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Evaluating the ototoxicity of topical piperacillin–tazobactam
International Journal of Pediatric Otorhinolaryngology (2008) 72, 1815—1821
www.elsevier.com/locate/ijporl
Evaluating the ototoxicity of topical piperacillin— tazobactam Chul-Ho Jang a,b,*, Haekyun Park c, Yong Bum Cho a, Cheol-Hee Choi b a
Department of Otolaryngology, Chonnam National University Medical School, Gwangju, South Korea Research Center for Resistant Cells, Chosun University, Gwangju, South Korea c Department of Biology, Chosun University, Gwangju, South Korea b
Received 6 March 2008; received in revised form 21 August 2008; accepted 26 August 2008 Available online 8 October 2008
KEYWORDS Ototoxicity; Piperacillin— tazobactam; Topical treatment
Summary Background and objective: With the increased use of ototopical ciprofloxacin solution, newly evolved bacterial fluoroquinolone resistance has also become more of a problem. The emergence of ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) has created a new therapeutic challenge in otology. We evaluated the ototoxicity of topical fortified piperacillin—tazobactam solution by performing experiments in young male albino guinea pigs (weight, 250—300 g each). Materials and methods: Antimicrobial assay by direct contact test using scanning electron microscope was performed. Twenty guinea pigs (250—300 g) were treated with fortified piperacillin—tazobactam solution. In experimental group 1 (n = 10), a gelfoam ball impregnated with 120 ml of fortified piperacillin—tazobactam was implanted on the round window membrane by a posterior approach. In experimental group 2 (n = 10), 20 ml of fortified piperacillin—tazobactam was injected into the middle ear cavity via silicone tube with a small hole placed in the superior aspect of the bulla by drilling. The drug application through the tube was performed for 7 consecutive days. In control group (n = 5), a gelfoam ball impregnated with 120 ml of gentamicin solution (80 mg/2 ml) was implanted on the round window membrane. Results: The fortified piperacillin—tazobactam 20 ml treated CRPAs showed destruction of their cell membrane in antimicrobial assay by direct contact test. No significant difference in the mean auditory brainstem response (ABR) thresholds before and after drug administration was found for the experimental group. However, significant elevation of the mean ABR thresholds was found for the control group. In experimental group, scanning electron microscopy showed almost normal sterociliary arrangements and surface structure on the inner and outer hair cells. However, significant destruction of outer hair cells was identified in control group.
* Corresponding author at: Department of Otolaryngology, Chonnam National University Hospital, Hak-dong 8, Dongku, Gwangju, South Korea. Tel.: +82 62 2206774. E-mail address: [email protected] (C.-H. Jang). 0165-5876/$ — see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2008.08.018
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C.-H. Jang et al. Conclusion: This present study’s data suggests that fortified piperacillin—tazobactam solution can be effectively used for topically treating CRPA otorrhea in those patients who suffer with chronic suppurative otitis media. # 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Purulent otorrhea is a common malady in patients suffering with chronic suppurative otitis media (CSOM). Patients with CSOM generally have a chronic inflammation of the middle ear and mastoid, with a persistent perforation of the tympanic membrane, which is associated with recurrent otorrhea. If purulent otorrhea persists, then the mucous membrane may become ulcerated, polypoid or granuomatous. Prolonged infection may be associated with resorption of the ossicles and mastoid bone by the process of demineralization and the osteoclastic activity. The most frequently isolated organisms are Pseudomonas aeruginosa and the anaerobes. Many otolaryngologists routinely use topical antibiotics for purulent otorrhea. Aminoglycosides such as neomycin sulphate, tobramycin and gentamicin are commonly used. Ciprofloxacin is one of the fluoroquinolones and it is available as a topical preparation; it has shown be effective against P. aeruginosa [1—3]. Ototopical ciprofloxacin in patients was curative for nearly 70% of the patients with otorrhea associated with P. aeruginosa and who were previously unresponsive to other antimicrobial agents [4,5]. As the clinical application of the ototopical ciprofloxacin solution has increased, newly evolved bacterial fluoroquinolone resistance has also become a challenge to treat. The resistance to fluoroquinolone is basically a reflection of both mutation and natural selection, which is the result of the selective pressure created by using fluoroquinolones [6]. Due to the fluoroquinolone resistance of P. aeruginosa and the development of fluoroquinolone resistance during therapy, selecting an optimal treatment for patients suffering with this pathogen is often complicated. We have previously reported on ciprofloxacin-resistant P. aeruginosa (CRPA) in patients with chronic suppurative otitis media [7]. Piperacillin—tazobactam contains two active ingredients, piperacillin, which is a penicillin-type antibiotic, and tazobactam, which is a medicine that prevents bacteria from inactivating piperacillin. The first ingredient piperacillin is an antibiotic that kills many types of bacteria. The second ingredient tazobactam belongs in the penicillin group,
but it does not have activity against bacteria. It helps piperacillin to overcome bacteria that have become resistant to piperacillin. Piperacillin—tazobactam provides excellent coverage against Grampositive, Gram-negative and anaerobic organisms [8]. A study by Nomura et al. [9] suggested that piperacillin—tazobactam has 4- to 64-fold stronger antimicrobial activity against all [beta]-lactamaseproducing bacteria compared with ampicillin—sulbactam. Piperacillin—tazobactam has greater activity than ampicillin—sulbactam (as measured by a higher percentage of susceptible bacteria) against Gram-negative organisms such as Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, P. aeruginosa and the Enterobacter species [10,11]. An otic preparation of piperacillin—tazobactam (currently unavailable commercially) results in a smaller amount of administered drug, compared with parenteral administration. To the best of our knowledge, there are no clinical studies in the Medline literature that have evaluated topical piperacillin— tazobactam for treating CRPA otorrhea. We designed the current study to investigate the ototoxic effect of topical piperacillin—tazobactam solution on the cochlea when the piperacillin—tazobactam solution was applied topically in the middle ear of guinea pigs.
2. Materials and methods 2.1. Antimicrobial assay by direct contact test The clinical CRPA (n = 12) bacteria samples were obtained from patients at our hospital. The CRPA samples were inoculated into 5 ml of Mueller-Hinton broth (MHB) media and incubated at 37 8C to obtain organisms in the mid-logarithmic growth phase. A 4.5 g vial of piperacillin—tazobactam powder (Wyeth Korea Pharmaceuticals Inc., Seoul, South Korea) was reconstituted with 30 ml of sterile distilled water. The resulting concentration of the piperacillin—tazobactam solution was 0.1125— 0.225 g/ml. The mid-logarithmic phase CRPAs were resuspended at 1 105 (CFU)/ml in MHB media and then they were inoculated at 37 8C with 10 or 20 ml of fortified piperacillin—tazobactam. After 1 h the
Ototoxicity of Topical Piperacillin-Tazobactam cells were fixed with an equal volume of 2% glutaraldehyde in 0.1 M sodium-cacodylate buffer, pH 7.4. After fixation for 4 h at 4 8C, the samples were extensively washed with 0.1 M cacodylate buffer, pH 7.4. The samples were then dehydrated through a graded series of ethanol solutions. After lyophilization and gold coating, the CRPA samples were examined in a scanning electron microscope (JSM 6400, JEOL Ltd. Tokyo, Japan).
2.2. Auditory brainstem response (ABR) measurement The experiments were performed in 25 young, male guinea pigs (weight, 250—300 g each) with normal tympanic membranes. The ear canals and the tympanic membranes of the guinea pigs were examined under an operating microscope. The presence of ear infection was excluded by examining each guinea pig’s external auditory canals and tympanic membranes. A (4.5 g) vial of piperacillin—tazobactam powder (Wyeth Korea Pharmaceuticals Inc., Seoul, South Korea) was reconstituted with 30 ml of sterile 5% dextrose in water. The resulting concentration of the piperacillin—tazobactam solution was 0.1125— 0.225 g/ml. Osmolarity was measured by a model 3300 osmometer (Advanced Instruments, Norwood, MA) The mean osmolarity was 664 mOsm. The pH was measured by pH indicator reagent strips and a laboratory micro-pH meter (Clinitek-200, Alpha Scientific Medical, La Verne, CA). The pH of fortified piperacillin—tazobactam solution was 5.08. The preparation was stored in a refrigerator following compounding and also during usage. The guinea pigs were anesthetized by an intraperitoneal injection of zolletil and xylazine hydrochloride. The guinea pigs received a baseline hearing assessment. In experimental group 1 (n = 10), a gelfoam ball impregnated with 120 ml of fortified piperacillin—tazobactam was implanted on the round window membrane by a posterior approach. In experimental group 2 (n = 10), 20 ml of fortified piperacillin—tazobactam was injected into the middle ear cavity via a small hole placed in the superior aspect of the bulla by drilling. A cut-down tube (polyvinyl chloride, Korea Medical Co., Seoul, Korea) was chosen for catheter insertion because it has a small diameter (i.d. = 0.6 mm, o.d. = 1 mm) and it was placed through the hole and fixed by cyanoacrylate (LOCTITE1, Ellsworth Adhesives, WA, USA). The drug application through the cut-down tube was performed for 7 consecutive days. In control group (n = 5), a gelfoam ball impregnated with 120 ml of gentamicin solution (80 mg/2 ml) (Kunhwa Pharmaceutical Co. Seoul, South Korea) was implanted on the round window membrane.
1817 The auditory brainstem responses were recorded under general anesthesia (ketamine 35 mg/kg and xylazine 15 mg/kg intramuscularly) with using an evoked potential system (Tucker-Davis Technologies, FL, USA) and a Samsung computer. The stimuli were digitally synthesized using Siggen# software, and they were presented through an insert earphone (ER-2, Etymotic Research, Inc., IL, USA). The acoustic stimuli, consisting of a click and 8, 16 and 32 kHz tone bursts, were then produced. The Intensity of the acoustic stimuli was expressed in decibels (dB). The animals were presented with a stimulus intensity series, which was initiated at 90 dB SPL and this reached a minimum of 10 dB SPL. The stimulus intensity was progressively lowered in 10 dB decrements. Each average consisted of 500 stimuli presentations, with a 10 ms analysis time. The electrical activity was recorded via a platinum needle electrode inserted into the scalp at the vertex, and it was referenced to another needle electrode in a deep neck muscle. A third needle electrode in the pinna serve as a ground. The intensities that appeared to be near threshold were repeated. The threshold was defined as the lowest intensity capable of producing a visually detectable, reproducible ABR response; the values were extrapolated to the nearest 5 dB assuming a log-linear fall off with decreasing intensity. At the threshold, the baselineto-peak amplitude of the largest ABR component was reduced to <10% of that evoked by the 90 dB stimuli. The ABRs were assessed preoperatively and at the third and seventh day after topical application of the drug. Repeated measure analysis of variance (ANOVA) was used to evaluate the differences among the groups. A P value <0.05 was considered to be statistically significant.
2.3. Scanning electron microscopic observation of the cochlea After the final ABR recordings, the anesthetized animals were perfused intracardially with 4% paraformaldehyde while they were under general anesthesia. The temporal bones were isolated and the perilymphatic spaces of the cochlear were gently perfused with 2.5% glutaraldehyde in 0.1 M phosphate-buffered solution (PBS) by performing cochleostomies at the round and oval windows. The bony capsules were removed to expose the organ of Corti, after which the specimens were post-fixed in 2.5% glutaraldehyde overnight at 4 8C. The specimens were washed three times in PBS and then post-fixed in 1% osmium tetroxide for 1 h at 4 8C. The organ of Corti specimens were then dehydrated through a graded series of ethanol solutions and then they were critical-point dried using
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liquid carbon dioxide. The critical-point dried specimens of the organ of Corti were attached to aluminum SEM stubs with aluminum paint and then sputter-coated with gold—palladium. The surfaces of the organs of Corti’s were examined in a scanning electron microscope (JSM6400, JEOL Ltd. Tokyo, Japan).
3. Results 3.1. Scanning electron microscopic observation of the CRPAs in vitro The piperacillin—tazobactam 10 ml treated CRPAs displayed a shortened cell length. The piperacillin—tazobactam 20 ml treated CRPAs showed destruction of their cell membrane (Fig. 1A—C).
3.2. Auditory brainstem responses The threshold shifts (decibels) between preoperative baseline and days 3 and 7, respectively, are shown in Fig. 2. No significant difference in the mean ABR thresholds before and after drug administration was found in the experimental group 1 (Fig. 2A) and group 2 (Fig. 2B). But statistically significant elevation of the mean ABR after gentamicin administration in the control group (Fig. 2C).
3.3. Scanning electron microscopic observation of the cochlea Fig. 3 shows the SEM micrographs of a guinea pig cochlea in experimental group 1, which showed an insignificantly elevated threshold at postoperative day 3. There was almost normal sterociliary arrangements and the surface structure were almost normal for the inner and outer hair cells and there was an almost a normal number of microvilli on the surface of the inner pillar cells. The SEM micrographs of a guinea pig cochlea in experimental group 2 show almost normal outer and inner hair cells (Fig. 4A). Fig. 4B shows the almost normal arrangements of stria vascularis of a guinea pig cochlea in experimental group 2. However, Fig. 5 shows that there was destruction of the outer hair cells in the control group.
Fig. 1 The SEM shows the CRPAs isolated from chronic suppurative otitis media (A). The CRPAs show a shortened cell length when they were treated with 10 ml of fortified piperacillin—tazobactam (B). The CRPAs show destruction of the cell membrane when they were treated with 20 ml of fortified piperacillin—tazobactam. SEM: scanning electron microscopy, CRPA: ciprofloxacin-resistant Pseudomonas aeruginosa.
4. Discussion It is well known that P. aeruginosa may become resistant to the antibiotic being used to treat an infection, and that prior use of a particular antibiotic predicts that P. aeruginosa will develop
resistance to that antibiotic. Barely 10 years after ciprofloxacin’s introduction into otology, the emergence of CRPA, when treating chronic suppurative otitis media, has been reported [7,12,13].
Ototoxicity of Topical Piperacillin-Tazobactam
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Fig. 2 The ABR shows a insignificant difference in the mean hearing threshold before and after topically applied fortified piperacillin—tazobactam by direct round window approach in experimental group 1 (A) or transtympanic instillation for 7 consecutive days in experimental group 2 (B). The ABR shows significant difference in the mean hearing threshold before and after topical application of diluted gentamicin in control group (C). Asterisk means the statistical significance. ABR: auditory brainstem response.
Just as news of aminoglycoside-resistant P. aeruginosa mandated the search for an alternative remedy, which led to ciprofloxacin’s introduction, CRPA now requires the development of more capable antibiotics that can properly deal with this scourge. Increasing resistance in P. aeruginosa to multiple antibiotics has been observed and is posing therapeutic dilemmas. Antibiotic utilization is one factor that has been associated with the emergence of antimicrobial resistance. CRPA is still sensitive to ceftazidime, cefepime, piperacillin, piperacillin— tazobactam and imipenem [7,12,14,15]. The tazobactam of piperacillin—tazobactam is known as a beta-lactamase inhibitor. Tazobactam is included in this medicine because it inhibits the action of the beta-lactamases produced by the defenses of certain bacteria. This prevents the bacteria from inactivating the piperacillin, and it leaves them susceptible to attack. Thus, piperacillin—tazobactam increases the range of bacteria that piperacillin can kill. This medicine is given by injection or infusion and it is generally used for severe infections. In the ophthalmologic field, the fortified
antibiotics for the treatment of P. aeruginosa are well established and their efficacy has been reported on [16]. However, no previous study has reported on the effect of topical piperacillin—tazobactam on the cochlea. In the present study, we administered the piperacillin—tazobactam by two approaches. There was no difference of electrophysiological and morphologic results between two groups. Transtympanic instillation of drug does not allow for consistent, reproducible amounts of drug to be presented to the round window [17]. And the administered drug can enter to the Eustachian tube. By Brown et al., focal hemorrhage and occasional middle ear effusion were found in the middle ears after transtympanic instillation [18]. Administration of drug via a local delivery system applied directly to the round window membrane may allow for a more reproducible damage pattern [17,19]. The damage accomplished with a direct application of gentamicin is qualitatively similar to that achieved with a systemic injection of gentamicin [17]. Hoffer et al. [20] examined the inner
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Fig. 3 The SEM shows almost normal inner and outer hair cells in experimental group 1 after topically applied fortified piperacillin—tazobactam by direct round window approach (A). (B) The magnified view of the outer hair cells. SEM: scanning electron microscopy.
Fig. 4 The SEM shows almost normal inner and outer hair cells in experimental group 2 after topically applied fortified piperacillin—tazobactam by transtympanic instillation for 7 consecutive days (A). The SEM shows almost normal arrangement of stria vascularis (B). SEM: scanning electron microscopy.
ear kinetics of transtympanic gentamicin and compare this with the kinetics of sustained-release delivery in a basic science model. The reliability of sustained-release delivery to the ear reduces functional and morphological variations between animals. Single-dose instillation of gentamicin induced greater hair cell loss than continuous administration at the same dose [21]. Sheppard et al. [17] evaluated the ototoxic damage sustained from drugs applied directly to the round window in varying delivery vehicles such as gelfoam, hyaluronic acid, and fibrin. The fibrin or gelfoam vehicle was the only delivery system to produce reliable damage. In our study, we evaluated the guinea pigs’ hearing using ABR and we evaluated their cochlear morphology using a SEM; these techniques have often been used in ototoxicity studies. In this study, we found that piperacillin—tazobactam destroyed the surface of the CRPA in vitro. We demonstrated that with topical application of piperacillin—tazobactam, the mean ABR threshold showed insignificant elevation compared to that at baseline. Further, the SEM showed almost normal outer and inner hair cells.
Topical otic solution treatment results in a smaller amount of drug administered, as compared with parenteral administration. In conclusion, the data derived from this present study suggests that tazobactam can be effectively used for topical treatment of CRPA otorrhea in patients who suffer from chronic suppurative otitis media.
Fig. 5 The SEM shows destruction of outer hair cells in control group after topically applied gentamicin by direct round window approach. SEM: scanning electron microscopy.
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Acknowledgements [10]
This study was supported by grants from the Ministry of Science and Technology, Korea, and from the Korea Science and Engineering Foundation through the Research Center for Resistant Cells (R13-2003009).
References
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[1] R.W. Force, M.C. Hart, S.A. Plummer, D.A. Powell, M.C. Nahata, Topical ciprofloxacin for otorrhea after tympanostomy tube placement, Arch. Otolaryngol. 121 (1995) 880— 884. [2] A. Tutken, A. Ozagar, A. Koc, C. Batman, U. Cuneyd, M.A. Schitoglu, Treatment of chronic ear disease: topical ciprofloxacin vs. topical gentamicin, Arch. Otolaryngol. 121 (1995) 1414—1416. [3] K. Ikeda, T. Takasaka, In vitro activity of ototopical drops against middle ear pathogens, Am. J. Otol. 14 (1993) 170—171. [4] S.M. Wintermeyer, M.C. Hart, M.C. Nahata, Efficacy of ototopical ciprofloxacin in pediatric patients with otorrhea, Otolaryngol. Head Neck Surg. 116 (1997) 450—453. [5] Miles Inc. Package Information, Cipro (Ciprofloxacin), Miles Inc., West Heaven, CT, 1993. [6] W.H. Sheang, Y.C. Chen, J.T. Wang, S.C. Chang, K.T. Luh, W.C. Hsieh, Emerging fluoroquinolone-resistance for common clinically important gram negative bacteria in Taiwan, Diagn. Microbiol. Infect. Dis. 43 (2002) 141—147. [7] C.H. Jang, S.Y. Park, Emergence of ciprofloxacin-resistant pseudomonas in chronic suppurative otitis media, Clin. Otolaryngol. Allied Sci. 29 (2004) 321—323. [8] R.N. Jones, M.A. Pfaller, P.C. Fuchs, K. Aldridge, S.D. Allen, E.H. Gerlach, Piperacillin/tazobactam (YTR 830) combination: comparative antimicrobial activity against 5889 recent aerobic clinical isolates and 60 Bacteroides fragilis group strains, Diagn. Microbiol. Infect. Dis. 12 (1989) 489—494. [9] S. Nomura, H. Hanaki, A. Nagayama, Tazobactam—piperacillin compared with sulbactam—ampicillin, clavulanic acid— ticarcillin, sulbactam—cefoperazone, and piperacillin for activity against beta-lactamase-producing bacteria isolated
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from patients with complicated urinary tract infections, J. Chemother. 9 (1997) 89—94. S. Gatermann, R. Marre, Comparative in vitro activities of amoxicillin—clavulanate, ampicillin—sulbactam and piperacillin—tazobactam against strains of Escherichia coli and Proteus mirabilis harbouring known [beta]-lactamases, Infection 19 (1991) 106—109. G.A. Pankey, R.B. Chambers, Activity of piperacillin/tazobactam compared to other antibiotics against 4,356 lowerrespiratory isolates of aerobic and facultative anaerobic gram-negative bacilli from hospitalized patients [Abstract], Chest 110 (1996) 163S. C.H. Jang, S.Y. Park, Emergence of ciprofloxacin-resistant pseudomonas in pediatric otitis media, Int. J. Pediatr. Otorhinolaryngol. 67 (2003) 313—316. C.H. Jang, Y.B. Cho, C.H. Choi, Structural features of tympanostomy tube biofilm formation in ciprofloxacin-resistant Pseudomonas otorrhea, Int. J. Pediatr. Otorhinolaryngol. 71 (2007) 591—595. M.K. Rhee, R.P. Kowalski, E.G. Romanowski, F.S. Mah, D.C. Ritterband, Y.J. Gordon, A laboratory evaluation of antibiotic therapy for ciprofloxacin-resistant Pseudomonas aeruginosa, Am. J. Ophthalmol. 138 (2004) 226—230. M.A. Aslam, Z. Ahmed, R. Azim, Microbiology and drug sensitivity patterns of chronic suppurative otitis media, J. Coll. Physicians Surg. Pak. 14 (2004) 459—461. P. Garg, S. Sharma, G.N. Rao, Ciprofloxacin-resistant Pseudomonas keratitis, Ophthalmology 106 (1999) 1319—1323. W.M. Sheppard, H.H. Wanamaker, A. Pack, S. Yamamoto, N. Slepecky, Direct round window application of gentamicin with varying delivery vehicles: a comparison of ototoxicity, Otolaryngol. Head Neck Surg. 131 (2004) 890—896. O.E. Brown, C.G. Wright, L.B. Edwards, W.L. Meyerhoff, The ototoxicity of ceftazidime in the chinchilla middle ear, Arch. Otolaryngol. Head Neck Surg. 115 (1989) 940—942. C.A. Bauer, T.J. Brozoski, Cochlear structure and function after round window application of ototoxins, Hear Res. 201 (2005) 121—131. M.E. Hoffer, K.A. Richard, P. Weisskopf, K. Gottshall, D. Wester, Transtympanic versus sustained-release administration of gentamicin: kinetics, morphology, and function, Laryngoscope 111 (2001) 1343—1357. N. Wagner, P. Caye-Thomasen, G. Laurell, D. Bagger-Sjoback, J. Thomsen, Cochlear hair cell loss in single-dose versus continuous round window administration of gentamicin, Acta Otolaryngol. (Stockh) 125 (2005) 340—345.
Available online at www.sciencedirect.com
www.elsevier.com/locate/ijporl
Evaluating the ototoxicity of topical piperacillin— tazobactam Chul-Ho Jang a,b,*, Haekyun Park c, Yong Bum Cho a, Cheol-Hee Choi b a
Department of Otolaryngology, Chonnam National University Medical School, Gwangju, South Korea Research Center for Resistant Cells, Chosun University, Gwangju, South Korea c Department of Biology, Chosun University, Gwangju, South Korea b
Received 6 March 2008; received in revised form 21 August 2008; accepted 26 August 2008 Available online 8 October 2008
KEYWORDS Ototoxicity; Piperacillin— tazobactam; Topical treatment
Summary Background and objective: With the increased use of ototopical ciprofloxacin solution, newly evolved bacterial fluoroquinolone resistance has also become more of a problem. The emergence of ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) has created a new therapeutic challenge in otology. We evaluated the ototoxicity of topical fortified piperacillin—tazobactam solution by performing experiments in young male albino guinea pigs (weight, 250—300 g each). Materials and methods: Antimicrobial assay by direct contact test using scanning electron microscope was performed. Twenty guinea pigs (250—300 g) were treated with fortified piperacillin—tazobactam solution. In experimental group 1 (n = 10), a gelfoam ball impregnated with 120 ml of fortified piperacillin—tazobactam was implanted on the round window membrane by a posterior approach. In experimental group 2 (n = 10), 20 ml of fortified piperacillin—tazobactam was injected into the middle ear cavity via silicone tube with a small hole placed in the superior aspect of the bulla by drilling. The drug application through the tube was performed for 7 consecutive days. In control group (n = 5), a gelfoam ball impregnated with 120 ml of gentamicin solution (80 mg/2 ml) was implanted on the round window membrane. Results: The fortified piperacillin—tazobactam 20 ml treated CRPAs showed destruction of their cell membrane in antimicrobial assay by direct contact test. No significant difference in the mean auditory brainstem response (ABR) thresholds before and after drug administration was found for the experimental group. However, significant elevation of the mean ABR thresholds was found for the control group. In experimental group, scanning electron microscopy showed almost normal sterociliary arrangements and surface structure on the inner and outer hair cells. However, significant destruction of outer hair cells was identified in control group.
* Corresponding author at: Department of Otolaryngology, Chonnam National University Hospital, Hak-dong 8, Dongku, Gwangju, South Korea. Tel.: +82 62 2206774. E-mail address: [email protected] (C.-H. Jang). 0165-5876/$ — see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2008.08.018
1816
C.-H. Jang et al. Conclusion: This present study’s data suggests that fortified piperacillin—tazobactam solution can be effectively used for topically treating CRPA otorrhea in those patients who suffer with chronic suppurative otitis media. # 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Purulent otorrhea is a common malady in patients suffering with chronic suppurative otitis media (CSOM). Patients with CSOM generally have a chronic inflammation of the middle ear and mastoid, with a persistent perforation of the tympanic membrane, which is associated with recurrent otorrhea. If purulent otorrhea persists, then the mucous membrane may become ulcerated, polypoid or granuomatous. Prolonged infection may be associated with resorption of the ossicles and mastoid bone by the process of demineralization and the osteoclastic activity. The most frequently isolated organisms are Pseudomonas aeruginosa and the anaerobes. Many otolaryngologists routinely use topical antibiotics for purulent otorrhea. Aminoglycosides such as neomycin sulphate, tobramycin and gentamicin are commonly used. Ciprofloxacin is one of the fluoroquinolones and it is available as a topical preparation; it has shown be effective against P. aeruginosa [1—3]. Ototopical ciprofloxacin in patients was curative for nearly 70% of the patients with otorrhea associated with P. aeruginosa and who were previously unresponsive to other antimicrobial agents [4,5]. As the clinical application of the ototopical ciprofloxacin solution has increased, newly evolved bacterial fluoroquinolone resistance has also become a challenge to treat. The resistance to fluoroquinolone is basically a reflection of both mutation and natural selection, which is the result of the selective pressure created by using fluoroquinolones [6]. Due to the fluoroquinolone resistance of P. aeruginosa and the development of fluoroquinolone resistance during therapy, selecting an optimal treatment for patients suffering with this pathogen is often complicated. We have previously reported on ciprofloxacin-resistant P. aeruginosa (CRPA) in patients with chronic suppurative otitis media [7]. Piperacillin—tazobactam contains two active ingredients, piperacillin, which is a penicillin-type antibiotic, and tazobactam, which is a medicine that prevents bacteria from inactivating piperacillin. The first ingredient piperacillin is an antibiotic that kills many types of bacteria. The second ingredient tazobactam belongs in the penicillin group,
but it does not have activity against bacteria. It helps piperacillin to overcome bacteria that have become resistant to piperacillin. Piperacillin—tazobactam provides excellent coverage against Grampositive, Gram-negative and anaerobic organisms [8]. A study by Nomura et al. [9] suggested that piperacillin—tazobactam has 4- to 64-fold stronger antimicrobial activity against all [beta]-lactamaseproducing bacteria compared with ampicillin—sulbactam. Piperacillin—tazobactam has greater activity than ampicillin—sulbactam (as measured by a higher percentage of susceptible bacteria) against Gram-negative organisms such as Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, P. aeruginosa and the Enterobacter species [10,11]. An otic preparation of piperacillin—tazobactam (currently unavailable commercially) results in a smaller amount of administered drug, compared with parenteral administration. To the best of our knowledge, there are no clinical studies in the Medline literature that have evaluated topical piperacillin— tazobactam for treating CRPA otorrhea. We designed the current study to investigate the ototoxic effect of topical piperacillin—tazobactam solution on the cochlea when the piperacillin—tazobactam solution was applied topically in the middle ear of guinea pigs.
2. Materials and methods 2.1. Antimicrobial assay by direct contact test The clinical CRPA (n = 12) bacteria samples were obtained from patients at our hospital. The CRPA samples were inoculated into 5 ml of Mueller-Hinton broth (MHB) media and incubated at 37 8C to obtain organisms in the mid-logarithmic growth phase. A 4.5 g vial of piperacillin—tazobactam powder (Wyeth Korea Pharmaceuticals Inc., Seoul, South Korea) was reconstituted with 30 ml of sterile distilled water. The resulting concentration of the piperacillin—tazobactam solution was 0.1125— 0.225 g/ml. The mid-logarithmic phase CRPAs were resuspended at 1 105 (CFU)/ml in MHB media and then they were inoculated at 37 8C with 10 or 20 ml of fortified piperacillin—tazobactam. After 1 h the
Ototoxicity of Topical Piperacillin-Tazobactam cells were fixed with an equal volume of 2% glutaraldehyde in 0.1 M sodium-cacodylate buffer, pH 7.4. After fixation for 4 h at 4 8C, the samples were extensively washed with 0.1 M cacodylate buffer, pH 7.4. The samples were then dehydrated through a graded series of ethanol solutions. After lyophilization and gold coating, the CRPA samples were examined in a scanning electron microscope (JSM 6400, JEOL Ltd. Tokyo, Japan).
2.2. Auditory brainstem response (ABR) measurement The experiments were performed in 25 young, male guinea pigs (weight, 250—300 g each) with normal tympanic membranes. The ear canals and the tympanic membranes of the guinea pigs were examined under an operating microscope. The presence of ear infection was excluded by examining each guinea pig’s external auditory canals and tympanic membranes. A (4.5 g) vial of piperacillin—tazobactam powder (Wyeth Korea Pharmaceuticals Inc., Seoul, South Korea) was reconstituted with 30 ml of sterile 5% dextrose in water. The resulting concentration of the piperacillin—tazobactam solution was 0.1125— 0.225 g/ml. Osmolarity was measured by a model 3300 osmometer (Advanced Instruments, Norwood, MA) The mean osmolarity was 664 mOsm. The pH was measured by pH indicator reagent strips and a laboratory micro-pH meter (Clinitek-200, Alpha Scientific Medical, La Verne, CA). The pH of fortified piperacillin—tazobactam solution was 5.08. The preparation was stored in a refrigerator following compounding and also during usage. The guinea pigs were anesthetized by an intraperitoneal injection of zolletil and xylazine hydrochloride. The guinea pigs received a baseline hearing assessment. In experimental group 1 (n = 10), a gelfoam ball impregnated with 120 ml of fortified piperacillin—tazobactam was implanted on the round window membrane by a posterior approach. In experimental group 2 (n = 10), 20 ml of fortified piperacillin—tazobactam was injected into the middle ear cavity via a small hole placed in the superior aspect of the bulla by drilling. A cut-down tube (polyvinyl chloride, Korea Medical Co., Seoul, Korea) was chosen for catheter insertion because it has a small diameter (i.d. = 0.6 mm, o.d. = 1 mm) and it was placed through the hole and fixed by cyanoacrylate (LOCTITE1, Ellsworth Adhesives, WA, USA). The drug application through the cut-down tube was performed for 7 consecutive days. In control group (n = 5), a gelfoam ball impregnated with 120 ml of gentamicin solution (80 mg/2 ml) (Kunhwa Pharmaceutical Co. Seoul, South Korea) was implanted on the round window membrane.
1817 The auditory brainstem responses were recorded under general anesthesia (ketamine 35 mg/kg and xylazine 15 mg/kg intramuscularly) with using an evoked potential system (Tucker-Davis Technologies, FL, USA) and a Samsung computer. The stimuli were digitally synthesized using Siggen# software, and they were presented through an insert earphone (ER-2, Etymotic Research, Inc., IL, USA). The acoustic stimuli, consisting of a click and 8, 16 and 32 kHz tone bursts, were then produced. The Intensity of the acoustic stimuli was expressed in decibels (dB). The animals were presented with a stimulus intensity series, which was initiated at 90 dB SPL and this reached a minimum of 10 dB SPL. The stimulus intensity was progressively lowered in 10 dB decrements. Each average consisted of 500 stimuli presentations, with a 10 ms analysis time. The electrical activity was recorded via a platinum needle electrode inserted into the scalp at the vertex, and it was referenced to another needle electrode in a deep neck muscle. A third needle electrode in the pinna serve as a ground. The intensities that appeared to be near threshold were repeated. The threshold was defined as the lowest intensity capable of producing a visually detectable, reproducible ABR response; the values were extrapolated to the nearest 5 dB assuming a log-linear fall off with decreasing intensity. At the threshold, the baselineto-peak amplitude of the largest ABR component was reduced to <10% of that evoked by the 90 dB stimuli. The ABRs were assessed preoperatively and at the third and seventh day after topical application of the drug. Repeated measure analysis of variance (ANOVA) was used to evaluate the differences among the groups. A P value <0.05 was considered to be statistically significant.
2.3. Scanning electron microscopic observation of the cochlea After the final ABR recordings, the anesthetized animals were perfused intracardially with 4% paraformaldehyde while they were under general anesthesia. The temporal bones were isolated and the perilymphatic spaces of the cochlear were gently perfused with 2.5% glutaraldehyde in 0.1 M phosphate-buffered solution (PBS) by performing cochleostomies at the round and oval windows. The bony capsules were removed to expose the organ of Corti, after which the specimens were post-fixed in 2.5% glutaraldehyde overnight at 4 8C. The specimens were washed three times in PBS and then post-fixed in 1% osmium tetroxide for 1 h at 4 8C. The organ of Corti specimens were then dehydrated through a graded series of ethanol solutions and then they were critical-point dried using
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liquid carbon dioxide. The critical-point dried specimens of the organ of Corti were attached to aluminum SEM stubs with aluminum paint and then sputter-coated with gold—palladium. The surfaces of the organs of Corti’s were examined in a scanning electron microscope (JSM6400, JEOL Ltd. Tokyo, Japan).
3. Results 3.1. Scanning electron microscopic observation of the CRPAs in vitro The piperacillin—tazobactam 10 ml treated CRPAs displayed a shortened cell length. The piperacillin—tazobactam 20 ml treated CRPAs showed destruction of their cell membrane (Fig. 1A—C).
3.2. Auditory brainstem responses The threshold shifts (decibels) between preoperative baseline and days 3 and 7, respectively, are shown in Fig. 2. No significant difference in the mean ABR thresholds before and after drug administration was found in the experimental group 1 (Fig. 2A) and group 2 (Fig. 2B). But statistically significant elevation of the mean ABR after gentamicin administration in the control group (Fig. 2C).
3.3. Scanning electron microscopic observation of the cochlea Fig. 3 shows the SEM micrographs of a guinea pig cochlea in experimental group 1, which showed an insignificantly elevated threshold at postoperative day 3. There was almost normal sterociliary arrangements and the surface structure were almost normal for the inner and outer hair cells and there was an almost a normal number of microvilli on the surface of the inner pillar cells. The SEM micrographs of a guinea pig cochlea in experimental group 2 show almost normal outer and inner hair cells (Fig. 4A). Fig. 4B shows the almost normal arrangements of stria vascularis of a guinea pig cochlea in experimental group 2. However, Fig. 5 shows that there was destruction of the outer hair cells in the control group.
Fig. 1 The SEM shows the CRPAs isolated from chronic suppurative otitis media (A). The CRPAs show a shortened cell length when they were treated with 10 ml of fortified piperacillin—tazobactam (B). The CRPAs show destruction of the cell membrane when they were treated with 20 ml of fortified piperacillin—tazobactam. SEM: scanning electron microscopy, CRPA: ciprofloxacin-resistant Pseudomonas aeruginosa.
4. Discussion It is well known that P. aeruginosa may become resistant to the antibiotic being used to treat an infection, and that prior use of a particular antibiotic predicts that P. aeruginosa will develop
resistance to that antibiotic. Barely 10 years after ciprofloxacin’s introduction into otology, the emergence of CRPA, when treating chronic suppurative otitis media, has been reported [7,12,13].
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Fig. 2 The ABR shows a insignificant difference in the mean hearing threshold before and after topically applied fortified piperacillin—tazobactam by direct round window approach in experimental group 1 (A) or transtympanic instillation for 7 consecutive days in experimental group 2 (B). The ABR shows significant difference in the mean hearing threshold before and after topical application of diluted gentamicin in control group (C). Asterisk means the statistical significance. ABR: auditory brainstem response.
Just as news of aminoglycoside-resistant P. aeruginosa mandated the search for an alternative remedy, which led to ciprofloxacin’s introduction, CRPA now requires the development of more capable antibiotics that can properly deal with this scourge. Increasing resistance in P. aeruginosa to multiple antibiotics has been observed and is posing therapeutic dilemmas. Antibiotic utilization is one factor that has been associated with the emergence of antimicrobial resistance. CRPA is still sensitive to ceftazidime, cefepime, piperacillin, piperacillin— tazobactam and imipenem [7,12,14,15]. The tazobactam of piperacillin—tazobactam is known as a beta-lactamase inhibitor. Tazobactam is included in this medicine because it inhibits the action of the beta-lactamases produced by the defenses of certain bacteria. This prevents the bacteria from inactivating the piperacillin, and it leaves them susceptible to attack. Thus, piperacillin—tazobactam increases the range of bacteria that piperacillin can kill. This medicine is given by injection or infusion and it is generally used for severe infections. In the ophthalmologic field, the fortified
antibiotics for the treatment of P. aeruginosa are well established and their efficacy has been reported on [16]. However, no previous study has reported on the effect of topical piperacillin—tazobactam on the cochlea. In the present study, we administered the piperacillin—tazobactam by two approaches. There was no difference of electrophysiological and morphologic results between two groups. Transtympanic instillation of drug does not allow for consistent, reproducible amounts of drug to be presented to the round window [17]. And the administered drug can enter to the Eustachian tube. By Brown et al., focal hemorrhage and occasional middle ear effusion were found in the middle ears after transtympanic instillation [18]. Administration of drug via a local delivery system applied directly to the round window membrane may allow for a more reproducible damage pattern [17,19]. The damage accomplished with a direct application of gentamicin is qualitatively similar to that achieved with a systemic injection of gentamicin [17]. Hoffer et al. [20] examined the inner
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Fig. 3 The SEM shows almost normal inner and outer hair cells in experimental group 1 after topically applied fortified piperacillin—tazobactam by direct round window approach (A). (B) The magnified view of the outer hair cells. SEM: scanning electron microscopy.
Fig. 4 The SEM shows almost normal inner and outer hair cells in experimental group 2 after topically applied fortified piperacillin—tazobactam by transtympanic instillation for 7 consecutive days (A). The SEM shows almost normal arrangement of stria vascularis (B). SEM: scanning electron microscopy.
ear kinetics of transtympanic gentamicin and compare this with the kinetics of sustained-release delivery in a basic science model. The reliability of sustained-release delivery to the ear reduces functional and morphological variations between animals. Single-dose instillation of gentamicin induced greater hair cell loss than continuous administration at the same dose [21]. Sheppard et al. [17] evaluated the ototoxic damage sustained from drugs applied directly to the round window in varying delivery vehicles such as gelfoam, hyaluronic acid, and fibrin. The fibrin or gelfoam vehicle was the only delivery system to produce reliable damage. In our study, we evaluated the guinea pigs’ hearing using ABR and we evaluated their cochlear morphology using a SEM; these techniques have often been used in ototoxicity studies. In this study, we found that piperacillin—tazobactam destroyed the surface of the CRPA in vitro. We demonstrated that with topical application of piperacillin—tazobactam, the mean ABR threshold showed insignificant elevation compared to that at baseline. Further, the SEM showed almost normal outer and inner hair cells.
Topical otic solution treatment results in a smaller amount of drug administered, as compared with parenteral administration. In conclusion, the data derived from this present study suggests that tazobactam can be effectively used for topical treatment of CRPA otorrhea in patients who suffer from chronic suppurative otitis media.
Fig. 5 The SEM shows destruction of outer hair cells in control group after topically applied gentamicin by direct round window approach. SEM: scanning electron microscopy.
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Acknowledgements [10]
This study was supported by grants from the Ministry of Science and Technology, Korea, and from the Korea Science and Engineering Foundation through the Research Center for Resistant Cells (R13-2003009).
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