Next Article 
Antimicrobial Agents and Chemotherapy, February 1999, p. 199-212, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
MINIREVIEW
Genetic Methods for Assessing Antimicrobial
Resistance
Franklin R.
Cockerill III*
Division of Clinical Microbiology, Mayo
Clinic and Foundation, Rochester, Minnesota 55905
 |
INTRODUCTION |
Over the past 2 decades,
antimicrobics have become increasingly available for a broad range of
pathogens. Due to the widespread use of these drugs, new
forms of antimicrobial resistance have emerged. Detection of this
resistance by conventional methods may not always be easy or possible.
The genetics of antimicrobial resistance has been elucidated completely
or in part for many organism-antimicrobial agent combinations. As a
result, genetic methods for assessing antimicrobial resistance have
been developed, and many of these are being used or soon will become
part of the standard testing menus in clinical microbiology laboratories. The intent of this review is to describe the principles and applications of the more frequently reported genetic methods for
evaluating antimicrobial resistance.
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CONVENTIONAL METHODS FOR SUSCEPTIBILITY TESTING |
Conventional antimicrobial susceptibility testing
methods require that pathogens are first isolated from
human specimens by culture methods. In separate assays, isolated
microorganisms are then exposed to various concentrations of
antimicrobial agents under specified growth conditions, and the ability
of these antimicrobics to inhibit growth is determined. Methods that
are frequently used for testing cultivated bacteria and yeasts include
disk diffusion, broth dilution, agar dilution, and gradient diffusion
(Epsilometer test). Isolated bacteria can also be screened for
antimicrobic-modifying enzymes. A commercially available chromogenic
disk method (Cefinase; Becton Dickinson Microbiology Systems,
Cockeysville, Md.) is frequently used as a rapid procedure to detect
-lactamase production in Staphylococcus spp.,
Haemophilus influenzae, and Bacteroides spp. This
method relies on the visualization of a colored product that results
from hydrolysis of the substrate -lactam molecule, nitrocefin, contained in a paper disk.
Tissue culture methods, with either human or primate cells, have been
used to determine the susceptibility of herpesviruses and human
immunodeficiency virus (HIV) to antiviral agents. For herpesviruses,
the effects of antiviral compounds are assessed by cytopathic (plaque)
reduction assays or the quantitation of specific herpesvirus DNA. For
HIV, inhibition of reverse transcriptase activity or p24 antigen
production by antiretroviral agents is assessed after exposure of virus
cultured in human lymphocytes.
Currently, published standards exist for media preparation,
incubation parameters, and the interpretation of results for
disk diffusion, broth dilution, and agar dilution methods.
These standards are provided by the National Committee for
Clinical Laboratory Standards (NCCLS) and are available and updated
periodically for selected aerobic (44) and anaerobic
bacteria (42) and yeasts (43). Tentative NCCLS
guidelines exist for mycobacteria (41). No published
standards have been developed for gradient diffusion or genetic testing
for any microorganisms.
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GENETIC METHODS FOR ASSESSING ANTIMICROBIAL RESISTANCE |
Potential advantages of genetic susceptibility testing
methods.
For several reasons, genetic methods, compared to
conventional susceptibility methods, have the potential to provide a
more rapid and reliable assessment of antimicrobial resistance. (i) Genetic susceptibility testing methods can be performed directly with
clinical specimens obviating the need for isolation of the organism by
culture. (ii) These methods assess the genotype of the organism,
whereas conventional susceptibility techniques asses the phenotype or
expression of the genotype under artificial or laboratory conditions.
Although debate exists among authorities as to which of these
assessments is more clinically relevant, it seems reasonable that the
lowest-risk approach for the patient is to determine the genotype. This
may be especially true if one is dealing with serious life-threatening
infections such as meningitis or bacteremia or infections such as
endocarditis or osteomyelitis, which require prolonged courses of
antimicrobial therapy. (iii) In some cases, genotypes may be discerned
long before phenotypes can be determined due to the slow growth of the
organism. (iv) Some organisms cannot be cultured or are not easily
cultured and so only genotypes can be determined in these cases. (v)
Genetic methods may lessen the biohazard risk which may occur with the propagation by culture of a microorganism, a requirement for
conventional test methods.
Disadvantages of genetic testing methods.
Several
disadvantages exist for genetic methods compared to conventional
phenotypic methods. (i) They may lack sensitivity when only a few
organisms are present in a sample. Refinements in techniques for
concentrating nucleic acids in large volumes of clinical specimens
hopefully will address this problem. (ii) Different assays are required
for each antimicrobial agent tested. (iii) Resistance of a
microorganism to a specific antimicrobial agent may occur via different
mechanisms associated with different resistance genes or a large array
of single or coincidental mutations. Pertinent to this and the prior
point, is the concept that "with genetic testing methods, one only
gets what one specifically is looking for." This is in contrast to
culture-based methods which are more comprehensive in assessing
antimicrobial resistance. That is, by using the same culture-based
assay, different forms of resistance can be detected. By virtue of the
capacity to survey for different forms of resistance, culture-based
methods also are useful for detecting emerging or new forms of
antimicrobial resistance. (iv) A genetic mechanism for resistance
for some antimicrobics may not have been defined. (v) Genetic methods
may detect resistance genotypes that are expressed at levels that may
not be clinically relevant. Examples of this include low-level
vancomycin resistance encoded by vanC genes and poorly
expressed extended-spectrum -lactamase resistance. (vi)
False-positive results may occur due to contamination of the test
sample with extraneous nucleic acid or residual nucleic acid from prior
samples. This problem is of particular concern when nucleic acid
amplification techniques such as PCR are used. However, the development
of enzymatic and chemical sterilization methods for nucleic acid
amplified by PCR has greatly enhanced the specificity for this
technique (14). (vii) Unlike for conventional culture-based
susceptibility test methods, no standards exist for performing genetic
testing methods. (viii) Multicenter, controlled clinical trials to
assess the accuracy, reproducibility, and clinical utility of these
methods have only been conducted for a few of these tests.
Evaluation of genetic testing methods for introduction into the
clinical laboratory.
Clinical microbiologists should critically
evaluate the advantages and disadvantages of genetic versus
conventional methods before changing test menus. Important in this
process are assessments of performance characteristics; turnaround time
for results, costs related to reagents, royalties or licensing fees;
and personnel time. Genetic susceptibility tests may be "add-ons,"
their introduction may not obviate conventional testing methods. An
example of this is mecA gene detection for staphylococci.
The mecA gene encodes a penicillin-binding protein,
designated 2a or 2', which has decreased affinity for methicillin and
related compounds. Because methicillin resistance may result from
mechanisms other than the mecA gene (e.g., hyperproduction
of -lactamase), conventional susceptibility testing is still
required for mecA-negative staphylococcal isolates. Also, if
the result for the mecA gene analysis is negative, testing for susceptibility to penicillin must still be performed, because for
-lactamase-negative strains, penicillin remains the therapy of
choice. In this situation, determination of penicillin susceptibility is important as this compound is more effective therapeutically and is
cheaper than -lactamase stable penicillins (nafcillin, oxacillin,
dicloxacillin, and cloxacillin). Thus, for each staphylococcal strain,
both a genetic and conventional testing method may ultimately be
required before a final susceptibility report can be issued.
The above is one example of how a genetic test for determining
antimicrobial resistance could be incorporated into the testing repertoire of a clinical microbiology laboratory. It is not yet possible to determine how or if any of the genetic tests covered in the
subsequent discussion in this review will fit into either routine or
reference laboratory test protocols. As the technology continues to
evolve, it is likely that more user-friendly testing formats based on
the principles of some of these tests will be developed. This should
make the technology more easily adaptable by small hospital-based
laboratories as well as large reference laboratories.
Genetic mechanisms of antimicrobial resistance.
Antimicrobial
resistance may occur as the result of intrinsic or acquired genetic
material. Furthermore, intrinsic or acquired genetic material may
undergo changes, usually single base mutations, which may affect the
spectrum of antimicrobial resistance.
In Table
1,
organism-antimicrobial agent combinations are shown for which genetic
mechanisms for or genetic associations
with antimicrobial resistance
have been defined. This is not meant
to be comprehensive summary,
rather it is limited to those organism-antimicrobial
agent combinations
for which conventional phenotypic susceptibility
test methods lack
accuracy, are technically difficult to perform,
and/or require
significant time periods before results are available.
Furthermore, for
these organism-antimicrobic combinations, specific
genetic testing
methods have been reported to be useful. These
methods are also
provided in Table
1. For general bacteria, these
combinations include
the assessment of methicillin resistance
in coaglase-negative
Staphylococcus species (
4,
18,
28,
29,
35,
36,
77,
78), low-level vancomycin resistance
in
Enterococcus
species (
46), and extended-spectrum
-lactamase
resistance
in gram-negative facultatively anaerobic bacteria such
as
Escherichia coli and
Klebsiella pneumonia
(
2,
45). For
Mycobacterium tuberculosis, genetic
methods have been reported
for detecting resistance to isoniazid
(
7,
19,
21,
34,
37,
39,
52,
61,
66,
69,
74,
76), rifampin
(
16,
39,
48,
51,
66,
67,
68,
79,
81,
82), streptomycin
(
39,
60) ethambutol (
59), pyrazinamide
(
58), and the fluoroquinolones
(
65). Detection of
mutations associated with resistance of herpes
simplex virus and herpes
zoster virus to acyclovir (
54,
56)
and of cytomegalovirus to
ganciclovir (
57) have also been described.
Assessments of
mutations in the reverse transcriptase (
31,
50,
64) and
protease (
8,
22,
75) genes of HIV have been reported
to be
convenient means by which resistance to reverse transcriptase
and
protease inhibitors can be determined. One must be cautious,
however, in attributing all mutations in these genes to antiretroviral
resistance; further clinical studies are required to validate
these
associations. Although a direct mechanistic link between
nucleotide
sequences in hepatitis C virus (HCV) genomes and the
effect of
immunotherapy (interferon) has not been proven, such
an association
appears to predict response to this therapy (
9,
10,
33,
62,
83) and so this combination is included in
the Table
1. In the
subsequent discussion, examples of genetic
testing methods which have
been developed for these purposes are
described.
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DESCRIPTIONS OF REPORTED GENETIC SUSCEPTIBILITY TESTING METHODS |
Most genotypic methods include an initial step where the
"target" nucleic acid is amplified. This is usually accomplished by
PCR. Other less frequently used methods which amplify either target
nucleic acid or nucleic acid contained in probes annealed to target
nucleic acid include self-sustaining sequence replication, strand
displacement amplification, ligase chain reaction, and Q replicase
amplification. The signal generated from probes annealed to target
nucleic acid can also be amplified; an application of this method,
branched DNA (bDNA) assay, is covered in subsequent discussion. The
reader is referred to a review by Podzorski and Persing (49)
which provides detailed descriptions of the nucleic acid amplification
techniques. The product of the PCR, referred to as an amplimer or
amplicon, can be confirmed as the desired target nucleic acid (i.e.,
part or all of a resistance-associated genetic material) by
electrophoretic mobility determinations, probe hybridization assays
(Southern blotting of electrophoretic gels, slot, dot blot,
enzyme-linked immunosorbent assay, or liquid hybridization formats),
restriction fragment length polymorphism (RFLP) analysis, or DNA
sequencing formats. Amplicons can also be assessed for specific
mutations associated with antibiotic resistance by direct DNA
sequencing methods and RFLP, single-strand conformation polymorphism
(SSCP), dideoxy fingerprinting (ddF), Cleavase fragment polymorphism
(CFLP), RNase cleavage, heteroduplex, line probe, molecular beacon, or
microchip oligonucleotide array assays.
Detection of antimicrobial resistance genes. (i) PCR amplification
of target DNA and amplicon confirmation by gel electrophoresis, probe
hybridization techniques, or DNA sequencing.
Standard PCRs with
amplicon sizing by gel electrophoresis are especially useful for
identifying genes which encode antimicrobial resistance (18, 77,
78). These assays are highly specific, especially if there are no
other nucleic sequences harbored by the organism which share
significant homology with the target genetic material and large
quantities of target nucleic acid are amplified. The latter condition
exists when organisms are first propagated by culture and then isolated
colonies are used for the PCR. Amplifying such large quantities of
target nucleic acid reduces the sensitivity requirements for such
assays, making it less likely that contamination with extraneous
nucleic acid will be a significant problem. Therefore, provided that
negative controls are used, specialized contained specimen processing
areas and/or amplicon "sterilization" may be unnecessary. Amplicons
can also be sterilized, that is, chemically or enzymatically modified, such that they cannot serve as a template for subsequent PCR assays which use the same oligonucleotide primers.
Figure
1 shows an example of a multiplex
PCR method used in our laboratory to detect the
mecA gene
from isolated staphylococcal
colonies which have been propagated by
standard culture methods
(
18). Two distinct PCR amplicons
are produced simultaneously
in the same reaction tube, hence the term
multiplex PCR. In one
reaction, a portion of the
mecA gene
is amplified. For the other
PCR, a nucleic acid sequence of the 16S
rRNA gene unique to staphylococci
is amplified. The second PCR assay
confirms that the bacterium
amplified is a staphylococcus. The PCR
amplicons are electrophorized
through an agarose gel, and their sizes
are determined by comparing
them to standard DNA fragments. If no bands
are produced, either
the organism tested is not a
Staphylococcus species or the PCR
did not occur. A positive
result should produce amplicons of the
appropriate size for
mecA and 16S ribosomal DNA (16S rDNA) sequences
based on the
oligonucleotide primers used for PCR. Additional
confirmatory steps not
shown in Figure
1 can be performed. The
mecA amplicon can by
hybridized to labeled robes (signal hybridization),
digested with
restriction endonucleases (RFLP analyses), or the
DNA sequence can be
determined. Clinical evaluations for
mecA by PCR and
Southern blot hybridization (
35,
36) are listed
in Table
1
and are not covered in further detail. Also indicated
in Table
1 are
clinical studies that have used a chemiluminescent
(
28) or
dot blot hybridization format (
4,
35) to detect
the
mecA from cultured staphylococci. The reader is referred to
comprehensive discussions on signal hybridization formats by Podzorski
and Persing (
49) and Tenover and colleagues (
70).
RFLP analyses
and DNA sequencing for other organism-antimicrobic
combinations
are discussed subsequently in greater detail.
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FIG. 1.
Multiplex PCR and gel electrophoresis for identifying
the mecA gene in Staphylococcus spp. Two distinct
PCRs were performed: one amplified a portion of 16S rDNA unique to
Staphylococcus spp., and the second amplified a segment of
the mecA gene. Lane 1, strain of methicillin-resistance
S. aureus; electrophoretic bands of the appropriate size
appear for the 16S rDNA and mecA. Lane 2, methicillin-susceptible S. aureus; an electrophoretic band
for 16S rDNA is present but there is no band for mecA. Lane
3, negative control (reagents only), lane 4, DNA fragment standards.
bp, base pair.
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Multiplex PCR assays, like that described for the
mecA gene,
are relatively easy to perform and can be completed in 6 to 8
h.
The approximate cost for reagents and supplies for each sample
is less
than $5. An initial capital outlay for a thermocycler
(~$5,000 to
$10,000) and gel electrophoresis equipment (~$3,000)
is required. The
multiplex
mecA assay is more sensitive than current
culture-based methods for detecting methicillin resistance related
to
the
mecA gene, especially for coagulase-negative
staphylococci
(
4,
18,
28,
29,
35,
36,
78). However, the
turnaround
time for results may be similar if this molecular assay is
performed
from isolated colonies of bacteria. Recently, Ubakata and
colleagues
(
72) and Kitagawa and colleagues (
26)
have performed
mecA assays by using PCR directly with broth
from blood culture bottles.
Direct detection of
mecA-associated resistance in blood cultures
should
appreciably decrease turnaround time compared to that of
culture-based
methods. Substances that inhibit PCR, which are
present in blood
culture medium, notably sodium polyanethol sulfonate
(SPS), may affect
the sensitivity of these assays and methods
to remove SPS should be
employed (
17).
(ii) bDNA assay.
The bDNA assay is a non-PCR-based signal
amplification method that is commercially produced by Chiron
Diagnostics (Norwood, Mass.). It has been used to determine blood
levels (viral loads) of HIV (25) and HCV (12).
Our laboratory has adapted this assay to detect the mecA
gene directly from blood cultures containing staphylococci
(84). The bDNA method depends on the amplification of a
signal molecule and not the target nucleic acid (Fig.
2). Signal generation is proportional to
the amount of mecA gene sequences in the sample, which in
turn is proportional to the number of staphylococcal cells analyzed. We
have found the mecA bDNA assay to be as accurate as
conventional PCR assays for detecting the mecA gene
(29, 84). The procedure is partly automated but requires
approximately 6 to 8 h to complete. Performing the test directly
from blood culture broth should permit a decrease in turnaround time
for methicillin susceptibility results for staphylococci. At this time,
the bDNA assay for mecA detection is not commercially available and therefore reagent and instrument costs remain unknown.
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FIG. 2.
bDNA method for identifying the mecA gene in
Staphylococcus spp. A generic representation of the bDNA
method, which has been used for detection of the mecA gene,
is shown. DNA is released from staphylococcal cells and hybridized to
both capture probes and target probes. bDNA molecules (amplifiers) are
then hybridized to target probes. Enzyme-labeled probes are
subsequently hybridized to the bDNA amplifier. A chemiluminsescent
substrate, dioxetone, is added and emitted light is measured. (Adapted
with permission from Chiron Diagnostics, Norwood, Mass.)
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(iii) PCR-RFLP analysis.
In RFLP analysis, the amplified DNA
is digested (fragmented) with restriction enzymes. These endonucleases
will only cleave DNA molecules at specific sites, i.e., unique short
sequences of nucleic acid. Therefore, if the sequence of the target DNA is known, RFLP analysis can be used to confirm the target DNA in its
amplified form.
Recently, we have reported the use of RFLP analysis to differentiate
among genes encoding vancomycin resistance (
46). Because
of
conserved DNA sequences which exist among vancomycin resistance
genes,
more than one vancomycin resistance gene may be amplified
with the same
PCR oligonucleotide primers. RFLP analysis then
can be used to
differentiate the vancomycin resistance gene amplicons,
providing that
sites for a restriction endonuclease are present
in one but not another
gene.
Figure
3A shows restriction fragments
produced with the enzyme
MspI for the vancomycin resistance
genes
vanA,
vanB,
vanC-1, and
vanC-2/3. Figure
3B shows the electrophoretic gel RFLP
patterns
obtained with
MspI and various control strain and
clinical isolates
of
Enterococcus spp. containing vancomycin
resistance genes. In
essence, the RFLP procedure results in "bar
codes" which specify
distinct genes associated with vancomycin
resistance. We have
found this technique to be particularly useful for
determining
vancomycin resistance associated with the
vanC
gene family as
this form of vancomycin resistance is not always
detectable by
conventional culture-based susceptibility methods
(
27). However,
caution must be taken when identifying
vanB and
vanC-2 by RFLP
analysis, as we have
noted significant DNA sequence variation
among these genes
(
47). Therefore, restriction sites may be
gained or lost or
PCR primers may be ineffective.
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FIG. 3.
PCR-RFLP analysis for identifying genes associated with
vancomycin resistance in Enterococcus spp. (A) Six primers
are used in a multiplex reaction to generate four PCR products.
Restriction fragment lengths predicted for vancomycin resistance genes
vanA, vanB, vanC-1, and
vanC-2/3 with the restriction enzyme (endonuclease)
MspI are shown. (B) RFLP electrophoretic patterns obtained
for various control strains and clinical isolates of
Enterococcus sp. containing vancomycin resistance-associated
genes. Lanes 2, 3, and 10, no restriction fragments produced; lanes 6 and 11, vanA pattern, lanes 1,4, 5, 7-9, 12, and 13 vanB patterns: lane 14, vanC-1 pattern; lane 15, vanC-2 pattern; lane 16, restriction fragment pattern
unrelated to vanA, vanB, and vanC;
lane 17, DNA fragment standards. (Adapted with permission from Patel et
al. [46].)
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A potential advantage of the RFLP analysis is that PCR amplicons may be
digested by a restriction enzyme to the extent that
full-length
amplicons are no longer available for subsequent PCR
assays. Therefore,
the amplicons are sterilized. The PCR-RFLP
procedure can be performed
in 6 to 8 h. The approximate cost for
reagents and supplies for
each test sample is less than $5. The
only additional reagent expense
beyond that required for simple
PCR-gel electrophoresis assays is the
cost of restriction enzymes.
Like PCR-gel electrophoresis assays, a
thermocycler and electrophoresis
equipment are
required.
Detection of mutations associated with antimicrobial resistance.
(i) PCR-RFLP analyses.
RFLP analysis is also useful for
identifying mutations associated with antimicrobial resistance. We and
others have shown that point mutations in the catalase-peroxidase
(katG) gene of M. tuberculosis are associated
with isoniazid resistance (5, 7, 19, 21, 34, 37, 39, 52, 69, 74,
76). Two of these mutations, which occur with significant
frequency, are located in codons 315 (serine 
threonine) and 463 (arginine leucine) and can be identified by using the restriction
endonuclease MspI (Fig. 4)
(19, 34, 39, 74). For each of these mutations, different
restriction fragments are produced by MspI digestion of
katG amplicons compared with those of the wild-type
katG amplicon. These fragments result from the gain of a
MspI restriction site at the 315 codon or the loss of an
MspI restriction site at the 463 codon in the respective
mutant alleles compared with the wild-type allele. Therefore, specific
DNA sequence interrogation in limited regions of katG is
possible by virtue of RFLP analysis.
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FIG. 4.
PCR-RFLP analysis for identifying isoniazid
resistance-associated mutations in M. tuberculosis. (A)
Restriction fragment length predicted for two mutations (codons 315 [serine threonine] and 463 [arginine leucine]) in the
katG gene of M. tuberculosis with the restriction
enzyme MspI. These mutations are associated with high- and
low-level isoniazid resistance, respectively. W+, wild
type. (Adapted with permission from Uhl et al. [74].)
(B) RFLP electrophoresis patterns obtained for M. tuberculosis strains. Lanes 1 and 20, DNA fragment standards;
lanes 2 and 10, DNA from strains with both the R463L and S315T
mutations; lanes 5, 9, 16, 18, and 19, DNA from strains with the R463L
mutation only; lanes 13 and 15, DNA from strains with the S315T
mutation only. The remaining lanes have neither of these mutations. bp,
base pair. (Adapted with permission from J. Uhl et al.
[74].)
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We have recently demonstrated that this technique is useful for
detecting
M. tuberculosis complex and isoniazid
resistance-associated
mutations directly from sputum specimens in 1 working day (
73).
RFLP analyses have also been demonstrated
to be useful for identifying
mutations associated with streptomycin
resistance which occur
in genes encoding 16S rRNA (
rrs) and
ribosomal protein S12 (
rspL)
in
M. tuberculosis
(
39), for classifying genes encoding TEM-
(
2) and
SHV-type (
45) extended-spectrum
-lactamases in
gram-negative
bacteria, for identifying mutations in the protease gene
of HIV
(
75), and for determining the genotype and subtype of
HCV (
9,
10).
Nucleic acid polymorphisms may be degenerate. That is, as part of a
codon, they may encode for the same amino acid and so
different base
pair changes may result in the same amino acid
change. In this case,
RFLP analyses using the same restriction
enzyme may not detect
different base pair changes yet the end
result is a resistance
genotype. Therefore, negative results for
RFLP analyses should be
backed up with other susceptibility test
methods.
(ii) PCR-SSCP analyses.
The technique of PCR-SSCP was first
used to detect single nucleotide substitutions in human genes
associated with hereditary disorders such as cystic fibrosis
(11) and phenylketonuria (13, 30). Several
investigators have reported on the utility of the PCR-SSCP method for
identifying mutations in genes encoding SHV-type extended-spectrum
-lactamases in the family Enterobacteriaceae (15,
38) and in genes of M. tuberculosis associated with
resistance to isoniazid (21, 52, 66, 69, 76), rifampin
(16, 55, 66, 67, 68, 79), ethambutol (59), and
the fluoroquinolones (65).
For this method, mobility shifts in high-resolution nondenaturing
polyacrylamide gels are discernible for single-stranded
mutated DNA
versus wild-type DNA. After amplification of the target
nucleic acid by
PCR, it is denatured to a single strand and then
electrophoresed.
Mutations are inferred by the appearance of bands
at positions
different to those observed with the wild-type strain
(Fig.
5). We have determined that the size of
the PCR product
is a critical factor for resolving differences of the
denatured
single-stranded DNA. In our hands, the optimal PCR product is
in the range of 310 to 320 bp (
69). When we limited the PCR
product to this size range, we noted that the sensitivity and
specificity of the PCR-SSCP method compared to those for PCR-RFLP
analysis for detection of the 463 codon
katG mutation
(arginine-leucine)
in
M. tuberculosis were 100%
(
69).
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FIG. 5.
PCR-SSCP analysis to identify isoniazid
resistance-associated mutations in M. tuberculosis.
Representative PCR-SSCP results for three M. tuberculosis
strains, one clinical strain with the R463L mutation (lane 1), one
clinical strain with the wild-type codon 463 (R463) (lane 2), and the
control strain H37Rv also containing the wild-type codon 463 (R463)
(lane 3). The arrows indicates the band position corresponding to the
R463L mutation. (Adapted with permission from Temesgen et al.
[69].)
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Sarkar and colleagues, using the PCR-SSCP method for mutation detection
in the factor 1X gene in humans, also noted a lack
of sensitivity,
especially when greater lengths of target DNA
were analyzed
(
53). They subsequently developed a hybrid technique
between
dideoxy (Sanger) sequencing and SSCP, termed ddF, which
provided a more
efficient detection of mutations independent of
the amplified product.
This technique identifies DNA sequence
changes by producing a ladder of
bands with one of the four standard
dideoxy sequencing reactions and
then resolving the products on
a nondenaturing polyacrylamide gel. In
our laboratory, Felmlee
and colleagues used this ddF method to
determine rifampin resistance-associated
mutations in clinical isolates
of
M. tuberculosis (
16). When
ddF results were
compared with those for SSCP analysis, they noted
that ddF results were
more easily interpreted and contained more
sequence-dependent
information that facilitated differentiation
of functionally important
versus silent mutations. The ddF method
requires the equipment costs
(purchase or lease) for an automated
DNA sequencer (purchase
~$100,000 to $120,000). The approximate
cost of reagents and supplies
per sample is less than $10 in addition
to baseline expenditures for a
thermocycler and electrophoresis
equipment.
A drawback of the conventional SSCP method has been the use of
radioactivity for identifying amplification products. This
has been
obviated by either silver-staining methods (
30,
69)
or
labeling PCR products with fluorescein (
68). Furthermore,
Telenti and colleagues have demonstrated that fluorescein-labeled
products can be evaluated on automated DNA sequencers, which should
make SSCP analysis a more acceptable testing method for the clinical
laboratory (
68). Another modification of the SSCP method by
Selvakumar and colleagues (
55) permits better visualization
of the shifts in migration of single strands. This is accomplished
by
biotinylation of one of the PCR primers. One biotinylated stand
of the
PCR product is produced which is separated from the unbiotinylated
strand by using streptavidin magnetic
beads.
A recent report by Victor and colleagues emphasizes the importance of
the positioning of PCR oligonucleotide primers used
to produce
amplified products for SSCP analysis (
76). In cases
where
multiple PCR products are analyzed by SSCP, overlap of amplified
sequences is essential to assure that mutations are not missed
which
are near the end of the PCR products, i.e., in the regions
where PCR
primers anneal. In this case, the S315T mutation in
katG
sequences of
M. tuberculosis strains was not detected due
to
primer placement. Although this example relates to SSCP analysis,
it
may have relevance to other PCR-based
assays.
The cost for reagents and supplies for the manual SSCP method is less
than $10 per sample, although expenses for a thermocycler
and
electrophoresis equipment must be considered. If a DNA sequencer
is
used for either the SSCP or ddF technique, savings in personnel
time
should be realized; however, capital outlay for this instrument
may be
substantial. Both the SSCP and ddF techniques, in our hands,
require
approximately 2 days for
completion.
(iii) PCR-CFLP analysis.
A genetically engineered
structure-specific endonuclease, Cleavase I, has been used to detect
mutations in the katG (5) and rpoB
(20) genes of M. tuberculosis and to determine
HCV genotypes (33). The Cleavase I enzyme recognizes the
folded structures which DNA strands form after denaturation and cleaves at the junction of the single-stranded and duplexed areas (Fig. 6). After the fragments generated by
Cleavase are separated by denaturing gel electrophoresis, unique
polymorphisms occur with different mutations.
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FIG. 6.
PCR-CFLP for identifying isoniazid resistance-associated
mutations in M. tuberculosis. (A) Schematic representation
of steps of CFLP pattern generation. Labeled fragments of DNA are
heated to separate complementary strands. When the samples are cooled,
the single strands of DNA assume folded hairpin-like structures; subtle
differences in the sequence of the fragments can cause formation of
different structures. The Cleavase I enzyme cleaves at the 5' side of
these structures, at the junction between duplexed and single-stranded
regions. Separation and detection of the resulting fragments create
signature banding patterns that can be compared to detect differences
between the test molecules. (Adapted with permission from Brow et al.
[5].) (B) Identification and positioning of mutations
associated with isoniazid resistance in M. tuberculosis. For
the gel on the left, amplicons generated with a 5' labeled primer and
an unlabeled primer were heat denatured for 15 s, rapidly cooled
to 60°C, and digested with 25 U of Cleavase I for 2 min. The results
are shown, with M indicating DNA size markers. Variant bands
distinguishing mutant from wild-type (WT) DNA are marked A and B. Shown
on the right is a second gel electrophoresis which was performed to
expand the 500- to 600-nt region. Bands C in the wild type and the
R463L mutant are similar, while the same band migrates faster in
variants containing the S315T mutation. (Adapted with permission from
Brow et al. [5].)
|
|
So far, this method has had limited use. According to the manufacturer,
it will be commercially available in the future in
kit form (Cleavase);
Third Wave Technologies, Inc., Madison, Wis.)
which should be easily
adaptable to most laboratories. The total
cost beyond that for a
thermocycler and electrophoresis equipment
is yet undetermined but the
turnaround time for results appears
to be less than 1 working
day.
(iv)PCR-RNA/RNA duplex RNase cleavage assay.
Nash and
Inderlied have recently used a PCR-RNA/RNA duplex RNase cleavage assay
to detect mutations in the 23S rRNA gene of Mycobacterium
avium complex which are associated with macrolide resistance
(40). The method is based on the ability of RNase 1 and
RNase T1 to cleave mismatches (Fig. 7).
Duplex RNA is transcribed from PCR products (test strain and wild-type
strain) generated with primers containing opposing phage RNA polymerase
promoters (T7/T7 or T7/Sp6). Following hybridization and RNase
treatment, cleavage products of RNA/RNA duplexes occur due to
mismatches. Mismatches are the result of mutation in the test strain
compared with the wild-type strain.
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|
FIG. 7.
PCR-nonisotopic RNase cleavage assay. Commercially
available as the Mutation Screener assay (Ambion, Inc.) the
PCR-nonisotopic RNase cleavage assay is based on the ability to
selectively cleave unpaired bases in RNA/RNA duplexes. Duplex RNA is
transcribed from PCR products generated with primers containing
opposing phage RNA polymerase promoters (T7/T7 or T7/Sp6). Following
hybridization and RNase treatment, the RNA/RNA cleavage products are
analyzed by nondenaturing gel electrophoresis and detected by ethidium
bromide staining. In the example shown, mismatches occur due to an
opposing uracil (U) and guanine (G) and an opposing cystosine (C) and
adenine (A) with the adenine representing a point mutation in the test
strain. (Adapted with permission from M. Goldrick, Ambion, Inc.)
|
|
This method is commercialized and is available in kit form (Mutation
Screener; Ambion, Inc., Austin, Tex.). It is relatively
easy to perform
but requires a longer time for completion than
a PCR alone. Nash and
Inderlied note that in their hands the assay
can be completed in about
1 working day and that the cost (including
reagent, supplies and
personnel time) is less than $50 if one
or more strains are tested
simultaneously. Of course, added to
this are expenditures for a
thermocycler and electrophoresis
equipment.
(v) PCR-universal heteroduplex generator analysis.
Williams
and colleagues have used PCR-universal heteroduplex generator analysis
to detect mutations in the rpoB genes of M. tuberculosis (80, 81, 82). DNA from a universal
heteroduplex generator (Fig. 8) and the
test strain are denatured simultaneously in the same reaction mixture
and then allowed to reanneal. The heteroduplex generator in the
hypothetical example in Fig. 8a has a 4-bp deletion. When separate
strands of DNA from the heteroduplex generator and the test strain
reanneal, four separate hybridizations can occur. Two of these
hybridizations (heteroduplexes) will result in double-stranded DNA with
a "bubble" occurring where no base-pair matches can hybridize.
These hybrids will migrate as a single band when electrophoresed in a
high-resolution gel. Two other bands will occur due to hybridization
reactions (homoduplexes) which result in the formation of the
double-stranded DNA of the heteroduplex generator and the test strain.
If mutations are present in the test strain, the positions of the
homoduplexes may vary compared to those of homoduplexes formed when no
mutations are present. This assay requires the same technical expertise
and time to complete (about 1 working day) as RFLP assays. Once a heteroduplex generator is produced, the cost for reagents and supplies
and instruments is similar to that for RFLP assays. A thermocycler and
gel electrophoresis equipment are required, and the approximate cost
for reagents and supplies for each test sample is less than $5.
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FIG. 8.
PCR-heteroduplex analysis to identify mutations
associated with rifampin resistance in M. tuberculosis. (A)
Hypothetical example of the universal heteroduplex generator technique.
Different bands are produced in the polyacrylamide gel electrophoresis
minigel depending on whether the test strain is susceptible or
resistant to rifampin. UHG, universal heteroduplex generator; RIF-S,
rifampin-susceptible test strain; RIF-R, rifampin-resistant test
strain; BP, base pair; ROP, RNA polymerase subunit gene.
(Adapted with permission from D. Williams.) (B) In this example, a
heteroduplex generator is used which contains four 3-bp deletions and
three 2-bp substitutions. The presence of rifampin-susceptible M. tuberculosis is indicated by a four-band pattern. Two homoduplexes
migrate in the gel at the equivalence of 181 (the universal
heteroduplex generator double-stranded DNA) and 193 bp (the test sample
PCR- generated double-stranded DNA). Two heteroduplexes migrate at the
equivalence of 470 and 519 bp (each a hybrid of complementary strands
of one strand of DNA from the heterodulex generator and on strand from
the test sample DNA). The first five lanes (from left to right) contain
rifampin-resistant strains, the next two lanes contain
rifampin-susceptible strains, and the last lane contains molecular
weight standards. (Adapted with permission from D. Williams.)
|
|
(vi) PCR-LiPA.
The PCR-line probe assay (LiPA) (Fig.
9) was first developed by Stuyer and
colleagues to discriminate types and subtypes of HCV (62). A
reverse transcriptase procedure followed by PCR is used to amplify HCV
genetic material. The LiPA assay, which is based on the reverse
hybridization principle, is then performed. Biotinylated PCR fragments
are hybridized to a selection of highly specific immobilized probes.
The biotin group in the hybridization complex is then revealed by
incubation with a streptavidin-alkaline phosphatase complex and the
appropriate chromogen compounds.
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FIG. 9.
PCR-LiPA. Biotinylated PCR fragments are hybridized to a
selection of highly specific probes immobilized on a nitrocellulose
strip. The biotin group in the hybridization complex is revealed by
incubation with a streptavidin-alkaline phosphatase complex and the
chromogen compounds 5-bromo-4-chloro-3-indolylphosphate (BCIP) and
nitroblue tetrazolium (NBT). (Figure provided by F. Shapiro,
Innogenetics.)
|
|
A second-generation LiPA assay was recently demonstrated to be highly
sensitive and specific for detecting signature sequence
motifs in HCV
(
63). The assay has also recently been used to
detect
mutations in the reverse transcriptase gene of HIV (
64)
and
rifampin resistance-associated mutations in
M. tuberculosis (
51).
According to the manufacturer (Innogenetic, Ghent, Belgium), the assay
can be performed in about 1 working day and should
be adaptable to most
clinical laboratories. The cost per test
sample is approximately $80.
Furthermore, a baseline expense for
a thermocycler is
required.
(vii) PCR-molecular beacon sequence analysis.
Piatek and
colleagues (48) have recently used PCR-molecular beacon
sequence analysis to detect mutations in the rpoB gene of
M. tuberculosis associated with rifampin resistance.
Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions (71) (Fig. 10). After these
oligonucleotides anneal to their targets, they undergo a conformational
change that restores the fluorescence of an internally quenched
fluorophore.
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FIG. 10.
Principles of molecular beacons. The molecular beacon
in its hairpin form shown on the left is nonfluorescent because the
stem hybrid keeps the fluorophore close to the quencher. When the probe
sequence in the loop hybridizes to its target, forming a rigid double
helix, a conformational reorganization occurs that separates the
quencher from the fluorophore, restoring fluorescence. (Adapted with
permission from Tyagi et al. [71].)
|
|
This method is not yet commercially available. The cost per test sample
is unknown; however, a thermocycler is required. Detection
of color
changes can be done manually, which make the assay particularly
appealing for use in developing
countries.
(viii) DNA oligonucleotide arrays on silicon microchips.
A promising new technology involves the synthesis of DNA probes
directly onto silicon microchips (Fig.
11) (6, 32). At present,
more than 50,000 oligonucleotide probes can be synthesized directly
onto these chips. In one method developed for HIV antiviral-associated resistance mutations, RNA is first reverse transcribed to cDNA. cDNA is
subsequently transcribed into RNA which is also biotin labeled. These
labeled RNA molecules are then hybridized to complementary probes which
have been synthesized directly onto silica microchips.
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FIG. 11.
Silicon microchip assay for detection of mutations in
the HIV genome associated with resistance to reverse transcriptase or
protease inhibitors. (a) A schematic of the commercially available
GeneChip HIV PRT Assay (Affymetrix, Santa Clara, Calif.) is shown.
Viral RNA is reversed transcribed to cDNA which is then transcribed
into RNA, labeled, and fragmented. These labeled RNA fragments are then
hybridized to complementary probes synthesized directly onto a
microchip. The hybridized array is then scanned to detect positive
hybridization signals in the form of emitted light. (Adapted with
permission from L. Constantine, Affymetrix.) (b) Example of DNA
microchip array image. (Adapted with permission from L. Constantine,
Affymetrix.)
|
|
Such matrix hybridization formats have great potential for rapid
comprehensive detection of resistance genes or mutations
associated
with antimicrobial resistance. This is especially true
if antimicrobial
resistance in a specific organism can be due
to multiple genes and/or
multiple mutations. Examples of this
include isoniazid resistance for
M. tuberculosis and resistance
to reverse transcriptase or
protease inhibitors for HIV, as illustrated
in Fig.
11. This technology
also has the potential for significant
automation and therefore should
be easily adaptable to most clinical
microbiology laboratories. The
cost for reagents and instrumentation
in addition to a thermocycler is
around $150,000.
(ix) Automated DNA sequencing.
DNA sequencing remains
the "gold standard" for identifying the products of amplification
reactions. DNA sequencing of PCR products has become a much cheaper and
faster method by virtue of automation (1, 3). Instruments
are now available for semi-automated running and analyzing of sequence
gels. Therefore, any resistance gene or resistance mutation can be
determined relatively easily and economically by direct DNA sequencing.
Musser and colleagues have published the results of a series of studies
which used automated DNA sequencing as the primary method to determine
antimicrobial resistance-associated mutations for a variety of drugs
and M. tuberculosis (24, 37, 58, 59, 60, 61). In
our institution, the cost for performing each test sample averages less
than $10, but the cost may vary significantly from one institution to
another. Initial expenditures for both a thermocycler and automated
sequencer are required.
| |
ACKNOWLEDGMENTS |
Members of the Division of Clinical Microbiology at the Mayo
Clinic (Rochester, Minn.) including David Persing, Glenn Roberts, Jon
Rosenblatt, and Thomas Smith are thanked for their critical review of
the manuscript. The following persons are recognized for their past or
ongoing collaborative efforts with conventional and genetic
antimicrobial susceptibility studies: David Persing, Glenn Roberts, Jon
Resenblatt, Peggy Kohner, and James Uhl of the Division of Clinical
Microbiology; Robin Patel, Zelalem Temesgen, and James Steckelberg of
the Division of Infectious Diseases; Bruce Kline, Frank Rusnak, and
Nancy Wengenack of the Department of Biochemistry and Molecular
Biology; Mayo Clinic; Diana Williams, Louisiana State University, Baton
Rouge, La; and Max Salfinger, New York State Department of Health,
Albany, N.Y. Finally, Roberta Kondert (Mayo Clinic) is thanked for her
efforts in preparing the manuscript.
| |
FOOTNOTES |
*
Mailing address: Division of Clinical Microbiology,
Mayo Clinic and Foundation, Rochester, MN 55905. Phone: (507) 284-2901. Fax: (507) 284-4272. E-mail:
cockerill.franklin{at}mayo.edu.
| |
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Introduction
Conventional methods for...
