Tiamulin Activity against Fastidious and Nonfastidious Veterinary and Human Bacterial Isolates: Initial Development of In Vitro Susceptibility Test Methods

Tiamulin is a pleuromutilin derivative used in veterinary practicefor the control and specific therapy of infections in swine.This report summarizes studies to establish standardized susceptibilitytesting methods, interpretive criteria, and reagent detailsfor use in veterinary methods recently developed by the NationalCommittee for Clinical Laboratory Standards (NCCLS) (standardsM31-A and M37-A, NCCLS, Wayne, Pa., 1999). A total of 636 fastidiousand nonfastidious animal and human pathogens were processedby using media and procedures described by the NCCLS. Tiamulindisk diffusion tests used a 30-µg disk concentration,and the proposed MIC breakpoints corresponding to levels achievablein animal target tissues (lung) were $<=$ 4 µg/ml for susceptibilityand $>=$ 32 µg/ml for resistance. Correlate zone diametersfor specific nonfastidious species were as follows: for Pasteurellamultocida and staphylococci tested on Mueller-Hinton agar, susceptibilityat $>=$ 19 mm and resistance at $<=$ 11 mm, and for Actinobacillus suis,Erysipelothrix rhusiopathiae, and Streptococcus suis testedon enriched chocolate Mueller-Hinton agar, susceptibility at $>=$ 16 mm and resistance at $<=$ 8 mm. When Actinobacillus pleuropneumoniaewas tested, a susceptibility breakpoint of $<=$ 16 µg/ml ( $>=$ 9mm) was suggested for veterinary fastidious medium broth andenriched chocolate Mueller-Hinton agar. Absolute categoricalagreement between NCCLS dilution and disk diffusion test resultswith these criteria ranged from 90.5 to 96.2%. Tiamulin susceptibilitytesting methods appear to be accurate in their categorical classificationfor indicated species, and their availability will allow immediatetesting of animal isolates to guide therapy via appropriatelevels of dosing and to monitor the development of resistancefor agents in this unique class.

INTRODUCTION

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Introduction

Tiamulin is a pleuromutilin derivative antimicrobial used inthe control and treatment of veterinary gram-positive and gram-negativepathogens, with a particular emphasis on infections in swine(11). It has exceptional activity (MIC, $<=$ 1 µg/ml) againstanaerobic bacterial species, intestinal spirochetes, and Mycoplasmaspp. (3, 12, 13). The present study was initiated in order todocument the sustained antimicrobial activity of tiamulin againstindicated bacterial species and rapidly growing and fastidiousanimal pathogens and to establish choices of diagnostic reagents.MIC and disk diffusion zone diameter correlates were determinedin order to ensure the precision and accuracy of the susceptibilitytesting procedures performed according to National Committeefor Clinical Laboratory Standards (NCCLS) guidelines for susceptibilitytesting of veterinary antimicrobial agents (5, 6).

MATERIALS AND METHODS

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Materials and Methods

Organisms tested. The organisms tested were isolates from various types of infectionsand included both human and veterinary pathogens. A total of636 strains were processed, each identified by routine methodsat the institution of initial isolation and confirmed by referencetesting at a referral location (CAST Laboratories, Iowa City,Iowa; Iowa State University, Ames). These strains included 392nonfastidious bacteria (Table 1) : Aerococcus spp. (3 strains),Enterococcus spp. (71 strains), Staphylococcus aureus (150 strains),coagulase-negative staphylococci (CoNS) (99 strains), Streptococcusspp. (4 strains), Enterobacteriaceae (24 strains; 8 species),Acinetobacter baumannii (2 strains), Pseudomonas aeruginosa(6 strains), Stenotrophomonas maltophilia (2 strains), and Pasteurellamultocida (31 strains). Also tested were 244 fastidious veterinarypathogens (Table 2) : Actinobacillus pleuropneumoniae (170 strains),Actinobacillus suis (21 strains), Erysipelothrix rhusiopathiae(11 strains), Haemophilus parasuis (18 strains), and Streptococcussuis (24 strains).

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TABLE 1. Tiamulin activity as demonstrated by MIC and zone diameter of inhibition results tested against 392 nonfastidious bacteria

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TABLE 2. Tiamulin activity as demonstrated by MICs and zone diameters of inhibition for 244 fastidious veterinary pathogens with three broth medium formulations

Susceptibility testing. Tiamulin, as hydrogen fumarate standard powder, was providedby Boehringer Ingelheim Vetmedica, Inc. (St. Joseph, Mo.). Brothmicrodilution testing of tiamulin was performed with four differentmedia formulations: nonfastidious bacteria were tested in cation-adjustedMueller-Hinton broth, whereas the fastidious veterinary pathogenswere each tested in veterinary fastidious medium (VFM), haemophilustest medium (HTM), and Mueller-Hinton broth supplemented with5% lysed horse blood (LHB) (5, 7). Tiamulin was dispensed intobroth microdilution trays in a serial twofold concentrationrange of 0.25 to 32 µg/ml. All trays were held at -20°Cor below until used. The trays were brought to room temperaturebefore inoculation (5 x 10⁵ CFU/ml), and the technical detailsof the veterinary NCCLS (5, 7) documents were followed.

The 30-µg tiamulin disks were produced by BD MicrobiologySystems (Cockeysville, Md.), and these reagents were used followingNCCLS methods M2-A7 (8) and M31-A (5). The nonfastidious isolateswere tested on Mueller-Hinton agar (MHA), and the fastidiousisolates were tested on chocolate MHA (CMHA). The fastidiousorganisms were tested using incubation conditions of 35°Cin 5 to 7% CO₂ for 20 to 24 h and the nonfastidious organismswere tested in ambient air at 35°C for 16 to 18 h. The MICendpoints and zone diameters of inhibition were read as definedby the NCCLS documents (5, 7, 8). The results were comparedby scattergram, regression statistics, and error-rate boundinganalysis. Breakpoints for susceptibility were proposed basedon available tiamulin pharmacokinetic data (Boehringer IngelheimVetmedica, Inc., data on file) and NCCLS (6, 9) guidelines.Quality control was performed with A. pleuropneumoniae ATCC27090 and S. aureus ATCC 25923 tested against enrofloxacin;all results were within MIC and zone diameter limits establishedby Marshall et al. (4), NCCLS (5), and the reagent manufacturer.

In addition, preliminary experiments were performed to determinethe optimal drug disk content by using investigator-prepareddisks containing 5, 15, 30, 60, and 90 µg of tiamulin.Each disk was tested (10 replicates) against the NCCLS qualitycontrol strains: Enterococcus faecalis ATCC 29212 and S. aureusATCC 25923, 29213, and 43300 (methicillin resistant). A 15-µgerythromycin disk was used as a control drug. Furthermore, Neo-Sensitabs(Rosco Diagnostica, Taastrup, Denmark) at 30 µg were testedagainst control strains with ranges 1 to 2 mm larger than thoserecorded for the corresponding disk diffusion method. This susceptibilitytesting method is often used in Europe.

RESULTS AND DISCUSSION

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Results and Discussion

Choices of disk diffusion testing reagents. Preliminary tiamulin disk content studies of tiamulin used diskdrug concentrations ranging from 5 to 90 µg. Each seriesof tests used four nonfastidious organisms, read by four technologistsfor a total of 10 replicates per strain and disk concentration.For tiamulin-susceptible strains of staphylococci, the meanzone diameters ranged from 22.4 to 31.7 mm (S. aureus) but nonsignificantincreases in zones were achieved at disk drug concentrationsgreater than 30 µg (data not shown). The 30-µg tiamulinand erythromycin control disks produced similar zones of inhibition(mean diameters of 25.7 and 25.3 mm, respectively, for the controlstrain S. aureus ATCC 25923). The 30-µg concentrationin disks or "tabs," used previously in Europe, was adopted forsubsequent experiments with disks produced commercially by BDMicrobiology Systems.

Previous experiments in Denmark (1) using A. pleuropneumoniaestrains (26 total) demonstrated average zone diameters of 20.3mm at pH 8.0 and smaller zones at pH 7.2 to 7.4. The averageMIC was 3.1 µg/ml (4 to 8 µg/ml at pH 7.4), anda susceptible breakpoint was suggested at $<=$ 8 µg/ml, witha correlate zone diameter of $>=$ 13 mm. However, these zones werebased on technical or methodological details which did not conformto NCCLS documents for antimicrobial susceptibility tests withbacterial strains of animal origin, including (i) a tablet drugdelivery system, (ii) semiconfluent growth inoculum (lighterthan NCCLS-recommended confluent growth), and (iii) modifiedor elevated pH due to not using CO₂ incubation, which favorsfastidious-pathogen growth (5, 8). All of these modificationsshould result in reduced zone diameters of inhibition around30-µg tiamulin disks used in the NCCLS procedure.

Susceptibility testing for tiamulin against nonfastidious pathogens. Organisms listed in Table 1 were also tested with the 30-µgtiamulin disk (5). Two distinct populations of susceptible organismswere identified based on relative tiamulin potency: staphylococci,for which the MICs at which 90% of strains were inhibited (MIC₉₀s)were 1 or 2 µg/ml and zone diameters were >20 mm, andP. multocida strains, for which MICs were 8 to 32 µg/mland zone diameters were 10 to 21 mm. All other organisms listedin Table 1, such as enterococci and enteric and nonfermentativegram-negative bacilli, were resistant to tiamulin, with MICsbeing >32 µg/ml and median zone diameters being 6 to12 mm.

To accurately categorize these two groups of tiamulin-inhibitedorganisms, the staphylococci were declared susceptible (MICbreakpoint, $<=$ 4 µg/ml) and P. multocida was categorizedas moderately susceptible or intermediate (MIC range, 8 to 16µg/ml). The correlate zone breakpoints were $>=$ 19 mm forsusceptibility and $<=$ 11 mm for resistance (Fig. 1). These criteriaproduce an intermethod accuracy rate of 96.2%, and errors wereas follows: very major (false susceptible) = 0.0%, major (falseresistant) = 0.0%, and minor = 3.8%. All error rates were acceptable,and the results should indicate the need for possible dosageadjustments to address infections caused by strains for whichtiamulin MICs are expected to be 8 or 16 µg/ml, such asP. multocida (see discussion of A. pleuropneumoniae below).The correlation coefficient was 0.98 (Fig. 1). Previously reportedin vitro experience confirms our results (1, 2). This potencywas similar to that noted for two fastidious species of Actinobacillus(Table 2).

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FIG. 1. Scattergram comparing reference tiamulin MICs to zone diameters around 30-µg tiamulin disks for 390 strains of nonfastidious, rapidly growing organisms. Horizontal lines show the proposed susceptibility ( $<=$ 4 µg/ml) and resistance ( $>=$ 32 µg/ml) MIC criteria. Vertical lines show the correlate interpretive zone diameters of $>=$ 19 and $<=$ 11 mm, respectively (96.2% intermethod agreement).

Susceptibility testing for tiamulin against five fastidious bacterial species. Like the rapidly growing nonfastidious species tested againsttiamulin by standardized NCCLS methods (5), the five fastidiousspecies groups segregate into two levels of tiamulin susceptibility.The species that were tiamulin susceptible by the broth MICresults in VFM or HTM were E. rhusiopathiae, H. parasuis (HTMonly), and the vast majority of S. suis isolates (MIC₅₀, 1 to2 µg/ml). The second, less-susceptible group includedA. pleuropneumoniae, A. suis, and a very small number of S.suis isolates (MIC₉₀, 16 µg/ml). The MICs for these speciesreported by the manufacturer and other investigators agree withthese findings (12).

To establish susceptibility testing methods and remain consistentwith the dose-related criteria suggested for nonfastidious species,the same MIC breakpoints ( $<=$ 4 µg/ml for susceptibility; $>=$ 32 µg/ml for resistance) were applied to these species.However, two distinct scattergram populations were also identified,where E. rhusiopathiae produced markedly larger zones around30-µg tiamulin disks (slow grower) than S. suis strains;MICs for the two species in VFM (5) broth were the same (Fig.2). Regardless of this phenomenon, a single set of interpretivecriteria could easily be established for the disk diffusionmethod: $>=$ 16 mm for susceptibility and $<=$ 8 mm for resistance.

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FIG. 2. Scattergram comparing reference tiamulin MICs in VFM broth compared to zone diameters around 30-µg tiamulin disks when testing the fastidious veterinary pathogens: A. pleuropneumoniae, A. suis, E. rhusiopathiae, and S. suis (226 total strains). Horizontal and vertical solid lines indicate the proposed MIC breakpoints of $<=$ 4 µg/ml ( $>=$ 16 mm) as susceptible and $>=$ 32 µg/ml as resistant for non-Actinobacillus species. Broken horizontal and vertical lines show the NCCLS-approved interpretive breakpoints for A. pleuropneumoniae (5). Circled numbers are non-Actinobacillus results.

Under these criteria, Actinobacillus spp. isolates, like P.multocida, would be categorized as moderately susceptible (intermediate)to tiamulin, requiring the appropriate (180 ppm in drinkingwater) dosing or intramuscular injections where indicated. However,we propose species-specific criteria for tiamulin as clinicallyindicated for A. pleuropneumoniae infections in swine: $<=$ 16 µg/ml( $>=$ 9 mm) for susceptibility and $>=$ 32 µg/ml ( $<=$ 8 mm) for resistance.Intermethod absolute categorical accuracy was 90.5% for the170 A. pleuropneumoniae strains and 90.1% for all 191 Actinobacillusstrains tested. A similar modification (no intermediate range)could be applied to P. multocida among the nonfastidious species.

H. parasuis isolates were tested by comparing reference tiamulinMICs (in HTM) (5) and the zone diameters around 30-µgdisks (on CMHA). Wide variations were noted in each test (datanot shown), but the criteria for other fastidious species allowedacceptable accuracy between methods (7, 8) for testing tiamulinagainst H. parasuis. The small number of isolates tested (18total; less than target numbers of $>=$ 100 strains), the wide zonerange variations at each MIC, and the use of HTM (not VFM) asthe broth medium require further test development before definitiveinterpretive criteria can be recommended for this agent or anyother.

In conclusion, tiamulin has a reputation as an effective veterinary-pathogen-specificantimicrobial that remains active against anaerobes, intestinalspirochetes, Actinobacillus spp., Pasteurella spp., and manycommon isolates of staphylococci and streptococci of animaland human origin (1-3, 12, 13). The proposed interpretive criteria(accepted by the NCCLS for A. pleuropneumoniae) for testingtiamulin against key indicated pathogens by NCCLS methods (5)will allow expanded testing of this agent and the accumulationof susceptibility statistics to monitor emerging resistanceto the pleuromutilin class. These interpretive criteria aresupplemented by recently published guidelines for quality controlof tiamulin dilution and disk diffusion tests (5, 10). Caremust be exercised to test the veterinary pathogens on appropriatemedia (5, 6) and to apply the species-specific breakpoint criteriaand quality control procedures.

ACKNOWLEDGMENTS

We express our appreciation to the following persons for theirassistance in the completion of the study and the manuscript:T. Anderegg, J. Kirby, W. J. Howard, K. L. Meyer, J. Jones,D. J. Biedenbach, M. E. Erwin, M. L. Beach, and M. G. Stilwell.

The study was funded, in part, by an educational/research grantfrom Boehringer Ingelheim Vetmedica, Inc.

FOOTNOTES

* Corresponding author. Mailing address: 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317. Phone: (319) 665-3370. Fax: (319) 665-3371. E-mail: ronald-jones{at}jmilabs.com.

REFERENCES

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