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Antimicrobial Agents and Chemotherapy, October 1998, p. 2661-2667, Vol. 42, No. 10
Hospital Infections Program, National Center
for Infectious Diseases, Centers for Disease Control and
Prevention, Atlanta, Georgia 30333
Received 9 April 1998/Returned for modification 19 June
1998/Accepted 28 July 1998
Fluoroquinolone resistance (FQ-R) in clinical isolates of
Enterobacteriaceae species has been reported with
increasing frequency in recent years. Two mechanisms of FQ-R have been
identified in gram-negative organisms: mutations in DNA gyrase and
reduced intracellular drug accumulation. A single point mutation in
gyrA has been shown to reduce susceptibility to
fluoroquinolones. To determine the extent of gyrA mutations
associated with FQ-R in enteric bacteria, one set of oligonucleotide
primers was selected from conserved sequences in the flanking regions
of the quinolone resistance-determining regions (QRDR) of
Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter
freundii, Enterobacter aerogenes, Enterobacter
cloacae, E. coli, K. pneumoniae,
Klebsiella oxytoca, Providencia stuartii, and
Serratia marcescens. Although similarity of
the nucleotide sequences of seven species ranged from 80.8 to 93.3%,
when compared with that of E. coli, the amino acid
sequences of the gyrA QRDR were highly conserved.
Conservative amino acid substitutions were detected in the QRDRs of the
susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2
µg/ml expressed amino acid substitutions primarily at the Gly-81,
Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly
for strains exhibiting identical gyrA mutations, indicating
that alterations outside gyrA contribute to resistance. The
type and position of amino acid alterations also differed among these
six genera. High-level FQ-R frequently was associated with single
gyrA mutations in all species of
Enterobacteriaceae in this study except E. coli.
Fluoroquinolones are
broad-spectrum antimicrobial agents effective in the treatment of a
wide range of infections. However, widespread use of this class of
agents has resulted in an increasing incidence of fluoroquinolone
resistance (13). Mechanisms of resistance to quinolones
include alterations in DNA gyrase and topoisomerase IV and decreased
intracellular accumulation of the antimicrobial agent due to
modifications of membrane proteins (3, 12).
The primary target of fluoroquinolones in gram-negative bacteria is DNA
gyrase, a type II topoisomerase required for DNA replication and
transcription (3). DNA gyrase, which is composed of two A
subunits and two B subunits, is encoded by the gyrA and
gyrB genes. In these organisms, resistance to
fluoroquinolones has been shown to be associated most frequently with
alterations in gyrA (7, 9, 29). The mutations are
localized at the 5' end of the gene (nucleotides 199 to 318 in the
Escherichia coli gene sequence) in an area designated as the
quinolone resistance-determining region, or QRDR, near Tyr-122, which
binds the transiently cleaved DNA (14, 28). Genetic
characterization of gyrA mutations associated with
fluoroquinolone resistance in E. coli has been well
defined by DNA sequence analysis of resistant strains of both clinical isolates and mutants selected in vitro (4, 11, 17, 24, 28).
These mutations are found most frequently in the Ser-83 and Asp-87
codons and in the corresponding codon positions of the gyrA
genes from several other organisms (6, 7, 9, 22, 25).
However, the mechanisms of quinolone resistance for many of the enteric
species associated with opportunistic infections have not been
well defined.
In this study, the DNA sequences of the gyrA QRDRs of
fluoroquinolone-resistant clinical isolates representing eight
species of Enterobacteriaceae were determined.
Oligonucleotide primers selected from conserved gyrA gene
sequences flanking the QRDR were used to amplify and sequence the
5' region of gyrA from the American Type Culture Collection
(ATCC) type strain for each species and from 60 fluoroquinolone-resistant strains. DNA and deduced amino acid
sequences were aligned, and the similarities and differences were
characterized. Within each species, the amino acid sequences of the
QRDRs from isolates with decreased fluoroquinolone susceptibilities were compared with those of the type strain, and amino acid
substitution profiles were analyzed for association with
fluoroquinolone resistance and for correlation with the ciprofloxacin
(CIP) MIC.
(This study was presented in part at the 37th Interscience Conference
on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997.)
Bacterial strains and determination of antimicrobial
susceptibility profiles.
Type strains of
Enterobacteriaceae were those designated by the ATCC.
Fluoroquinolone-resistant and -susceptible clinical isolates were
obtained during the Intensive Care Antimicrobial Resistance
Epidemiology (ICARE) study, in which isolates were collected from 39 hospitals across the United States between June 1994 and April 1997 (1). Strains were selected from among the ICARE isolates to
represent a range of CIP MICs and geographical locations for each
species group. Duplicate isolates from the same patient were excluded.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
gyrA Mutations Associated with
Fluoroquinolone Resistance in Eight Species of
Enterobacteriaceae
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Amplification of the 5' region of gyrA. Oligonucleotide primers were designed on the basis of homologous regions of gyrA from E. coli (21) and the gyrA sequence reported for Klebsiella pneumoniae (8). Primer gyrA6 (5'-CGACCTTGCGAGAGAAAT-3') corresponds to nucleotides 6 to 23 and gyrA631R (5'-GTTCCATCAGCCCTTCAA-3') is complementary to nucleotides 631 to 614 of the E. coli gyrA sequence.
The gyrA gene fragments were amplified from chromosomal DNA present in crude cell lysates prepared by the method of Conrad et al. (4). Using a GeneAmp 9600 PCR System (Perkin-Elmer, Applied Biosystems Division [PE-ABI], Foster City, Calif.), amplifications were carried out in 50-µl volumes containing 50 pmol of each primer, 200 µM deoxynucleoside triphosphates, 1× reaction buffer with 1.5 mM MgCl2 (PE-ABI), 1 U of native Taq polymerase (PE-ABI), and 10 µl of cell lysate, containing approximately 100 ng of chromosomal DNA. An initial 4-min period of denaturation at 94°C was followed by 30 cycles of denaturation (1 min at 94°C), annealing (30 s at 55°C), and extension (45 s at 72°C) and then a final cycle of 72°C for 10 min. Amplification products were visualized by agarose gel electrophoresis and ethidium bromide staining to confirm the sizes of the gene fragments. PCR products were purified on QIAquick spin columns (QIAGEN, Chatsworth, Calif.).DNA sequencing and analysis. Oligonucleotides gyrA6 and gyrA631R were also used as primers for direct sequencing of the amplified gyrA gene fragments. The DNA sequences were determined with ABI Prism Dye Terminator or dRhodomine Terminator Cycle Sequencing reactions, using an ABI 377 automated sequencer (PE-ABI). Products from sequencing reactions were purified on Centri-Sep spin columns (Princeton Separations, Adelphia, N.J.). To eliminate errors caused by amplification artifacts, the forward and reverse sequences of each QRDR were determined for products from at least two independent PCRs. The GCG analysis programs (Genetics Computer Group, Madison, Wis.) were used for DNA and amino acid sequence alignments.
Nucleotide sequence accession numbers. The partial sequences of the gyrA genes reported in this study were assigned the following GenBank accession numbers: Citrobacter freundii (ATCC 8090), AF052253; E. coli (ATCC 11775), AF052254; Enterobacter aerogenes (ATCC 13048), AF052255; Enterobacter cloacae (ATCC 13047), AF052256; Klebsiella oxytoca (ATCC 13182), AF052257; K. pneumoniae (ATCC 13883), AF052258; Providencia stuartii (ATCC 29914), AF052259; and Serratia marcescens (ATCC 13880), AF052260.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Amplification of the gyrA genes from eight species of Enterobacteriaceae. PCRs with oligonucleotide primers gyrA6 and gyrA631R amplified the expected 626-bp DNA fragments from the type strains of C. freundii, E. aerogenes, E. cloacae, E. coli, K. oxytoca, K. pneumoniae, P. stuartii, and S. marcescens (Fig. 1) and from 60 fluoroquinolone-resistant clinical isolates of these eight species. No amplification products were detected for Proteus mirabilis or Morganella morganii, indicating that the sequences of the 5' region of gyrA in these two organisms diverge from the conserved sequences shared by the other species listed.
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Genetic analysis of the gyrA QRDR. The DNA sequences of the gyrA gene fragments for the type strains and clinical isolates extended from nucleotides 24 to 613 (590 bp), including the QRDR (nucleotides 199 to 319 of the corresponding E. coli sequence) and excluding the primer sequences. Alignment of DNA sequences for the QRDRs from the type strains of eight enterobacterial species with the E. coli sequence revealed numerous nucleotide substitutions in the former (Fig. 2). However, 87 of the 120 nucleotides comprising the QRDR (72.5%) were conserved. The greatest degree of similarity to the gyrA QRDR sequence from E. coli was found in the sequence of the E. cloacae type strain (93.3% identity) (Table 1), while P. stuartii (80.8% identity) displayed the lowest degree of similarity. Comparisons of all eight species revealed that the gyrA QRDRs of K. pneumoniae and P. stuartii, with 75.8% identical nucleotides, were the least similar. Significant diversity of gyrA QRDR sequences was also noted among species of the same genus. The E. aerogenes and E. cloacae sequences exhibited 88.3% identity, while K. pneumoniae and K. oxytoca showed 87.5% identity in this region, which is a lower degree of similarity than was noted among several species from different genera.
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Amino acid sequence analysis. The deduced amino acid sequences of the QRDRs for the eight enterobacterial species were highly conserved (Fig. 3). The QRDR amino acid sequences of E. cloacae, K. pneumoniae, and S. marcescens were identical to the E. coli QRDR. Sequence analysis of C. freundii, E. aerogenes, and K. oxytoca strain QRDRs revealed one conservative substitution, Ser-83 to Thr, which was identified in all fluoroquinolone-susceptible isolates of these species as well as the type strains. Only P. stuartii exhibited more than one amino acid substitution in this region. In this organism, two conservative changes were detected: Val-69 to Ile and Asp-87 to Glu. In addition, all P. stuartii strains exhibited Leu-92-to-Met and Met-98-to-Leu substitutions when compared with the other members of the Enterobacteriaceae family included in this study.
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Detection of mutations in gyrA sequences amplified from clinical isolates. After the DNA and amino acid sequences of the QRDRs from the quinolone-susceptible type strains were determined, the 5' regions of gyrA from ciprofloxacin-resistant and -susceptible clinical isolates were amplified, sequenced, and analyzed for mutations leading to amino acid changes associated with fluoroquinolone resistance. Clinical isolates from each species were selected to represent a range of CIP MICs. The codon and amino acid alterations detected in fluoroquinolone-resistant and -susceptible clinical isolates and the CIP, OFLX, and SPAR MICs are summarized in Table 2 (amino acid positions refer to the corresponding positions in the E. coli GyrA protein).
In E. coli, a C-to-T substitution at the second position of the codon, resulting in a Ser-83-to-Leu mutation, was consistent for all fluoroquinolone-resistant isolates. In strains with double mutations, the second alteration involved the codon for Asp-87 and the amino acid substitution was Gly, Tyr, or Asn. A single mutation in codon 83 of gyrA was associated with decreased susceptibility or low levels of resistance to fluoroquinolones, and double mutations (codons 83 and 87) were associated with high levels of resistance. All clinical isolates of C. freundii with reduced susceptibility to fluoroquinolones exhibited Thr-83-to-Ile mutations resulting from C-to-T substitutions at nucleotide position 248. Two isolates, C. freundii 1958 and 5757, had double mutations of Thr-83 to Ile and Asp-87 to Gly. CIP MICs of
16 µg/ml
in C. freundii were associated with both single and
double mutations.
Also, in all E. aerogenes isolates examined, mutations
in codon 83 resulted in Thr-83-to-Ile substitutions. No double
mutations were detected in gyrA genes from seven strains of
E. aerogenes with reduced susceptibility to
fluoroquinolones. However, CIP MICs for isolates with the single
mutation ranged from 2 to 16 µg/ml.
The gyrA gene fragments from clinical isolates of
E. cloacae exhibited numerous nucleotide
substitutions, resulting in changes of Ser-83 to Phe, Ile, or
Tyr. The only substitution detected in E. cloacae 3529 (CIP MIC = 4) was Ser-83 to Thr, which would not be expected to
result in decreased susceptibility since Thr-83 is found in susceptible
strains of K. oxytoca, C. freundii, and E. aerogenes. In E. cloacae 1524, the only
GyrA alteration was a change of Asp-87 to Asn. There was no alteration
of Ser-83, and only a marginal decrease in susceptibility to
fluoroquinolones (CIP MIC = 0.25 µg/ml) was detected for this
strain.
K. pneumoniae isolates exhibited either single or
double mutations involving Ser-83 and Asp-87. All single mutations
involved either a C-to-T or C-to-A change at the second position in
codon 83, resulting in alteration of Ser to either Phe or Tyr,
respectively. In two double mutants, K. pneumoniae 1775 and 1565, a nucleotide substitution at the first or second position of
codon 87 resulted in a change of Asp to Gly or Asn. CIP MICs ranged
from 1 to 16 µg/ml, and double mutations were not required for
high-level resistance. In the group of K. pneumoniae
strains with single mutations, no specific mutation (Ser-83 to Phe or
Tyr) was associated with low or high levels of fluoroquinolone
resistance.
K. oxytoca mutations were confined to the Thr-83
codon and were consistently C-to-T substitutions in the second
position, resulting in Thr-to-Ile alterations, similar to C. freundii and E. aerogenes. CIP MICs associated
with this alteration ranged from 0.5 to 16 µg/ml.
Changes in the QRDR of P. stuartii gyrA were also
confined to codon 83; however, the nucleotide substitutions
differed. Single nucleotide substitutions included A to C at the
first position and C to G at the third position, both resulting in
Ser-to-Arg mutations, and G to T in the second position, resulting in a
Ser-to-Ile mutation. CIP MICs ranged from 2 to 16 µg/ml for
P. stuartii isolates with alterations of Ser-83.
The fluoroquinolone-resistant clinical isolates of S. marcescens displayed the greatest diversity in mutations,
including Gly-81 to Cys, Ser-83 to Ile or Arg, and Asp-87 to Asn.
No double mutations were detected in the gyrA QRDRs
from the six isolates examined. The GyrA sequences from clinical
isolates S. marcescens 1221 (CIP MIC = 4)
and 1570 (CIP MIC = 8) revealed only Gly-81 to Cys substitutions,
and Asp-87 to Asn was the only alteration detected in strain 1969.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The association of DNA gyrase A mutations with fluoroquinolone resistance has been established for both gram-negative and gram-positive organisms (3). In the family Enterobacteriaceae, E. coli is the only species for which mutations in gyrA leading to fluoroquinolone resistance have been well characterized (4, 10, 23, 28). In E. coli, a single point mutation in gyrA results in decreased susceptibility to fluoroquinolones (27), and high-level resistance is associated with double amino acid substitutions in the GyrA protein (4, 10, 24). Although additional factors, such as mutations in the ParC subunit of topoisomerase IV and decreased intracellular drug accumulation, have been shown to play a complementary role by increasing the level of resistance (17, 26), in vitro studies with E. coli suggest that the first step in selection for decreased susceptibility to fluoroquinolones is an alteration of Ser-83 (11).
The similarity of the N-terminal region of the gyrA sequence of E. coli (21) to that reported for K. pneumoniae (8) was exploited to design oligonucleotide primers that amplified 590-bp gene fragments, including the QRDRs, from type strains of 8 of the 10 Enterobacteriaceae species tested. No amplification products were detected for M. morganii or Proteus mirabilis. Based on DNA relatedness studies, species of Proteus, Providencia, and Morganella are less than 25% related to other genera of Enterobacteriaceae (2). Therefore, the failure of the primers to amplify the gene fragments from Proteus mirabilis and M. morganii was less remarkable than the successful amplification of the fragment from P. stuartii and served to emphasize the highly conserved nature of the 5' region of gyrA sequences among the Enterobacteriaceae.
Although the G+C content of the Enterobacteriaceae genomes ranges from 38 to 60 mol% (2) and the diversity of the DNA sequences for the gyrA QRDRs from the type strains, when compared with the corresponding region in the E. coli gyrase gene, ranged from 6.7% for E. cloacae to 19.2% for P. stuartii, the amino acid sequence of GyrA was highly conserved among the eight species in this study. Nucleotide substitutions, which were found primarily at the third codon position, may reflect the preferential codon usage required to preserve both the amino acid sequence of the gyrase A subunit and the characteristic moles percent G+C of each species. When compared with the corresponding region of the E. coli parC gene, nucleotide sequence similarities of the amplified gene fragments decreased to 60 to 62% for all species examined. Based on these findings, it was concluded that the gene fragments represented sequences from the gyrA gene.
The detection of Ser-83 in susceptible strains of K. pneumoniae is consistent with the gyrA gene
fragment sequence reported by Deguchi et al. (6). The
presence of Thr instead of Ser at position 83, evident in C. freundii, E. aerogenes, and K. oxytoca, is also noted in gyrA genes from susceptible
strains of Pseudomonas aeruginosa (16) and
Campylobacter jejuni (25). Previous studies of
Pseudomonas aeruginosa and Campylobacter jejuni
led to the suggestion that the intrinsically lower quinolone
susceptibilities of these two organisms might be due to the presence of
Thr at position 83 (25). The susceptibility profiles of
fluoroquinolone-susceptible strains of C. freundii and
K. oxytoca (CIP MICs, 
0.12 µg/ml) do not support
this premise. However, the gyrA sequence from one strain
of E. cloacae, 3529, revealed a deduced Ser-83-to-Thr
substitution associated with fluoroquinolone resistance (CIP MIC = 4), a conservative mutation that would not be expected to lead to
a significant decrease in susceptibility. Similarly, Nishino et al.
(19) have analyzed several clinical isolates of
C. freundii, exhibiting decreased susceptibility to
fluoroquinolones (MICs, 1.56 µg/ml), in which no mutations were found
in gyrA or parC. As with these strains, further
genetic analysis will be required to determine the mechanism(s) of
resistance in E. cloacae 3529.
Each of the four amino acid substitutions found in the type strain of P. stuartii is present in the GyrA protein of at least one other organism. Alignment of 13 microbial GyrA QRDR sequences by Tankovic et al. (22) revealed that the Val-69-to-Ile substitution is common to both gram-positive and gram-negative species. Glu-87 is typical of gram-positive organisms such as Streptococcus pneumoniae, Enterococcus faecalis, Bacillus subtilis, and Staphylococcus aureus but has not been described previously for a gram-negative organism. The combined substitutions of Leu for Met-92 and Met for Leu-98 are present in the GyrA sequence of Aeromonas salmonicida (20).
When the amino acid alterations of the fluoroquinolone-resistant clinical isolates and the corresponding MICs for CIP, OFLX, and SPAR were evaluated, no specific amino acid alterations were associated with low- or high-level resistance profiles. Consistent with previous studies (10, 24, 27), low-level fluoroquinolone resistance in E. coli was associated with single mutations in the GyrA protein and high-level resistance required double mutations. However, for all other species in this study, there were strains for which high-level fluoroquinolone resistance was associated with a gyrA QRDR sequence exhibiting a single mutation. The association of low- and high-level resistance in clinical isolates of the same species with identical gyrA mutations is consistent with recent reports of gyrA mutations associated with fluoroquinolone resistance in clinical isolates of C. freundii (19), E. cloacae (5), and S. marcescens (15).
Unlike the consistent single mutation resulting in the alteration of Thr-83 to Ile in GyrA of C. freundii, E. aerogenes, and K. oxytoca, several different nucleotide substitutions resulting in diverse amino acid changes were associated with fluoroquinolone resistance in S. marcescens. For the six clinical isolates of S. marcescens having CIP MICs of >2 µg/ml, five different nucleotide substitutions resulted in four distinct amino acid alterations at three codon positions, Gly-81, Ser-83, and Asp-87. In contrast with other enterobacterial species, mutation of Ser-83 was not required for high-level fluoroquinolone resistance in S. marcescens.
In summary, comparison of the gyrA gene sequences and amino acid alterations associated with fluoroquinolone resistance in the Enterobacteriaceae revealed numerous differences as well as similarities among the eight species examined. The amino acid sequences of these Enterobacteriaceae species were highly conserved, although the nucleotide sequences differed by as much as 19.2% from that of E. coli gyrA. E. coli was the only species in which double mutations of gyrA were required for high-level fluoroquinolone resistance. With the exception of S. marcescens, high-level resistance was associated primarily with alterations of the Ser or Thr at position 83. These data extend our understanding of the molecular mechanisms of fluoroquinolone resistance associated with gyrA mutations to include E. aerogenes, K. oxytoca, and P. stuartii and provide additional data on both type strain gyrA sequences and mutations associated with fluoroquinolone resistance in clinical isolates of E. coli, E. cloacae, K. pneumoniae, S. marcescens, and C. freundii.
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ACKNOWLEDGMENTS |
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We thank Bertha Hill for MIC susceptibility testing, Caroline O'Hara for species identification, and Kamile Rasheed and George Killgore for helpful discussions. We also thank John McGowan, Jr., Lennox Archibald, Robert Gaynes, Scott Fridkin, and all of the personnel and hospitals of Project ICARE for providing clinical isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Nosocomial Pathogens Laboratory Branch (G-08), Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404) 639-1497. Fax: (404) 639-1381. E-mail: lew9{at}cdc.gov.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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