Sexually Transmitted Diseases:
Multiclonal Increase in Ciprofloxacin-Resistant Neisseria gonorrhoeae, Thailand, 1998–1999
TREES, DAVID L. PhD§; SIRIVONGRANGSON, PACHARA BSc, MD*; SCHULTZ, ABBIGAIL J. BS, MPH§; BUATIANG, ANUKUL BEd*; NEAL, SANDRA W. MS§; KNAPP, JOAN S. PhD§; KILMARX, PETER H. MD†‡
From the *Venereal Disease Division, Ministry of Public Health, Bangkok, Thailand; †HIV/AIDS Collaboration, Nonthaburi, Thailand; and ‡Division of STD Prevention and §Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
The authors thank Dr. Khanchit Limpakarnjanarat and the clinic staff and laboratory technicians at Bangrak Hospital for their assistance.
Reprint requests: David L. Trees, PhD, Gonorrhea Research Branch, Mailstop G-39, DASTLR, NCID, Centers for Disease Control and Prevention, Atlanta, GA 30333. E-mail: email@example.com
Received for publication October 17, 2001,
revised January 25, 2002, and accepted January 25, 2002.
Background: Neisseria gonorrhoeae isolates exhibiting clinically significant resistance to fluoroquinolones have been isolated most frequently in Asian and western Pacific countries, including Thailand. In Bangkok, Thailand, ciprofloxacin has been used to treat gonorrhea since 1987.
Goal: Our goal was to determine the prevalence of isolates of N gonorrhoeae that exhibit resistance to ciprofloxacin in Bangkok and to characterize these strains with regard to ciprofloxacin MICs, auxotype/serovar (A/S) classification, gyr A and par C mutations responsible for ciprofloxacin resistance, and outer membrane lipoprotein (Lip) subtype analysis.
Study Design: MICs of gonococcal isolates from consecutive patients attending the Bangrak Hospital STD Clinic in Bangkok were determined by agar dilution. A/S class was determined by established procedures. Mutations within gyr A and par C were determined by DNA sequencing. Lip subtypes were determined by PCR and DNA sequencing.
Results: In 1998 and 1999, 115 of 168 isolated strains of N gonorrhoeae exhibited decreased susceptibility or resistance to ciprofloxacin, and three cases of possible ciprofloxacin treatment failure were identified. Ciprofloxacin-resistant (CipR) strains increased from 13.8% (8/58) in 1998 to 25.4% (28/110) in 1999 (P = 0.08). Ciprofloxacin MICs of CipR isolates ranged from 1.0 μg/ml to 32.0 μg/ml. CipR strains belonged to a number of A/S classes and Lip subtypes. Different CipR strains contained alterations at both amino acid 91 and amino acid 95 of gyr A and also contained an amino acid alteration in par C. These alterations are known to be involved in gonococcal resistance to ciprofloxacin.
Conclusions: The prevalence of CipR strains of N gonorrhoeae isolated in Bangkok increased substantially in the 1990s. Characterization of the CipR isolates revealed a number of different strain subtypes, indicating that CipR isolates in Bangkok are not from a single clonal source and therefore result from multiple cases of importation or local emergence. Because of the high level of CipR isolates at Bangrak Hospital, in 2000 the Thai Ministry of Public Health issued recommendations against the use of fluoroquinolones for the treatment of gonococcal infection in Thailand.
SINCE 1993, the Centers for Disease Control and Prevention (CDC) has recommended that broad-spectrum cephalosporins or fluoroquinolones be used to treat uncomplicated gonorrhea, except in Hawaii and for cases acquired in the western Pacific 1 (also unpublished data). However, strains exhibiting decreased susceptibility to ciprofloxacin have been reported from many countries; strains exhibiting clinically significant resistance to CDC-recommended doses of ciprofloxacin (500 mg, single dose, PO) or ofloxacin (400 mg, single dose, PO) have been reported from Asia, Australia, the United Kingdom, the United States, and Canada. 2,3Neisseria gonorrhoeae isolates exhibiting clinically significant resistance to fluoroquinolones have been isolated most frequently in Asian and western Pacific countries, where resistant strains may account for more than 10% of all gonococcal isolates. 3–5
Studies of the genetic mutations resulting in resistance of gonococcal isolates to fluoroquinolones have shown that mutations within the quinolone resistance–determining regions (QRDRs) of gyr A and par C are responsible for resistance to these agents. 6–8 These mechanisms are analogous to those observed in Escherichia coli and other bacteria. 9,10 However, unlike gyr B mutations in E coli, mutations in the N gonorrhoeae gyrB gene do not appear to have an impact on fluoroquinolone resistance. 11 The order in which mutations and the resulting amino acid alterations occur and result in increased fluoroquinolone MICs in gonococcal strains follows a fairly predictable pattern in N gonorrhoeae: mutations which confer alterations at Ser-91 and/or Asp-95 of GyrA result in intermediate resistance to ciprofloxacin (CipI; MICs of ciprofloxacin, 0.125–0.5 μg/ml). On the other hand, clinically significant resistance to CDC-recommended fluoroquinolone regimens (CipR; MIC of ciprofloxacin, ≥1.0 μg/ml) appears to require one or more mutations in par C in addition to both gyr A mutations. However, this order of mutations is not absolute, and a number of GyrA/ParC protein “alteration patterns” have been described. 5,7,8,12,13
In this study, 115 strains of N gonorrhoeae exhibiting decreased susceptibility or resistance to ciprofloxacin were isolated in Bangkok, Thailand, where ciprofloxacin has been used to treat gonorrhea since 1987. These isolates were characterized to determine their diversity by examination of their A/S class, GyrA/ParC alterations, penicillin/tetracycline phenotype, and Lip subtype.
Materials and Methods
One hundred sixty-eight isolates of N gonorrhoeae were recovered, including 58 isolates from the period of January through September 1998 and 110 from the period of May through September 1999. Of 117 isolates collected in 1998, only 58 remained viable upon reculture at the Bangrak Hospital Laboratory in 1999. Isolates were from symptomatic male patients and all female patients seeking services at the Bangrak Hospital Sexually Transmitted Disease (STD) Clinic. Also included in the set were 7 CipR isolates (1 from 1998 and 6 from 1999), which were recovered from 6 patients during follow-up examinations. Isolates were identified in Bangkok by gram stain and were frozen in trypticase soy broth containing 20% glycerol, stored at −70 °C, and shipped on dry ice to the CDC.
Isolates were characterized by auxotype, serovar, and plasmid content as described previously. 14–16 Agar dilution susceptibilities of isolates to penicillin G, tetracycline, spectinomycin, and ciprofloxacin were determined and interpreted by National Committee for Clinical Laboratory Standards (NCCLS)–recommended methods, as described previously. 17,18 Isolates were assigned to penicillin/tetracycline resistance phenotypes as described previously. 19–21 Lip subtyping of CipR isolates was performed as previously described. 22
GyrA/ParC Sequencing Analysis
Primers used for GyrA were a forward-synthesis 21-base oligonucleotide with the sequence 5′ GCT ATC TCG ACT ACG CCA TGA 3′ and a reverse-synthesis 21-base oligonucleotide with the sequence 5′ CAT GCG GAT TTC GGT ATA GCG 3′. Primers used for ParC were a forward-synthesis 21-base oligonucleotide with the sequence 5′ GTT TCA GAC GGC CAA AAG CCC 3′ and a reverse-synthesis 21-base oligonucleotide with the sequence 5′ GGA CAA CAG CAA TTC CGC AAT 3′. 23,24 Sequencing reactions and analysis were performed as described previously. 12
A total of 168 N gonorrhoeae isolates from 1998 and 1999 were tested by MIC analysis; 79 exhibited decreased susceptibility (CipI) and 36 exhibited clinically significant resistance (CipR) to ciprofloxacin. In 1998, 58.6% (34/58) of the isolates were CipI and 13.8% (8/58) of the isolates were CipR. In 1999, 40.9% (45/110) of the isolates were CipI and 25.4% (28/110) of the isolates were CipR. These CipI and CipR isolates were further analyzed by A/S classification and GyrA/ParC alterations in order to determine their strain profiles. A/S classification indicated that there were 14 A/S classes among the 1998 CipI isolates, with Proto/IB-3 being represented by 5 isolates (Table 1). In 1999, CipI isolates belonged to 19 A/S classes, with Proto/IB-1 (8 isolates) and Proto/IB-3 (6 isolates) being observed most often (Table 1). CipR isolates demonstrated 4 A/S classes in 1998 and 9 classes in 1999, with Pro/IB-3 (5/9 isolates) and Proto/IB-10 (10/28 isolates) being the predominant classes, respectively (Table 1).
The mutations within the QRDRs of gyr A and par C of 115 isolates of N gonorrhoeae are shown by ciprofloxacin resistance category (CipI or CipR), GyrA and ParC QRDR alteration, and year of isolation in Table 2. CipI isolates exhibited six alteration patterns: three patterns with alterations only in GyrA and three patterns with alterations in both GyrA and ParC. Most CipI isolates (40/79; 50.6%) exhibited alterations only at amino acid 91 in GyrA. CipR isolates exhibited five alteration patterns: one pattern in GyrA alone and four patterns involving both GyrA and ParC. Most CipR isolates (21/36) exhibited GyrA-91,95/ParC-Asp-86→Asn (91,95/Asp-86→Asn) alterations (Table 2). The relationship between alteration patterns in the QRDRs of GyrA and ParC and susceptibilities to ciprofloxacin in the 1998 and 1999 isolates is shown in Figure 1. CipI isolates generally contained alterations only in GyrA. When a ParC alteration was detected in a CipI strain, only a single GyrA alteration was present, except for one strain in 1999, which had both. In contrast, CipR strains, with the exception of 3 isolates, had both the 91 and 95 GyrA alterations and at least 1 ParC alteration. One CipR isolate contained two alterations in both GyrA and ParC.
To further characterize the CipR isolates, Lip subtyping was performed. A total of 9 different subtypes were seen, with 16b being the most prominent; it was found in 3 different A/S classes. The Lip subtyping also identified a group of CipR isolates that consisted of 12 strains (2 from 1998 and 10 from 1999) that had the same A/S class (Proto IB-10), GyrA/ParC alterations (91,95/Asp-86), and Lip type (16b). However, when the penicillin–tetracycline phenotypes of the isolates were examined, eight of the isolates were penicillinase-producing N gonorrhoeae (PPNG), three were chromosomally resistant N gonorrhoeae (CMRNG), and one was penicillinase-producing/plasmid-mediated tetracycline-resistant N gonorrhoeae (PP/TR).
Among the CipR isolates were six sets (five pairs and one triplet) of isolates from patients who were culture-positive on their follow-up visit to the clinic. These sets were examined by A/S classification, Lip subtype, GyrA/ParC alterations, and penicillin/tetracycline susceptibilities. In four of the sets (Table 3, patients 1, 3, 4, and 5), the pretreatment and posttreatment isolate pairs had identical strain phenotypes. The other two sets, patients 2 and 6, had identical A/S class, GyrA/ParC alterations, and Lip subtype but differed in their penicillin–tetracycline phenotype. Patient 2’s initial isolate was CMRNG, and the follow-up isolate was PPNG. Patient 6 had 3 isolates; the first 2 isolates were PPNG and the third isolate was PP/TR.
In this study we determined the phenotypes in CipI and CipR strains of N gonorrhoeae isolated in Bangkok. The percentage of isolates exhibiting decreased susceptibilities remained relatively constant from 1998 (72.4%) to 1999 (66.3%). However, the number of isolates with clinically significant resistance increased from 13.8% to 25.4%. The MICs of ciprofloxacin for the CipR isolates ranged from 1.0 to 32.0 μg/ml. The number of GyrA/ParC QRDR alteration patterns among the CipR isolates also increased between 1998 and 1999. Although strains exhibiting the 91,95/Asp-86→Asn alteration pattern accounted for 63.6% of the CipR isolates, four additional GyrA/ParC QRDR alteration patterns were observed. This alteration pattern has previously been seen in isolates from Thailand. 13 CipI isolates accounted for a majority of the isolates exhibiting decreased susceptibilities to ciprofloxacin. The majority of CipI isolates had a GyrA-91 alteration, either alone or in combination with another alteration.
Resistance to ciprofloxacin has increased dramatically since 1994–1995, when only one CipR isolate was detected among a total of 101 isolates. 25 In 1998–1999, decreased susceptibility to ciprofloxacin occurred in a large variety of strains as defined by A/S class, Lip subtype, and GyrA/ParC alterations. After initial examination of the results, there appeared to be two strain clusters of CipR isolates, on the basis of A/S class, that contained four or more isolates. In 1998, a cluster of four Pro/IB-3 CipR isolates was present. However, GyrA/ParC alteration analysis revealed that two of the isolates were 91,95/none and two isolates were 91,95/Ser-87. In 1999, a cluster of 10 Proto IB-10/Lip 16b/91,95/Asp-86 CipR isolates appeared. However, upon examination of the isolates’ penicillin–tetracycline phenotype, eight isolates were found to be PPNG, three were CMR, and one was PP/TR. The presence of the possible strain clusters reinforces the need to use multiple methods when subtyping N gonorrhoeae.
The sets of isolates from the six patients who had positive cultures at the follow-up visit appear to represent varying scenarios. The CipR isolates from patients 3, 4, and 5 appear to represent treatment failures, in that all 3 patients were treated with ciprofloxacin and the dates of their follow-up cultures were in the 7 to 10–day range, which would be consistent with treatment failure. Isolates from patient 1 could also represent a treatment failure since both isolates possess the same strain phenotype, but no treatment data were available for this patient. Isolates from patient 2 appear to indicate a reinfection since the original and posttreatment isolates have different penicillin–tetracycline phenotypes, the patient was treated with ceftriaxone, and the initial isolate was susceptible to this drug. The time period between the initial and follow-up culture, 30 days, would also be consistent with the opportunity for reinfection. Patient 6 had 3 isolates cultured, the first 2 of which had the same strain phenotype. However, the patient was treated with doxycycline, so this could not be considered a ciprofloxacin treatment failure. The third isolate appears to be the result of a reinfection, because this isolate was PP/TR, which contrasts with the first and second isolates, which were PPNG.
This study supports previous results 13 indicating that strains containing identical GyrA/ParC QRDR alteration patterns frequently exhibit different MICs to ciprofloxacin. Examples in the current study are the 21 isolates that contained the 91,95/Asp-86→Asn alteration pattern and had a ciprofloxacin MIC range of 2.0 to 8.0 μg/ml and the 30 isolates with the 91/none alteration pattern with MICs of 0.125 to 0.5 μg/ml. We speculate that the presence of ParC QRDR alterations facilitates high ciprofloxacin MICs, but the actual MIC may be influenced by other characteristics of the strain, such as reduced intracellular drug accumulation, as described by Tanaka et al. 5
The epidemiology of gonorrhea in Thailand has changed dramatically in recent years. Reported cases decreased from 227,451 in 1985 to only 5,382 in 1999, a 42-fold decline. 26 This decline was likely due to decreases in commercial sex patronage and increases in condom use during commercial sex in response to the HIV/AIDS epidemic in Thailand. 27 The wide availability of single-dose, oral treatment of urethritis with fluoroquinolones may also have contributed to the decrease in reported cases. From 1999 to 2000, cases increased 4.5% to 5,622. 26 This increase is likely to be due in part to the emergence of gonococcal resistance to fluoroquinolones as described here, although behavioral changes, including an increase in casual, noncommercial sex, may also have had a role. 28
In summary, these results strongly suggest that the continuing emergence of CipR gonococci is not due to expansion of a single or a few strains but to the emergence of many different CipR gonococcal strains that continue to spread. The importation of CipR strains from other Asian countries and de novo generation of ciprofloxacin resistance in endemic ciprofloxacin-susceptible strains may also have contributed to the increase in CipR isolates in the 1990s. Because of this high level of CipR isolates at Bangrak Hospital, in 2000 the Thai Ministry of Public Health recommended not using fluoroquinolones for the treatment of gonococcal infection in Thailand. Furthermore, the emergence of additional isolates with high ciprofloxacin MICs illustrates the need for continued monitoring throughout the world of the susceptibilities of N gonorrhoeae isolates to fluoroquinolones.
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