Development of Resistant Bacteria Isolated from Dogs with Otitis Externa or Urinary Tract Infections after Exposure to Enrofloxacin In Vitro

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1 A. M. Brothers, P. S. Gibbs, and R. E. Wooley Development of Resistant Bacteria Isolated from Dogs with Otitis Externa or Urinary Tract Infections after Exposure to Enrofloxacin In Vitro Amy M. Brothers, MS Penelope S. Gibbs, PhD Richard E. Wooley, DVM, PhD Department of Medical Microbiology and Parasitology College of Veterinary Medicine University of Georgia Athens, GA ABSTRACT Minimum inhibitory concentrations for enrofloxacin were determined for 63 bacterial isolates from dogs with otitis externa or urinary tract infections. Development of resistant mutants was determined after exposing the isolates to enrofloxacin in vitro for up to five serial passages. Results indicated that Pseudomonas aeruginosa and Enterococcus spp isolates exposed to enrofloxacin developed resistance rapidly, whereas Klebsiella, Proteus, and Streptococcus spp were less likely to develop resistance. Despite the presence of enrofloxacin pressure, no resistant bacteria developed in the Escherichia coli and staphylococcal isolates. In many isolates, susceptibility patterns changed from susceptible to intermediate. INTRODUCTION Enrofloxacin, a fluoroquinolone, was developed exclusively for use in veterinary practice. 1 Fluoroquinolones are totally synthetic antimicrobial compounds that share low toxicity and broad-spectrum activity, and they are easily absorbed by host cells and tissues. 2 In gram-negative organisms, the primary antimicrobial activity of the fluoroquinolones blocks bacterial DNA replication by inhibition of bacterial DNA gyrase. DNA gyrase is responsible for the negative supercoiling of DNA that controls replication, transcription, and recombination. 3 7 Specifically, a single mutation in the gene gyra, which encodes the A subunit of DNA gyrase, results in fluoroquinolone resistance Another target of fluoroquinolones is topoisomerase IV, an enzyme responsible for ATP-dependent relaxation of DNA Mutations in parc and pare, genes that encode for topoisomerase IV, have been shown to contribute to fluoroquinolone resistance in Escherichia coli. 9,11 Resistance resulting from decreases in permeability of the bacterial cell wall and drug accumulation have also been reported. 8,11,12 Reduced expression of the porin protein OmpF and overexpression of active efflux systems are associated with the multiple antibiotic resistance (Mar) mutants and with increased fluoroquinolone resistance by E. coli. 9,13 The development of antimicrobial resistance has paralleled the use of antimicrobial agents. 14 Many researchers hypothesize that the use of fluoroquinolones in companion animals will enhance the emergence of drug-resistant organisms. 493

2 Veterinary Therapeutics Vol. 3, No. 4, Winter 2002 Of primary concern is the potential for transmission of these organisms to other animals and to humans. To this end, it is important to note that these mutations bestowing fluoroquinolone resistance can occur even in the absence of environmental fluoroquinolone pressure. The presence of a fluoroquinolone in the environment encourages the multiplication of the mutant (and thus resistant) bacteria that are present. The purpose of this study was to determine the antimicrobial resistances in bacteria isolated from dogs with otitis externa or urinary tract infection at the time of culture and to determine if resistance developed following in vitro exposure to enrofloxacin. Organisms studied included Staphylococcus intermedius, Staphylococcus aureus, Streptococcus spp, Proteus mirabilis, Pseudomonas aeruginosa, E. coli, Klebsiella spp, and Enterococcus spp. MATERIALS AND METHODS Bacterial Isolates The Athens Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia supplied multiple isolates of bacteria (S. intermedius, S. aureus, Streptococcus spp, P. mirabilis, P. aeruginosa, E. coli, Klebsiella spp, and Enterococcus spp) from dogs with either otitis externa or urinary tract infections. Reference Strains Reference strains (American Type Culture Collection [ATCC], Rockville, MD) were used to monitor the in vitro procedures as recommended by the National Committee for Clinical Laboratory Standards. 15 Strains included E. coli (ATCC# 25922), P. aeruginosa (ATCC# 27853), Enterococcus faecalis (ATCC# 29212), and S. aureus (ATCC# 29213). TABLE 1. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Pseudomonas aeruginosa Isolated from Dogs with Otitis Externa Isolate Passage 0 Passage 1 Passage > > Control Values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml); values in bold are resistant ( 4.0 µg/ml). Minimum Inhibitory Concentrations The minimum inhibitory concentrations (MICs) for enrofloxacin were determined by the broth dilution susceptibility test. 15 The criteria used to evaluate the MICs for enrofloxacin were as follows: 0.5 µg/ml or less was designated susceptible, more than 0.5 but less than 4.0 µg/ml was designated as having intermediate resistance, and an MIC 4.0 µg/ml or higher was resistant. 15 Approximately colony-forming units (CFU)/ml ( CFU/well) of the test organism was added to microtiter wells containing serial twofold dilutions of the antimicrobial agent in Mueller-Hinton (M-H) broth. The MICs were determined after incubation at 35 C for 18 to 48 hours. Resistant Mutants A stepwise selection of resistant mutants was accomplished by inoculating 10 6 CFU of the test organism into a set of microtiter plates containing twofold dilutions of enrofloxacin in MH broth. The microtiter plates were incubated at 37 C for 18 to 48 hours. Microtiter wells containing the highest concentration of enrofloxacin that showed growth were used to inoculate another set of microtiter plates also containing 494

3 A. M. Brothers, P. S. Gibbs, and R. E. Wooley twofold dilutions of enrofloxacin in M-H broth. Serial passages were repeated until a resistant mutant ( 4.0 µg enrofloxacin/ml) was obtained or five serial passages had been completed. 16 RESULTS Pseudomonas aeruginosa Two of the nine isolates of P. aeruginosa originating from cases of otitis externa were resistant to enrofloxacin, having an MIC 4.0 µg/ml or higher on initial isolation. The remaining seven isolates were classified as having intermediate resistance. After one passage under enrofloxacin exposure, five of these isolates were resistant to enrofloxacin, and the remaining two isolates became resistant after the second passage (Table 1). Enterococcus spp Ten Enterococcus spp isolates were obtained from nine dogs with urinary tract infections and one with otitis externa. One isolate was susceptible to enrofloxacin, and the remaining nine were initially classified as having intermediate resistance. Three isolates became resistant to enrofloxacin on the first passage, and individual isolates became resistant on passages two, three, four, or five. Three isolates remained classified as having intermediate resistance to enrofloxacin after five passages (Table 2). Staphylococcus spp Six isolates were determined to be S. intermedius, including three from dogs with otitis externa and three from dogs with urinary tract infections. Four isolates were S. aureus, including three from cases of otitis externa and one from a urinary tract infection. Seven of these 10 isolates were classified as susceptible to enrofloxacin and three as having intermediate resistance on initial isolation. After five serial passages, three of the isolates remained susceptible and seven demonstrated intermediate resistance to enrofloxacin (Table 3). TABLE 2. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Enterococcus spp Isolated from Dogs with Otitis Externa and Urinary Tract Infections 1* * > > * * * > * * 1.4 > * * Control Values in plain type are susceptible ( 0.5 µg/ml); values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml); values in bold are resistant ( 4.0 µg/ml). 495

4 Veterinary Therapeutics Vol. 3, No. 4, Winter 2002 TABLE 3. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Staphylococcus spp Isolated from Dogs with Otitis Externa and Urinary Tract Infections 1 S. intermedius* S. intermedius* S. intermedius S. aureus* S. aureus S. aureus* S. intermedius S. intermedius S. intermedius* S. aureus* Control Values in plain type are susceptible ( 0.5µg/ml); values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml). Klebsiella spp Seven Klebsiella spp isolates from dogs with urinary tract infections were initially susceptible to enrofloxacin. On the first passage, six of the seven were classified as having intermediate resistance. By the second passage, all seven isolates were of intermediate resistance. One isolate was resistant to enrofloxacin in the third passage, and the remaining six isolates continued to be of intermediate resistance through passages four and five (Table 4). Streptococcus spp Three of seven Streptococcus spp isolates were from dogs with urinary tract infections and four were from dogs with otitis externa. Initially, all seven isolates were classified as having intermediate resistance to enrofloxacin. One isolate (otitis externa) developed resistance to enrofloxacin after five serial passages (Table 5). Proteus spp Eight of 10 P. mirabilis isolates came from dogs with urinary tract infections and two were isolated from dogs with otitis externa. Nine of the isolates were susceptible to enrofloxacin and one was classified as having intermediate resistance on initial isolation. Enrofloxacin sensitivity did not change for these isolates until the second passage when one isolate became resistant, seven became intermediate, and two remained susceptible. Further changes in sensitivity did not occur until the fifth passage when one isolate became resistant and one developed intermediate resistance to enrofloxacin (Table 6). Escherichia coli Ten isolates of E. coli were evaluated, including six that were β-hemolytic. Nine isolates were from dogs with urinary tract infections and one was from a dog with otitis externa. All 10 isolates were susceptible to enrofloxacin on initial isolation. On the second passage, two isolates showed intermediate resistance. On the fifth passage, five isolates were of intermediate resistance and five were susceptible to enrofloxacin (Table 7). 496

5 A. M. Brothers, P. S. Gibbs, and R. E. Wooley TABLE 4. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Klebsiella spp Isolated from Dogs with Urinary Tract Infections 1 K. pneumoniae K. pneumoniae Klebsiella spp K. oxytoca K. pneumoniae K. pneumoniae K. pneumoniae Control Values in plain type are susceptible ( 0.5 µg/ml); values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml); values in bold are resistant ( 4.0 µg/ml). DISCUSSION Nalidixic acid, the parent bicyclic 4-quinolone molecule, is active primarily against aerobic gram-negative bacteria. Modifications of the chemical structure resulted in the fluoroquinolones, which have an increased spectrum of activity to include gram-positive bacteria, mycobacteria, Chlamydia spp, and Mycoplasma spp. 2 Enrofloxacin is a synthetic antibacterial agent of the fluoroquinolone class with activity against a broad antibacterial spectrum. The enrofloxacin molecule, which is almost identical to its metabolite ciprofloxacin except for the presence of an ethyl group on the piperazinyl ring for enrofloxacin that is not present in ciprofloxacin, was developed exclusively for use in veterinary medicine. In dogs, enrofloxacin is deethylated to ciprofloxacin. 17 TABLE 5. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Streptococcus spp Isolated from Dogs with Otitis Externa and Urinary Tract Infections MIC µg/ml 1* * * * Control Values in plain type are susceptible ( 0.5 µg/ml); values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml); values in bold are resistant ( 4.0 µg/ml). 497

6 Veterinary Therapeutics Vol. 3, No. 4, Winter 2002 A study of the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in dogs indicated that a considerable part of the antimicrobial activity is produced by ciprofloxacin plasma concentrations exceeding the MIC for several different organisms when enrofloxacin is administered orally or intravenously at 5 mg/kg body weight. 17 Enrofloxacin is recommended for the treatment of respiratory, dermal, and urinary infections in companion animals. In dermal and urinary tract infections, enrofloxacin is reported to have a broad antibacterial spectrum against Staphylococcus spp, E. coli, and P. mirabilis. In respiratory infections, antibacterial activity is claimed for E. coli, Staphylococcus spp, Bordetella bronchiseptica, and Pasteurella multocida. In the present study, resistance to enrofloxacin developed rapidly for P. aeruginosa when exposed to enrofloxacin pressure. Initially, two of nine isolates were resistant to enrofloxacin, and the remaining isolates were resistant by the third passage under antibiotic pressure. Such resistance has also been reported recently by other laboratories. 18,19 Resistance to enrofloxacin developed for the Enterococcus spp isolates. None of the isolates was initially resistant to enrofloxacin, but nine of 10 were classified as having intermediate resistance. After five serial passes, eight of 10 isolates developed resistance. The development of resistant P. mirabilis was less dramatic. None were resistant to enrofloxacin initially and only two of 10 became resistant in the fifth serial passage. The seven isolates of Klebsiella spp were initially susceptible to enrofloxacin, but by the fifth passage, one was classified as resistant and six were of intermediate resistance. All seven isolates of Streptococcus spp were initially classified as having intermediate resistance to enrofloxacin, TABLE 6. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Proteus mirabilis Isolated from Dogs with Otitis Externa and Urinary Tract Infections * * Control Values in plain type are susceptible ( 0.5 µg/ml); values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml); values in bold are resistant ( 4.0 µg/ml). 498

7 A. M. Brothers, P. S. Gibbs, and R. E. Wooley TABLE 7. Minimum Inhibitory Concentrations (MICs) for Enrofloxacin with Serial Passages of Escherichia coli Isolated from Dogs with Otitis Externa and Urinary Tract Infections β β β β β β * Control Values in plain type are susceptible ( 0.5 µg/ml); values in italics are of intermediate resistance (>0.5 and <4.0 µg/ml). β = beta hemolytic. and only one isolate became resistant after five serial passages. None of the E. coli or Staphylococcus spp isolates were initially resistant to enrofloxacin nor did they develop resistance in five serial passages in enrofloxacin. However, seven of the staphylococcal and five of the E. coli isolates were classified as having intermediate resistance. The possibility of organisms developing resistance with any antimicrobial agent is a reality. However, when choosing a treatment plan for bacterial infections, it is important that the antimicrobial agent selected is one that has the least propensity to create resistance in the specific causative organism(s). Data generated in this study suggest that P. aeruginosa and Enterococcus spp isolated from canine urinary tract infections and otitis externa exposed to enrofloxacin produce resistance rapidly, whereas Klebsiella, Proteus, and Streptococcus spp are less likely to develop resistance. No resistance to enrofloxacin was noted for E. coli and the staphylococcal isolates. Therefore, it is imperative that veterinarians isolate and correctly identify the causative organism(s) to develop a rational plan for therapy. 20 REFERENCES 1. Mengozzi G, Intorre L, Bertini S, et al: Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and intramuscular administrations in sheep. Am J Vet Res 57: , Walker RD: Antimicrobial Therapy in Veterinary Medicine, ed 3. Ames, Iowa State University Press, 2000, pp Hooper DC, Wolfson JS: Quinolone Antimicrobial Agents, ed 2. Washington DC, American Society for Microbiology, 1993, pp 53 76, Reece RJ, Maxwell A: DNA gyrase: Structure and function. Crit Rev Biochem Mol Biol 26: , Sanzey B: Modulation of gene expression by drugs affecting deoxyribonucleic acid gyrase. J Bacteriol 138:40 47, Smith GR: DNA super coiling: Another level for regulating gene expression. Cell 24: ,

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