved. doi:10.1016/j.jiph.2008.10.004 106 W.F. ElKhatib et al. Pseudomonas aeruginosa biofilms account for a prepared according to the Clinical Laboratory significant proportion of infections among catheter- Standards Institute (CLSI) guidelines M7-A7 and associated urinary tract infection (UTI) in long-term M100-16. care facilities, diabetic foot infections and lung Initial inocula of 1—3 × 106 CFU/mL P.aeruginosa infections of cystic fibrosis and other immunocom- cultures in the log phase were seeded into two promised patients [3—7]. The biofilm matrix affords 100-well polystyrene honeycomb plates. Biofilms the bacteria protection from antibiotic penetration were allowed to develop in an incubator over 6 h leading to suboptimal treatment levels at the site at 37 ◦C. Wells were washed carefully, once with of infection and potentially inducing outgrowth of 150 ␮L sterile saline, to remove planktonic cells. resistant strains [8—10]. Cation-adjusted Mueller—Hinton II medium (MH) Fluoroquinolones, ␤-lactams and aminoglyco- containing diltiazem alone at 10 mM or diltiazem sides are the three classes of antibiotics used plus a fluoroquinolone at two fold serial dilutions for treatment of P. aeruginosa infections. Unfor- were added to the biofilm cultures (250 ␮L/well tunately, the multi-drug and pan-drug resistant final volume). Cell growth was monitored using the isolates of P. aeruginosa which patients are often Bioscreen C (Growth Curves USA, Piscataway, NJ). exposed to in the hospital setting are resis- The Bioscreen C monitors microorganism growth by tant to one or more these three drug classes measuring total turbidity of the liquid growth media [7,11—15]. Selective pressure due to excessive in each well. Changes in turbidity due to growth exposure of bacteria to antibiotics is generally or lysis of microorganisms are measured kinetically cited as the cause for this high incidence of resis- with a vertical photometrical technology through tance in a hospital environment. ICU and long-term the bottom of the well. A wide-band filter was used care facilities are also notorious worldwide for to measure the turbidity. The wide-band filter is a harboring multi- and pan-resistant P. aeruginosa white filter for turbidity measurement and its spec- strains [6,15,16]. However, the epidemiology of trum range is 420—580 nm. It is used in microbiology community-acquired resistant strains is less clear research because its sensitivity is not affected by [7]. The causes or selective pressures behind any color change of the medium. the generation of resistant strains become an Plates containing biofilms treated with dilti- important question as our population ages and as azem (10 mM) and fluoroquinolones were placed more immunocompromised patients survive and are in the preheated incubating chamber of Bioscreen cared for in a community setting [17—20]. It is com- C which was programmed to maintain a temper- mon for frail elderly patients to be on multiple drug ature of 37 ◦C. Optical density (OD) values were therapies including antibiotics. Detailed studies obtained at 1 h intervals over 96 h, without shaking. regarding the induction of resistance by combina- Growth control wells contained biofilms treated tions of antibiotics with other common medications with Mueller—Hinton II broth only. Biofilm was also are rare to non-existent [21—23]. This work was treated with MH medium spiked with increasing part of a large project designed to study the effect concentrations of D(+)-glucose (0.1—25 mM) with- of the co-administration of calcium channel block- out diltiazem or antibiotics. Negative controls ers on fluoroquinolone treatment of P. aeruginosa containing MH alone and MH containing diltiazem biofilm. (10 mM) were involved in the experiments to check for the sterility and absence of any precipita- tions, respectively, within 96 h of incubation period. Materials and methods Each biofilm growth curve was repeated three times. The diltiazem concentration (10 mM) was Purchases were made from the following suppliers: chosen as the highest concentration that did not levofloxacin, ciprofloxacin, norfloxacin, ofloxacin inhibit biofilm growth and did not precipitate from and lomefloxacin from Sigma—Aldrich (St. Louis, the medium. Fluoroquinolone concentrations were MO). Diltiazem (MP Biomedicals, Solon, OH) within the standard range used in CLSI protocols was purchased from Thermo-Fisher Scientific Inc. to determine the minimum inhibitory concentration (Waltham, MA). Moxifloxacin was obtained from (MIC) for each fluoroquinolone against planktonic Schering-Plough (Woodlands, TX). P. aeruginosa P. aeruginosa. Data were automatically collected ATCC27853 was purchased from the American Type by the Growth Curves software (EZExperiment) and Culture Collection (Manassas, VA). Cultures were exported to an Excel spread sheet for processing. prepared according to ATCC guidelines. All cell MIC is defined as the concentration of fluoro- culture media and supplements were purchased quinolone at which the OD remains at or returns to from Thermo Fisher Scientific Inc. (Waltham, MA). baseline at the 24 h time point. Mutant prevention All antibiotic stock solutions and dilutions were concentration (MPC) was reported as the antibi- “Induction of resistant P. aeruginosa biofilm to fluoroquinolones by diltiazem” 107 otic value at 96 h that prevented the development Results of resistant organisms [24]. Baseline OD readings taken for all experiments was at approximately 0.2. The fluoroquinolone ciprofloxacin is a well-known For visualization of P. aeruginosa biofilm by con- anti-pseudomonal that is rather commonly used for focal scanning laser microscope (CSLM), the method UTI in the elderly as well as for other P. aerug- described by Walker et al. [41] was applied with inosa infections [25,26]. CLSI expected range for some modifications. Briefly, biofilm of P. aerug- P. aeruginosa ATCC27853 in the planktonic form inosa was grown as described for the control is 0.25—1 ␮g/mL. Biofilm MIC values are gener- in MH medium within 4-chambers culture slides ally expected to be anywhere from two fold to a (SuperCellTM Culture Slides, Fisher Scientific Inc., 1000× higher [10]. However, ciprofloxacin, in our Waltham, MA). At 0 h and 12 h time points of biofilm experiments, showed a low MIC against P. aerug- incubation, MH medium was carefully aspirated and inosa biofilm. In this study, we found an MIC of culture slides were immersed in 5% (v/v) glutaralde- 0.2—0.4 ␮g/mL depending on the time point, with hyde (Fisher Scientific) in normal saline for 24 h for 0.4 ␮g/mL at 96 h reported as the MPC (Fig. 1A). fixation and imparting fluorescence for the biofilm However, when diltiazem (10 mM) was added to the without distortion of the biofilm architecture. The medium with ciprofloxacin, an unusual growth pat- incubating chambers of the culture slides were tern developed. While the MIC might still have been carefully removed and each slide was rinsed with reported as 0.2 ␮g/mL at 24 h as the plate had been 10 mL of PBS (Fisher Scientific). The biofilms were read by the naked eye, in fact, real-time monitor- mounted with Citifluor antifading (Sigma—Aldrich, ing with the Bioscreen C allowed us to discern that St. Louis). Each experiment was repeated twice the culture is about to enter a log phase growth. with three replicates each and the slides were kept In the presence of the ciprofloxacin—diltiazem at −20 ◦C till visualization using CSLM.