Abstract
Surveillance of antibiotic resistance in Neisseria gonorrhoeae showed a decrease in the percentage of β-lactamase-producing isolates but an increase in intermediately penicillin-resistant strains and strains resistant to a high level of tetracycline. MICs for the ciprofloxacin-resistant isolates that emerged increased, and these isolates had mutations in gyrA and parC similar to those observed in the Far East.
Since 1991 five sentinel laboratories in The Netherlands have participated in a surveillance of antibiotic resistance in Neisseria gonorrhoeae. The laboratories are located in Amsterdam, Rotterdam, and The Hague and account for approximately 70% of the gonococcal isolates from The Netherlands (6). They forwarded all isolates of N. gonorrhoeae to the National Institute of Public Health and the Environment 1 month per quarter. Here we report the results of this survey in the period 1991 to 1998.
β-Lactamase production was detected using nitrocefin (Oxoid, Basingstroke, United Kingdom). The susceptibility to antibiotics was determined by the agar dilution method using Iso-Sensitest agar (Oxoid) with 5% horse blood and multipoint inoculation. The inoculum was 106 CFU/spot. The plates were incubated for 18 h at 35°C and 5% CO2. N. gonorrhoeae ATCC 49226 was used as the reference strain. The MICs for this strain were 0.5 μg of penicillin, 1 μg of tetracycline, 0.002 μg of ciprofloxacin, 0.008 μg of ceftriaxone, and 0.25 μg of azithromycin/ml.
Resistance to quinolones is known to be related to specific point mutations in the quinolone resistance-determining regions of genes gyrA and parC. The DNA regions of gyrA (GenBank accession no. U08817) encoding amino acids 44 to 156 and of parC (GenBank accession no. U08907) encoding amino acids 54 to 239 were amplified by PCR and sequenced directly using the ABI PRISM Big Dye cycle sequencing ready reaction kit on a 377 DNA sequencer (Perkin-Elmer, Applied Biosystems, Foster City, Calif.).
The total number of gonococcal isolates forwarded by the participating laboratories decreased from 967 in 1991 to 1992 to 449 in 1997 to 1998 (Table 1). This decrease may have been due to the implementation of prevention measures against human immunodeficiency virus infection (2). The MIC distributions of azithromycin (MIC at which 50% of isolates tested are inhibited [MIC50], 0.12 μg/ml; MIC90, 0.25 μg/ml) and ceftriaxone (MIC50, 0.002 μg/ml; MIC90, 0.008 μg/ml) did not change in the investigated period. The majority (362 of 374) of the isolates resistant to high levels of penicillin produced β-lactamase. In contrast 1,124 of 1,183 isolates intermediately resistant to penicillin were β-lactamase negative. Although the frequency of β-lactamase-producing strains declined from a maximum of 24% in 1993 to 1994 to 11% in 1997 to 1998, the percentage of strains with intermediate resistance to penicillin increased steadily from 40% in 1991 to 1992 to 68% in 1997 to 1998. High-level tetracycline resistance (MIC ≥ 16 μg/ml) rose from 7% in 1991 to 1992 to 18% in 1997 to 1998.
TABLE 1.
β-Lactamase production and antimicrobial susceptibility of N. gonorrhoeae
| Drug or enzyme | MIC (μg/ml) | % of isolates in:
|
|||
|---|---|---|---|---|---|
| 1991–1992 (n = 967) | 1993–1994 (n = 500) | 1995–1996 (n = 436) | 1997–1998 (n = 449) | ||
| β-Lactamase | 16 | 24 | 22 | 11 | |
| Penicillin | 0.12–1 | 40 | 47 | 58 | 68 |
| ≥2 | 15 | 21 | 19 | 10 | |
| Tetracycline | 0.5–1 | 34 | 41 | 39 | 40 |
| 2–8 | 27 | 25 | 27 | 20 | |
| ≥16 | 7 | 10 | 18 | 18 | |
| Ciprofloxacin | 0.12–0.5 | 0.83 | 0.60 | 0.92 | 0.67 |
| ≥1 | 0.00 | 0.00 | 0.23 | 1.56 | |
Isolates intermediately resistant to ciprofloxacin (MIC, 0.12 to 0.5 μg/ml) had been observed in our survey since the mid-1980s. In 1996 the first highly ciprofloxacin-resistant strains were isolated (MIC ≥ 1 μg/ml), and in 1998 two strains for which the ciprofloxacin MIC was 16 μg/ml emerged. The buildup of mutations in the quinolone resistance-determining region of the gyrA and parC genes followed a course similar to that found in mutants obtained by sequential selection in vitro (1). Most of the early isolates resistant to low levels of ciprofloxacin showed single mutations in gyrA (Table 2). Since 1996 such strains have been largely replaced by triple mutants which were resistant to ciprofloxacin (MIC ≥ 1 μg/ml) and which carried an identical pattern of two mutations in gyrA and the Asp86Asn mutation in ParC. Isolates carrying this pattern of mutations were received from six laboratories all over the country. This was also the most prominent pattern of mutations observed in gonococci from the Republic of the Philippines (5). One of these patients had been infected in that country. In contrast, the most frequent mutation in ParC of gonococci from Japan was the Ser88Pro substitution (3), which seems to be absent in gonococci from The Netherlands. This indicates that the strains obtained from The Netherlands are more related to the strains from the Republic of the Philippines than to those from Japan. The range of MICs of the triple mutants (1 to 16 μg/ml) was rather wide. So other resistance mechanisms may have played a role (7). The MIC for one strain carrying a Ser91Phe mutation in GyrA and an Asp86Asn mutation in ParC was intermediate, 0.5 μg/ml. This pattern was also observed in quinolone-resistant gonococci from the Far East (4, 5). One isolate for which the ciprofloxacin MIC was 16 μg/ml showed two mutations in gyrA as well as two mutations in parC. It originated from a patient who had returned from the Republic of the Philippines, where this pattern of mutations was observed before (5).
TABLE 2.
Amino acid substitutions within GyrA and ParC of N. gonorrhoeae and associated nucleotide substitutions
| No. of isolates | Period of isolation | Ciprofloxacin MIC (μg/ml) | Substitution at indicated positiona in:
|
||||
|---|---|---|---|---|---|---|---|
| GyrA
|
ParC
|
||||||
| 91, Ser (TCC) | 95, Asp (GAC) | 86, Asp (GAC) | 87, Ser (AGT) | 91, Glu (GAG) | |||
| 5 | 1992–1996 | 0.06–0.25 | — (—) | — (—) | — (—) | — (—) | — (—) |
| 8 | 1985–1996 | 0.03–0.25 | — (—) | Asn (AAC) | — (—) | — (—) | — (—) |
| 2 | 1996 | 0.03–0.12 | — (—) | Gly (GGC) | — (—) | — (—) | — (—) |
| 6 | 1985–1998 | 0.06–0.12 | Phe (TTC) | — (—) | — (—) | — (—) | — (—) |
| 1 | 1998 | 0.5 | Phe (TTC) | — (—) | Asn (AAC) | — (—) | — (—) |
| 10 | 1996–1998 | 1–16 | Phe (TTC) | Gly (GGC) | Asn (AAC) | — (—) | — (—) |
| 1 | 1998 | 16 | Phe (TTC) | Gly (GGC) | — (—) | Asn (AAT) | Lys (AAG) |
The numbering of amino acids is from reference 1. The wild-type amino acids and associated codons (parentheses) are indicated. —, sequence homology.
We detected hitherto-unreported, silent mutations in codon 131 (CTC to CTG) of the parC gene in 21 strains and in codon 138 (CTG to CTA) in 11 strains. The latter mutation did not occur in the absence of the former, but no relation was found between these mutations and the pattern of mutations in the quinolone resistance-determining region of either gyrA or parC.
This study indicates that fluoroquinolone resistance in N. gonorrhoeae has emerged in The Netherlands due to the sequential accumulation of point mutations in the gyrA and parC genes. Strains with a high level of resistance may have been imported from the Far East, where strains with identical mutations in gyrA and parC were observed.
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