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Journal of and Public (2008) 1, 105—112

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Unexpected induction of resistant Pseudomonas aeruginosa biofilm to fluoroquinolones by diltiazem: A new perspective of microbiological —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 therapy, including flu- aeruginosa; oroquinolones. We address a previously unstudied question as to the effect of a Biofilm; commonly prescribed (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- 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 ’’. 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 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 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 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 are measured kinetically cited as the cause for this high 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 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 . 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. combination, all cultures showed an increase in A series of images for P. aeruginosa biofilm in growth up to four times that of the control at 96 h the z direction (z series) were digitized in selected (Fig. 1B). optical planes with a CLSM Nikon TE2000 inverted Most notable, however, was the unusual growth confocal laser scanning microscope equipped with pattern exhibited by P. aeruginosa biofilm in the Nikon C1 laser scanning unit that provides laser presence of diltiazem alone. In the presence of beams including Solid State Laser (405 nm), 480 nm diltiazem, the growth of P. aeruginosa biofilm dichroic long pass filter, 450/35 nm band pass fil- greatly exceeded the control, showing an OD value ter, Spectra Physics argon laser (488 nm), 545 nm of about 2.4. This extraordinary growth pattern dichroic long pass filter, 515/30 nm band pass filter, occurred consistently throughout all experiments and HeNe Laser (543 nm), 505/75 nm band pass fil- whenever diltiazem was added and regardless of ter (both instruments from Nikon Instruments Inc., the fluoroquinolone tested (Fig. 1). The growth Melville, NY, USA). The system used a motorized promoting effect of diltiazem on P. aeruginosa computer-assisted device to control the vertical biofilm was noticeable within the concentration positioning during optical sectioning of the biofilm. range of 1—10 mM (data not shown) in a dose Image scanning with the above mentioned lasers dependant manner. Nevertheless, we selected the was processed but the best images were obtained higher concentration level (10 mM) to facilitate with argon laser (488 nm), 545 nm dichroic long pass monitoring the effect of diltiazem using Bioscreen filter, 515/30 nm band pass filter using 1003/1.3 C technology. Although higher concentrations of NA Plan-Neofluar lens. Images were generated by fluoroquinolones delayed the growth of P. aerug- applying automated microscope image acquisition. inosa biofilm but none of the tested levels could The morphological processor software (Nikon EZ- inhibit biofilm growth by the end of the experi- C1 software) on the attached computer enhanced ments. Furthermore, this growth was maintained images, discarded unwanted details, and acceler- in MH medium that otherwise might be consid- ated the colored-scale processing. Computerized ered depleted in nutrients. The growth controls pixel calibration and image setup were defined enter log phase growth then drop to a plateau relative to the calibration value used for image gen- of around OD 0.6 while the cultures contain- eration on the CLSM system. In this system, the size ing diltiazem maintained plateau at OD 2.4. At and position of the ‘‘area of interest’’ for manip- time points >24 h, MICs for all fluoroquinolones ulation were specified manually. The area covered were increased over published CLSI mentioned by microbial growth was scanned for each confo- resistance levels when diltiazem was incorpo- cal plane relative to the thickness of the biofilm rated to the MH medium and no MPC values and subsequently visualized at a 90◦ angle on the could be reported for any fluoroquinolone at 96 h obtained images. (Table 1). 108 W.F. ElKhatib et al.

Figure 1 (A—L) represent real-time data for each set of experiments comparing treatment of Pseudomonas aeruginosa biofilm with a fluoroquinolone alone and its companion experiment where diltiazem was present in addition to the fluoroquinolone. The MICs and MPCs for fluoroquinolones are reported in ␮g/mL. Figures (A, C, E, G, I and K) show MIC at 24 h and MPC at 96 h for fluoroquinolones alone against Pseudomonas aeruginosa biofilm. Figures (B, D, F, H, J and L) show MIC for each fluoroquinolone with diltiazem against Pseudomonas aeruginosa biofilm. No MPC could be reported. Diltiazem concentration level in all experiments was 10 mM. For clarity, some curves that overlay the growth control were omitted. “Induction of resistant P. aeruginosa biofilm to fluoroquinolones by diltiazem” 109

Figure 1 (Continued)

Table 1 Fluoroquinolone MICs at 24 h and MPCs at 96 h (␮g/mL) with and without diltiazem (10 mM) added to Mueller—Hinton medium for Pseudomonas aeruginosa ATCC27853 biofilm. Fluoroquinolone CLSI expected MIC Without diltiazem With diltiazem (10 mM) Planktonic MIC Biofilm Biofilm

24 h MIC 96 h MPC 24 h MIC 96 h MPC Ciprofloxacin 0.25—1 0.2 0.4 0.2 >1.6 Levofloxacin 0.5—4 2 4 2 >8 Lomefloxacin 1—4 3.2 >3.2 3.2 >3.2 Moxifloxacin 1—8 2 8 2 >16 Norfloxacin 1—4 1.25 2.5 5 >10 Ofloxacin 1—8 3.2 6.4 3.2 >12.8

Regarding the unusual growth curves, we won- dered if diltiazem might be acting as a surrogate carbon source in depleted MH medium. To deter- mine if simply providing increased nutrients could have such an effect on the growth curve, cultures were prepared as for the diltiazem and fluoro- quinolone experiments but adding only increasing concentrations of glucose (0.1—25 mM) to the MH medium. Although increased glucose level in the MH medium slightly increased the OD at which the growth curve reached a plateau, no significant Figure 2 The effect of increasing concentrations of changes in the OD of the curve peak was observed glucose (0.1—25 mM) added to the MH medium on the even with the highest tested glucose concentration growth of Pseudomonas aeruginosa biofilm. There was (25 mM). Furthermore, the maximum growth was no significant effect on the growth curve of Pseudomonas maintained at OD 1.3 or less (Fig. 2). aeruginosa biofilm. For visualization of the growth for the estab- lished P. aeruginosa biofilm, CSLM technique was applied and the results (Fig. 3a and b) showed Discussion marked increase in the biofilm growth (cell count and biofilm depth) within 12 h as compared to 0 h There is a pressing need for a deeper and more incubation. thorough understanding of the mechanisms that 110 W.F. ElKhatib et al.

scribed to ‘‘preselect’’ drug resistant strains, induce changes in growth rate and/or encour- age resistance. In this instance, we assessed diltiazem—fluoroquinolones combinations in vitro using real-time monitoring of effects of such com- binations on the biofilm growth using Bioscreen C. Although the CLSM images showed marked growth for P. aeruginosa biofilm, but it has to be taken into consideration that OD readings obtained from Bioscreen C represent the overall growth of both biofilm and the shedding planktonic cells from that biofilm. While there are rare references on the potential antibacterial activities of verapamil and amlodipine on planktonic bacteria [34—36], there is no previous evidence, of which we are aware, that describes the effect of diltiazem on P. aerug- inosa biofilms and the resultant resistance of this organism to the most important antibiotic class, flu- oroquinolones, used for its treatment. The results revealed that the diltiazem inducing resistance for P. aeruginosa biofilm against fluoroquinolones was not through carbon source enrichment for the medium. Eickelberg et al. [42] demonstrated that diltiazem is capable of activating expression of some genes and it may directly regulate cellular functions by affecting the activity of transcrip- tion factors independently of intracellular calcium concentrations. Since divalent cations, particularly calcium, are important bridging ions for bacte- rial polysaccharides, biofilm formation and they play regulatory roles in bacterial gene expres- sion [43], the agents that affect calcium binding may also influence the gene expression. Therefore, the observed diltiazem effect in this study might Figure 3 (a) Optical sectioning produced by CLSM, illus- be through transcription up-regulation of some P. trating the horizontal colonization of P. aeruginosa at aeruginosa genes including parC, gyrA, quorum different biofilm depths at the beginning of the exper- sensing system, or alteration in size and expression iment (0 h of incubation). Scale bar = 10 ␮m. In some of different porins. Consequently, further studies cases, images were affected by light reflections at the are required to elucidate the possible mechanism(s) position closest to the substratum. (b) Optical sectioning of resistance. produced by CLSM, illustrating the horizontal coloniza- The study of biofilms as they relate to chronic tion of P.aeruginosa at different biofilm depths at 12 h of incubation. Scale bar = 10 ␮m. infections is a burgeoning field. The founders of biofilm study began by studying biofilms of P.aerug- inosa in the environment [37,38]. As years passed, lie behind the selection and development of MDR it became clear that biofilms must also play a sig- and PDR pathogens especially within biofilms. P. nificant role in human infection as well as to the aeruginosa is a fierce environmental organism, well emergence of resistant bacterial diseases [39—41]. designed to withstand attacks by other bacte- Even as we are only beginning to understand this ria, fungi, amoebas and [4]. Since it form of growth and its attendant survival mech- comes pre-equipped with the machinery for pan- anisms that relate to antibiotic resistance, we , selection of resistant strains occurs must broaden the scope of our view on induc- quickly and often as evidenced by outbreaks of MDR tion of resistance. We need to reappraise many and PDR P.aeruginosa in health facilities around the drugs as potential catalysts for the selection of globe [6,11,14,19,27—33]. resistance. We must begin to reevaluate commonly Here we present an argument for detailed prescribed drugs like calcium channel blockers for studies regarding the potential for commonly pre- its potential microbiological interaction. It might “Induction of resistant P. aeruginosa biofilm to fluoroquinolones by diltiazem” 111 be essential to reassess several drug-antibiotic co- [3] Blickle JF. Management of hypertension in elderly diabetic administration scenarios that we once thought to patients. Diabetes Metab 2005;2, 5S82—91. be safe because, for example, there is no interfer- [4] Palmer Jr RJ, Stoodley P.Biofilms 2007: broadened horizons and new emphases. J Bacteriol 2007;189(22):7948—60. ence through metabolic mechanisms in the human [5] Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as body. We must currently reappraise the effect of complex differentiated communities. 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