Antibiotic Resistence: Methods and Protocols

279 Pages·2001·5.09 MB·English

by  Robin Patel

Antibiotic Resistance
M E T H O D S IN M O L E C U L A R MEDICINE™
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M E T H O D S IN MOLECULAR MEDICINE'^
Antibiotic Resistance
Methods and Protocols
Edited by
Stephen H. Gillespie
Department of Medical Microbiology.
Royal Free and University College Medical School,
University College London, London, UK
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Antibiotic resistance: methods and protocols / edited by Stephen H. Gillespie.
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I. Drug resistance in microorganisms—laboratory manuals. 2. Antibiotics—Laboratory
manuals, 1, Gillespie. S. H. II. Series.
[DNLM: 1. Drug Resistance. Microbial—physiology. 2. Anti-Infective
AgenIS—therapeutic use. 3. Antibiotics—therapeutic use. 4. Clinical Protocol!.. WB 330
A629 2000]
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Preface
The discovery of antibiotics and their introduction into medical practice
was a revolution that has transformed the health of the human race. From its
first beginnings under Paul Erhlich, v/ho experimented with arsenical dyes to
produce drugs active against trypanosomes and spirochetes, anti-infective
chemotherapy is now available against almost ail hxunan pathogens. Even
viruses, which for 50 years had proved a much more difficult target, are nov/
treatable with chemotherapeutic agents.
From the start of the antibiotic era resistance was noted to be a problem.
Erhlich observed that trypanosomes cou!d become resistant to the arsenical
dyes that he was using and that the resistant organisms never spontaneously
reverted to susceptibility (1). Fleming carefully identified and listed the bac
teria that were not susceptible to the culture filtrate of his Penicillium mold
(2). Once antibiotics were introduced into clinical practice, acquired resis
tance was quickly noted: Neisseria gonorrhoeae to sulfonamides being one of
the earliest examples. At first, resistance did not appear to be such a problem
as the flood of drug discoveries— chloramphenicol, tetracyclines, erythromy
cin, cephalosporins, and aminoglycosides—appeared in time to widen the num
ber of bacterial species becoming susceptible to antibiotics and to overcome
developing resistance problems. Often resistance among bacteria seemed to
be limited to organisms isolated in hospitals, mainly from patients with other
serious conditions. For example, when resistance to penicillin through the elabo
ration of a p-lactamase became more common among Staphylococcus aureus.
the infections were ahnost exclusively found in hospitals. Indeed the popular
name for a S. aureus expressing p-lactamase was "the hospital Staphvlococ-
cus"(3).
The pharmaceutical industry was able to solve the problem of p-lactamase
with the discovery and chemical raodif cation of the amido-penicillin nucleus
leading to a new range of semisynthetic penicillins including methicillin,
flucloxacillin, and ampicillin (4). The difficulfies encountered in overcoming
P-lactamase-producing staphylococci v^ere a harbinger of future problems. In
succeeding years Gram-negative bacilli developed increasing resistance acquir
ing and transmitting new resistance detenninants with unfailing regularity
viaplasraids, transposons, and bacteriophages (5—7). It was only with mas-
V
vt Preface
sive investment in drug discovery and scientific success that kept medicine
ahead of the microbes.
In recent years there has been a significant change in the pattern of anti
microbial resistance and public and scientific perceptions of it. Although mul
tiple drug resistance was previously identified in Salmonella typhi (8), multiple
drug resistance has emerged in primary respiratory pathogens, such as Mycv-
baclerium tuberculosis and Streptococcus pneumoniae, spreading widely and
rapidly throughout the world (9,10). This has been coupled with a fall in the
number of new compounds coming to the market, at a time when drug devel
opment costs have increased dramatically. With the description of glycopcp-
tide intermediate resistant staphylococci, the last vestige of wishful thinking
was torn away (11). Although S. aureus had always had a talent for acquiring
resistance, it had always remained susceptible to the glycopeptides. The pros
pect of untreatable tuberculosis and pneumococcal and S. aureus infections
has generated dramatic and often hysterical headlines in the lay and medical
press (12). In many countries government investigations and reports were com
missioned f'iJ^.
At one time antibiotic drug development was considered the cutting edge
of science. Erhlich. Fleming, Florey, Chain, and Waxsman all collected Nobel
prizes for their efforts in developing successful antibiotics. With increasing
success came complacency and research in antibiotics became mere routine
and the natural area for industry not academia. All of this has now changed—
antibiotic research has returned to the forefront of medical science once again.
This volume strives to provide scientists with the critical techniques to enter
antibiotic resistance research.
From the point of view of patients and clinicians, rapid diagnosis must
be a priority for research. More rapic diagnosis of resistant organisms will
allow patients with resistant organi.sms to be isolated and the correct therapy
initiated with minimal delay. This will not only improve treatment outcomes,
but will reduce the risk of transmission of resistant organisms, in this volume
many different teciiniques are described that address rapid diagnosis, ranging
from DNA amplification techniques to phage display.
It is imperative that scientists be able to plot the transmission of resistant
organisms; thus, molecular biological techniques have been harnessed to inves
tigate their epidemiology. For tuberculosis, monitoring tt-eatment has always been
problematical and many patients fail to complete an inadequate course of
therapy, allowing drug resistant strains to emerge and be transmitted. Several
innovative techniques are described and could be readily applied to other
organisms.
The complex problems posed by pathogenic microorganisms in causing
disease and in developing antibiotic resistance mean that the ^proach to over-
Preface vii
coming them must be sophisticated and multifuceted. We also need to under
stand the mechanisms of resistance, the biology of resistant organisms, and
the way in which they evolve and to quantify the evolutionary barriers to resis
tance.
In this volume a wide range of techniques have been brought together
that will be useful in developing new antibiotic research programs. There is
strong emphasis on M. tuberculosis and S. pneumoniae, and this reflects cur
rent research questions and concerns. Although many of the techniques are
designed for one organism, they are readily adaptable to other problem organ
isms. I hope that this volume will be useful to microbiologists developing and
implementing new diagnostic and cpidemioiogicai techniques, and to basic
scientists trying to understand the basic biology of antimicrobial resistance. In
whatever .sphere of antibiotic research you are in, I hope that you will find this
volume of value.
I gratefully acknowiedgethe assistance provided by Dr. Janet P. Gillespie
in managing this project and Ms. Therese Donnelly fur her secretarial assistance.
Stephen H. Gillespie
References
1. Erlicli P, (1909) Ubcr modcrne Chemotherapie. Beitrdge zur experimentalleii
Paiholagie and Chemotherapie 167-2D2.
2. Fleming A. (1929) On the antibacterial action of cultures of 3. Penicillium, with
special reference to their use in the isolation of B. influenzae. Brit. J. Exp. Paihol.
10,226-236.
3. Haley RW., Highlower AW., Kabbatz Rf'., Thornsberry C, Martone WJ., .Mien
JR., Hughes JM. (1982) The emergence of methicillin rcsiytanl Staphylococcm
aureus infection in United States hospitals. Ann. Intern. Med. 97, 297—308.
4. Ratchelor FR., Doyle FP., Naylor JHC, Roiinson GN. (1959) Synthesis of peni
cillin: 6-amidopenicillanic acid in penicillin femientations. Nature 183,257.
5. I^nski RE. Simpson SC. Nguyen TT. (1994) Genetic analysis of a plasmid-encoded,
host genotype-specific enhancement of bacterial fitness. J. Bacieriol. 176,
3140-3147.
6. Hyder SL, Steitfeld MM. (1978) Transfer of erythromycin resistance trom clini
cally isolated lysogenic strains of^ Streptococcus pyogenes via their endogenous
phage. / Infect Dis. 138, 281-286.
7. PoyiUl C, Pierre C, Quesne G, Pron B, Berche P, Trien-Cust P. (1997) Emergence
of vancomycin resistance in the genus Streptococcus: characterization of a vanB
transferable determinant in Streptococcus bovis. Antimicrob. Agent Chemother.
41,24-29.
8. Goldstein FW,, Chumpitaz JC, Guevara JM., PapadopoulouB., Acar JF., Vieu
wj Preface
.TF. (1986) PIasmid-mediated resisiance to multiple antibioitics in Salmonella
typhi../. Infect. Di.s: 153, 261-266
9. Muftos R., Coffey TJ., Daniels M., et al (1991) Intercontinental spread of sero
type 23F Streptococcus pneumoniae. J. Infect. Dis. 164, 302—306.
10. The WHO/IUALTD. Anti-tubiirculosis drug resistance in the world. World Health
Organ, Geneva. 1997.
11. Hiramatsu K., H. Hatiaki, T. Ino, K_. Yabuta, T. Oguri, and F. C. Tenover. (1997).
MetbieiUin-resistant Staphylococcus aureus elinicai strain witti reduced vaneo-
mycin susceptibility../. Antimicrob. Chemother. 40,135—136.
12. Bloom BR, Murray CJL. (1992)Tubercu!<]sis: commentary on a reemergent killer.
Science 257,1055-1064.'
13. Standing Medical Advisory Committee. 1998. The Path of Least Resistance. Deparl-
raem of Healtli. London.
Contents
Preface v
Contributors x//V
PART I. DIAGNOSIS OF RESISTANCE
1 Multiplex Polymerase Chain Reaction Detection of yanA vanB,
vanC-1, and vanC-2/3 Genes in Enlemcoccl
Robin Patel, Jim R. Ulil, and Franitlin R. Cockerill, III 3
2 Drug Susceptibility of Mycobacterium tuberculosis Through
tfie Mycolic Acid Index
Jose M. Viader-Salvado, Martha Guerrero-Olazaran,
Elvira Garza-Gonzaiez, and Rolando Tijerina-Mencttaca 13
3 Micro-Well Ptiage Replication Assay for Screening Mycobacteria
for Resistance to Rifampin and Streptomycin
Ruth H/JcNerney 21
4 Application of 3SCP to Identification of Resislanus Mulaliuiis
Timothy D. McHugh 31
5 Quantitative, Single-Tube, Nested, PCR (QSTN-PCR)
for Determining ttie Antibiotic Susceptibility
of Mycobacterium tuberculosis
Nainn-Tsyr Jou and l\/lichael R. Liebling 39
6 Rapid Rjfamycin Susceptibility Testing of Small-Volume
Mycobacterium tuberculosis Cultures by Detection
of Precursor rRNA
William H. Brabant and Gerard A. Cangelosi. 55
7 Detection of Penicillin Resistance in Streptococcus pneumoniae
by a Seminested PCR Strategy
Mignon du Plessis, Anthony M. Smith, and Keith P. Klugman 65
8 Diagnosis of Penicillin Resistance by PCR-RFLP
Gail C. Whiting 77
9 Detection Methods of Glycopeptide-Restraint Staphylococcus
aureus I: Susceptibility Testing
Hedeald Hanaki and Keiichi Hiramatsu 85
iX
X Contents
10 Detection Methods for Glycopeptide-Resistant Staphylococcus
aureus II: Cell Wall Analysis
Hedeaki Hanaki and Keiichi Hiramatsu 93
11 Multiplex PCR for the Rapid Simultaneous Speciation and Detection
of Methicillin-Resistance and Genes Encoding Toxin Production
in Staphylococcus aureus
Mark E. Jones, Karl Kohrer, and Franz-Josef Schmitz 103
12 Chromogenic Detection of Aminoglycoside Phosphotransferases
Ana M. Amoroso and Gabriel 0. Gutkind 113
PART 11. MONITORING RESPONSE TO TREATMENT
13 Quantification of M. tuberculosis DNA in Sputum During
the Treatment of Pulmonary Tuberculosis
Lucy E. DesJardin and Gery L. Hehman 121
14 isolation of M. tuberculosis RNA from Sputum
LucyE. DesJardin 133
15 Detection of Viable Mycobacterium tuberculosis by Reverse
Transcriptase-Strand Displacement of mRNA
Tobin J. Hellyer 141
PART III. MOLECULAR EPIDEMIOLOGY
16 BOX PCR Fingerprinting for Molecular Typing
of Streptococcus pneumoniae
Alex van Belkum and Peter W. M. Hermans 159
17 Restriction Fragment End Labeling Analysis: High-Resolution
Genomic Typing of Streptococcus pneumoniae Isolates
Peter W. M. Hermans, Marcel Sluljter, and Alex van Belkum 169
18 Pulse-field Gel Electrophoresis for Epidemiological Studies
of Streptococcus pneumoniae
Anne-Marie Gasc 181
19 Outer Membrane Profiles of Clonally Related Klebsiella pneumoniae
Vicente J. Benediand Luis Martinez -Martinez 189
20 Atomic Force Microscopy: Theory and Practice in Bacteria
Morphostructural Analysis
Pier Carlo Braga and Davide Ricci 199
PART IV. BIOLOGY OF RESISTANCE
21 Assessing the Activity of Bacterisl Multidrug Efflux Pumps
Keitli Poole and Ramakrishnan Srikumar 211