Stephen Gillespie and a panel of leading clinical and diagnostic microbiologists describe a series of detailed molecular and physical methods designed to study the growing problem of antibiotic resistance, as well as facilitate new antibiotic research programs for its effective redress. The techniques range widely from those that provide rapid diagnosis via DNA amplification and phage display, to those for plotting the transmission of resistant organisms and investigating their epidemiology. The methods are readily adaptable to a wide range of resistant bacterial organisms. Practical and wide-ranging, Antibiotic Resistance: Methods and Protocols provides a collection of indispensable techniques not only for illuminating the basic biology of antimicrobial resistance, but also for developing and implementing new diagnostic and epidemiological tools.

Preface

v

Contributors

xiii

Part I. Diagnosis of Resistance

Multiplex Polymerase Chain Reaction Detection of vanA, vanB, vanC-1, and vanC-2/3 Genes in Enterococci

3

(10)

Robin Patel

Jim R. Uhl

Franklin R. Cockerill, III

Drug Susceptibility of Mycobacterium tuberculosis Through the Mycolic Acid Index

13

(8)

Jose M. Viader-Salvado

Martha Guerrero-Olazaran

Elvira Garza-Gonzalez

Rolando Tijerina-Menchaca

Micro-Well Phage Replication Assay for Screening Mycobacteria for Resistance to Rifampin and Streptomycin

21

(10)

Ruth McNerney

Application of SSCP to Identification of Resistance Mutations

31

(8)

Timothy D. McHugh

Quantitative, Single-Tube, Nested, PCR (QSTN-PCR) for Determining the Antibiotic Susceptibility of Mycobacterium tuberculosis

39

(16)

Nainn-Tsyr Jou

Michael R. Liebling

Rapid Rifamycin Susceptibility Testing of Small-Volume Mycobacterium tuberculosis Cultures by Detection of Precursor rRNA

55

(10)

William H. Brabant

Gerard A. Cangelosi

Detection of Penicillin Resistance in Streptococcus pneumoniae by a Seminested PCR Strategy

65

(12)

Mignon du Plessis

Anthony M. Smith

Keith P. Klugman

Diagnosis of Penicillin Resistance by PCR-RFLP

77

(8)

Gail C. Whiting

Detection Methods of Glycopeptide-Restraint Staphylococcus aureus I: Susceptibility Testing

85

(8)

Hedeaki Hanaki

Keiichi Hiramatsu

Detection Methods for Glycopeptide-Resistant Staphylococcus aureus II: Cell Wall Analysis

93

(10)

Hedeaki Hanaki

Keiichi Hiramatsu

Multiplex PCR for the Rapid Simultaneous Speciation and Detection of Methicillin-Resistance and Genes Encoding Toxin Production in Staphylococcus aureus

103

(10)

Mark E. Jones

Karl Kohrer

Franz-Josef Schmitz

Chromogenic Detection of Aminoglycoside Phosphotransferases

113

(8)

Ana M. Amoroso

Gabriel O. Gutkind

Part II. Monitoring Response to Treatment

Quantification of M. tuberculosis DNA in Sputum During the Treatment of Pulmonary Tuberculosis

121

(12)

Lucy E. DesJardin

Gery L. Hehman

Isolation of M. tuberculosis RNA from Sputum

133

(8)

Lucy E. DesJardin

Detection of Viable Mycobacterium tuberculosis by Reverse Transcriptase-Strand Displacement of mRNA

141

(18)

Tobin J. Hellyer

Part III. Molecular Epidemiology

BOX PCR Fingerprinting for Molecular Typing of Streptococcus pneumoniae

159

(10)

Alex van Belkum

Peter W. M. Hermans

Restriction Fragment End Labeling Analysis: High-Resolution Genomic Typing of Streptococcus pneumoniae Isolates

169

(12)

Peter W. M. Hermans

Marcel Sluijter

Alex van Belkum

Pulse-field Gel Electrophoresis for Epidemiological Studies of Streptococcus pneumoniae

181

(8)

Anne-Marie Gasc

Outer Membrane Profiles of Clonally Related Klebsiella pneumoniae

189

(10)

Vicente J. Benedi

Luis Martinez-Martinez

Atomic Force Microscopy: Theory and Practice in Bacteria Morphostructural Analysis

199

(12)

Pier Carlo Braga

Davide Ricci

Part IV. Biology of Resistance

Assessing the Activity of Bacterial Multidrug Efflux Pumps

211

(4)

Keith Poole

Ramakrishnan Srikumar

The Use of a Continuous Culture System to Study the Antimicrobial Susceptibility of Bacteria in Biofilm

215

(12)

J. Keith Struthers

Estimation of Mutation Rates in Antibiotic Research

227

(6)

Owen J. Billington

Stephen H. Gillespie

In Vitro Assessment of the Fitness of Resistant M. tuberculosis Bacteria by Competition Assay

233

(4)

Stephen H. Gillespie

Purification of DNA Topoisomerases and Inhibition by Fluoroquinolones

237

(8)

John T. George

Ian Morrissey

Site-Directed Mutagenesis to Determine Structure Function Relationships in Streptococcus pneumoniae Penicillin-Binding Protein Genes

245

(20)

Victoria A. Barcus

Christopher G. Dowson

Detection of Low Affinity Penicillin-Binding Protein Variants in Streptococcus pneumoniae

265

(10)

Regine Hakenbeck

Part V. Transmission of Resistance

Mobilization of Transposons: Rationale and Techniques for Detection

275

(10)

Louis B. Rice

Index

285

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