- Email: [email protected]
Susceptibility of rapidly growing mycobacteria isolated from cats and dogs, to ciprofloxacin, enrofloxacin and moxifloxacin
Veterinary Microbiology 147 (2011) 113–118
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Research article
Susceptibility of rapidly growing mycobacteria isolated from cats and dogs, to ciprofloxacin, enrofloxacin and moxifloxacin M. Govendir a,* , T. Hansen b, B. Kimble a, J.M. Norris a, R.M. Baral c, D.I. Wigney a, S. Gottlieb d, R. Malik e a
Faculty of Veterinary Science, McMaster building, B14, The University of Sydney, NSW 2006, Australia Pathology Queensland, Mycobacterium Reference Laboratory, Herston Hospitals Complex, Herston, QLD 4029, Australia Paddington Cat Hospital, Oxford Street, Paddington, NSW 2021, Australia d The Cat Clinic, Creek Road, Mt Gravatt, QLD 4122, Australia e Centre for Veterinary Education, The University of Sydney, NSW 2006, Australia b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 November 2009 Received in revised form 9 June 2010 Accepted 14 June 2010
Rapidly growing mycobacteria (RGM) cause infections in cats and dogs which require prolonged antibacterial medication for resolution. In Australia, pathogens from the Mycobacterium fortuitum and Mycobacterium smegmatis clusters are responsible for most of the RGM infections in cats and dogs. As fluoroquinolones are often recommended for treating such infections, 14 M. fortuitum isolates, 51 isolates from the M. smegmatis cluster and 2 M. mageritense isolates, collected from feline and canine patients, underwent susceptibility testing to the second generation fluoroquinolones ciprofloxacin and enrofloxacin and the newer generation fluoroquinolone moxifloxacin. Using microbroth dilution, the MIC90 of ciprofloxacin, enrofloxacin, and moxifloxacin that inhibited growth of M. fortuitum isolates were 0.500, 0.250 and 0.063 mg/mL respectively. For the M. smegmatis cluster isolates the corresponding MIC90 was 0.500, 0.250 and 0.125 mg/mL respectively. E-test results showed similar trends but MICs were lower than those determined by microbroth dilution. Additionally, moxifloxacin was administered to 10 clinically normal cats (50 mg per cat, once daily for 4 days). The plasma moxifloxacin concentration 2 h after the last dose was determined by liquid chromatography as 2.2 0.6 mg/mL. The plasma concentration at 2 h:MIC90 ratios for moxifloxacin for M. fortuitum and M. smegmatis cluster was 34.9 and 17.6 respectively which exceeded the recommended threshold of 10, indicating that moxifloxacin has good theoretical efficacy for treatment of those M. fortuitum and M. smegmatis infections in cats and dogs that have become refractory to other antibacterial drug classes. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Mycobacterium fortuitum Mycobacterium smegmatis Fluoroquinolone Moxifloxacin Cat Dog
1. Introduction RGM are occasional pathogens of people and some animal species. RGM are Gram-positive bacilli with a cell wall rich in complex fatty acids and waxes that generally produce visible colonies within 5 days when cultured at
* Corresponding author. Tel.: +61 02 93515442; fax: +61 02 93513056. E-mail address: [email protected] (M. Govendir). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.06.011
room temperature on routine laboratory media (Malik et al., 2000; Brown-Elliott and Wallace, 2002). The most common clinical manifestation of RGM infection in immunocompetent cats and dogs is a chronic panniculitis whereby pathogens are introduced into subcutaneous fatty tissue (e.g. the inguinal fat pad) and less commonly, systemic disease. Treatment of RGM infections in cats and dogs requires prolonged antimicrobial therapy and sometimes surgical debulking. The newer generation 8-methoxy fluoroquinolone moxifloxacin, has been documented
114
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
to inhibit RGM growth at lower concentrations than ciprofloxacin, a prototypical fluoroquinolone drug of the previous generation (Brown-Elliott et al., 2002). RGM are classified into six phylogenetic clusters (Adekambi and Drancourt, 2004). Those responsible for chronic infections in cats and dogs in Australia are predominantly, but not exclusively, from the Mycobacterium fortuitum cluster and Mycobacterium smegmatis cluster. The aim of this investigation was to compare the minimum inhibitory concentrations (MIC) of ciprofloxacin, enrofloxacin and moxifloxacin by microbroth dilution and concentration gradient E-tests, to inhibit growth of RGM isolates from Australian feline and canine patients. This investigation also determined the presumptive steady state plasma concentration of moxifloxacin 2 h after oral administration in normal cats by liquid chromatography, as pharmacokinetic data for this agent is lacking for cats. 2. Materials and methods 2.1. Bacterial isolates Feline and canine RGM isolates from clinical cases were available from the freeze-dried collection of Veterinary Pathology Diagnostic Services, Faculty of Veterinary Science, The University of Sydney. Further testing was conducted at the Queensland Mycobacteria Reference Laboratory for species identification by phenotypic and molecular methods. These isolates were collected from clinical cases encountered over the period 1983–2009. The preferred method for collecting isolates from clinical cases has been described previously (Malik et al., 2000, 2004). Two M. smegmatis senso stricto isolates were from cats that had been previously treated for their infection with a course of enrofloxacin. 2.2. Strain identification 2.2.1. Biochemical identification of strains Isolates were initially identified by a range of standard biochemical tests, including isolate morphology and degree of acid-fastness in Ziehl–Neelsen stained smears of growth taken from Lowenstein–Jensen medium, colonial morphology (rough or smooth), pigmentation both in light and dark, rate of growth at room temperature and 37 8C, ability to grow at 42 and 52 8C, iron uptake, pamino salicylic acid degradation, nitrate reduction, bgalactosidase activity, acid production from carbohydrates (glucose, inositol and mannitol), utilisation of compounds (glucose, fructose, inositol, mannitol and citrate) as the sole carbon source, tolerance to 5% sodium chloride in Lowenstein–Jensen medium and susceptibility to polymixin B, trimethoprim and tobramycin (Malik et al., 2000, 2004; Brown-Elliott and Wallace, 2002). Presence of nitrate reductase activity identified isolates from the M. fortuitum and M. smegmatis clusters. Presence of arylsulphatase activity distinguished M. fortuitum, while its absence identified M. smegmatis isolates (Kubica and Beam, 1961; Kubica and Vestal, 1961; Tsukamura, 1984). Further strain identification required molecular techniques.
2.2.2. Preparation of DNA template and amplification of 16S rRNA and heat shock protein (hsp) loci Chromosomal DNA from these isolates was prepared by adding a loop full of colonies to 300 mL of sterile water in a micro-centrifuge tube containing sterile glass beads. Tubes were placed in a dry water bath at 95 8C for 30 min, sonicated for 10 min and then centrifuged for 10 min to remove cellular debris. PCR amplification of 16S rRNA and hsp65 loci was performed using previously described primer sets illustrated in Table 1. Each 50 mL PCR reaction contained 2 mL of DNA template, 500 mM of each dNTP, 2.5 mM of MgCl2, 1 U of Taq DNA polymerase (Fisher Biotech, Thebarton, South Australia) and 2 mM of each primer. The PCR was performed using an Applied Biosystems Gene-Amp 2700 Thermal Cycler (Applied Biosystems, Scoresby, VIC) and involved an initial denaturation at 94 8C for 5 min, followed by 35 cycles of denaturation at 94 8C for 1 min, annealing at 55 8C for 1 min and extension at 72 8C for 1 min, with a final extension at 72 8C for a further 10 min. 2.2.3. DNA sequencing PCR products were purified using the enzyme EXOSAPIT (Amersham Biosciences, Rydalmere, NSW), and sequenced using an automatic DNA sequencer (ABI 3700, Applied Biosystems, Scoresby, VIC), using the two forward primers (BF and TB11) described in Table 1. DNA sequences were compared to published sequences in GenBank via the BLAST network service. 2.3. Minimum inhibitory concentration testing MICs were determined using the microbroth dilution method in accordance with guidelines M24-A (CLSI, 2003). Fourteen two-fold serial dilutions of ciprofloxacin, enrofloxacin (both obtained from Sigma–Aldrich, Castle Hill, NSW) and moxifloxacin (Bayer HealthCare, Berlin, Germany) ranging in concentration from 64 to 0.008 mg/mL, were pipetted into microtitre wells (Nunc 16620, Noble Park, VIC). MICs were also determined by concentration gradient tests (E-test1 strips, AB Biodisk, Solna, Sweden) used according to the manufacturer’s instructions. Most, but not all isolates underwent susceptibility testing by both methods. A recommended quality control strain, Staphylococcus aureus ATCC 29213 was tested concurrently with each fluoroquinolone (CLSI, 2003). The MIC required to inhibit growth of the S. aureus strain was determined 24 h after incubation at 35 8C, while the MIC for RGM isolates were determined at 30 8C after 72 h.
Table 1 Primer sequences used in the amplification of 16S and sp65 loci for organism identification. Name
Sequence a
BF (16S Fwd) R2 (16S Rev)a TB11 (hspFwd)b TB12 (hspRev)b a b
Rogall et al. (1990). Telenti et al. (1993).
50 -AGAGTTGGATCCTGGCTCAG-30 50 -CCTACGAGCTCTTTACG-30 50 -ACCAACGATGGTGTGTCCAT-30 50 -CTTGTCGAACCGCATACCCT-30
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
115
2.4. Moxifloxacin dosing of cats
3. Statistical analysis
Ten normal mature cats (7 female and 3 male, all neutered; 5 Burmese and 5 domestic short-hair), with a mean weight of 4.8 0.6 kg, had their baseline biochemical analytes determined and were dosed for 4 days with compounded moxifloxacin capsules (50 mg once daily; BOVA Chemist, Caringbah, NSW). Patients were dosed first thing in the morning and then fed their usual diet. Two hours after the last dose, 3 mL of blood was collected by cephalic venipuncture into a lithium heparin tube, the plasma separated and frozen. All samples were batched to detect plasma moxifloxacin concentration.
As RGM are saprophytic organisms that infect mammalian hosts by accident, with host factors having minimal impact on the biology of these bacteria, feline and canine isolates of the same RGM group were combined for statistical analysis and graphical representation. The median MIC (MIC50), MIC90 and MIC range for each drug were determined for both M. fortuitum and M. smegmatis cluster isolates. As MICs were not normally distributed, the MIC50 of each drug to inhibit the isolates of each group were compared by Mann–Whitney’s two-tailed tests. Spearman’s correlation coefficient (rs) was determined to establish whether there was a relationship between the MICs obtained by microbroth dilution and Etests. For this latter analysis and graphical representation, data were converted to nearest log2 dilution MIC. The level of significance for all analyses was accepted at p < 0.05.
2.4.1. Determination of plasma moxifloxacin concentration Determination of moxifloxacin concentrations in feline plasma was performed by reversed-phase, high performance liquid chromatography (HPLC) method. The HPLC system (Shimadzu, Rydalmere, NSW) consisted of a pump (LC20AT), vacuum degassing solvent delivery unit (DGU-20As), autosampler (SIL 20AC), column oven (CTO 20AC) maintained at 25 8C, ultra-violet visible light detector (SPD-20A), system controller (CBM 20A) and Class VP software (version 7.4). Separation was performed using an Apollo C-18, 5 mm (4.6 mm 250 mm) column (Alltech, Dandenong South, VIC, Australia) coupled with a 1 mm Optiguard C-18 guard column (Optimize Technologies, Oregon City, OR, USA). The isocratic mobile phase consisted of 25 mM citric acid (Sigma–Aldrich, Castle Hill, NSW) and 10 mM sodium dodecyl sulfate (Boehringer Mannheim, Indianapolis, USA) with 50% acetonitrile (Ajax Finechem, Taren Point, NSW). The flow rate was 1 mL/min. Absorption was detected at 296 nm. Orbifloxacin was the internal standard and retention times for orbifloxacin and moxifloxacin were 9.03 and 10.01 min respectively. The stock solution of HPLC analytical grade moxifloxacin was prepared at a concentration of 1 mg/mL in purified water and further diluted into working solutions ranging from 0.315 to 50 mg/mL. Calibration standards of moxifloxacin were prepared by adding 50 mL of working solutions to 200 mL of blank pooled feline plasma. Clinical and calibration samples underwent solid-phase extraction (SPE) using an Oasis HLB 1 cm3 30 mg column filter (Waters, Milford, MA, USA) via a vacuum manifold, according to manufacturer’s instructions. After complete dryness of methanol–TFA by speed vacuum concentrator SPD121P (Thermo Scientific) at 35 8C for 2 h, samples were reconstituted with 250 mL of mobile phase, vortexed and 10 mL of supernatant was injected into the HPLC system (as duplicates). Eight calibration concentrations, ranging from 0.0625 to 10 mg/mL were used to construct the calibration curve which had a correlation coefficient (r2) >0.98. The intraday precision and accuracy were determined by repeated analysis of plasma samples (n = 4) spiked with 1, 2 and 5 mg/mL moxifloxacin determined on 4 consecutive days. The intra- and inter-day values of precision and accuracy were <4.55% (RSD) and >96% respectively. The moxifloxacin ‘recovery’ percentage from the SPE procedure was 89.3% with a RSD of 6.4%.
4. Results A total of 67 isolates were available for this study. Two isolates from dogs and 12 from cats were M. fortuitum. Of the M. smegmatis cluster, 3 dog and 39 cat isolates were M. smegmatis sensu stricto and an additional 9 were M. goodii, all from cats. There were two M. mageritense isolates, one each from a dog and cat. The MIC ranges of all three drugs that inhibited the growth of the isolates by microbroth dilution, are presented in Fig. 1. The MIC50 (and MIC90) for moxifloxacin for the M. fortuitum isolates were 0.063 (and 0.063) mg/mL, which were significantly lower compared to ciprofloxacin 0.250 (0.500) mg/mL (p = 0.0002) and enrofloxacin 0.125 (0.250) mg/mL (p = 0.0003). The MIC50 (and MIC90) for moxifloxacin for the M. smegmatis cluster were 0.063 (0.125) mg/mL which was significantly lower compared to ciprofloxacin 0.250 (0.500) mg/mL and enrofloxacin 0.125 (0.250) mg/mL (both p < 0.0001). The MIC50 of each drug to inhibit growth of both M. fortuitum versus M. smegmatis isolates were compared and there were no significant differences. The two M. mageritense (cluster V) isolates had a MIC range for ciprofloxacin, enrofloxacin and moxifloxacin of
[(Fig._1)TD$IG]
Fig. 1. MIC (mg/mL) of ciprofloxacin, enrofloxacin and moxifloxacin to inhibit the growth of M. fortuitum and M. smegmatis cluster isolates determined by microbroth dilution. The dashed line represents the susceptibility breakpoint of ciprofloxacin for RGM in people (<1 mg/mL). t Both cats from which these two isolates were obtained had received a prior course of enrofloxacin as monotherapy.
[(Fig._2)TD$IG]
116
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
Fig. 2. Comparison of log2 MICs determined by microbroth dilution (x-axes) versus MICs determined by E-test (y-axes) for the same M. fortuitum and M. smegmatis cluster isolates (n = 65) for ciprofloxacin, enrofloxacin and moxifloxacin.
0.125 (both isolates), 0.125 and 0.25, 0.031 and 0.063 mg/ mL respectively. All agents inhibited the growth of the QC strain S. aureus ATCC 29213 after 24 h by microbroth dilution within the expected ranges of 0.12–0.5 mg/mL for ciprofloxacin (CLSI, 2007), 0.03–0.12 mg/mL for enrofloxacin (CLSI, 2008) and 0.015–0.12 mg/mL for moxifloxacin (CLSI, 2007). The MIC50 determined by E-test for ciprofloxacin, enrofloxacin and moxifloxacin for M. fortuitum were 0.016, 0.047 and 0.012; and for the M. smegmatis cluster were 0.064, 0.125 and 0.016 mg/mL respectively. The log2 dilution MICs determined by microbroth dilution and E-test for the identical isolate for each drug were compared and presented in Fig. 2. The Spearman coefficient for ciprofloxacin, enrofloxacin and moxifloxacin was 0.13, 0.28 and 0.55 respectively with a p-value of <0.0001 for moxifloxacin. The mean SD plasma moxifloxacin concentration 2 h after the fourth consecutive daily dose (50 mg per cat per morning) was 2.2 0.6 mg/mL. Moxifloxacin administration resulted in mild diarrhoea in two cats and medication had to be stopped in one cat due to vomiting soon after administration. The cat that vomited was excluded from the trial, leaving data from 10 others.
5. Discussion Reviews are available on RGM taxonomy, biology (Brown-Elliott and Wallace, 2002; Adekambi and Drancourt, 2004) and clinical management of RGM infections in cats (Malik et al., 2000; Horne and Kunkle, 2009) and in dogs (Malik et al., 2004). The key finding was that for all isolates, moxifloxacin had significantly lower MICs than ciprofloxacin or enrofloxacin which is in accordance with similar studies using human isolates (Brown-Elliott et al., 2002). Enrofloxacin was more active against the majority of the isolates than ciprofloxacin. On the whole, the vast majority of isolates were susceptible to all fluoroquinolones. This reflects in part, the rareness of the M. chelonaeabscessus group strains in Australia, as members of this complex tend to be inherently resistant to the fluoroquinolones (Brown-Elliott and Wallace, 2002; Malik et al., 2004). Only two resistant M. smegmatis cluster isolates were encountered, and interestingly, both were obtained from cats that had received a prior course of enrofloxacin as monotherapy and appeared to have developed cross resistance to all fluoroquinolones. This suggests two things: (1) mutational resistance to fluoroquinolones can occur during a course of therapy (Escribano et al., 2007;
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
Rodriguez et al., 2007; Wang et al., 2007) and (2) there is a need for susceptibility testing at the outset, or if there is an unsatisfactory response to therapy, or if relapse occurs. Microbroth dilution is recommended as the ‘gold standard’ for susceptibility testing of RGM (CLSI, 2003) as results of other susceptibility tests are regarded as more variable (Woods et al., 2000; Brown-Elliott and Wallace, 2002; Esteban et al., 2002; Martin-de-Hijas et al., 2008). Microbroth dilution is not without limitations as the precise test end-point can be difficult to read (Woods et al., 1999; CLSI, 2003), is laborious and personnel require experience with the in vitro growth characteristics of these pathogens (Esteban et al., 2002; Martin-de-Hijas et al., 2008). In order to find an easier method to generate susceptible versus resistant information for these pathogens in a veterinary laboratory, the results of E-tests were compared to those determined by microbroth dilution tests resulting in a poor correlation between microbroth dilution and E-test MICs. The factors for this poor correlation currently remain unknown. An indication of antibacterial agents’ in vivo antibacterial efficacy is the maximal concentration achieved in plasma after a conventional dose of drug (Cmax) considered in relation to MIC (i.e. Cmax:MIC). As fluoroquinolones are classified as concentration dependent antibacterial agents, a Cmax:MIC ratio of at least 10:1 is recommended (Walker and Dowling, 2007). We were unable to obtain serial blood samples to determine the moxifloxacin Cmax, although a presumptive steady state 2 h plasma concentration of 2.2 0.6 mg/mL in 10 clinically normal cats after administration of 50 mg per cat daily (9.6–12.5 mg/kg per day) for 4 days was determined. Thus, the C2h:MIC90 ratios for moxifloxacin for M. fortuitum and the M. smegmatis cluster were calculated as 34.9 and 17.6 respectively, indicating that moxifloxacin should theoretically have good clinical efficacy at a dose of 50 mg per adult cat, once daily. By way of contrast, 10 mg/kg of ciprofloxacin administered orally to cats with a Cmax of 1.26 0.67 mg/mL at 1.30 0.67 h (Albarellos et al., 2004) resulted in a Cmax:MIC90 of 2.52 for M. fortuitum and 5.04 for the M. smegmatis cluster isolates. Likewise when 5 mg/kg of enrofloxacin was administered orally to dogs for 5 days resulting in a Cmax of 2.01 0.18 mg/ mL (Frazier et al., 2000), the Cmax:MIC50 was calculated as 16.08 for both RGM clusters. Moxifloxacin is registered for use in people only, although it has been administered to horses, resulting in adverse gastrointestinal effects (Gardner et al., 2004), rabbits (Carceles et al., 2006) and sheep (Carceles et al., 2009). Moxifloxacin’s antibacterial properties are distinguished from second generation fluoroquinolones (e.g. ciprofloxacin, enrofloxacin, marbofloxacin and difloxacin) by increased efficacy against Gram-positive bacteria (including Enterococcus species) as well as aerobic and obligate anaerobes (Stass et al., 2001). Activity of fourth generation fluoroquinolones is reported to be less effected overall by mutations responsible for fluoroquinolone resistance (Brown-Elliott et al., 2002). Available as tablets and as an intravenous formulation, moxifloxacin was initially developed to treat respiratory tract infections in people (Ball et al., 2004). It is worth noting that although uncommon, it is not unknown, for people to acquire a RGM
117
infection from their pets by the pathogen penetrating skin via a laceration or abrasion (McKinsey et al., 1995). In Australia, fluoroquinolones are registered for use in companion animals only; however potential does exist for the excreta of fluoroquinolone treated animals to contaminate the environment and enter both animal and human food chains, to further select for fluoroquinolone resistant bacteria. Use of elevated dosages of enrofloxacin in cats has been associated with mild to severe retinopathy in cats, especially at doses markedly higher than the label approved dosage of 5 mg/kg or if the drug was given intravenously to critical patients (Gelatt et al., 2001; Wiebe and Hamilton, 2002). For this reason, enrofloxacin is not recommended for treating RGM infections in cats because a clinical cure can take months to achieve and there is sometimes a requirement to obtain higher than usual plasma concentrations by increasing drug dosage to ‘drive’ the agent into marginally perfused tissues. One of the authors (RM) has used moxifloxacin administration at a dose of 50 mg per cat once daily with food as a component of therapy in four cats with mycobacterial panniculitis including some patients that were refractory to other antibiotics with uniform success. As moxifloxacin is not registered for use in cats or dogs, the authors suggest that it should be considered for treatment only if the infection has become refractory to those drugs formulated and registered for animal use. Further studies to characterise moxifloxacin’s pharmacokinetic properties and safety in both dogs and cats and to explore its potential synergistic effects with other antibacterial agents are warranted. Role of funding source This study was financially supported by Australian Companion Animal Health Foundation. Acknowledgements Bayer Healthcare AG provided the analytical grade moxifloxacin gratis. Thanks to Ms. Robyn Carter and Dr. Chris Coulter of the Mycobacterium Reference Laboratory, QLD, for their collaboration in this project, the owners who consented to their cats participating in the oral dosing of moxifloxacin and BOVA Chemist, Caringbah, NSW, who compounded and provided the moxifloxacin capsules gratis. The authors thank Dr. Thomas Gottlieb who alerted us to potential value of using moxifloxacin in cats. We acknowledge the forethought of the late Associate Professor Daria Love in initiating the archiving rapidly growing mycobacteria isolates. Richard Malik is supported by the Valentine Charlton Bequest. Ethical approval: This study was approved by The University of Sydney Animal Ethics Committee. References Adekambi, T., Drancourt, M., 2004. Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int. J. Syst. Evol. Microbiol. 54, 2095–2105.
118
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
Albarellos, G.A., Kreil, V.E., Landoni, M.F., 2004. Pharmacokinetics of ciprofloxacin after single intravenous and repeated oral administration to cats. J. Vet. Pharmacol. Ther. 27, 155–162. Ball, P., Stahlmann, R., Kubin, R., Choudhri, S., Owens, R., 2004. Safety profile of oral and intravenous moxifloxac: cumulative data from clinical trials and postmarketing studies. Clin. Ther. 26, 940–950. Brown-Elliott, B.A., Wallace, R.J., 2002. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmented rapidly growing mycobacteria. Clin. Microbiol. Rev. 15, 716–746. Brown-Elliott, B.A., Wallace, R.J., Crist, C.J., Mann, L., Wilson, R.W., 2002. Comparison of in vitro activities of gatifloxacin and ciprofloxacin against four taxa of rapidly growing mycobacteria. Antimicrob. Agents Chemother. 46, 3283–3285. Carceles, C.M., Escudero, E., Fernandez-Varon, E., Marin, P., 2009. Pharmacokinetics after intravenous, intramuscular and subcutaneous administration of moxifloxacin in sheep. Vet. J. 180, 343–347. Carceles, C.M., Serrano, J.M., Marin, P., Escudero, E., Fernandez-Varon, E., 2006. Pharmacokinetics of moxifloxacin in rabbits after intravenous, subcutaneous and a long-acting poloxamer 407 gel formation administration. J. Vet. Med. A 53, 300–304. CLSI, 2003. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved Standard. Clinical and Laboratory Standards Institute, Wayne, PA. CLSI, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Information Supplement. Clinical and Laboratory Standards Institute, Wayne, PA. CLSI, 2008. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved standard – Third Edition. Clinical and Laboratory Standards Institute, Wayne, PA. Escribano, I., Rodriguez, J., Llora, B., Garcia-Panchon, E., Ruiz, M., Royo, G., 2007. Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid. Chemotherapy 53, 397–401. Esteban, J., Gadea, I., Torres, M.-V., Cabria, F., Rollan, E., Santos-O’Connor, F., Fernandez-Roblas, R., Soriano, F., 2002. A comparison between disk diffusion and microdilution for susceptibility testing of Mycobacterium fortuitum complex. J. Chemother. 14, 547–553. Frazier, D.L., Thompson, L., Trettien, A., Evans, E.I., 2000. Comparison of fluoroquinolone pharmacokinetic parameters after treatment with marbofloxacin, enrofloxacin and dofloxacin in dogs. J. Vet. Pharmacol. Ther. 23, 293–302. Gardner, S.Y., Davis, J.L., Jones, S.L., Lafevers, D.H., Hoskins, M.S., McArver, E.M., Papich, M.G., 2004. Moxifloxacin pharmacokinetics in horses and disposition into phagocytes after oral dosing. J. Vet. Pharmacol. Ther. 27, 57–60. Gelatt, K.N., Van-Der-Woerdt, A., Ketring, K.L., Andrew, S.E., Brooks, D.E., Biros, D.J., Denis, H.M., Cutler, T.J., 2001. Enrofloxacin-associated retinal degeneration in cats. Vet. Ophthalmol. 4, 99–106. Horne, K.S., Kunkle, G.A., 2009. Clinical outcome of cutaneous rapidly growing mycobacterial infections in cats in the south-eastern United States: a review of 10 cases (1996–2006). J. Feline Med. Surg.. Kubica, G.P., Beam, R.E., 1961. The arylsulfatase activity of acid-fast bacilli. The differentiation of Mycobacterium avium from the unclassified group III nonphotochromogenic mycobacterium. Am. Rev. Respir. Dis. 83, 733–736.
Kubica, G.P., Vestal, A.L., 1961. The arylsulfatase activity of acid-fast bacilli. Investigation of stock cultures of acid-fast bacilli. Am. Rev. Respir. Dis. 83, 728–732. Malik, R., Shaw, S.E., Griffin, C., Stanley, B., Burrows, A.K., Bryden, S.L., Titmarsh, J., Stutsel, M.J., Carter, S.A., Warner, A., Martin, P., Wigney, D.I., Gilpin, C., 2004. Infections of the subcutis and skin of dogs caused by rapidly growing mycobacteria. J. Small. Anim. Pract. 45, 485–494. Malik, R., Wigney, D.I., Dawson, D., Martin, P., Hunt, G.B., Love, D.N., 2000. Infection of the subcutis and skin or cats with rapidly growing mycobacteria: a review of microbiological and clinical findings. J. Feline Med. Surg. 2, 35–48. Martin-de-Hijas, N.Z., Fernandez-Roblas, R., Fernandez-Martinez, A.I., Gadea, I., Esteban, J., 2008. Usefulness of the Etest for studying tigecycline susceptibility of non-pigmented rapidly growing mycobacteria. Int. J. Antimicrob. Agents 32, 366–367. McKinsey, D., Dykstra, M., Smith, D., 1995. The terrier and tendinitis. N. Engl. J. Med. 332, 338. Rodriguez, J., Garcia-Panchon, E., Flores, E., Escribano, I., Ruiz, M., Royo, G., 2007. Generation of resistant mutants of Mycobacterium chelonae and Mycobacterium fortuitum after exposure to subinhibitory concentrations of clarithromycin and moxifloxacin. J. Chemother. 19, 599–601. Rogall, T., Flohr, T., Bottger, E.C., 1990. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J. Gen. Microbiol. 136, 1915–1920. Stass, H., Kubitza, D., Schuhly, U., 2001. Pharmacokinetics, safety and tolerability of moxifloxacin, a novel 8-methoxyfluoroquinolone, after repeated oral administration. Clin. Pharmacokinet. 40 (Suppl.), 1–9. Telenti, A., Marchesi, F., Balz, M., Bally, F., Bottger, E.C., Bodmer, T., 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31, 175–178. Tsukamura, M., 1984. Identification of Mycobacteria. Mycobacteriosis Research Laboratory of the National Chubu Hospital, Obu, Aichi, Japan. Walker, R.D., Dowling, P.M., 2007. Fluoroquinolones. In: Giguere, S., Prescott, J., Baggot, J., Walker, R., Dowling, P. (Eds.), Antimicrobial Therapy in Veterinary Medicine. Blackwell Publishing, Ames, IA, pp. 263–283. Wang, J.-Y., Lee, L.-N., Lai, H.-C., Wang, S.-K., Yang, P.-C., 2007. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J. Antimicrob. Chemother. 59, 860–865. Wiebe, V., Hamilton, P., 2002. Fluoroquinolone-induced retinal degeneration in cats. JAVMA 221, 1568–1571. Woods, G.L., Bergmann, J.S., Witebsky, F.G., Fahle, G.A., Boulet, B., Plaunt, M., Brown, B.A., Wallace, R.J., Wanger, A., 2000. Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. J. Clin. Microbiol. 38, 656–661. Woods, G.L., Bergmann, J.S., Witebsky, F.G., Fahle, G.A., Wanger, A., Boulet, B., Plaint, M., Brown, B.A., Wallace, R.J., 1999. Multisite reproducibility of results obtained by the broth microdilution method for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum. J. Clin. Microbiol. 37, 1676–1682.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Research article
Susceptibility of rapidly growing mycobacteria isolated from cats and dogs, to ciprofloxacin, enrofloxacin and moxifloxacin M. Govendir a,* , T. Hansen b, B. Kimble a, J.M. Norris a, R.M. Baral c, D.I. Wigney a, S. Gottlieb d, R. Malik e a
Faculty of Veterinary Science, McMaster building, B14, The University of Sydney, NSW 2006, Australia Pathology Queensland, Mycobacterium Reference Laboratory, Herston Hospitals Complex, Herston, QLD 4029, Australia Paddington Cat Hospital, Oxford Street, Paddington, NSW 2021, Australia d The Cat Clinic, Creek Road, Mt Gravatt, QLD 4122, Australia e Centre for Veterinary Education, The University of Sydney, NSW 2006, Australia b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 November 2009 Received in revised form 9 June 2010 Accepted 14 June 2010
Rapidly growing mycobacteria (RGM) cause infections in cats and dogs which require prolonged antibacterial medication for resolution. In Australia, pathogens from the Mycobacterium fortuitum and Mycobacterium smegmatis clusters are responsible for most of the RGM infections in cats and dogs. As fluoroquinolones are often recommended for treating such infections, 14 M. fortuitum isolates, 51 isolates from the M. smegmatis cluster and 2 M. mageritense isolates, collected from feline and canine patients, underwent susceptibility testing to the second generation fluoroquinolones ciprofloxacin and enrofloxacin and the newer generation fluoroquinolone moxifloxacin. Using microbroth dilution, the MIC90 of ciprofloxacin, enrofloxacin, and moxifloxacin that inhibited growth of M. fortuitum isolates were 0.500, 0.250 and 0.063 mg/mL respectively. For the M. smegmatis cluster isolates the corresponding MIC90 was 0.500, 0.250 and 0.125 mg/mL respectively. E-test results showed similar trends but MICs were lower than those determined by microbroth dilution. Additionally, moxifloxacin was administered to 10 clinically normal cats (50 mg per cat, once daily for 4 days). The plasma moxifloxacin concentration 2 h after the last dose was determined by liquid chromatography as 2.2 0.6 mg/mL. The plasma concentration at 2 h:MIC90 ratios for moxifloxacin for M. fortuitum and M. smegmatis cluster was 34.9 and 17.6 respectively which exceeded the recommended threshold of 10, indicating that moxifloxacin has good theoretical efficacy for treatment of those M. fortuitum and M. smegmatis infections in cats and dogs that have become refractory to other antibacterial drug classes. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Mycobacterium fortuitum Mycobacterium smegmatis Fluoroquinolone Moxifloxacin Cat Dog
1. Introduction RGM are occasional pathogens of people and some animal species. RGM are Gram-positive bacilli with a cell wall rich in complex fatty acids and waxes that generally produce visible colonies within 5 days when cultured at
* Corresponding author. Tel.: +61 02 93515442; fax: +61 02 93513056. E-mail address: [email protected] (M. Govendir). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.06.011
room temperature on routine laboratory media (Malik et al., 2000; Brown-Elliott and Wallace, 2002). The most common clinical manifestation of RGM infection in immunocompetent cats and dogs is a chronic panniculitis whereby pathogens are introduced into subcutaneous fatty tissue (e.g. the inguinal fat pad) and less commonly, systemic disease. Treatment of RGM infections in cats and dogs requires prolonged antimicrobial therapy and sometimes surgical debulking. The newer generation 8-methoxy fluoroquinolone moxifloxacin, has been documented
114
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
to inhibit RGM growth at lower concentrations than ciprofloxacin, a prototypical fluoroquinolone drug of the previous generation (Brown-Elliott et al., 2002). RGM are classified into six phylogenetic clusters (Adekambi and Drancourt, 2004). Those responsible for chronic infections in cats and dogs in Australia are predominantly, but not exclusively, from the Mycobacterium fortuitum cluster and Mycobacterium smegmatis cluster. The aim of this investigation was to compare the minimum inhibitory concentrations (MIC) of ciprofloxacin, enrofloxacin and moxifloxacin by microbroth dilution and concentration gradient E-tests, to inhibit growth of RGM isolates from Australian feline and canine patients. This investigation also determined the presumptive steady state plasma concentration of moxifloxacin 2 h after oral administration in normal cats by liquid chromatography, as pharmacokinetic data for this agent is lacking for cats. 2. Materials and methods 2.1. Bacterial isolates Feline and canine RGM isolates from clinical cases were available from the freeze-dried collection of Veterinary Pathology Diagnostic Services, Faculty of Veterinary Science, The University of Sydney. Further testing was conducted at the Queensland Mycobacteria Reference Laboratory for species identification by phenotypic and molecular methods. These isolates were collected from clinical cases encountered over the period 1983–2009. The preferred method for collecting isolates from clinical cases has been described previously (Malik et al., 2000, 2004). Two M. smegmatis senso stricto isolates were from cats that had been previously treated for their infection with a course of enrofloxacin. 2.2. Strain identification 2.2.1. Biochemical identification of strains Isolates were initially identified by a range of standard biochemical tests, including isolate morphology and degree of acid-fastness in Ziehl–Neelsen stained smears of growth taken from Lowenstein–Jensen medium, colonial morphology (rough or smooth), pigmentation both in light and dark, rate of growth at room temperature and 37 8C, ability to grow at 42 and 52 8C, iron uptake, pamino salicylic acid degradation, nitrate reduction, bgalactosidase activity, acid production from carbohydrates (glucose, inositol and mannitol), utilisation of compounds (glucose, fructose, inositol, mannitol and citrate) as the sole carbon source, tolerance to 5% sodium chloride in Lowenstein–Jensen medium and susceptibility to polymixin B, trimethoprim and tobramycin (Malik et al., 2000, 2004; Brown-Elliott and Wallace, 2002). Presence of nitrate reductase activity identified isolates from the M. fortuitum and M. smegmatis clusters. Presence of arylsulphatase activity distinguished M. fortuitum, while its absence identified M. smegmatis isolates (Kubica and Beam, 1961; Kubica and Vestal, 1961; Tsukamura, 1984). Further strain identification required molecular techniques.
2.2.2. Preparation of DNA template and amplification of 16S rRNA and heat shock protein (hsp) loci Chromosomal DNA from these isolates was prepared by adding a loop full of colonies to 300 mL of sterile water in a micro-centrifuge tube containing sterile glass beads. Tubes were placed in a dry water bath at 95 8C for 30 min, sonicated for 10 min and then centrifuged for 10 min to remove cellular debris. PCR amplification of 16S rRNA and hsp65 loci was performed using previously described primer sets illustrated in Table 1. Each 50 mL PCR reaction contained 2 mL of DNA template, 500 mM of each dNTP, 2.5 mM of MgCl2, 1 U of Taq DNA polymerase (Fisher Biotech, Thebarton, South Australia) and 2 mM of each primer. The PCR was performed using an Applied Biosystems Gene-Amp 2700 Thermal Cycler (Applied Biosystems, Scoresby, VIC) and involved an initial denaturation at 94 8C for 5 min, followed by 35 cycles of denaturation at 94 8C for 1 min, annealing at 55 8C for 1 min and extension at 72 8C for 1 min, with a final extension at 72 8C for a further 10 min. 2.2.3. DNA sequencing PCR products were purified using the enzyme EXOSAPIT (Amersham Biosciences, Rydalmere, NSW), and sequenced using an automatic DNA sequencer (ABI 3700, Applied Biosystems, Scoresby, VIC), using the two forward primers (BF and TB11) described in Table 1. DNA sequences were compared to published sequences in GenBank via the BLAST network service. 2.3. Minimum inhibitory concentration testing MICs were determined using the microbroth dilution method in accordance with guidelines M24-A (CLSI, 2003). Fourteen two-fold serial dilutions of ciprofloxacin, enrofloxacin (both obtained from Sigma–Aldrich, Castle Hill, NSW) and moxifloxacin (Bayer HealthCare, Berlin, Germany) ranging in concentration from 64 to 0.008 mg/mL, were pipetted into microtitre wells (Nunc 16620, Noble Park, VIC). MICs were also determined by concentration gradient tests (E-test1 strips, AB Biodisk, Solna, Sweden) used according to the manufacturer’s instructions. Most, but not all isolates underwent susceptibility testing by both methods. A recommended quality control strain, Staphylococcus aureus ATCC 29213 was tested concurrently with each fluoroquinolone (CLSI, 2003). The MIC required to inhibit growth of the S. aureus strain was determined 24 h after incubation at 35 8C, while the MIC for RGM isolates were determined at 30 8C after 72 h.
Table 1 Primer sequences used in the amplification of 16S and sp65 loci for organism identification. Name
Sequence a
BF (16S Fwd) R2 (16S Rev)a TB11 (hspFwd)b TB12 (hspRev)b a b
Rogall et al. (1990). Telenti et al. (1993).
50 -AGAGTTGGATCCTGGCTCAG-30 50 -CCTACGAGCTCTTTACG-30 50 -ACCAACGATGGTGTGTCCAT-30 50 -CTTGTCGAACCGCATACCCT-30
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
115
2.4. Moxifloxacin dosing of cats
3. Statistical analysis
Ten normal mature cats (7 female and 3 male, all neutered; 5 Burmese and 5 domestic short-hair), with a mean weight of 4.8 0.6 kg, had their baseline biochemical analytes determined and were dosed for 4 days with compounded moxifloxacin capsules (50 mg once daily; BOVA Chemist, Caringbah, NSW). Patients were dosed first thing in the morning and then fed their usual diet. Two hours after the last dose, 3 mL of blood was collected by cephalic venipuncture into a lithium heparin tube, the plasma separated and frozen. All samples were batched to detect plasma moxifloxacin concentration.
As RGM are saprophytic organisms that infect mammalian hosts by accident, with host factors having minimal impact on the biology of these bacteria, feline and canine isolates of the same RGM group were combined for statistical analysis and graphical representation. The median MIC (MIC50), MIC90 and MIC range for each drug were determined for both M. fortuitum and M. smegmatis cluster isolates. As MICs were not normally distributed, the MIC50 of each drug to inhibit the isolates of each group were compared by Mann–Whitney’s two-tailed tests. Spearman’s correlation coefficient (rs) was determined to establish whether there was a relationship between the MICs obtained by microbroth dilution and Etests. For this latter analysis and graphical representation, data were converted to nearest log2 dilution MIC. The level of significance for all analyses was accepted at p < 0.05.
2.4.1. Determination of plasma moxifloxacin concentration Determination of moxifloxacin concentrations in feline plasma was performed by reversed-phase, high performance liquid chromatography (HPLC) method. The HPLC system (Shimadzu, Rydalmere, NSW) consisted of a pump (LC20AT), vacuum degassing solvent delivery unit (DGU-20As), autosampler (SIL 20AC), column oven (CTO 20AC) maintained at 25 8C, ultra-violet visible light detector (SPD-20A), system controller (CBM 20A) and Class VP software (version 7.4). Separation was performed using an Apollo C-18, 5 mm (4.6 mm 250 mm) column (Alltech, Dandenong South, VIC, Australia) coupled with a 1 mm Optiguard C-18 guard column (Optimize Technologies, Oregon City, OR, USA). The isocratic mobile phase consisted of 25 mM citric acid (Sigma–Aldrich, Castle Hill, NSW) and 10 mM sodium dodecyl sulfate (Boehringer Mannheim, Indianapolis, USA) with 50% acetonitrile (Ajax Finechem, Taren Point, NSW). The flow rate was 1 mL/min. Absorption was detected at 296 nm. Orbifloxacin was the internal standard and retention times for orbifloxacin and moxifloxacin were 9.03 and 10.01 min respectively. The stock solution of HPLC analytical grade moxifloxacin was prepared at a concentration of 1 mg/mL in purified water and further diluted into working solutions ranging from 0.315 to 50 mg/mL. Calibration standards of moxifloxacin were prepared by adding 50 mL of working solutions to 200 mL of blank pooled feline plasma. Clinical and calibration samples underwent solid-phase extraction (SPE) using an Oasis HLB 1 cm3 30 mg column filter (Waters, Milford, MA, USA) via a vacuum manifold, according to manufacturer’s instructions. After complete dryness of methanol–TFA by speed vacuum concentrator SPD121P (Thermo Scientific) at 35 8C for 2 h, samples were reconstituted with 250 mL of mobile phase, vortexed and 10 mL of supernatant was injected into the HPLC system (as duplicates). Eight calibration concentrations, ranging from 0.0625 to 10 mg/mL were used to construct the calibration curve which had a correlation coefficient (r2) >0.98. The intraday precision and accuracy were determined by repeated analysis of plasma samples (n = 4) spiked with 1, 2 and 5 mg/mL moxifloxacin determined on 4 consecutive days. The intra- and inter-day values of precision and accuracy were <4.55% (RSD) and >96% respectively. The moxifloxacin ‘recovery’ percentage from the SPE procedure was 89.3% with a RSD of 6.4%.
4. Results A total of 67 isolates were available for this study. Two isolates from dogs and 12 from cats were M. fortuitum. Of the M. smegmatis cluster, 3 dog and 39 cat isolates were M. smegmatis sensu stricto and an additional 9 were M. goodii, all from cats. There were two M. mageritense isolates, one each from a dog and cat. The MIC ranges of all three drugs that inhibited the growth of the isolates by microbroth dilution, are presented in Fig. 1. The MIC50 (and MIC90) for moxifloxacin for the M. fortuitum isolates were 0.063 (and 0.063) mg/mL, which were significantly lower compared to ciprofloxacin 0.250 (0.500) mg/mL (p = 0.0002) and enrofloxacin 0.125 (0.250) mg/mL (p = 0.0003). The MIC50 (and MIC90) for moxifloxacin for the M. smegmatis cluster were 0.063 (0.125) mg/mL which was significantly lower compared to ciprofloxacin 0.250 (0.500) mg/mL and enrofloxacin 0.125 (0.250) mg/mL (both p < 0.0001). The MIC50 of each drug to inhibit growth of both M. fortuitum versus M. smegmatis isolates were compared and there were no significant differences. The two M. mageritense (cluster V) isolates had a MIC range for ciprofloxacin, enrofloxacin and moxifloxacin of
[(Fig._1)TD$IG]
Fig. 1. MIC (mg/mL) of ciprofloxacin, enrofloxacin and moxifloxacin to inhibit the growth of M. fortuitum and M. smegmatis cluster isolates determined by microbroth dilution. The dashed line represents the susceptibility breakpoint of ciprofloxacin for RGM in people (<1 mg/mL). t Both cats from which these two isolates were obtained had received a prior course of enrofloxacin as monotherapy.
[(Fig._2)TD$IG]
116
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
Fig. 2. Comparison of log2 MICs determined by microbroth dilution (x-axes) versus MICs determined by E-test (y-axes) for the same M. fortuitum and M. smegmatis cluster isolates (n = 65) for ciprofloxacin, enrofloxacin and moxifloxacin.
0.125 (both isolates), 0.125 and 0.25, 0.031 and 0.063 mg/ mL respectively. All agents inhibited the growth of the QC strain S. aureus ATCC 29213 after 24 h by microbroth dilution within the expected ranges of 0.12–0.5 mg/mL for ciprofloxacin (CLSI, 2007), 0.03–0.12 mg/mL for enrofloxacin (CLSI, 2008) and 0.015–0.12 mg/mL for moxifloxacin (CLSI, 2007). The MIC50 determined by E-test for ciprofloxacin, enrofloxacin and moxifloxacin for M. fortuitum were 0.016, 0.047 and 0.012; and for the M. smegmatis cluster were 0.064, 0.125 and 0.016 mg/mL respectively. The log2 dilution MICs determined by microbroth dilution and E-test for the identical isolate for each drug were compared and presented in Fig. 2. The Spearman coefficient for ciprofloxacin, enrofloxacin and moxifloxacin was 0.13, 0.28 and 0.55 respectively with a p-value of <0.0001 for moxifloxacin. The mean SD plasma moxifloxacin concentration 2 h after the fourth consecutive daily dose (50 mg per cat per morning) was 2.2 0.6 mg/mL. Moxifloxacin administration resulted in mild diarrhoea in two cats and medication had to be stopped in one cat due to vomiting soon after administration. The cat that vomited was excluded from the trial, leaving data from 10 others.
5. Discussion Reviews are available on RGM taxonomy, biology (Brown-Elliott and Wallace, 2002; Adekambi and Drancourt, 2004) and clinical management of RGM infections in cats (Malik et al., 2000; Horne and Kunkle, 2009) and in dogs (Malik et al., 2004). The key finding was that for all isolates, moxifloxacin had significantly lower MICs than ciprofloxacin or enrofloxacin which is in accordance with similar studies using human isolates (Brown-Elliott et al., 2002). Enrofloxacin was more active against the majority of the isolates than ciprofloxacin. On the whole, the vast majority of isolates were susceptible to all fluoroquinolones. This reflects in part, the rareness of the M. chelonaeabscessus group strains in Australia, as members of this complex tend to be inherently resistant to the fluoroquinolones (Brown-Elliott and Wallace, 2002; Malik et al., 2004). Only two resistant M. smegmatis cluster isolates were encountered, and interestingly, both were obtained from cats that had received a prior course of enrofloxacin as monotherapy and appeared to have developed cross resistance to all fluoroquinolones. This suggests two things: (1) mutational resistance to fluoroquinolones can occur during a course of therapy (Escribano et al., 2007;
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
Rodriguez et al., 2007; Wang et al., 2007) and (2) there is a need for susceptibility testing at the outset, or if there is an unsatisfactory response to therapy, or if relapse occurs. Microbroth dilution is recommended as the ‘gold standard’ for susceptibility testing of RGM (CLSI, 2003) as results of other susceptibility tests are regarded as more variable (Woods et al., 2000; Brown-Elliott and Wallace, 2002; Esteban et al., 2002; Martin-de-Hijas et al., 2008). Microbroth dilution is not without limitations as the precise test end-point can be difficult to read (Woods et al., 1999; CLSI, 2003), is laborious and personnel require experience with the in vitro growth characteristics of these pathogens (Esteban et al., 2002; Martin-de-Hijas et al., 2008). In order to find an easier method to generate susceptible versus resistant information for these pathogens in a veterinary laboratory, the results of E-tests were compared to those determined by microbroth dilution tests resulting in a poor correlation between microbroth dilution and E-test MICs. The factors for this poor correlation currently remain unknown. An indication of antibacterial agents’ in vivo antibacterial efficacy is the maximal concentration achieved in plasma after a conventional dose of drug (Cmax) considered in relation to MIC (i.e. Cmax:MIC). As fluoroquinolones are classified as concentration dependent antibacterial agents, a Cmax:MIC ratio of at least 10:1 is recommended (Walker and Dowling, 2007). We were unable to obtain serial blood samples to determine the moxifloxacin Cmax, although a presumptive steady state 2 h plasma concentration of 2.2 0.6 mg/mL in 10 clinically normal cats after administration of 50 mg per cat daily (9.6–12.5 mg/kg per day) for 4 days was determined. Thus, the C2h:MIC90 ratios for moxifloxacin for M. fortuitum and the M. smegmatis cluster were calculated as 34.9 and 17.6 respectively, indicating that moxifloxacin should theoretically have good clinical efficacy at a dose of 50 mg per adult cat, once daily. By way of contrast, 10 mg/kg of ciprofloxacin administered orally to cats with a Cmax of 1.26 0.67 mg/mL at 1.30 0.67 h (Albarellos et al., 2004) resulted in a Cmax:MIC90 of 2.52 for M. fortuitum and 5.04 for the M. smegmatis cluster isolates. Likewise when 5 mg/kg of enrofloxacin was administered orally to dogs for 5 days resulting in a Cmax of 2.01 0.18 mg/ mL (Frazier et al., 2000), the Cmax:MIC50 was calculated as 16.08 for both RGM clusters. Moxifloxacin is registered for use in people only, although it has been administered to horses, resulting in adverse gastrointestinal effects (Gardner et al., 2004), rabbits (Carceles et al., 2006) and sheep (Carceles et al., 2009). Moxifloxacin’s antibacterial properties are distinguished from second generation fluoroquinolones (e.g. ciprofloxacin, enrofloxacin, marbofloxacin and difloxacin) by increased efficacy against Gram-positive bacteria (including Enterococcus species) as well as aerobic and obligate anaerobes (Stass et al., 2001). Activity of fourth generation fluoroquinolones is reported to be less effected overall by mutations responsible for fluoroquinolone resistance (Brown-Elliott et al., 2002). Available as tablets and as an intravenous formulation, moxifloxacin was initially developed to treat respiratory tract infections in people (Ball et al., 2004). It is worth noting that although uncommon, it is not unknown, for people to acquire a RGM
117
infection from their pets by the pathogen penetrating skin via a laceration or abrasion (McKinsey et al., 1995). In Australia, fluoroquinolones are registered for use in companion animals only; however potential does exist for the excreta of fluoroquinolone treated animals to contaminate the environment and enter both animal and human food chains, to further select for fluoroquinolone resistant bacteria. Use of elevated dosages of enrofloxacin in cats has been associated with mild to severe retinopathy in cats, especially at doses markedly higher than the label approved dosage of 5 mg/kg or if the drug was given intravenously to critical patients (Gelatt et al., 2001; Wiebe and Hamilton, 2002). For this reason, enrofloxacin is not recommended for treating RGM infections in cats because a clinical cure can take months to achieve and there is sometimes a requirement to obtain higher than usual plasma concentrations by increasing drug dosage to ‘drive’ the agent into marginally perfused tissues. One of the authors (RM) has used moxifloxacin administration at a dose of 50 mg per cat once daily with food as a component of therapy in four cats with mycobacterial panniculitis including some patients that were refractory to other antibiotics with uniform success. As moxifloxacin is not registered for use in cats or dogs, the authors suggest that it should be considered for treatment only if the infection has become refractory to those drugs formulated and registered for animal use. Further studies to characterise moxifloxacin’s pharmacokinetic properties and safety in both dogs and cats and to explore its potential synergistic effects with other antibacterial agents are warranted. Role of funding source This study was financially supported by Australian Companion Animal Health Foundation. Acknowledgements Bayer Healthcare AG provided the analytical grade moxifloxacin gratis. Thanks to Ms. Robyn Carter and Dr. Chris Coulter of the Mycobacterium Reference Laboratory, QLD, for their collaboration in this project, the owners who consented to their cats participating in the oral dosing of moxifloxacin and BOVA Chemist, Caringbah, NSW, who compounded and provided the moxifloxacin capsules gratis. The authors thank Dr. Thomas Gottlieb who alerted us to potential value of using moxifloxacin in cats. We acknowledge the forethought of the late Associate Professor Daria Love in initiating the archiving rapidly growing mycobacteria isolates. Richard Malik is supported by the Valentine Charlton Bequest. Ethical approval: This study was approved by The University of Sydney Animal Ethics Committee. References Adekambi, T., Drancourt, M., 2004. Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int. J. Syst. Evol. Microbiol. 54, 2095–2105.
118
M. Govendir et al. / Veterinary Microbiology 147 (2011) 113–118
Albarellos, G.A., Kreil, V.E., Landoni, M.F., 2004. Pharmacokinetics of ciprofloxacin after single intravenous and repeated oral administration to cats. J. Vet. Pharmacol. Ther. 27, 155–162. Ball, P., Stahlmann, R., Kubin, R., Choudhri, S., Owens, R., 2004. Safety profile of oral and intravenous moxifloxac: cumulative data from clinical trials and postmarketing studies. Clin. Ther. 26, 940–950. Brown-Elliott, B.A., Wallace, R.J., 2002. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmented rapidly growing mycobacteria. Clin. Microbiol. Rev. 15, 716–746. Brown-Elliott, B.A., Wallace, R.J., Crist, C.J., Mann, L., Wilson, R.W., 2002. Comparison of in vitro activities of gatifloxacin and ciprofloxacin against four taxa of rapidly growing mycobacteria. Antimicrob. Agents Chemother. 46, 3283–3285. Carceles, C.M., Escudero, E., Fernandez-Varon, E., Marin, P., 2009. Pharmacokinetics after intravenous, intramuscular and subcutaneous administration of moxifloxacin in sheep. Vet. J. 180, 343–347. Carceles, C.M., Serrano, J.M., Marin, P., Escudero, E., Fernandez-Varon, E., 2006. Pharmacokinetics of moxifloxacin in rabbits after intravenous, subcutaneous and a long-acting poloxamer 407 gel formation administration. J. Vet. Med. A 53, 300–304. CLSI, 2003. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved Standard. Clinical and Laboratory Standards Institute, Wayne, PA. CLSI, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Information Supplement. Clinical and Laboratory Standards Institute, Wayne, PA. CLSI, 2008. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved standard – Third Edition. Clinical and Laboratory Standards Institute, Wayne, PA. Escribano, I., Rodriguez, J., Llora, B., Garcia-Panchon, E., Ruiz, M., Royo, G., 2007. Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid. Chemotherapy 53, 397–401. Esteban, J., Gadea, I., Torres, M.-V., Cabria, F., Rollan, E., Santos-O’Connor, F., Fernandez-Roblas, R., Soriano, F., 2002. A comparison between disk diffusion and microdilution for susceptibility testing of Mycobacterium fortuitum complex. J. Chemother. 14, 547–553. Frazier, D.L., Thompson, L., Trettien, A., Evans, E.I., 2000. Comparison of fluoroquinolone pharmacokinetic parameters after treatment with marbofloxacin, enrofloxacin and dofloxacin in dogs. J. Vet. Pharmacol. Ther. 23, 293–302. Gardner, S.Y., Davis, J.L., Jones, S.L., Lafevers, D.H., Hoskins, M.S., McArver, E.M., Papich, M.G., 2004. Moxifloxacin pharmacokinetics in horses and disposition into phagocytes after oral dosing. J. Vet. Pharmacol. Ther. 27, 57–60. Gelatt, K.N., Van-Der-Woerdt, A., Ketring, K.L., Andrew, S.E., Brooks, D.E., Biros, D.J., Denis, H.M., Cutler, T.J., 2001. Enrofloxacin-associated retinal degeneration in cats. Vet. Ophthalmol. 4, 99–106. Horne, K.S., Kunkle, G.A., 2009. Clinical outcome of cutaneous rapidly growing mycobacterial infections in cats in the south-eastern United States: a review of 10 cases (1996–2006). J. Feline Med. Surg.. Kubica, G.P., Beam, R.E., 1961. The arylsulfatase activity of acid-fast bacilli. The differentiation of Mycobacterium avium from the unclassified group III nonphotochromogenic mycobacterium. Am. Rev. Respir. Dis. 83, 733–736.
Kubica, G.P., Vestal, A.L., 1961. The arylsulfatase activity of acid-fast bacilli. Investigation of stock cultures of acid-fast bacilli. Am. Rev. Respir. Dis. 83, 728–732. Malik, R., Shaw, S.E., Griffin, C., Stanley, B., Burrows, A.K., Bryden, S.L., Titmarsh, J., Stutsel, M.J., Carter, S.A., Warner, A., Martin, P., Wigney, D.I., Gilpin, C., 2004. Infections of the subcutis and skin of dogs caused by rapidly growing mycobacteria. J. Small. Anim. Pract. 45, 485–494. Malik, R., Wigney, D.I., Dawson, D., Martin, P., Hunt, G.B., Love, D.N., 2000. Infection of the subcutis and skin or cats with rapidly growing mycobacteria: a review of microbiological and clinical findings. J. Feline Med. Surg. 2, 35–48. Martin-de-Hijas, N.Z., Fernandez-Roblas, R., Fernandez-Martinez, A.I., Gadea, I., Esteban, J., 2008. Usefulness of the Etest for studying tigecycline susceptibility of non-pigmented rapidly growing mycobacteria. Int. J. Antimicrob. Agents 32, 366–367. McKinsey, D., Dykstra, M., Smith, D., 1995. The terrier and tendinitis. N. Engl. J. Med. 332, 338. Rodriguez, J., Garcia-Panchon, E., Flores, E., Escribano, I., Ruiz, M., Royo, G., 2007. Generation of resistant mutants of Mycobacterium chelonae and Mycobacterium fortuitum after exposure to subinhibitory concentrations of clarithromycin and moxifloxacin. J. Chemother. 19, 599–601. Rogall, T., Flohr, T., Bottger, E.C., 1990. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J. Gen. Microbiol. 136, 1915–1920. Stass, H., Kubitza, D., Schuhly, U., 2001. Pharmacokinetics, safety and tolerability of moxifloxacin, a novel 8-methoxyfluoroquinolone, after repeated oral administration. Clin. Pharmacokinet. 40 (Suppl.), 1–9. Telenti, A., Marchesi, F., Balz, M., Bally, F., Bottger, E.C., Bodmer, T., 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31, 175–178. Tsukamura, M., 1984. Identification of Mycobacteria. Mycobacteriosis Research Laboratory of the National Chubu Hospital, Obu, Aichi, Japan. Walker, R.D., Dowling, P.M., 2007. Fluoroquinolones. In: Giguere, S., Prescott, J., Baggot, J., Walker, R., Dowling, P. (Eds.), Antimicrobial Therapy in Veterinary Medicine. Blackwell Publishing, Ames, IA, pp. 263–283. Wang, J.-Y., Lee, L.-N., Lai, H.-C., Wang, S.-K., Yang, P.-C., 2007. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J. Antimicrob. Chemother. 59, 860–865. Wiebe, V., Hamilton, P., 2002. Fluoroquinolone-induced retinal degeneration in cats. JAVMA 221, 1568–1571. Woods, G.L., Bergmann, J.S., Witebsky, F.G., Fahle, G.A., Boulet, B., Plaunt, M., Brown, B.A., Wallace, R.J., Wanger, A., 2000. Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. J. Clin. Microbiol. 38, 656–661. Woods, G.L., Bergmann, J.S., Witebsky, F.G., Fahle, G.A., Wanger, A., Boulet, B., Plaint, M., Brown, B.A., Wallace, R.J., 1999. Multisite reproducibility of results obtained by the broth microdilution method for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum. J. Clin. Microbiol. 37, 1676–1682.