National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Public Health Service,
U.S. Department of Health and Human Services, Atlanta, Georgia
 |
INTRODUCTION |
The National Antimicrobial
Resistance Monitoring System (NARMS) was initiated on 1 January 1996 as
a collaborative study conducted by the Centers for Disease Control and
Prevention (CDC), the Food and Drug Administration Center for
Veterinary Medicine, and 14 state and local health departments to
prospectively monitor the antimicrobial resistance of human
nontyphoidal Salmonella and Escherichia coli O157
isolates. In 1997, the study was expanded to include
Campylobacter isolates from five state health departments.
Two to four million persons are infected with Campylobacter
each year in the United States. Most of these infections are caused by
Campylobacter jejuni. Most Campylobacter
infections cause gastroenteritis, but invasive disease (e.g.,
bacteremia and meningitis) may also occur. While gastroenteritis caused
by C. jejuni usually is self-limiting, treatment with
antibiotics is often necessary for young children, pregnant women, or
immunosuppressed patients because of the possibility of greater
severity or duration of infection (11). Furthermore, antibiotics are essential for patients with invasive disease, which
occurs in approximately 1% of culture-confirmed infections. Fluoroquinolones, particularly ciprofloxacin, have been widely used for
the treatment of Campylobacter infections and for empiric treatment of patients with gastroenteritis, including traveler's diarrhea. In recent years, however, an increased proportion of Campylobacter isolates, both in the United States and other
countries, have been reported to be resistant to ciprofloxacin (2,
4, 8, 13, 15). In 1997, 13% of Campylobacter isolates
received at the CDC via the NARMS were ciprofloxacin resistant
(18).
In Campylobacter, E. coli, and other
gram-negative bacteria, fluoroquinolones work by interfering with DNA
gyrase, a type II topoisomerase that catalyzes the negative
supercoiling of relaxed or positively supercoiled, double-strand,
covalently closed circular DNA (5, 10, 14). Mutations in the
gyrA gene of gram-negative bacteria cause resistance to
fluoroquinolones by altering the amino acid sequence near the putative
active site of the GyrA protein (3). In E. coli,
the quinolone resistance determining region (QRDR) of the
gyrA gene, including codons 67 to 106, near the Tyr-122
catalytic site of DNA gyrase (16, 17), is the principal
location for mutations leading to quinolone resistance. A similar QRDR
exists in the C. jejuni gyrA gene, but the gyrA Tyr-125 residue appears to be the catalytic site involved in the transient, covalent DNA-protein bridge that forms during the DNA strand
passage process of DNA topoisomerization (3).
The purpose of this study was to investigate the type and frequency of
gyrA mutations in a sample of ciprofloxacin-resistant C. jejuni isolates, particularly those obtained in NARMS in
1997. Furthermore, we describe a mismatch amplification mutation assay (MAMA), which utilizes PCR technology to allow the rapid and specific characterization of gyrA mutations without performing DNA sequencing.
| |
MATERIALS AND METHODS |
Strains, culture conditions, and antimicrobial susceptibility
testing.
Twenty-five NARMS 1997 C. jejuni isolates, 12 ciprofloxacin resistant and 13 ciprofloxacin susceptible by E test,
were chosen for analysis; their CS (Campylobacter Study)
numbers were 5, 7, 8, 10, 17, 18, 34, 40, 42, 50, 54, 57, 58, 59, 61, 64, 71, 82, 89, 120, 133, 143, 161, 165, and 166. In addition, isolate
D4344, a CDC ciprofloxacin-resistant C. jejuni isolate from
1992, was included. Seven additional quinolone-susceptible control
Campylobacter spp. employed in this study were
Campylobacter jejuni subsp. jejuni ATCC 33560 (CDC number D133), D125, D135, and D2589 and Campylobacter jejuni subsp. doylei ATCC 49349 (CDC number D2295),
D3820, and D3836. The NARMS isolates and isolate D4344 were identified
to species level by the methods below. Quality control strains used for
antimicrobial susceptibility testing were E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923.
Campylobacter isolates for NARMS antibiotic resistance
screening were streaked for isolation on plates of Trypticase soy agar containing 5% sheep blood at 37°C in an atmosphere of ~5%
O2 for 48 h. Microaerophilic growth conditions were
created in a model A143 vacuum incubator (VWR Scientific Products,
Bridgeport, N.J.) by drawing a vacuum to 21 in. of mercury then
flushing the system with an anaerobic gas mixture (5% H2,
10% CO2, and 85% N2) until the vacuum was
lowered to, and maintained at, 5 in. of mercury. After incubation, the
plates were examined for characteristic morphology and motility by
dark-field microscopy, followed by biochemical tests for catalase,
oxidase, and hippurate hydrolysis. Heavy growth from each isolate was
suspended in Trypticase soy broth containing 20% glycerol and was
stored at 
70°C.
NARMS screening for antimicrobial resistance was performed by the use
of E-test strips (AB Biodisk, Solna, Sweden). Overnight growth of
campylobacters isolated on Trypticase soy agar containing 5% sheep
blood was suspended in sterile saline and was adjusted to a turbidity
matching a 0.5 McFarland standard. Sterile swabs were used to inoculate
plates of Mueller-Hinton agar containing 5% lysed horse blood. E-test
strips were placed on the plates, the plates were incubated at 37°C
for 48 h under the microaerobic conditions described above, and
the results were read according to the manufacturer's instructions.
With every test run, the American Type Culture Collection standard
strains were inoculated with E-test strips into duplicate sets of
Mueller-Hinton plates containing 5% lysed horse blood. One set was
incubated aerobically at 37°C and read at 18 h, while the other
set was incubated microaerobically at 37°C and read at 48 h.
The nalidixic acid and ciprofloxacin antibiotypes of all
Campylobacter isolates chosen for molecular characterization
were confirmed by agar dilution testing as described by the National Committee for Clinical Laboratory Standards (12). Overnight growth on Trypticase soy agar containing 5% sheep blood was suspended in sterile saline and adjusted to a turbidity matching a 0.5 McFarland standard. Aliquots (450 µl) of each saline suspension were pipetted into the seeding wells of a Cathra replicator (Oxoid, Inc., Nepean, Ontario, Canada). Freshly prepared plates of Mueller-Hinton agar containing 5% sheep blood and doubling dilutions of nalidixic acid
(Sigma Chemical Co., St. Louis, Mo.) or ciprofloxacin (Bayer Corp.,
Pharmaceutical Division, West Haven, Conn.) were then inoculated by
using 3-mm pins in the inoculating head of the replicator. The
inoculated plates were incubated at 37°C for 48 h in the
microaerobic environment generated by the use of three BBL CampyPaks in
one BBL Gas Pack 150 anaerobic jar (Becton Dickinson, Cockeysville, Md.).
DNA isolation, PCR, DNA sequencing, and nucleotide sequence
analysis.
Chromosomal DNA was isolated by using a Puregene DNA
isolation kit (Gentra Systems, Minneapolis, Minn.) according to the
manufacturer's instructions. The QRDRs of the gyrA genes of
the Campylobacter isolates were amplified by PCR. Primers
GZgyrA5 and GZgyrA6 (Table 1) were chosen for PCR amplification of a
673-bp product containing the QRDR of the gyrA gene of
quinolone-resistant and -susceptible isolates of C. jejuni
after analysis of the L04566 C. jejuni gyrA gene sequence in
GenBank (15). PCRs (100 µl each) contained 5 µl of
Puregene purified chromosomal DNA (~75 ng in sterile, deionized
H2O), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, a 200 µM
concentration of each deoxynucleoside triphosphate, 1.5 mM
MgCl2, 2.5 U of AmpliTaq DNA polymerase (The
Perkin-Elmer Corp., Foster City, Calif.), and 20 pmol of each primer.
PCR cycling conditions were as follows: initial denaturation at 94°C
for 3 min, followed by 30 cycles of 94°C for 1 min, 50°C for 1 min,
and 72°C for 1 min, with a final step at 72°C for 5 min. Primers
were synthesized at the CDC Biotechnology Core Facility.
Five-microliter aliquots of each PCR mixture were loaded onto
horizontal agarose gels and stained with ethidium bromide for analysis
after electrophoresis.
The remaining 673-bp gyrA PCR products were purified by the
use of QIAquick purification columns (QIAGEN, Inc., Hilden, Germany) for use in sequencing reactions. Taq dye terminator cycle
sequencing was performed with the ABI Prism dRhodamine Ready Reaction
Kit (Applied Biosystems). Nested primers GZgyrA7 and
GZgyrA8 (Table 1), which are internal to the 673-bp
gyrA PCR product, were used for sequencing. Labeled
sequencing reactions were purified by using CENTRI-SEP columns
(Princeton Separations, Adelphia, N.J.) before analysis on an Applied
Biosystems 377 automated sequencer.
Conserved primers were chosen with OLIGO Primer Analysis Software
version 5.0 (National Biosciences, Inc., Plymouth, Minn.). Mutation
primers were chosen after a manual analysis of the DNA sequence near
codon 86 of the C. jejuni gyrA gene of
fluoroquinolone-susceptible and -resistant isolates and the
C. jejuni L04566 gyrA DNA sequence in
GenBank (1, 15). DNA sequences were analyzed with DNASIS version 2.5 (Hitachi Software Engineering Co., Ltd., San Francisco, Calif.).
MAMA PCR protocol.
A conserved, forward primer,
CampyMAMAgyrA1, and a reverse, mutation detection primer,
CampyMAMAgyrA5 (Table 1), were used together in a PCR to
generate a 265-bp PCR product that was a positive indication of the
presence of the Thr-86-to-Ile (ACA
ATA) mutation in the C. jejuni gyrA gene. Primer GZgyrA4, a conserved reverse
primer, was used in conjunction with primer CampyMAMAgyrA1 to produce a positive PCR control product of 368 bp with any C. jejuni gyrA gene. PCRs (100 µl each) were the same as above
except the PCR cycling conditions were as follows: initial denaturation was at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 20 s. Twenty-microliter
aliquots of each PCR product were loaded onto horizontal, 2.0%, 0.5×
Tris-borate-EDTA agarose gels and were stained with ethidium bromide
for analysis after electrophoresis.
| |
RESULTS |
Detection of gyrA QRDR mutations associated with
quinolone resistance.
An examination of the gyrA DNA
sequence data, some of which are shown in Fig.
1, enabled us to determine the presence
or absence of amino acid mutations in the QRDR and to compare this
information with the nalidixic acid and ciprofloxacin MICs for the 1997 NARMS isolates (Table 2). All 13 ciprofloxacin-resistant isolates had a substitution at amino acid
position 86 of the GyrA protein due to a mutation of the DNA codon from
ACA (threonine) to ATA (isoleucine). Furthermore, one isolate had an
additional GyrA proline-to-serine mutation at amino acid position 104, but this mutation did not appear to dramatically alter the
ciprofloxacin or nalidixic acid MICs for this isolate when compared to
other ciprofloxacin-resistant isolates.
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FIG. 1.
Comparison of C. jejuni gyrA QRDR DNA
sequences. Sequence L04566 is a portion of the gyrA QRDR of
C. jejuni UA580 (GenBank no. L04566). 1997 NARMS isolates
CS5, CS34, and CS50 (bold type) have a Thr-86-to-Ile (ACAATA)
gyrA mutation frequently associated with fluoroquinolone
resistance. Isolate CS50 has an additional Pro-104-to-Ser (CCATCA)
mutation. Silent mutations were noted at amino acid positions 78, 81, 82, 92, 110, 117, 119, and 120. *, C. jejuni subsp.
jejuni ATCC 33560 type strain; **, C. jejuni
subsp. doylei ATCC 49349 type strain.
|
|
Detection of the Thr-86-to-Ile mutations (ACAATA) by use of MAMA
PCR.
A rapid PCR method was developed for the detection of the
Thr-to-Ile (ACA-to-ATA) mutation at amino acid position 86 in the DNA
sequence of C. jejuni isolates. Isolates with the
wild-type amino acid 86 codon (ACA, ciprofloxacin susceptible) were not amplified with the reverse mutation primer
CampyMAMAgyrA5, whereas the isolates with the mutated amino
acid 86 codon (ATA, ciprofloxacin resistant) generated a 265-bp
PCR product with the CampyMAMAgyrA5 reverse mutation primer
and the CampyMAMAgyrA1 forward conserved primer (Fig.
2). Conserved primers GZgyrA4
and CampyMAMAgyrA1 generated a 368-bp gyrA
PCR product with DNA isolated from all of the isolates listed in
this study. Primers used for MAMA PCR are listed in Table 1.
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FIG. 2.
Agarose gel of C. jejuni MAMA PCR products.
Lanes contain the following: A, 0.8 µg of a 100-bp DNA ladder
(Bethesda Research Laboratories, Inc., Gaithersburg, Md.); B (control
lane), no DNA plus CampyMAMAgyrA1 and GZgyrA4
conserved primers; C through E, DNAs from wild-type,
ciprofloxacin-sensitive C. jejuni subsp. jejuni
ATCC 33560, C. jejuni subsp. doylei ATCC 49349, and CS18, respectively, with conserved primer CampyMAMAgyrA1
and mutation primer CampyMAMAgyrA5; F (positive control
lane), C. jejuni subsp. jejuni ATCC 33560 DNA and
conserved primers GZgyrA4 and CampyMAMAgyrA1 (a
368-bp PCR product is generated); and G through K, DNAs from
Thr-86-to-Ile (ACAATA) gyrA ciprofloxacin-resistant
mutants CS5, CS8, CS34, CS143, and CS166, respectively, with conserved
primer CampyMAMAgyrA1 and mutation primer
CampyMAMAgyrA5 (a 265-bp PCR product is generated).
|
|
| |
DISCUSSION |
Fluoroquinolone resistance among Campylobacter isolates
tested in this study appears to have been caused by a
threonine-to-isoleucine mutation of amino acid 86 of the GyrA protein.
Although other mechanisms, such as changes in membrane permeability or
mutations in the parC and parE genes, may be
involved, this point mutation appears to be sufficient to cause
fluoroquinolone resistance. The relationship of the Thr-86-to-Ile
mutation to the acquisition of fluoroquinolone resistance in
Campylobacter isolates has been previously characterized
(2, 4, 13, 15). This mutation appears to be equivalent to
the serine-to-alanine mutation of amino acid 83 in the E. coli GyrA protein (2, 4, 7, 8, 13, 15). Other mutations
in the gyrA gene have also been reported to cause
fluoroquinolone resistance in C. jejuni isolates. Wang et
al. (15) reported C. jejuni Asp-90-to-Asn
(GAT-to-AAT) and Ala-70-to-Thr (GCC-to-TCC) gyrA mutations
in two laboratory mutants with MICs of 4 and 1 µg of ciprofloxacin
per ml, respectively. Ruiz et al. (13) reported a unique
C. jejuni Thr-86-to-Lys gyrA mutation from a
clinical isolate with a ciprofloxacin MIC of 
16 µg/ml and a
nalidixic acid MIC of 128 µg/ml. Eleven other C. jejuni
isolates from the same study (13) had Thr-86-to-Ile
gyrA mutations resulting in similar ciprofloxacin and
nalidixic acid MICs, but the actual DNA sequence was not included for
either type of Thr-86 mutation. In addition, Ruiz et al. mention
"transitions" at codon 119 of the gyrA gene, but since
they were present in eight fluoroquinolone-resistant strains and nine
fluoroquinolone-susceptible strains, they appear to be irrelevant to
the matter of fluoroquinolone resistance phenotypes (13).
Factors other than gyrA QRDR mutations, such as mutations in
gyrB, parC, and parE and efflux pumps,
permeability factors, or detoxification (3), may be
responsible for some of the variability in nalidixic acid and
ciprofloxacin MICs among groups of resistant and susceptible isolates
in our study.
In addition to the Thr-86-to-Ile mutation, several silent mutations
were evident in the gyrA QRDR of fluoroquinolone-resistant and -susceptible C. jejuni isolates. While most of the
silent mutations appear to be somewhat random, the silent mutations at gyrA codons 82, 110, and 117 may be conserved, because all
of the isolates with these mutations were C. jejuni subsp.
doylei isolates. Also, 11 of the 12 ciprofloxacin-resistant
C. jejuni isolates from the NARMS study shared the same DNA
sequence in the QRDRs of their gyrA genes as NARMS isolates
CS5 and CS34, a sequence which is unlike the DNA sequence of any of the
other C. jejuni isolates of either subspecies examined.
Isolates with this particular gyrA QRDR DNA sequence were
received from five state health laboratories, indicating the
distribution of isolates with this DNA sequence is not localized.
Although the number of isolates examined in this study is too small to
draw any definite conclusions, comparison of gyrA QRDR
sequences suggests the possibility that many of the
ciprofloxacin-resistant C. jejuni isolates are derived from
the same clone. However, it is also possible that C. jejuni
isolates with this particular gyrA DNA sequence developed the quinolone resistance mutation at amino acid position 86 due to
separate environmental events. If the second possibility is true,
perhaps certain C. jejuni strains are more prone than other strains to develop gyrA quinolone resistance mutations.
NARMS isolate CS50 has a gyrA QRDR DNA sequence unlike that
of the other ciprofloxacin-resistant NARMS isolates but which is
identical to the DNA sequences of C. jejuni isolates UA580,
ATCC 33560, and D135, except for mutations at amino acid positions 86 and 104. It is likely that these additional mutations arose in isolate CS50 following exposure to quinolone or fluoroquinolone antibiotics. Unique DNA sequences found in the gyrA QRDRs of
ciprofloxacin-resistant isolates may be useful during an outbreak
investigation for determining if isolates are related.
Since the Thr-86-to-Ile (ACAATA) mutation in codon 86 of the
C. jejuni gyrA gene was the most commonly encountered
mutation leading to fluoroquinolone resistance (for which the DNA
sequence has been reported [references 4, 8, and 15
and this study]) and since many laboratories do not have the
equipment, time, or expertise to sequence genes for the investigation
of mutations relevant to antibiotic resistance, we felt it was
worthwhile to develop a MAMA protocol for the specific detection of
this mutation. When the MAMA protocol is used, however, isolates should
be confirmed as being C. jejuni by hippurate hydrolysis or
PCR assay (6, 9). This is important because the 3' end of
the CampyMAMAgyrA5 mutation detection primer, designed for
the Thr-86-to-Ile mutation (ACAATA) in codon 86 of the C. jejuni gyrA gene, is homologous to codon 86 of the wild-type
C. coli gyrA gene and will generate a false-positive PCR
product. The 3'-terminal nucleotide of primer CampyMAMAgyrA5
pairs correctly with the thymine in codon 86 of the
ciprofloxacin-resistant C. jejuni gyrA mutants from this
study, but a mismatch in the primer base immediately 5' to the base at the 3' end reduces amplification efficiency to about 70% of what could
be expected with perfectly conserved primers (1). Neither the nucleotide at the 3' end nor the nucleotide immediately 5' to it
pairs with the wild-type, fluoroquinolone-susceptible C. jejuni
gyrA DNA sequence, and thus no PCR product is amplified (1).
While the MAMA PCR assay described here is undoubtedly simpler than DNA
sequencing to determine the presence of mutations relevant to
fluoroquinolone resistance, it does have some disadvantages. Although
the majority of fluoroquinolone-resistant C. jejuni isolates have the Thr-86-to-Ile mutation, which the
mutation primer in this assay was developed to detect, we have not yet
developed additional primers to detect other reported mutations in the
C. jejuni gyrA gene (15). However, it will be a
simple matter to develop additional MAMA PCR mutation detection primers
as isolates with other mutations become more widely available to test.
We also concede that, for the present time, many research laboratories will still wish to sequence the gyrA genes of any
fluoroquinolone-resistant C. jejuni isolates they obtain in
order to detect new or additional mutations. In addition, clinical
laboratories will undoubtedly still prefer to perform phenotypic
susceptibility tests as a means for detecting resistant strains. The
most relevant use for this assay, at this point in time, will be as a
quick screening method for public health laboratories interested in
quickly characterizing the resistance profiles of resistant outbreak
isolates or in public health laboratories with little access to DNA
sequencing equipment or the funds to perform sequencing.
Fluoroquinolone-resistant C. jejuni isolates that test
negative by the MAMA PCR assay can be further examined by DNA
sequencing of the gyrA, gyrB, parC, and parE genes (3). Other resistance
mechanisms, such as efflux pumps, permeability factors, or
detoxification, could be studied if no other obvious mechanism
appears to be causing fluoroquinolone resistance in the isolates being
examined (3). We believe the MAMA PCR method is a simple,
specific, rapid, inexpensive, and portable alternative to the
nonradioisotopic single-strand conformation polymorphism method
previously published (2) and DNA sequencing for the
detection of this important fluoroquinolone resistance mutation.
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Abstract
Introduction
