- Email: [email protected]
Characterization of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli by DNA sequence analysis and MAMA PCR
FEMS Microbiology Letters 190 (2000) 1^7
www.fems-microbiology.org
Characterization of gyrA mutations associated with £uoroquinolone resistance in Campylobacter coli by DNA sequence analysis and MAMA PCR Gerald Zirnstein a
a;
*, Leta Helsel a , Yu Li a , Bala Swaminathan a , John Besser
b
National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Mail Stop C03, 1600 Clifton Road, Atlanta, GA 30333, USA b Minnesota Department of Health, Minneapolis, MN, USA Received 2 March 2000 ; received in revised form 26 June 2000; accepted 29 June 2000
Abstract Increasing numbers of fluoroquinolone-resistant Campylobacter coli isolates received at the Minnesota State Public Health Laboratory and at the Centers for Disease Control and Prevention have been a cause for concern. The gyrA quinolone resistance-determining regions of several fluoroquinolone-resistant isolates were sequenced to examine the mechanism of resistance. Ciprofloxacin-resistant C. coli isolates examined by DNA sequencing had a Thr-86 to Ile (ACTCATT) gyrA mutation, leading to resistance to fluoroquinolone antibiotics. A mismatch amplification mutation assay polymerase chain reaction protocol was developed to detect this gyrA mutation. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Campylobacter coli ; Cipro£oxacin resistance; Mismatch polymerase chain reaction; gyrA mutation
1. Introduction Diarrhea caused by Campylobacter coli and Campylobacter jejuni is generally a self-limiting disease. However, early antibiotic treatment is recommended to reduce severity and duration. Fluoroquinolones are the treatment of choice for adults and are also prescribed for prophylactic treatment against traveller's diarrhea [1^6]. The existence of £uoroquinolone-resistant Campylobacter isolates has been reported in various countries for several years, including the Netherlands [6], Sweden [7], Thailand [8], UK [9] and USA [10,11]. The use of £uoroquinolones in agriculture to heal diseases of poultry [11,12] is thought to be a contributing factor for the emergence of £uoroquinolone-resistant enteric bacteria. Reports of the emergence of quinolone-resistant Campylobacter isolates in vivo after quinolone therapy [13,14] lend additional support to the contention that resistant isolates may develop after therapeutic treatment with quinolone antibiotics. For these reasons it is of interest to us to investigate the
* Corresponding author. Tel. : +1 (404) 639 0272; Fax: +1 (404) 639 3333; E-mail : [email protected]
mechanisms by which C. coli isolates are becoming resistant to quinolone antibiotics. DNA gyrase, a type II topoisomerase which introduces negative supercoils into DNA and which is essential for bacterial viability, is a major target for quinolone antibiotics in bacteria. The binding of quinolones to the GyrA subunits to prevent gyrase function is the primary mechanism [15,16]. Several studies have shown that mutations in the gyrA gene of several Campylobacter species are primarily responsible for the resistance of most Campylobacter clinical isolates to quinolone antibiotics [9,10,17,18]. In Escherichia coli, most mutations resulting in £uoroquinolone resistance map between codons 67 and 106, near the Tyr-122 catalytic site of DNA gyrase. Thus, this region has been termed the quinolone resistance-determining region (QRDR) [19]. An analogous QRDR region exists in the C. jejuni gyrA gene, but the gyrA Tyr-125 residue appears to be the catalytic site [18,20]. A single mutation of Ser-83 to Ala in the GyrA protein of E. coli allows DNA gyrase activity to become resistant to the action of cipro£oxacin and nalidixic acid in vitro [21]. Mutations in the QRDR region of the C. coli gyrA gene analogous to known quinolone resistance mutations in C. jejuni and E. coli could reasonably be expected to be re-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 0 6 - 2
FEMSLE 9530 10-8-00
2
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
sponsible for quinolone antibiotic resistance in C. coli isolates. Several £uoroquinolone-resistant C. coli isolates were recently received by the Minnesota State Public Health Laboratory (SPHL) and the Centers for Disease Control and Prevention (CDC). This study was initiated to investigate the type and frequency of gyrA mutations in C. coli isolates with resistance to nalidixic acid and cipro£oxacin, and to develop a rapid polymerase chain reaction (PCR) protocol for the detection of gyrA mutations relevant to these resistance phenotypes that could serve as an alternative to previously reported rapid methods [22]. In addition, we wished to determine a greater amount of the DNA sequence surrounding the gyrA QRDR region of C. coli isolates than had been previously submitted to GenBank (Everett and Piddock, accession number U63413) in order to allow for greater £exibility in the choice of primers for PCR ampli¢cation, direct sequencing of the PCR ampli¢cation products, and rapid analysis of the actual DNA point mutations themselves. To our knowledge, the DNA sequence of C. coli gyrA quinolone-resistance point mutations has never been published or submitted to GenBank. Fluoroquinolone antibiotics are currently one of the most e¡ective antimicrobial agents for the elimination of C. coli infections and a rapid, speci¢c, inexpensive, molecular method for the detection of quinolone-resistance mutations should be able to serve as an aid to determine if £uoroquinolone antibiotics are appropriate treatment for speci¢c clinical cases of the infection or for epidemiological studies. 2. Materials and methods 2.1. Strains, culture conditions and antibiotic susceptibility testing Quinolone-susceptible C. coli isolates used in this study included nine isolates from the CDC and Minnesota SPHL collections. Fifteen quinolone-resistant isolates examined in this study were isolated in Minnesota and provided by the Minnesota SPHL ; two were from CDC. Culture media, growth environment and biochemical tests were performed as previously reported [10]. PCR ampli¢cation for the identi¢cation of the genus Campylobacter was performed according to the method of Linton et al. [23]. Further characterization to the species level was
performed by using a PCR test based on the ceuE gene developed by Gonzalez et al. [24]. Screening for antimicrobial resistance was performed with E-test strips (AB Biodisk, Solna, Sweden) as previously published [10]. The nalidixic acid and cipro£oxacin antibiotypes were con¢rmed by agar dilution testing, as described by the National Committee for Clinical Laboratory Standards (NCCLS) [25], with modi¢cations as previously published [10]. The interpretive breakpoints for the minimum inhibitory concentrations (MICs) to nalidixic acid were 9 8 Wg ml31 as susceptible and v 32 Wg ml31 as resistant. The interpretive breakpoints for the MICs to cipro£oxacin were 9 1 Wg ml31 as susceptible and v 4 Wg ml31 as resistant [26]. 2.2. DNA isolation, PCR, DNA sequencing and nucleotide sequence analysis Chromosomal DNA was puri¢ed, as previously published [10]. Primers GZgyrA5 and GZgyrA6 (Table 1) were originally chosen for PCR ampli¢cation of a 673bp product containing the QRDR region of the gyrA gene of quinolone-resistant and -sensitive C. jejuni [10] after analysis of the C. jejuni gyrA gene sequence published by Wang et al. [18]. In this study, primers GZgyrA5 and GZgyrA6 were used for gyrA PCR, which resulted in DNA bands of approximately 673 bp from the majority of C. coli isolates from this study. However, direct sequencing of these C. coli gyrA PCR products only yielded reliable DNA sequence data with primer GZgyrA6 from three of the C. coli isolates. Good gyrA DNA sequence data from C. coli type strain ATCC 33559 using primer GZgyrA6 was used to select new primers for further PCR and sequencing. Primers GZgyrACcoli3F and GZgyrACcoli4R (Table 1) were chosen for the ¢nal PCR and sequencing of the gyrA QRDR region of the C. coli isolates, producing a PCR product 505 bp in length. PCR reactions (100 Wl each) contained 75 ng of puri¢ed chromosomal DNA and were performed under conditions previously described [10]. Primers were synthesized by the CDC Biotechnology Core Facility. After ampli¢cation, 5 Wl of the product was loaded onto a 2.0%, 0.5UTBE, horizontal agarose gel and stained with ethidium bromide for analysis after electrophoresis at 150 V for 1 h. The remaining gyrA PCR products were puri¢ed and used in Taq dye terminator cycle sequencing, as previously described [10].
Table 1 Primers used in this study Name
Sequence (5P to 3P)
GZgyrA5 GZgyrA6 GZgyrACcoli3F GZgyrACcoli4R CampyMAMAgyrA8
ATT CCA TAT GTC TAA
TTT TAA GAG CAT GGC
AGC ATT CGT CTA ATC
AAA ATT TAT CAA GTA
GAT CCA TAT GCT AAC
TCT CCT CGG CGT AGC
GAT GT TC TA CA
Direction, purpose
Reference
forward, C. jejuni gyrA primer reverse, C. jejuni gyrA primer forward, speci¢c for C. coli gyrA reverse, speci¢c for C. coli gyrA reverse, C. coli putative codon 86 gyrA mutation detection primer
[10] [10] this study this study this study
FEMSLE 9530 10-8-00
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
DNA sequence analysis and primer selection for the mismatch ampli¢cation mutation assay (MAMA) PCR [27] were performed in a manner similar to that previously described [10]. Brie£y, conserved primers were chosen with OLIGO Primer Analysis Software version 5.0 (National Biosciences, Plymouth, MN, USA) and mutation primers were chosen after a manual analysis of the DNA sequence near putative codon 86 of the C. coli gyrA gene of £uoroquinolone-susceptible and -resistant isolates. DNA sequences were analyzed with DNASIS version 2.5 (Hitachi Software Engineering Co., San Francisco, CA, USA).
3
except only 10 pmol of each primer was used. PCR cycling conditions were as follows : initial denaturation was at 94³C for 3 min, followed by 30 cycles of 94³C for 30 s, 50³C for 30 s and 72³C for 20 s. Eight-Wl aliquots of each PCR product were loaded onto horizontal, 2.0%, 0.5UTBE agarose gels and stained with ethidium bromide for analysis after electrophoresis. 3. Results
2.3. MAMA PCR protocol
3.1. Detection of gyrA mutations associated with quinolone resistance
The mutation primer was chosen after a visual analysis of the DNA sequences near putative codon 86 of the C. coli gyrA genes. A conserved, forward primer, GZgyrACcoli3F, and a reverse, mutation detection primer, CampyMAMAgyrA8 (Table 1), were used together in a PCR reaction to generate a 192-bp PCR product that was a positive indication of the presence of the Thr-86 to Ile (ACTCATT) mutation in the C. coli gyrA gene. Primer GZgyrACcoli4R, a conserved reverse primer, was used in conjunction with primer GZgyrACcoli3F to produce a positive PCR control product of 505 bp from the gyrA gene of all C. coli DNA preparations used in this study. PCR reactions (100 Wl each) were performed as above,
We used primers based on the C. jejuni sequence [10] to generate PCR fragments for preliminary sequencing of C. coli gyrA. Only primer GZgyrA6 produced reliable gyrA sequence data with DNA from only a few C. coli isolates. Reliable gyrA DNA sequence data from C. coli type strain ATCC 33559, using primer GZgyrA6, was employed to select new primers for PCR and sequencing (Table 1 and Fig. 1). The identity between the 633 bases of the gyrA gene of C. coli ATCC 33559 and the analogous region of the gyrA gene of C. jejuni UA580 was 82%, with some areas much more conserved than others (Fig. 1). An examination of the gyrA DNA sequence data, some of which are shown in Fig. 1, enabled us to determine the presence
Table 2 Mutations in the gyrA QRDR region of C. coli isolates and MICs to nalidixic acid and cipro£oxacin CDC No.
Health laboratory/source
Putative gyrA aa 86 mutationa
Nalidixic acid MIC (Wg ml31 )
Cipro£oxacin MIC (Wg ml31 )
MAMA PCR result
D126 D134 D145b D730 D2591 D2592 D2595 D2596 D5416 D5403 D5404 D5413 D5414 D5415 D5417 D5418 D5419 D5420 D5423 D5427 D5428 D5429 D5430 D5431 D5472 D5496
Canada, animal Canada animal WA USA Australia Belgium animal MN MN MN MN MN MN MN MN MN MN MN MN MN MN MN MN MA MA
NA NA no NA NA NA NA NA no yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes NA NA
16 8 16 8 8 8 16 32 16 256 128 128 128 256 256 128 256 256 128 256 256 256 256 128 128 128
0.25 0.06 0.25 0.03 0.06 0.125 0.125 0.125 0.25 8 4 8 8 16 16 8 16 16 4 8 16 16 16 16 16 32
negative negative negative negative negative negative negative negative negative positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive
a b
Mutants have a substitution of Thr-86 to Ile (ACT to ATT). NA indicates isolates were not sequenced. C. coli ATCC 33559 type strain.
FEMSLE 9530 10-8-00
4
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
Fig. 1. Nucleotide sequence alignment of partial gyrA gene sequences of C. coli isolates from this study compared to other Campylobacter and E. coli gyrA sequences from the GenBank database. The sequences of E. coli KL-16 (accession number x06744), Campylobacter fetus ATCC 27374 (accession number u25640) and C. jejuni UA580 (accession number l04566) were compared to the partial gyrA gene sequences of C. coli NCTC 11366 (accession number u63413), C. coli ATCC 33559 (accession number af092101, this study) and C. coli isolates D5416, D5428 and D5429 from Minnesota. Codon numbers are based on codon positions of the complete C. jejuni UA580 gyrA gene, and are only putative for C. coli sequences, since the ¢rst few bases of the C. coli gyrA genes were not sequenced.
or absence of non-synonymous mutations in the QRDR region of the Minnesota isolates and compare this information to the nalidixic acid and cipro£oxacin MICs for the C. coli isolates (Table 2). The mutation at putative amino acid (aa) position 86 of the gyrA gene correlates with resistance to cipro£oxacin and nalidixic acid in the C. coli isolates examined. The relationship of the Thr-86 to
Ile mutation to the acquisition of quinolone resistance in other Campylobacter isolates has been previously characterized [9,10,18]. With the exception of isolates D5404 and D5413, all cipro£oxacin-resistant Minnesota isolates possessed identical gyrA DNA sequences with isolates D5428 and D5429 (Fig. 1). Isolate D5404 shared identical 39 and 99 gyrA
FEMSLE 9530 10-8-00
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
DNA codons as the C. coli ATCC 33559 type strain, and isolate D5413 shared the same 39 and 157 gyrA DNA codons as the C. coli ATCC 33559 type strain, while the remainder of the D5404 and D5413 gyrA DNA sequences were identical to D5428 and D5429 (refer to Fig. 1). 3.2. Detection of the Thr to Ile mutation (ACTCATT) by use of MAMA PCR A rapid PCR method was developed for the detection of the Thr to Ile (ACT to ATT) mutation at the putative aa position 86 in the gyrA DNA sequence of C. coli isolates. Isolates with the wild-type aa 86 codon (ACT, quinolonesusceptible) were not ampli¢ed with the reverse mutation primer, CampyMAMAgyrA8 (Fig. 1), whereas the isolates with the mutated aa 86 codon (ATT, quinolone-resistant) generated a 192-bp PCR product with the CampyMAMAgyrA8 reverse mutation primer and the GZgyrACcoli3F forward conserved primer (Table 2, Fig. 1). Conserved primers GZgyrACcoli3F and GZgyrACcoli4R generated a 505-bp gyrA PCR product with DNA isolated from all of the isolates listed in this study. Seventeen of the strains in Table 2 were examined by direct DNA sequencing of gyrA gene PCR products. CDC isolate D145 (the ATCC 33559 type strain) and isolate D5416 (from Minnesota) were both cipro£oxacin-susceptible, had wild-type gyrA DNA sequences and had negative results with the MAMA PCR mutation detection primer. Isolates D5403, D5404, D5413, D5414, D5415, D5417, D5418, D5419, D5420, D5423, D5427, D5428, D5429, D5430 and D5431 were all from Minnesota, were all cipro£oxacin-resistant, all had the Thr to Ile (ACT to ATT) mutation at the putative aa 86 position of the gyrA gene, and all had positive results with the MAMA PCR mutation detection primer. To determine if the MAMA PCR assay was valid with other CDC C. coli isolates from varied sources and locations, spanning a number of years, nine more CDC isolates, D126, D134, D730, D2591, D2592, D2595, D2596, D5472 and D5496, were examined by MAMA PCR alone to verify whether or not the MAMA PCR assay could serve as a replacement for sequencing data when the antibiotype of the isolate is taken into account (Table 2). Results indicate the putative aa position 86 cipro£oxacin-resistance mutation was correctly identi¢ed in all cipro£oxacin-resistant isolates and that the MAMA PCR never gave a positive PCR response with any cipro£oxacin-sensitive isolates from this group. We were satis¢ed that the MAMA PCR assay worked well for all isolates tested. 4. Discussion The e¡ectiveness of £uoroquinolone antibiotics for the treatment of traveller's diarrhea [3], the use of £uoroqui-
5
nolones in agriculture to heal diseases of poultry [12], and the common practice of prescribing £uoroquinolone antibiotics for gastroenteritis for the purpose of shortening absences from work [2] have led to exposure to this class of antibiotics. The incidence of quinolone-resistant Campylobacter infection has increased recently, and evidence suggests that a signi¢cant number of human isolates are acquired from poultry products [11]. Reports of the emergence of quinolone-resistant Campylobacter isolates in vivo after quinolone therapy [13,14] can also be found in the literature. Several £uoroquinolone-resistant C. coli isolates received by the SPHL and the CDC prompted us to perform this study to investigate the type and frequency of gyrA mutations in C. coli isolates with resistance to nalidixic acid and cipro£oxacin, and to develop a rapid PCR protocol for the detection of gyrA mutations relevant to these resistance phenotypes that could serve as an alternative to previously reported rapid methods [22]. Another goal of this study was to obtain a greater amount of the DNA sequence surrounding the gyrA QRDR region of C. coli isolates than had been previously submitted to GenBank (Everett and Piddock, accession number U63413) in order to allow for greater £exibility in the choice of primers for PCR ampli¢cation, direct sequencing of the PCR ampli¢cation products and rapid analysis of the actual DNA point mutations themselves. A single mutation of Ser-83 to Ala in the GyrA protein of E. coli allows DNA gyrase activity to become resistant to the action of cipro£oxacin and nalidixic acid in vitro [21]. A C. jejuni Thr-86 to Ile gyrA mutation, in a position equivalent to the E. coli Ser-83 to Ala mutation, has been reported as responsible for high resistance to £uoroquinolones in C. jejuni in vivo [9,18]. We reported that other US isolates of C. jejuni that were resistant to nalidixic acid (MIC v32 Wg ml31 ) were also resistant to cipro£oxacin (MIC v4 Wg ml31 ), and all had the Thr-86 to Ile (ACA to ATA) mutation [10]. The same mechanism, a Thr-86 to Ile mutation (ACT to ATT) in the QRDR region of the gyrA gene, appears to be responsible for the increased resistance to £uoroquinolone antibiotics noted for the C. coli isolates, since isolates without this mutation were sensitive to cipro£oxacin (Table 1). C. coli isolates tested that were highly resistant to nalidixic acid (MIC v128 Wg ml31 ) were also resistant to cipro£oxacin (MIC v4 Wg ml31 ). Only isolate D2596 had a nalidixic acid MIC of 32 Wg ml31 , which is at the breakpoint for resistance, while being susceptible to cipro£oxacin (Table 1). Factors other than gyrA QRDR mutations, such as mutations in gyrB, parC and parE, e¥ux pumps, permeability factors or detoxi¢cation [20], may be responsible for some of the variability in nalidixic acid and cipro£oxacin MICs among resistant C. coli isolates in Table 1. Since the Thr to Ile (ACT to ATT) mutation in putative codon 86 of the C. coli gyrA gene appears to be a commonly encountered mutation leading to £uoroquinolone resistance and one for which we have reported DNA se-
FEMSLE 9530 10-8-00
6
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
quence data (this study), we developed a MAMA protocol for the speci¢c detection of this mutation. The design of this type of mismatch primer for mutation detection has been explained in detail previously [10,27]. The scienti¢c basis for the ability of the mutation detection primer, CampyMAMAgyrA8, to generate a PCR product with mutant DNAs and not wild-type DNAs is based on the fact that the 3P end of the mutation detection primer has only one mismatch with the mutant, £uoroquinolone-resistant, gyrA sequences, and is able to generate a PCR product, while two mismatches occur between the extreme 3P end of the mutation detection primer and the wild-type, £uoroquinolone-susceptible, gyrA sequences, and no PCR product is generated (see Fig. 1) [27]. The primary variable used to eliminate false positive PCR signals between the mutation detection primer and wild-type DNAs was to increase the annealing temperature of the PCR reaction. With a PCR annealing temperature of 50³C, no PCR DNA bands were observed when wild-type DNA was used with the mutation detection primer, but a very strong PCR DNA band was always observed on the gel when the mutant DNA was included with the mutation detection primer in the PCR reaction. The likelihood of false negative reactions can be greatly reduced by careful addition of measured amounts of DNA template and by including PCR controls with each experiment which include conserved primers (GZgyrACcoli3F and GZgyrACcoli4R) to help determine if the DNA template is degraded. It appears that most of the C. coli cipro£oxacin-resistant isolates are more highly related to one another than to the C. coli ATCC 33559 type strain or isolates D5404 and D5413 and may be from a related source. However, the number of isolates studied is very limited, and they come from a geographically restricted area, which makes it di¤cult to generalize the ¢ndings to other parts of the USA or the world. The analysis of a larger number of isolates from di¡erent areas and di¡erent periods in time would enable a better evaluation of the frequencies of silent mutations within gyrA and would more thoroughly explain the di¡erences between the MN isolates and the reference strain. Cipro£oxacin-susceptible isolate D5416 appears to be similar to the parent strain from which the majority of the cipro£oxacin-resistant isolates apparently arose, since the only di¡erence is the lack of a gyrA mutation at codon 86. However, since there is little data on the stability of the mutations in the gyrA gene of C. coli, then it is also theoretically possible that isolate D5416 may have reverted from CipR to CipS , particularly if antibiotics were no longer present in the growth environment. The MAMA PCR method developed for the C. coli gyrA Thr-86 to Ile mutation (ACT to ATT) should serve as a simple, speci¢c, rapid, inexpensive and portable alternative to methods such as the nonradioisotopic, singlestrand conformation polymorphism method previously published [22] or DNA sequencing for detection of this important £uoroquinolone resistance mutation and may
prove useful for clinical diagnosis or epidemiological studies.
References [1] Nachamkin, I. (1993) Campylobacter infections. Curr. Opin. Infect. Dis. 6, 72^76. [2] Piddock, L.J.V. (1995) Review : quinolone resistance and Campylobacter spp.. J. Antimicrob. Chemother. 36, 891^898. [3] Taylor, D.N., Sanchez, J.L., Candler, W., Thornton, S., McQueen, C. and Escheverria, P. (1991) Treatment of travellers diarrhea: cipro£oxacin plus loperamide compared with cipro£oxacin alone. Ann. Intern. Med. 114, 731^734. [4] Gilbert, D.N., Moellering, Jr., R.C. and Sande, M.A. (1999) In: The Sandford Guide to Antimicrobial Therapy, p. 12 and p. 48. Antimicrobial Therapy, Hyde Park, VT. [5] Abramowicz, M. (Ed.) (1998) In: The Medical Letter Handbook of Antimicrobial Therapy, p. 43. The Medical Letter, New Rochelle, NY. [6] Koenraad, P.M.F.J., Jacobs-Reitsma, W.F., Van der Laan, T., Beumer, R.R. and Rombouts, F.M. (1995) Antibiotic susceptibility of Campylobacter isolates from sewage and poultry abattoir drain water. Epidemiol. Infect. 115, 475^483. [7] Sjo«gren, E., Lindblom, G.B. and Kaijser, B. (1997) Nor£oxacin resistance in Campylobacter jejuni and Campylobacter coli isolates from Swedish patients. J. Antimicrob. Chemother. 40, 257^261. [8] Kuschner, R.A., Trofa, A.F., Thomas, R.J., Hoge, C.W., Pitarangsi, C. and Amato, C. et al. (1995) Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where cipro£oxacin resistance is prevalent. Clin. Infect. Dis. 21, 536^541. [9] Gaunt, P.N. and Piddock, L.J.V. (1996) Cipro£oxacin resistant Campylobacter spp. In humans: an epidemiological and laboratory study. J. Antimicrob. Chemother. 37, 747^757. [10] Zirnstein, G.W., Li, Y., Swaminathan, B. and Angulo, F. (1999) Cipro£oxacin resistance in Campylobacter jejuni isolates : detection of gyrA resistance mutations by MAMA PCR and DNA sequence analysis. J. Clin. Microbiol. 37, 3276^3280. [11] Smith, K., Besser, J.M., Hedberg, C.W., Leano, F.T., Bender, J.B. and Wicklund, J.H. et al. (1999) Quinolone-resistant Campylobacter jejuni infections in Minnesota, 1992^1998. Investigation Team (see comments). N. Engl. J. Med. 340, 1525^1532. [12] Establishment of a National Surveillance Program for Antimicrobial Resistance in Salmonella (1996) Centers for Disease Control and Prevention, Morbidity and Mortality Weekly Report, February 9, 45(05), pp. 110^111. [13] Adler Mosca, H., Lu«thy-Hottenstein, J., Martinetti, G., Burnens, A.P. and Alwegg, M. (1991) Development of resistance to quinolones in ¢ve patients with campylobacteriosis treated with nor£oxacin or cipro£oxacin. Eur. J. Clin. Microbiol. Infect. Dis. 10, 953^957. [14] Segreti, J., Gootz, T.D., Goodman, L.J., Parkhurst, G.W., Quinn, J.P., Martin, B.A. and Trenholme, G.M. (1992) High-level quinolone resistance in clinical isolates of Campylobacter jejuni. J. Infect. Dis. 165, 667^670. [15] Maxwell, A. (1992) The molecular basis of quinolone action. J. Antimicrob. Chemother. 30, 409^414. [16] Sugino, A., Peebles, C.L., Kreuzer, K.N. and Cozzarelli, N.R. (1977) Mechanism of action of nalidixic acid: puri¢cation of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc. Natl. Acad. Sci. USA 74, 4767^ 4771. [17] Taylor, D.E. and Chau, A. (1997) Cloning and nucleotide sequence of the gyrA gene from Campylobacter fetus subsp. fetus ATCC 27374 and characterization of cipro£oxacin-resistant laboratory and clinical isolates. Antimicrob. Agents Chemother. 28, 708^710.
FEMSLE 9530 10-8-00
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7 [18] Wang, Y., Huang, W.M. and Taylor, D.E. (1993) Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations. Antimicrob. Agents Chemother. 37, 457^463. [19] Yoshida, H., Bogaki, M., Nakamura, M. and Nakamura, S. (1990) Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob. Agents Chemother. 34, 1271^ 1272. [20] Drlica, K. and Zhao, X. (1997) DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61, 377^392. [21] Hallett, P. and Maxwell, A. (1991) Novel quinolone resistance mutations of the E. coli DNA gyrase A protein; enzymatic analysis of the mutant proteins. Antimicrob. Agents Chemother. 35, 335^340. [22] Charvalos, E., Peteinaki, E., Spyridaki, I., Manetas, S. and Tselentis, Y. (1996) Detection of cipro£oxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing. J. Clin. Lab. Anal. 10, 129^133. [23] Linton, D., Owen, R.J. and Stanley, J. (1996) Rapid identi¢cation by PCR of the genus Campylobacter and of ¢ve Campylobacter species
[24]
[25]
[26]
[27]
7
enteropathogenic for man and animals. Res. Microbiol. 147, 707^ 718. Gonzalez, I.., Grant, K.A., Richardson, P.T., Park, S.F. and Collins, M.D. (1997) Speci¢c identi¢cation of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol. 35, 759^763. National Committee for Clinical Laboratory Standards (1997) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow Aerobically, 4th edn. Approved Standard M7-A4, 4th edn., National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania. National Committee for Clinical Laboratory Standards (1998) Performance Standards for Antimicrobial Susceptibility Testing; 8th Informational Suppl.. NCCLS document M100-S8. National Committee for Clinical Laboratory Standards, Wayne, PA. Cha, R.S., Zarbl, H., Keohavong, P. and Thilly, W.G. (1992) Mismatch ampli¢cation mutation assay (MAMA): application to the c-H-ras gene. In: PCR Methods and Applications, pp. 14^20. Cold Spring Harbor Laboratory Press, Plainview, NY.
FEMSLE 9530 10-8-00
www.fems-microbiology.org
Characterization of gyrA mutations associated with £uoroquinolone resistance in Campylobacter coli by DNA sequence analysis and MAMA PCR Gerald Zirnstein a
a;
*, Leta Helsel a , Yu Li a , Bala Swaminathan a , John Besser
b
National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Mail Stop C03, 1600 Clifton Road, Atlanta, GA 30333, USA b Minnesota Department of Health, Minneapolis, MN, USA Received 2 March 2000 ; received in revised form 26 June 2000; accepted 29 June 2000
Abstract Increasing numbers of fluoroquinolone-resistant Campylobacter coli isolates received at the Minnesota State Public Health Laboratory and at the Centers for Disease Control and Prevention have been a cause for concern. The gyrA quinolone resistance-determining regions of several fluoroquinolone-resistant isolates were sequenced to examine the mechanism of resistance. Ciprofloxacin-resistant C. coli isolates examined by DNA sequencing had a Thr-86 to Ile (ACTCATT) gyrA mutation, leading to resistance to fluoroquinolone antibiotics. A mismatch amplification mutation assay polymerase chain reaction protocol was developed to detect this gyrA mutation. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Campylobacter coli ; Cipro£oxacin resistance; Mismatch polymerase chain reaction; gyrA mutation
1. Introduction Diarrhea caused by Campylobacter coli and Campylobacter jejuni is generally a self-limiting disease. However, early antibiotic treatment is recommended to reduce severity and duration. Fluoroquinolones are the treatment of choice for adults and are also prescribed for prophylactic treatment against traveller's diarrhea [1^6]. The existence of £uoroquinolone-resistant Campylobacter isolates has been reported in various countries for several years, including the Netherlands [6], Sweden [7], Thailand [8], UK [9] and USA [10,11]. The use of £uoroquinolones in agriculture to heal diseases of poultry [11,12] is thought to be a contributing factor for the emergence of £uoroquinolone-resistant enteric bacteria. Reports of the emergence of quinolone-resistant Campylobacter isolates in vivo after quinolone therapy [13,14] lend additional support to the contention that resistant isolates may develop after therapeutic treatment with quinolone antibiotics. For these reasons it is of interest to us to investigate the
* Corresponding author. Tel. : +1 (404) 639 0272; Fax: +1 (404) 639 3333; E-mail : [email protected]
mechanisms by which C. coli isolates are becoming resistant to quinolone antibiotics. DNA gyrase, a type II topoisomerase which introduces negative supercoils into DNA and which is essential for bacterial viability, is a major target for quinolone antibiotics in bacteria. The binding of quinolones to the GyrA subunits to prevent gyrase function is the primary mechanism [15,16]. Several studies have shown that mutations in the gyrA gene of several Campylobacter species are primarily responsible for the resistance of most Campylobacter clinical isolates to quinolone antibiotics [9,10,17,18]. In Escherichia coli, most mutations resulting in £uoroquinolone resistance map between codons 67 and 106, near the Tyr-122 catalytic site of DNA gyrase. Thus, this region has been termed the quinolone resistance-determining region (QRDR) [19]. An analogous QRDR region exists in the C. jejuni gyrA gene, but the gyrA Tyr-125 residue appears to be the catalytic site [18,20]. A single mutation of Ser-83 to Ala in the GyrA protein of E. coli allows DNA gyrase activity to become resistant to the action of cipro£oxacin and nalidixic acid in vitro [21]. Mutations in the QRDR region of the C. coli gyrA gene analogous to known quinolone resistance mutations in C. jejuni and E. coli could reasonably be expected to be re-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 0 6 - 2
FEMSLE 9530 10-8-00
2
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
sponsible for quinolone antibiotic resistance in C. coli isolates. Several £uoroquinolone-resistant C. coli isolates were recently received by the Minnesota State Public Health Laboratory (SPHL) and the Centers for Disease Control and Prevention (CDC). This study was initiated to investigate the type and frequency of gyrA mutations in C. coli isolates with resistance to nalidixic acid and cipro£oxacin, and to develop a rapid polymerase chain reaction (PCR) protocol for the detection of gyrA mutations relevant to these resistance phenotypes that could serve as an alternative to previously reported rapid methods [22]. In addition, we wished to determine a greater amount of the DNA sequence surrounding the gyrA QRDR region of C. coli isolates than had been previously submitted to GenBank (Everett and Piddock, accession number U63413) in order to allow for greater £exibility in the choice of primers for PCR ampli¢cation, direct sequencing of the PCR ampli¢cation products, and rapid analysis of the actual DNA point mutations themselves. To our knowledge, the DNA sequence of C. coli gyrA quinolone-resistance point mutations has never been published or submitted to GenBank. Fluoroquinolone antibiotics are currently one of the most e¡ective antimicrobial agents for the elimination of C. coli infections and a rapid, speci¢c, inexpensive, molecular method for the detection of quinolone-resistance mutations should be able to serve as an aid to determine if £uoroquinolone antibiotics are appropriate treatment for speci¢c clinical cases of the infection or for epidemiological studies. 2. Materials and methods 2.1. Strains, culture conditions and antibiotic susceptibility testing Quinolone-susceptible C. coli isolates used in this study included nine isolates from the CDC and Minnesota SPHL collections. Fifteen quinolone-resistant isolates examined in this study were isolated in Minnesota and provided by the Minnesota SPHL ; two were from CDC. Culture media, growth environment and biochemical tests were performed as previously reported [10]. PCR ampli¢cation for the identi¢cation of the genus Campylobacter was performed according to the method of Linton et al. [23]. Further characterization to the species level was
performed by using a PCR test based on the ceuE gene developed by Gonzalez et al. [24]. Screening for antimicrobial resistance was performed with E-test strips (AB Biodisk, Solna, Sweden) as previously published [10]. The nalidixic acid and cipro£oxacin antibiotypes were con¢rmed by agar dilution testing, as described by the National Committee for Clinical Laboratory Standards (NCCLS) [25], with modi¢cations as previously published [10]. The interpretive breakpoints for the minimum inhibitory concentrations (MICs) to nalidixic acid were 9 8 Wg ml31 as susceptible and v 32 Wg ml31 as resistant. The interpretive breakpoints for the MICs to cipro£oxacin were 9 1 Wg ml31 as susceptible and v 4 Wg ml31 as resistant [26]. 2.2. DNA isolation, PCR, DNA sequencing and nucleotide sequence analysis Chromosomal DNA was puri¢ed, as previously published [10]. Primers GZgyrA5 and GZgyrA6 (Table 1) were originally chosen for PCR ampli¢cation of a 673bp product containing the QRDR region of the gyrA gene of quinolone-resistant and -sensitive C. jejuni [10] after analysis of the C. jejuni gyrA gene sequence published by Wang et al. [18]. In this study, primers GZgyrA5 and GZgyrA6 were used for gyrA PCR, which resulted in DNA bands of approximately 673 bp from the majority of C. coli isolates from this study. However, direct sequencing of these C. coli gyrA PCR products only yielded reliable DNA sequence data with primer GZgyrA6 from three of the C. coli isolates. Good gyrA DNA sequence data from C. coli type strain ATCC 33559 using primer GZgyrA6 was used to select new primers for further PCR and sequencing. Primers GZgyrACcoli3F and GZgyrACcoli4R (Table 1) were chosen for the ¢nal PCR and sequencing of the gyrA QRDR region of the C. coli isolates, producing a PCR product 505 bp in length. PCR reactions (100 Wl each) contained 75 ng of puri¢ed chromosomal DNA and were performed under conditions previously described [10]. Primers were synthesized by the CDC Biotechnology Core Facility. After ampli¢cation, 5 Wl of the product was loaded onto a 2.0%, 0.5UTBE, horizontal agarose gel and stained with ethidium bromide for analysis after electrophoresis at 150 V for 1 h. The remaining gyrA PCR products were puri¢ed and used in Taq dye terminator cycle sequencing, as previously described [10].
Table 1 Primers used in this study Name
Sequence (5P to 3P)
GZgyrA5 GZgyrA6 GZgyrACcoli3F GZgyrACcoli4R CampyMAMAgyrA8
ATT CCA TAT GTC TAA
TTT TAA GAG CAT GGC
AGC ATT CGT CTA ATC
AAA ATT TAT CAA GTA
GAT CCA TAT GCT AAC
TCT CCT CGG CGT AGC
GAT GT TC TA CA
Direction, purpose
Reference
forward, C. jejuni gyrA primer reverse, C. jejuni gyrA primer forward, speci¢c for C. coli gyrA reverse, speci¢c for C. coli gyrA reverse, C. coli putative codon 86 gyrA mutation detection primer
[10] [10] this study this study this study
FEMSLE 9530 10-8-00
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
DNA sequence analysis and primer selection for the mismatch ampli¢cation mutation assay (MAMA) PCR [27] were performed in a manner similar to that previously described [10]. Brie£y, conserved primers were chosen with OLIGO Primer Analysis Software version 5.0 (National Biosciences, Plymouth, MN, USA) and mutation primers were chosen after a manual analysis of the DNA sequence near putative codon 86 of the C. coli gyrA gene of £uoroquinolone-susceptible and -resistant isolates. DNA sequences were analyzed with DNASIS version 2.5 (Hitachi Software Engineering Co., San Francisco, CA, USA).
3
except only 10 pmol of each primer was used. PCR cycling conditions were as follows : initial denaturation was at 94³C for 3 min, followed by 30 cycles of 94³C for 30 s, 50³C for 30 s and 72³C for 20 s. Eight-Wl aliquots of each PCR product were loaded onto horizontal, 2.0%, 0.5UTBE agarose gels and stained with ethidium bromide for analysis after electrophoresis. 3. Results
2.3. MAMA PCR protocol
3.1. Detection of gyrA mutations associated with quinolone resistance
The mutation primer was chosen after a visual analysis of the DNA sequences near putative codon 86 of the C. coli gyrA genes. A conserved, forward primer, GZgyrACcoli3F, and a reverse, mutation detection primer, CampyMAMAgyrA8 (Table 1), were used together in a PCR reaction to generate a 192-bp PCR product that was a positive indication of the presence of the Thr-86 to Ile (ACTCATT) mutation in the C. coli gyrA gene. Primer GZgyrACcoli4R, a conserved reverse primer, was used in conjunction with primer GZgyrACcoli3F to produce a positive PCR control product of 505 bp from the gyrA gene of all C. coli DNA preparations used in this study. PCR reactions (100 Wl each) were performed as above,
We used primers based on the C. jejuni sequence [10] to generate PCR fragments for preliminary sequencing of C. coli gyrA. Only primer GZgyrA6 produced reliable gyrA sequence data with DNA from only a few C. coli isolates. Reliable gyrA DNA sequence data from C. coli type strain ATCC 33559, using primer GZgyrA6, was employed to select new primers for PCR and sequencing (Table 1 and Fig. 1). The identity between the 633 bases of the gyrA gene of C. coli ATCC 33559 and the analogous region of the gyrA gene of C. jejuni UA580 was 82%, with some areas much more conserved than others (Fig. 1). An examination of the gyrA DNA sequence data, some of which are shown in Fig. 1, enabled us to determine the presence
Table 2 Mutations in the gyrA QRDR region of C. coli isolates and MICs to nalidixic acid and cipro£oxacin CDC No.
Health laboratory/source
Putative gyrA aa 86 mutationa
Nalidixic acid MIC (Wg ml31 )
Cipro£oxacin MIC (Wg ml31 )
MAMA PCR result
D126 D134 D145b D730 D2591 D2592 D2595 D2596 D5416 D5403 D5404 D5413 D5414 D5415 D5417 D5418 D5419 D5420 D5423 D5427 D5428 D5429 D5430 D5431 D5472 D5496
Canada, animal Canada animal WA USA Australia Belgium animal MN MN MN MN MN MN MN MN MN MN MN MN MN MN MN MN MA MA
NA NA no NA NA NA NA NA no yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes NA NA
16 8 16 8 8 8 16 32 16 256 128 128 128 256 256 128 256 256 128 256 256 256 256 128 128 128
0.25 0.06 0.25 0.03 0.06 0.125 0.125 0.125 0.25 8 4 8 8 16 16 8 16 16 4 8 16 16 16 16 16 32
negative negative negative negative negative negative negative negative negative positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive
a b
Mutants have a substitution of Thr-86 to Ile (ACT to ATT). NA indicates isolates were not sequenced. C. coli ATCC 33559 type strain.
FEMSLE 9530 10-8-00
4
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
Fig. 1. Nucleotide sequence alignment of partial gyrA gene sequences of C. coli isolates from this study compared to other Campylobacter and E. coli gyrA sequences from the GenBank database. The sequences of E. coli KL-16 (accession number x06744), Campylobacter fetus ATCC 27374 (accession number u25640) and C. jejuni UA580 (accession number l04566) were compared to the partial gyrA gene sequences of C. coli NCTC 11366 (accession number u63413), C. coli ATCC 33559 (accession number af092101, this study) and C. coli isolates D5416, D5428 and D5429 from Minnesota. Codon numbers are based on codon positions of the complete C. jejuni UA580 gyrA gene, and are only putative for C. coli sequences, since the ¢rst few bases of the C. coli gyrA genes were not sequenced.
or absence of non-synonymous mutations in the QRDR region of the Minnesota isolates and compare this information to the nalidixic acid and cipro£oxacin MICs for the C. coli isolates (Table 2). The mutation at putative amino acid (aa) position 86 of the gyrA gene correlates with resistance to cipro£oxacin and nalidixic acid in the C. coli isolates examined. The relationship of the Thr-86 to
Ile mutation to the acquisition of quinolone resistance in other Campylobacter isolates has been previously characterized [9,10,18]. With the exception of isolates D5404 and D5413, all cipro£oxacin-resistant Minnesota isolates possessed identical gyrA DNA sequences with isolates D5428 and D5429 (Fig. 1). Isolate D5404 shared identical 39 and 99 gyrA
FEMSLE 9530 10-8-00
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
DNA codons as the C. coli ATCC 33559 type strain, and isolate D5413 shared the same 39 and 157 gyrA DNA codons as the C. coli ATCC 33559 type strain, while the remainder of the D5404 and D5413 gyrA DNA sequences were identical to D5428 and D5429 (refer to Fig. 1). 3.2. Detection of the Thr to Ile mutation (ACTCATT) by use of MAMA PCR A rapid PCR method was developed for the detection of the Thr to Ile (ACT to ATT) mutation at the putative aa position 86 in the gyrA DNA sequence of C. coli isolates. Isolates with the wild-type aa 86 codon (ACT, quinolonesusceptible) were not ampli¢ed with the reverse mutation primer, CampyMAMAgyrA8 (Fig. 1), whereas the isolates with the mutated aa 86 codon (ATT, quinolone-resistant) generated a 192-bp PCR product with the CampyMAMAgyrA8 reverse mutation primer and the GZgyrACcoli3F forward conserved primer (Table 2, Fig. 1). Conserved primers GZgyrACcoli3F and GZgyrACcoli4R generated a 505-bp gyrA PCR product with DNA isolated from all of the isolates listed in this study. Seventeen of the strains in Table 2 were examined by direct DNA sequencing of gyrA gene PCR products. CDC isolate D145 (the ATCC 33559 type strain) and isolate D5416 (from Minnesota) were both cipro£oxacin-susceptible, had wild-type gyrA DNA sequences and had negative results with the MAMA PCR mutation detection primer. Isolates D5403, D5404, D5413, D5414, D5415, D5417, D5418, D5419, D5420, D5423, D5427, D5428, D5429, D5430 and D5431 were all from Minnesota, were all cipro£oxacin-resistant, all had the Thr to Ile (ACT to ATT) mutation at the putative aa 86 position of the gyrA gene, and all had positive results with the MAMA PCR mutation detection primer. To determine if the MAMA PCR assay was valid with other CDC C. coli isolates from varied sources and locations, spanning a number of years, nine more CDC isolates, D126, D134, D730, D2591, D2592, D2595, D2596, D5472 and D5496, were examined by MAMA PCR alone to verify whether or not the MAMA PCR assay could serve as a replacement for sequencing data when the antibiotype of the isolate is taken into account (Table 2). Results indicate the putative aa position 86 cipro£oxacin-resistance mutation was correctly identi¢ed in all cipro£oxacin-resistant isolates and that the MAMA PCR never gave a positive PCR response with any cipro£oxacin-sensitive isolates from this group. We were satis¢ed that the MAMA PCR assay worked well for all isolates tested. 4. Discussion The e¡ectiveness of £uoroquinolone antibiotics for the treatment of traveller's diarrhea [3], the use of £uoroqui-
5
nolones in agriculture to heal diseases of poultry [12], and the common practice of prescribing £uoroquinolone antibiotics for gastroenteritis for the purpose of shortening absences from work [2] have led to exposure to this class of antibiotics. The incidence of quinolone-resistant Campylobacter infection has increased recently, and evidence suggests that a signi¢cant number of human isolates are acquired from poultry products [11]. Reports of the emergence of quinolone-resistant Campylobacter isolates in vivo after quinolone therapy [13,14] can also be found in the literature. Several £uoroquinolone-resistant C. coli isolates received by the SPHL and the CDC prompted us to perform this study to investigate the type and frequency of gyrA mutations in C. coli isolates with resistance to nalidixic acid and cipro£oxacin, and to develop a rapid PCR protocol for the detection of gyrA mutations relevant to these resistance phenotypes that could serve as an alternative to previously reported rapid methods [22]. Another goal of this study was to obtain a greater amount of the DNA sequence surrounding the gyrA QRDR region of C. coli isolates than had been previously submitted to GenBank (Everett and Piddock, accession number U63413) in order to allow for greater £exibility in the choice of primers for PCR ampli¢cation, direct sequencing of the PCR ampli¢cation products and rapid analysis of the actual DNA point mutations themselves. A single mutation of Ser-83 to Ala in the GyrA protein of E. coli allows DNA gyrase activity to become resistant to the action of cipro£oxacin and nalidixic acid in vitro [21]. A C. jejuni Thr-86 to Ile gyrA mutation, in a position equivalent to the E. coli Ser-83 to Ala mutation, has been reported as responsible for high resistance to £uoroquinolones in C. jejuni in vivo [9,18]. We reported that other US isolates of C. jejuni that were resistant to nalidixic acid (MIC v32 Wg ml31 ) were also resistant to cipro£oxacin (MIC v4 Wg ml31 ), and all had the Thr-86 to Ile (ACA to ATA) mutation [10]. The same mechanism, a Thr-86 to Ile mutation (ACT to ATT) in the QRDR region of the gyrA gene, appears to be responsible for the increased resistance to £uoroquinolone antibiotics noted for the C. coli isolates, since isolates without this mutation were sensitive to cipro£oxacin (Table 1). C. coli isolates tested that were highly resistant to nalidixic acid (MIC v128 Wg ml31 ) were also resistant to cipro£oxacin (MIC v4 Wg ml31 ). Only isolate D2596 had a nalidixic acid MIC of 32 Wg ml31 , which is at the breakpoint for resistance, while being susceptible to cipro£oxacin (Table 1). Factors other than gyrA QRDR mutations, such as mutations in gyrB, parC and parE, e¥ux pumps, permeability factors or detoxi¢cation [20], may be responsible for some of the variability in nalidixic acid and cipro£oxacin MICs among resistant C. coli isolates in Table 1. Since the Thr to Ile (ACT to ATT) mutation in putative codon 86 of the C. coli gyrA gene appears to be a commonly encountered mutation leading to £uoroquinolone resistance and one for which we have reported DNA se-
FEMSLE 9530 10-8-00
6
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7
quence data (this study), we developed a MAMA protocol for the speci¢c detection of this mutation. The design of this type of mismatch primer for mutation detection has been explained in detail previously [10,27]. The scienti¢c basis for the ability of the mutation detection primer, CampyMAMAgyrA8, to generate a PCR product with mutant DNAs and not wild-type DNAs is based on the fact that the 3P end of the mutation detection primer has only one mismatch with the mutant, £uoroquinolone-resistant, gyrA sequences, and is able to generate a PCR product, while two mismatches occur between the extreme 3P end of the mutation detection primer and the wild-type, £uoroquinolone-susceptible, gyrA sequences, and no PCR product is generated (see Fig. 1) [27]. The primary variable used to eliminate false positive PCR signals between the mutation detection primer and wild-type DNAs was to increase the annealing temperature of the PCR reaction. With a PCR annealing temperature of 50³C, no PCR DNA bands were observed when wild-type DNA was used with the mutation detection primer, but a very strong PCR DNA band was always observed on the gel when the mutant DNA was included with the mutation detection primer in the PCR reaction. The likelihood of false negative reactions can be greatly reduced by careful addition of measured amounts of DNA template and by including PCR controls with each experiment which include conserved primers (GZgyrACcoli3F and GZgyrACcoli4R) to help determine if the DNA template is degraded. It appears that most of the C. coli cipro£oxacin-resistant isolates are more highly related to one another than to the C. coli ATCC 33559 type strain or isolates D5404 and D5413 and may be from a related source. However, the number of isolates studied is very limited, and they come from a geographically restricted area, which makes it di¤cult to generalize the ¢ndings to other parts of the USA or the world. The analysis of a larger number of isolates from di¡erent areas and di¡erent periods in time would enable a better evaluation of the frequencies of silent mutations within gyrA and would more thoroughly explain the di¡erences between the MN isolates and the reference strain. Cipro£oxacin-susceptible isolate D5416 appears to be similar to the parent strain from which the majority of the cipro£oxacin-resistant isolates apparently arose, since the only di¡erence is the lack of a gyrA mutation at codon 86. However, since there is little data on the stability of the mutations in the gyrA gene of C. coli, then it is also theoretically possible that isolate D5416 may have reverted from CipR to CipS , particularly if antibiotics were no longer present in the growth environment. The MAMA PCR method developed for the C. coli gyrA Thr-86 to Ile mutation (ACT to ATT) should serve as a simple, speci¢c, rapid, inexpensive and portable alternative to methods such as the nonradioisotopic, singlestrand conformation polymorphism method previously published [22] or DNA sequencing for detection of this important £uoroquinolone resistance mutation and may
prove useful for clinical diagnosis or epidemiological studies.
References [1] Nachamkin, I. (1993) Campylobacter infections. Curr. Opin. Infect. Dis. 6, 72^76. [2] Piddock, L.J.V. (1995) Review : quinolone resistance and Campylobacter spp.. J. Antimicrob. Chemother. 36, 891^898. [3] Taylor, D.N., Sanchez, J.L., Candler, W., Thornton, S., McQueen, C. and Escheverria, P. (1991) Treatment of travellers diarrhea: cipro£oxacin plus loperamide compared with cipro£oxacin alone. Ann. Intern. Med. 114, 731^734. [4] Gilbert, D.N., Moellering, Jr., R.C. and Sande, M.A. (1999) In: The Sandford Guide to Antimicrobial Therapy, p. 12 and p. 48. Antimicrobial Therapy, Hyde Park, VT. [5] Abramowicz, M. (Ed.) (1998) In: The Medical Letter Handbook of Antimicrobial Therapy, p. 43. The Medical Letter, New Rochelle, NY. [6] Koenraad, P.M.F.J., Jacobs-Reitsma, W.F., Van der Laan, T., Beumer, R.R. and Rombouts, F.M. (1995) Antibiotic susceptibility of Campylobacter isolates from sewage and poultry abattoir drain water. Epidemiol. Infect. 115, 475^483. [7] Sjo«gren, E., Lindblom, G.B. and Kaijser, B. (1997) Nor£oxacin resistance in Campylobacter jejuni and Campylobacter coli isolates from Swedish patients. J. Antimicrob. Chemother. 40, 257^261. [8] Kuschner, R.A., Trofa, A.F., Thomas, R.J., Hoge, C.W., Pitarangsi, C. and Amato, C. et al. (1995) Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where cipro£oxacin resistance is prevalent. Clin. Infect. Dis. 21, 536^541. [9] Gaunt, P.N. and Piddock, L.J.V. (1996) Cipro£oxacin resistant Campylobacter spp. In humans: an epidemiological and laboratory study. J. Antimicrob. Chemother. 37, 747^757. [10] Zirnstein, G.W., Li, Y., Swaminathan, B. and Angulo, F. (1999) Cipro£oxacin resistance in Campylobacter jejuni isolates : detection of gyrA resistance mutations by MAMA PCR and DNA sequence analysis. J. Clin. Microbiol. 37, 3276^3280. [11] Smith, K., Besser, J.M., Hedberg, C.W., Leano, F.T., Bender, J.B. and Wicklund, J.H. et al. (1999) Quinolone-resistant Campylobacter jejuni infections in Minnesota, 1992^1998. Investigation Team (see comments). N. Engl. J. Med. 340, 1525^1532. [12] Establishment of a National Surveillance Program for Antimicrobial Resistance in Salmonella (1996) Centers for Disease Control and Prevention, Morbidity and Mortality Weekly Report, February 9, 45(05), pp. 110^111. [13] Adler Mosca, H., Lu«thy-Hottenstein, J., Martinetti, G., Burnens, A.P. and Alwegg, M. (1991) Development of resistance to quinolones in ¢ve patients with campylobacteriosis treated with nor£oxacin or cipro£oxacin. Eur. J. Clin. Microbiol. Infect. Dis. 10, 953^957. [14] Segreti, J., Gootz, T.D., Goodman, L.J., Parkhurst, G.W., Quinn, J.P., Martin, B.A. and Trenholme, G.M. (1992) High-level quinolone resistance in clinical isolates of Campylobacter jejuni. J. Infect. Dis. 165, 667^670. [15] Maxwell, A. (1992) The molecular basis of quinolone action. J. Antimicrob. Chemother. 30, 409^414. [16] Sugino, A., Peebles, C.L., Kreuzer, K.N. and Cozzarelli, N.R. (1977) Mechanism of action of nalidixic acid: puri¢cation of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc. Natl. Acad. Sci. USA 74, 4767^ 4771. [17] Taylor, D.E. and Chau, A. (1997) Cloning and nucleotide sequence of the gyrA gene from Campylobacter fetus subsp. fetus ATCC 27374 and characterization of cipro£oxacin-resistant laboratory and clinical isolates. Antimicrob. Agents Chemother. 28, 708^710.
FEMSLE 9530 10-8-00
G. Zirnstein et al. / FEMS Microbiology Letters 190 (2000) 1^7 [18] Wang, Y., Huang, W.M. and Taylor, D.E. (1993) Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations. Antimicrob. Agents Chemother. 37, 457^463. [19] Yoshida, H., Bogaki, M., Nakamura, M. and Nakamura, S. (1990) Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob. Agents Chemother. 34, 1271^ 1272. [20] Drlica, K. and Zhao, X. (1997) DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61, 377^392. [21] Hallett, P. and Maxwell, A. (1991) Novel quinolone resistance mutations of the E. coli DNA gyrase A protein; enzymatic analysis of the mutant proteins. Antimicrob. Agents Chemother. 35, 335^340. [22] Charvalos, E., Peteinaki, E., Spyridaki, I., Manetas, S. and Tselentis, Y. (1996) Detection of cipro£oxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing. J. Clin. Lab. Anal. 10, 129^133. [23] Linton, D., Owen, R.J. and Stanley, J. (1996) Rapid identi¢cation by PCR of the genus Campylobacter and of ¢ve Campylobacter species
[24]
[25]
[26]
[27]
7
enteropathogenic for man and animals. Res. Microbiol. 147, 707^ 718. Gonzalez, I.., Grant, K.A., Richardson, P.T., Park, S.F. and Collins, M.D. (1997) Speci¢c identi¢cation of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol. 35, 759^763. National Committee for Clinical Laboratory Standards (1997) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow Aerobically, 4th edn. Approved Standard M7-A4, 4th edn., National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania. National Committee for Clinical Laboratory Standards (1998) Performance Standards for Antimicrobial Susceptibility Testing; 8th Informational Suppl.. NCCLS document M100-S8. National Committee for Clinical Laboratory Standards, Wayne, PA. Cha, R.S., Zarbl, H., Keohavong, P. and Thilly, W.G. (1992) Mismatch ampli¢cation mutation assay (MAMA): application to the c-H-ras gene. In: PCR Methods and Applications, pp. 14^20. Cold Spring Harbor Laboratory Press, Plainview, NY.
FEMSLE 9530 10-8-00