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VERSION 2: JCM 1439-12 1

Standardized Methods and Quality Control Limits for Agar and Broth 2

Microdilution Susceptibility Testing of Mycoplasma pneumoniae, Mycoplasma 3

hominis, and Ureaplasma urealyticum 4

Ken B. Waites, 1 Lynn B. Duffy, 1 Cécile M. Bébéar, 2 Anne Matlow, 3 Deborah F. Talkington, 4 5

George E. Kenny, 5 Patricia A. Totten, 5 Donald J. Bade, 6 Xiaotian Zheng, 7 Maureen K. 6

Davidson, 8 Virginia D. Shortridge, 9 * Jeffrey L. Watts, 10 and Steven D. Brown 11 7

8

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA; 2 USC 9

Mycoplasmal and Chlamydial Infections in Humans. Université Bordeaux, Bordeaux Cedex, 10

France; 3 The Hospital for Sick Children, Toronto, Ont, Canada; 4 Centers for Disease Control 11

and Prevention, Atlanta, GA, USA; 5 Department of Medicine, School of Public Health, 12

University of Washington, Seattle, WA, USA; 6 Microbial Research, Inc., Fort Collins, CO, USA; 13

7 Children's Memorial Hospital, Chicago, IL, USA; 8 FDA Center for Veterinary Medicine, Laurel, 14

MD, USA; 9 bioMérieux, Inc., Hazewood, MO., USA; 10 Pfizer Animal Health, Kalamazoo, MI, 15

USA; 11 Clinical Microbiology Institute, Wilsonville, OR, USA 16

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Running Head: Susceptibility Testing of Mycoplasma and Ureaplasma 18

Correspondence: 19

Ken B. Waites, M.D. F(AAM) 20

Department of Pathology 21

WP 230 22

619 19th Street South 23

University of Alabama at Birmingham 24

Birmingham, AL 35249 25

Telephone: (205) 934-4960 26

Fax: (205) 975-4468 27

Email: [email protected] 28

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01439-12 JCM Accepts, published online ahead of print on 22 August 2012

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Abstract 30

An international multi-laboratory collaborative study was conducted to develop 31

standard media and consensus methods for performance and quality control of 32

antimicrobial susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, 33

and Ureaplasma urealyticum using broth microdilution and agar dilution techniques. A 34

reference strain from the American Type Culture Collection was designated for each 35

species to be used for quality control purposes. Repeat testing of replicate samples of 36

each reference strain by participating laboratories utilizing both methods and different 37

lots of media enabled a 3 to 4 dilution MIC range to be established for drugs in several 38

different classes, including tetracyclines, macrolides, ketolides, lincosamides, and 39

fluoroquinolones. This represents the first multi-laboratory collaboration to standardize 40

susceptibility testing methods and designate quality control parameters to ensure 41

accurate and reliable assay results for mycoplasmas and ureaplasmas that infect 42

humans. 43

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Key Words: Mycoplasma, Ureaplasma, Minimal Inhibitory Concentration, Antimicrobial 47

Susceptibility Testing, Quality Control, Reference Strains 48

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Introduction 51

Methods for in vitro antimicrobial susceptibility testing of mycoplasmas were first 52

described in the 1960s (6). Despite numerous publications during the ensuing years 53

that have reported activities of antimicrobial agents against these organisms, there 54

have been no universally accepted, standardized, broth dilution or agar based methods 55

designating the optimum testing conditions, pH, media, length of incubation, quality 56

control (QC) minimum inhibitory concentration (MIC) reference ranges, or reference 57

strains. Lack of a consensus method for MIC determination coupled with complex 58

cultivation requirements has resulted in considerable confusion regarding antimicrobial 59

activities of various drugs against these fastidious organisms. 60

To address the needs for a standard method for performing and validating in vitro 61

susceptibility tests for human mycoplasmas and ureaplasmas, the Clinical and 62

Laboratory Standards (CLSI) Subcommittee on Antimicrobial Susceptibility Testing of 63

Human Mycoplasmas devised a series of studies involving a total of 10 laboratories 64

from 3 different countries representing academia, industry, and government. Sequential 65

evaluations of both broth and agar-based methods were done with Mycoplasma 66

hominis, Mycoplasma pneumoniae, and Ureaplasma species. Methods included 67

commercial and individual laboratory-produced media and testing of multiple reference 68

strains for evaluation as QC strains. QC strains and their respective MIC reference 69

ranges were designated for several drug classes including macrolides, ketolides, 70

lincosamides, tetracyclines, and fluoroquinolones for each organism. 71

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Methods 73

The CLSI mandates specific protocols and numbers of participating laboratories 74

for determining MIC reference ranges for QC purposes and for the actual measurement 75

of MICs. These requirements have evolved over the years and have now become quite 76

stringent (2). However, the fastidious nature, complex media and incubation 77

requirements, and relatively slow growth for some mycoplasmal species necessitated 78

some modifications in the protocols that were used in the present studies. These 79

modifications were performed with CLSI knowledge and approval. Even though a total 80

of 10 laboratories contributed data to these investigations, some individual laboratories 81

were unable to participate in every one of the agar and broth dilution studies for all 3 82

species. 83

The CLSI Human Mycoplasma Susceptibility Testing Subcommittee reviewed 84

various protocols for broth microdilution and agar dilution and developed a consensus 85

method for each technique, including rigorous QC parameters. The actual testing 86

protocols that were used are described in the Supplementary Data. Once the actual 87

methodology was approved, it was possible to plan studies to investigate the optimal 88

media for use in the respective assays, to determine assay reproducibility within and 89

among participating laboratories, and to designate reference QC strains with the tightest 90

MIC ranges for the greatest number of drugs. 91

Selection of broth and agar media for performing MIC assays. 92

Broth Media. A preliminary study performed at the University of Alabama at 93

Birmingham (UAB) tested 3 M. hominis reference strains against 8 drugs using a single 94

lot of laboratory-prepared SP4 broth supplemented with arginine and a single batch of 95

laboratory-prepared Modified Hayflick's Mycoplasma Broth (MHMB) with arginine. See 96


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Supplementary Data for media formulations. No differences in the number of colony 97

forming units (CFUs) recovered or MICs were detected between these two media, so 98

they were deemed equivalent. A second study performed in 6 laboratories tested the 99

same 3 reference strains against 8 drugs using a single lot of commercially prepared 100

SP4 glucose broth plus arginine (Remel, Lenexa, KS, available by special order) and 6 101

batches of MHMB with arginine prepared individually in each laboratory using assay 102

conditions described in the Supplementary Data. Due to problems in detection of a 103

sharp color-change endpoint with commercial SP4 broth plus arginine in some 104

participating laboratories, MHMB, with its sharper MIC endpoints, simpler composition, 105

and lower cost than SP4 was chosen as the primary medium for performing broth 106

microdilution assays for M. hominis. For M. pneumoniae, 5 laboratories tested 3 107

reference strains against 7 drugs using a single lot of commercial SP4 glucose broth 108

(Remel) and 5 batches of MHMB with glucose prepared individually in each laboratory. 109

No differences in MIC color change endpoints were detected between these media for 110

any of the antimicrobial agents. Testing to determine MIC QC ranges in the present 111

investigation was performed using Remel SP4 broth with glucose due its commercial 112

availability. For Ureaplasma spp., 5 laboratories tested 3 reference strains against 7 113

drugs using a single lot of commercially prepared 10 B broth (Remel) and 6 batches of 114

10 B broth prepared individually in each laboratory. Due to problems with detection of 115

sharp MIC color-change endpoints in laboratory-prepared 10 B broth, commercial 10 B 116

broth was chosen for Ureaplasma spp. However, since the formulation of the 117

commercial 10B broth is essentially the same as the laboratory-prepared 10 B broth, 118

either may be used if definitive color-change endpoints can be detected. 119


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Agar Media. Agar dilution MIC assays must use non-commercially prepared agars to 120

which antimicrobials are added because it is not possible to purchase such materials 121

commercially. Furthermore, the generally accepted 72 hour shelf life for media 122

containing antibiotics requires that such media be made in the individual testing 123

laboratory. Modified Hayflick's Mycoplasma Agar (MHMA) with arginine was chosen for 124

testing M. hominis since the broth equivalent gave satisfactory performance in the 125

preliminary evaluation for broth microdilution. For M. pneumoniae, MHMA with glucose 126

was used for QC reproducibility testing, but SP4 glucose agar is also acceptable for 127

MIC determination since growth of the organisms and MICs are equivalent based on 128

data described previously using the corresponding broths and direct comparisons 129

performed at the University of Alabama at Birmingham (data not shown). A 8 agar is the 130

primary agar medium used for cultivation of ureaplasmas, so it was used for agar 131

dilution MIC assays. Individual formulations for non-commercial media used in broth 132

and agar dilution assays are provided in the Supplementary Data. 133

Preparation of organisms for MIC assays. Since the inoculum can influence MIC 134

values, it is important to accurately quantify the numbers of organisms used for a broth 135

or agar-based MIC system. Due to their small cellular dimensions, mycoplasmas and 136

ureaplasmas do not produce turbidity in liquid media, so use of the turbidity method for 137

determination of organism concentrations is not possible. In order to quantify these 138

organisms for use in MIC determinations, it is first necessary to grow them in the 139

appropriate broth medium until color change occurs due to pH changes brought about 140

by hydrolysis of urea (Ureaplasma spp.), arginine (M. hominis), or glucose (M. 141

pneumoniae) using phenol red as a pH indicator. The culture is then frozen in multiple 142

aliquots at - 80 o C overnight. Afterwards, an aliquot is thawed and the CFU per ml 143


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determined by performing serial 10-fold dilutions in broth, plating each dilution on agar, 144

incubating, and then counting colonies as described in the Supplementary Data. Those 145

results can be used to determine the proper dilution that must be made in the 146

appropriate broth to yield 104 - 105 CFU/ml for the MIC assay. The volume of inoculum 147

required for MIC assays will depend on the number of drugs being tested in duplicate 148

and the range of dilutions of each drug to be tested. The MIC inoculum is incubated at 149

37oC for 2 hours to allow organisms to become metabolically active. Ureaplasma spp. 150

should be incubated for only one hour prior to setting up the MIC assay due to their 151

more rapid growth rate. 152

Preparation of antimicrobial agent stock solutions. Reference standard powders of 153

each drug were obtained from the manufacturers or from Sigma Chemical (St. Louis, 154

MO) for non-proprietary compounds. The same lot number of each drug was used by all 155

of the participating laboratories. Drugs tested included clindamycin, erythromycin, 156

tetracycline, azithromycin (Pfizer), telithromycin (Sanofi-Aventis), levofloxacin (Ortho-157

McNeil), and moxifloxacin (Bayer). Powdered drugs were weighed and dissolved 158

according to manufacturer instructions as described in the Supplementary Data, taking 159

into account the purity of the drugs. Stock solutions for each drug were prepared on the 160

days the MIC assays were performed in each participating laboratory in accordance 161

with CLSI procedures (3,4). Dilutions of the stock solutions for use in the individual MIC 162

assays were also prepared in accordance with published CLSI procedures (5). 163

Organisms considered for designation as QC reference strains. Organisms 164

considered for designation as reference strains for QC purposes included type strains 165

obtained from the American Type Culture Collection (ATCC) and clinical isolates 166

derived from patient cultures in Birmingham, Alabama. Three different strains of each of 167


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the organisms underwent reproducibility testing for inclusion as the type strain for MIC 168

determination for each respective species. The following strains were tested: M. 169

hominis: American Type Culture Collection (ATCC) 43521 (M132), ATCC 23114 170

(PG21), and clinical isolate, MH 5155; M. pneumoniae ATCC 29342 (M129), ATCC 171

15531 (FH), and clinical isolate MPN 834; U. parvum serovar 3 ATCC 27815, U. 172

urealyticum serovar 8 ATCC 27618, and U. urealyticum serovar 9 ATCC 33175. 173

Broth microdilution MIC procedure. The broth microdilution MIC assay employed a 174

96-well microdilution plate into which a defined inoculum of the organism to be tested 175

was added to wells containing doubling dilutions of antimicrobial agents using a 176

multichannel pipette. Microdilution plates were incubated in ambient air at 37 o C until 177

the positive growth control well changed color due to the phenol red pH indicator. 178

Growth of Ureaplasma spp. in 10B broth will cause a color change from yellow to pink, 179

Growth of M. hominis in MHMB or SP4 broth will cause a color change from pink to 180

deep red, while growth of M. pneumoniae will cause MHMB or SP4 broth to change 181

from pink to yellow. For Ureaplasma spp., this color change occurs in approximately 16-182

18 hours of incubation, whereas M. hominis typically requires 48-72 hours, and M. 183

pneumoniae may require 4 to 6 days to show a color change in broth. The MIC end 184

point was then determined as the lowest concentration of antimicrobial that did not show 185

any color change at the time the growth control showed a color change. It is particularly 186

important to read the MIC end point at the first appearance of a color change in the 187

growth control well due to the tendency of the MIC to shift after longer incubation, thus 188

giving a falsely elevated MIC. Details of the broth microdilution procedure and its 189

interpretation along with the following agar dilution procedure are described in the 190

Supplementary Data. 191


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Agar dilution MIC procedure. The agar dilution method for determination of MICs was 192

based on the incorporation of doubling dilutions of antimicrobial agents into molten agar 193

plates, with each plate containing a different concentration. Antimicrobial dilutions were 194

prepared for the entire concentration range desired for each drug using sterile ultrapure 195

clinical laboratory reagent water (CLRW) as the diluent. There were typically 8 dilutions 196

of each drug tested. Instructions for weighing out drug powder, accounting for purity, 197

and preparing stock solutions are the same as for broth microdilution. Appropriate 198

dilutions of the drugs were dispensed in 2-ml volumes in 50-ml sterile polystyrene 199

culture screw-cap tubes to facilitate mixing of the antibiotics with the molten agar. After 200

agar plates solidified, 10 µl of a defined organism inoculum of 104-105 CFU/ml prepared 201

in the appropriate broth was added to the agar plates using a Steers replicator. Plates 202

were incubated in ambient air plus 5% CO2 at 37 o C. The MIC was read as the lowest 203

concentration of the antimicrobial agent that prevented colony formation when 204

examined under a stereomicroscope at the same time the antimicrobial-free control 205

plate demonstrated growth of approximately 30 to 300 CFUs per spot of inoculum. 206

Length of time until MICs can be read is similar to what is typically observed with the 207

broth microdilution assay. Due to difficulties sometimes encountered in obtaining the 208

correct inoculum, it may be useful to perform the MIC assay using an undilute, a 1:10 209

and 1:100 dilution of the stock inoculum and use data from the dilution that provides the 210

desired number of CFUs on the antimicrobial-free control plate. 211

Establishment of broth and agar dilution assay reproducibility. Six laboratories 212

tested 10 replicates of the 3 reference strains of M. hominis against 8 drugs prepared 213

from separate inocula using 3 separate batches of MHMB with arginine prepared in 214

each laboratory. Tests were performed over a minimum of 3 days with a maximum of 4 215


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replicates of each strain tested per day. For M. pneumoniae, testing was performed 216

similarly to what was done for M. hominis, except MICs were determined for 7 drugs 217

and SP4 glucose broth was used in 7 participating laboratories. For Ureaplasma spp., 5 218

laboratories tested 7 drugs using 10 B broth. 219

Agar dilution testing was performed with M. hominis being tested against 8 drugs 220

by 6 laboratories using MHMA with arginine. M. pneumoniae was tested by 8 221

laboratories using MHMA with glucose; and testing of Ureaplasma spp. was performed 222

by 6 laboratories using A8 agar. 223

Establishment of MIC reference ranges and designation of QC strains. All of the 224

MIC data points obtained for the 10 replicates of each reference strain from each 225

laboratory were reviewed in aggregate, so a total of 60-80 replicate MICs were reviewed 226

for each organism/drug combination. The reference strain of each species that had the 227

most MIC data points that occurred within a 3 to 4 two-fold dilution range for the 228

greatest number of drugs was selected as the designated QC strain. The QC ranges for 229

individual drugs for the designated reference strains were calculated using the methods 230

established by the CLSI (2). This method includes tabulating the MIC mode for each 231

antimicrobial/strain combination and expanding the range to include 1 log2 dilution on 232

each side of the mode, giving a 3 dilution QC range. If there was a bi-modal distribution 233

of the MICs, then a 4 dilution range was selected. In both cases, the QC range must 234

include > 95% of the MIC replicate data points determined in aggregate for all 235

participating laboratories, excluding outliers as determined by the method of Turnidge 236

and Bordash (7). No QC range was proposed for drugs for which MIC ranges exceeded 237

4 dilutions. 238

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Results 240

M. hominis ATCC 23114 (PG21), M. pneumoniae ATCC 29342 (M129) and U. 241

urealyticum ATCC 29342 (serovar 9) demonstrated the most reproducible MIC data 242

points and tightest ranges for the most drugs (data not shown) and were designated as 243

the reference strains to be used for QC purposes for both agar and broth microdilution 244

assays. Therefore, reproducibility data and MIC ranges derived from replicate testing 245

are shown only for these strains. 246

Tables 1-3 show the combined results for the MIC data points obtained by 247

replicate testing for the designated reference strains of M. hominis, M. pneumoniae, 248

and U. urealyticum, respectively. Table 4 shows the MIC QC ranges for several drugs 249

that were derived from these data. These QC ranges and reference strains were 250

approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing and have 251

recently been published in a CLSI document (3). Overall, reproducibility for MIC 252

determinations within individual laboratories and among the laboratories was best for 253

the mycoplasmacidal fluoroquinolones and worst for the macrolides, telithromycin, and 254

clindamycin. Despite the efforts of the participating laboratories to determine MIC 255

ranges for the respective reference strains for all currently available antimicrobial 256

agents relevant for testing against these organisms, there was lack of consensus for 257

assignment of ranges for some drugs by agar and/or broth methods due to excessive 258

variation in the MICs obtained among the participating laboratories (Tables 1-3). It was 259

not possible to designate a 3 to 4 dilution range for azithromycin for any organism by 260

either MIC method. However, given the importance of azithromycin for treatment of M. 261

pneumoniae infections, and increasing prevalence of macrolide resistance in this 262

organism, a single cutoff value of < 0.06 µg/ml was chosen for broth microdilution 263


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tested against M. pneumoniae ATCC 29342. This cutoff readily distinguishes 264

macrolide-susceptible from resistant strains for which azithromycin MICs may exceed 265

32 µg/ml (1). A similar decision was made for tetracycline tested by agar dilution 266

against U. urealyticum ATCC 33175. Even though no 4 dilution range could be 267

established, a single cutoff MIC of > 8 µg/ml was selected for this tetracycline-resistant 268

strain known to contain the tet(M) transposon that mediates tetracycline resistance in 269

this organism (1). Tetracycline-susceptible strains usually have MICs < 2 µg/ml (1,9). 270

Discussion 271

Standardized antimicrobial susceptibility testing methods and designated QC 272

parameters for human mycoplasmas and ureaplasmas are needed because culture is 273

seldom performed for diagnostic purposes and in vitro testing of individual isolates is 274

even more rarely obtained. Antimicrobial susceptibilities can vary geographically and in 275

response to selective antimicrobial pressure. Moreover, clinically significant acquired 276

drug resistance, potentially affecting multiple antimicrobial classes can occur in all of 277

the mycoplasmal and ureaplasmal human pathogens (1). Since most mycoplasmal or 278

ureaplasmal infections are treated empirically, accurate and reproducible antimicrobial 279

resistance surveillance data for currently available drugs and to new investigational 280

agents is important. In the event of systemic infection, particularly in an 281

immunosuppressed host, an individual who has failed previous treatment, or someone 282

known to harbor a resistant organism, susceptibility testing can be valuable for patient 283

management (8,9). As new antimicrobials are developed, it is also important to have 284

knowledge of their antimycoplasmal activities relative to existing drugs. 285

The data described in this publication represent the first systematic multi-286

laboratory project to standardize test methodology and demonstrate inter-laboratory 287


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reproducibility for MIC determinations for human mycoplasmas and ureaplasmas, thus 288

allowing designation of specific type strains for M. pneumoniae, M. hominis and U. 289

urealyticum with defined QC reference MIC ranges for several antimicrobial agents, and 290

using study design and methods approved by the CLSI. The fastidious nature of these 291

organisms and the relative difficulty in obtaining an accurate and reproducible inoculum 292

were probably at least partially responsible for the inability to get the sufficient 293

consensus of results necessary to generate 3 to 4 dilution QC ranges for some drugs. 294

Fluoroquinolones gave the tighter QC ranges overall than the other drug classes, 295

perhaps because they are bactericidal against mycoplasmas while the other drug 296

classes are bacteriostatic and are known to have shifting MICs, especially for broth 297

microdilution (8). 298

Standard methods, media formulations, and QC parameters have been 299

described for both broth microdilution and agar dilution methods. The relative 300

advantages and disadvantages of each technique for testing human mycoplasmas and 301

ureaplasmas have been discussed elsewhere (8), and either method can be performed, 302

depending on the preferences and needs of individual laboratories. 303

Due to inherent differences in their cultivation requirements and growth rates, no 304

single procedure, pH, or medium was considered sufficient for testing all of the clinically 305

important species and the differences in the proposed methods and test conditions 306

reflect this observation. For example, the slow growth of M. pneumoniae mandates that 307

several days of incubation must take place before MICs can be determined. 308

Additionally, the low media pH (6.0) required to detect growth of ureaplasmas can affect 309

MIC results since activity of some drugs such as macrolides are highly pH dependent 310

(8). It is also important to note that there has been no attempt to generalize these 311


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methods for application to other mycoplasmal species of human or animal origin, which 312

may have very different growth and testing requirements. Therefore, these procedures 313

and reference ranges should be limited to testing only the species for which they are 314

described. 315

Individual laboratories can prepare their own SP4 glucose and 10B media for 316

broth microdilution MIC determinations for M. pneumoniae and Ureaplasma species, 317

respectively, using the formulations provided in the Supplementary Data as long as the 318

QC strains yield MICs with a sufficiently sharp endpoint to allow visual interpretation 319

and the values fall within the designated reference ranges. Alternatively, laboratories 320

can purchase these media from commercial sources, though it may require special 321

order. Reproducibility testing and determination of QC ranges for reference strains by 322

agar dilution for M. pneumoniae was performed using MHMA with glucose. However, it 323

is also acceptable to perform these assays using SP4 glucose agar prepared as 324

described in the Supplementary Data as long as the QC reference strain MICs fall 325

within the designated ranges. 326

Performance of the studies described here took place in accordance with 327

procedures approved by the CLSI, so it is anticipated that these procedures and MIC 328

ranges for reference strains will be widely adopted for future work. Use of these CLSI-329

approved methods for broth microdilution MIC determination has recently been proven 330

accurate for detection of resistance to macrolides and fluoroquinolones in organisms in 331

which resistance markers have been genotypically characterized (10,11). Detailed 332

protocols for performance of antimicrobial susceptibilities, QC, and interpretive MIC 333

breakpoints for several drugs are included in the Methods for Antimicrobial 334


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Susceptibility Testing for Human Mycoplasmas; Approved Guideline M43-A published 335

by the CLSI (3). 336

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Acknowledgments 338 339

This work was performed by the Clinical and Laboratory Standards (CLSI) 340

Subcommittee on Antimicrobial Susceptibility Testing of Human Mycoplasmas. 341

Financial support was provided by the following companies: Abbott Laboratories, 342

AstraZeneca Pharmaceuticals, Bayer Pharmaceuticals, Bristol-Myers-Squibb, Ortho-343

McNeil Pharmaceuticals, Pharmacia-Upjohn, Pfizer Animal Health and Sanofi-Aventis 344

Pharmaceuticals. Remel Laboratories provided commercially prepared SP4 and 10B 345

broth media. Technical and administrative support from the following persons and 346

organizations is gratefully acknowledged: Tracy Dooley and the CLSI, Donna Crabb, 347

Danuta Kovach, Dena Hensey-Rudloff, Hélène Renaudin, Jeanette Jones, W. Lanier 348

Thacker, Bill Kabat, and Laura Houston. 349


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350 351

References 352

353

1. Bébéar, C. 2010. Mycoplasma, Ureaplasma, p. 1-11. In P. Courvalin, R. Leclerq, and L. 354

B. Rice (ed.), Antibiogram. ASM Press, Washington, D.C. 355

356

2. Clinical & Laboratory Standards Institute. 2008. Development of in vitro susceptibility 357

testing criteria and quality control parameters. Approved guideline M23-A3. Clinical & 358

Laboratory Standards Institute, Wayne, PA. 359

360

3. Clinical and Laboratory Standards Institute. 2011. Methods for antimicrobial 361

susceptibility testing of human mycoplasmas. Approved Guideline M43-A. Clinical and 362

Laboratory Standards Institute, Wayne, PA. 363

364

4. Clinical and Laboratory Standards Institute. 2003. Methods for dilution antimicrobial 365

susceptibility tests for bacteria that grow aerobically. Approved standard M07-A6. 366

Clinical and Laboratory Standards Institute, Wayne, PA. 367

368

5. Clinical and Laboratory Standards Institute. 2009. Performance standards for 369

antimicrobial susceptibility testing. Nineteenth informational supplement M100-S19. 370

Clinical and Laboratory Standards Institute, Wayne, PA. 371

372

6. Jao, R. L. 1967. Susceptibility of Mycoplasma pneumoniae to 21 antibiotics in vitro. Am 373

J Med Sci 253:639-650. 374

375

376


http://jcm.asm

.org/D

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http://jcm.asm.org/

18

7. Turnidge, J. and G. Bordash. 2007. Statistical methods for establishing quality control 377

ranges for antibacterial agents in Clinical and Laboratory Standards Institute 378

susceptibility testing. Antimicrob Agents Chemother 51:2483-2488. 379

380

8. Waites, K. B., C. M. Bébéar , J. A. Robertson, D. F. Talkington, and G. E. Kenny. 381

2001. Cumitech 34, Laboratory diagnosis of mycoplasmal infections. ASM Press, 382

Washington, D.C. 383

384

9. Waites, K. B., and D. Taylor-Robinson. 2011. Mycoplasma and Ureaplasma, p. 970-385

985. In J. Versalovic, K. C. Carroll, G. Funke, J. Jorgensen, M. L. Landry, and D. W. 386

Warnock (ed.), Manual of Clinical Microbiology, 10th Ed. ASM Press, Washington, D.C. 387

388

10. Xiao, L., D.M. Crabb, L.B. Duffy, V. Paralanov, V., J.I. Glass, and K.B. 389

Waites. 2011.Characterization of mutations in ribosomal proteins and 23S rRNA 390

that confer resistance to macrolides in Ureaplasma species. Int J Antimicrob 391

Agents, 37:377-379. 392

11. Xiao, L., D.M. Crabb, L.B. Duffy, J.I. Glass, and K.B. Waites. 2012. 393

Chromosomal mutations responsible for fluoroquinolone resistance in 394

Ureaplasma species in the United States. Antimicrob Agents Chemother. 395

56:2780-2783. 396

397

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Table 1. MIC ranges of M. hominis ATCC 23114 by broth microdilution

and agar dilution a,b

Broth microdilution data

Drug No. of occurrences at indicated MIC (µg/ml)

< 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 8

% in

range c

Azithromycin -- -- -- -- -- -- -- -- -- -- -- --

Erythromycin -- -- -- -- -- -- -- -- -- -- -- --

Clindamycin 0 2 61 66 30 17 0 1 0 0 0 98.3

Levofloxacin -- -- -- -- -- -- -- -- -- -- -- --

Moxifloxacin 0 33 93 45 6 0 0 0 0 0 0 100

Telithromycin -- -- -- -- -- -- -- -- -- -- -- --

Tetracycline -- -- -- -- -- -- -- -- -- -- -- --

Agar dilution data


< 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 8 % in range c

Azithromycin d -- -- -- -- -- -- -- -- -- -- -- --

Erythromycin d -- -- -- -- -- -- -- -- -- -- -- --

Clindamycin 0 0 0 1 104 74 0 1 0 0 0 99.4

Levofloxacin 0 0 0 0 22 85 61 12 0 0 0 100

Moxifloxacin 0 0 0 12 168 0 0 0 0 0 0 100

Telithromycin -- -- -- -- -- -- -- -- -- -- -- --

Tetracycline 0 0 0 0 37 65 30 27 1 0 0 99.4

a Recommended QC ranges are represented by boldface characters in shaded areas.

b No QC range could be established for antimicrobials that do not have MIC data points shown.

c Percentage of data points which fall within the recommended range (acceptable limit > 95%).

d M.hominis is intrinsically resistant to 14 and 15-membered macrolides and azalides.


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Table 2. MIC ranges of M. pneumoniae ATCC 29342 by broth microdilution




< 0.004 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4

% in range c

Azithromycin 160 0 3 14 3 0 0 0 0 0 0 100 d

Erythromycin 29 70 51 29 0 1 0 0 0 0 0 99.4

Clindamycin -- -- -- -- -- -- -- -- -- -- -- --

Levofloxacin 0 0 0 0 0 1 72 97 10 0 0 100

Moxifloxacin 2 2 0 64 49 51 12 0 0 0 0 97.7

Telithromycin -- -- -- -- -- -- -- -- -- -- -- --

Tetracycline 0 0 0 0 13 75 61 24 7 0 0 96.1

Agar dilution data


< 0.004 0.008 0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 % in range c

Azithromycin -- -- -- -- -- -- -- -- -- -- -- --

Erythromycin -- -- -- -- -- -- -- -- -- -- -- --

Clindamycin -- -- -- -- -- -- -- -- -- -- -- --

Levofloxacin 0 0 0 0 30 e 17 40 56 6 0 0 96

Moxifloxacin 0 0 0 18 75 49 8 0 0 0 0 100

Telithromycin -- -- -- -- -- -- -- -- -- -- -- --

Tetracycline 0 0 0 0 44 45 59 1 0 0 0 100a Recommended QC ranges are represented by boldface characters in shaded areas.

b No QC range could be established for antimicrobials that do not have MIC data points shown.


d No 3 to 4 dilution range could be established, but all MICs were < 0.06 and this upper limit cutoff was

recommended for broth microdilution since azithromycin is an important drug for M. pneumoniae.

e All values of 0.06 µg/ml for levofloxacin occurred in a single laboratory, which was excluded as a

statistical outlier by the method of Turnidge & Bordash (7).


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Table 3. MIC ranges of U. urealyticum ATCC 33175 by broth microdilution




0.016

0.03 0.06 0.12 0.25 0.5 1 2 4 8 > 8 % in range c

Azithromycin -- -- -- -- -- -- -- -- -- -- -- --

Erythromycin 0 0 0 0 0 3 47 63 18 16 2 96.6

Clindamycin d -- -- -- -- -- -- -- -- -- -- -- --

Levofloxacin 0 0 0 0 0 10 92 43 4 0 0 97.3

Moxifloxacin 0 0 0 0 5 37 87 19 1 0 0 96

Telithromycin 0 0 0 2 98 29 16 2 0 0 0 98.6

Tetracycline -- -- -- -- -- -- -- -- -- -- -- --

Agar dilution data


0.016 0.03 0.06 0.12 0.25 0.5 1 2 4 8 > 8

% in range c

Azithromycin -- -- -- -- -- -- -- -- -- -- -- --

Erythromycin -- -- -- -- -- -- -- -- -- -- -- --

Clindamycin d -- -- -- -- -- -- -- -- -- -- -- --

Levofloxacin 0 0 0 0 0 0 61 89 0 0 0 100

Moxifloxacin 0 0 0 0 90 26 34 0 0 0 0 100

Telithromycin 0 0 0 26 29 95 0 0 0 0 0 100

Tetracycline 0 0 0 0 0 0 0 0 0 0 150 100 e

a Recommended QC ranges are represented by boldface characters in shaded areas.

b No QC range could be established for antimicrobial agents that do not have MIC data points shown.


d Ureaplasma spp. are intrinsically resistant to clindamycin and other lincosamides.


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e No 3 to 4 dilution range could be established, but all MICs were > 8 µg/ml and this cutoff was

recommended for agar dilution because tetracycline is an important drug for U urealyticum and this strain

is known to carry tet(M), a determinant of tetracycline resistance (1).


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Table 4. Minimal Inhibitory Concentration (µg/ml): Quality Control Ranges for

Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum for

Broth Microdilution and Agar Dilution Methods a

Antimicrobial Agent

M. hominis b

ATCC 23114

M. pneumoniae c

ATCC 29342

U. urealyticum d,e

ATCC 33175

Broth

Azithromycin − ≤ 0.06 −

Erythromycin − 0.004–0.03 1–8

Clindamycin 0.03–0.25 − −

Levofloxacin − 0.12–1 0.5–2

Moxifloxacin 0.016–0.12 0.03–0.25 0.5–2

Telithromycin − − 0.12–1

Tetracycline − 0.06–0.5 −

Agar

Azithromycin − − −

Erythromycin − − −

Clindamycin 0.06–0.5 − −

Levofloxacin 0.12–1 0.12–1 0.5–4

Moxifloxacin 0.06–0.25 0.03–0.25 0.25–2

Telithromycin − − 0.12–1

Tetracycline 0.12–1 0.06–0.5 ≥ 8


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a Drugs for which no MIC values are given denote that although tested, no range could

be established.

b Broth microdilution testing performed using Modified Hayflick's Mycoplasma Broth

(MHMB) with arginine; agar dilution testing performed using Modified Hayflick's

Mycoplasma Agar (MHMA) with arginine.

c Broth microdilution testing performed using SP4 glucose broth-; agar dilution testing

performed using Modified Hayflck's Mycoplasma Agar (MHMA) with glucose.

d Broth microdilution testing performed using 10 B broth; agar dilution testing performed

using A 8 agar.

e This strain is known to have the tet(M) transposon mediating tetracycline resistance

1

2


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