THE USE OF ANTIBIOTICS to treat gonorrhea and other infections has caused the emergence of Neisseria gonorrhoeae strains with increased resistance to one or more antibiotics. This increased resistance has resulted in reduced effectiveness of penicillin and tetracycline and increased costs of treatment when quinolones and third-generation cephalosporins are used. The most recent recommendations for treatment of N. gonorrhoeae in Canada do not include penicillin and tetracycline. However, these antibiotics are used in some countries, and the prevalence of gonococci resistant to these 2 antibiotics remains high worldwide. 1 Therefore, global surveillance of N. gonorrhoeae to these antibiotics remains important. Antimicrobial resistance in N. gonorrhoeae is either plasmid or chromosomally mediated, and strains can often have a multiple resistance phenotype. Penicillin resistance seen in penicillinase-producing N. gonorrhoeae (PPNG) is caused by the production of TEM-1 β-lactamase encoded on a family of plasmids. 2 Also, tetracycline-resistant N. gonorrhoeae (TRNG) have high-level resistance to tetracycline (minimum inhibitory concentration [MIC] ≥16 μg/mL) and possess a 25.2-MDa tetM-conjugative plasmid. Chromosomally mediated resistance in N. gonorrhoeae (CMRNG) can encompass a variety of antimicrobial agents, including penicillin, tetracycline, erythromycin, and ciprofloxacin. Mutations in different genes have been identified, and the effects of different mutations simultaneously can act together to produce stepwise increases in resistance that are additive. 3
We describe the antimicrobial susceptibilities and the occurrence of CMRNG strains isolated in Canada from 1994 to 1999 as well as phenotypic characteristics (auxotype [A], serovar [S], and plasmid profiles [P]) of these strains.
Materials And Methods
Neisseria gonorrhoeae Strains
Between 1994 and 1999, an estimated 27,798 N. gonorrhoeae isolates were collected and tested by sexually transmitted disease (STD) clinics and provincial public health laboratories throughout the country. The National Laboratory for Sexually Transmitted Diseases (NLSTD) received 7280 of these N. gonorrhoeae strains as part of the national surveillance program. Isolates were obtained from both males and females, and sometimes more than one isolate was obtained from different sites of the same patient (rectal, pharyngeal, urethral, vaginal, or cervical). If strain information was available, duplicate strains from the same patient were not included in the study. Information on the gonococcal infection rates were derived from an analysis of data from the Health Canada, Population and Public Health Branch web site. 4 Identification of the isolates took place in regional laboratories, and the antibiotic susceptibilities of the strains were determined by either the NLSTD or provincial public health laboratories in British Columbia, Alberta, and Quèbec. The resistant strains and the MIC results of these strains were submitted to NLSTD. To standardize the susceptibility testing between laboratories, proficiency surveys are conducted 3 times a year. Once strains were received at the NLSTD, they were checked for purity and identification reconfirmed if necessary. Strains were subcultured on GC medium base (Difco Laboratories, Detroit, MI) containing 0.2% BioX 5 (QueLab, Montreal, PQ) and incubated for 24 hours at 35°C in a 5% CO2 atmosphere with or without antibiotics and maintained in brain heart infusion broth containing 20% glycerol and stored at -80°C.
Strains were received from the following provincial public health or reference laboratories: the British Columbia Center for Disease Control, Vancouver; the Provincial Laboratory of Public Health for Northern Alberta, Edmonton; the Provincial Laboratory of Public Health for Southern Alberta, Calgary; the Laboratory and Disease Control Services Branch, Saskatchewan Health, Saskatoon; the Cadham Provincial Laboratory, Manitoba Health, Winnipeg; the Laboratories Branch, Ontario Ministry of Health and Long Term Care, Etobicoke; Laboratorie de santé publique du Québec, Ste-Anne-de-Bellevue; the Queen Elizabeth II Health Science Center, Halifax, Nova Scotia; the Saint John Regional Hospital, Saint John, New Brunswick; the Queen Elizabeth Hospital, Charlottetown; and the Newfoundland Public Health Laboratory, St. John's, Newfoundland. Only resistant isolates (as determined by agar dilution using current NCCLS breakpoints) were submitted by Ontario as part of the surveillance program.
Strain Characterization and Antimicrobial Susceptibility Testing
Antimicrobial susceptibilities of N. gonorrhoeae to azithromycin (compliments of Pfizer, Pointe-Claire/Dorval, Quèbec), cefixime (Wyeth-Ayerst Laboratories, Mason, MI), ceftriaxone (Roche, Laval, Quèbec), ciprofloxacin (compliments of Bayer, Etobicoke, Ontario), erythromycin (compliments of Lilly, Indianapolis, IN), penicillin (compliments of Novopharm, Scarborough, Ontario), spectinomycin (compliments of Pharmacia & Upjohn, Kalamazoo, MI), and tetracycline (Sigma-Aldrich Canada Ltd., Oakville, Ontario) were determined using the agar dilution method with a GC medium base containing 1% Kellogg's defined supplement 6 and 2-fold dilutions of antibiotic and interpreted using criteria of the National Committee for Clinical Laboratory Standards 7 except for erythromycin and azithromycin. The breakpoint for erythromycin and azithromycin resistance were both MIC ≥2.0 μg/mL. 8–10 N. gonorrhoeae American Type Culture Collection (ATCC) 49226 and the World Health Organization strains WHO III, WHO V, WHO VII were used as controls. CMRNG are defined as strains chromosomally resistant to 3 antibiotics: penicillin (MIC ≥2.0 μg/mL), tetracycline (MIC ≥2.0 μg/mL and ≤8.0 μg/mL), and erythromycin (MIC ≥2.0 μg/mL).
Auxotyping of the N. gonorrhoeae isolates was performed as previously described on a chemically defined medium for their nutritional requirements for leucine (L), ornithine (O), citrulline (C), proline (P), arginine (A), hypoxanthine (H), uracil (U), and methionine (M). 11 Strains that did not have requirements for the amino acid supplements used were designated NR (nonrequiring). Serotyping 12 and plasmid profiles 13 of the N. gonorrhoeae isolates were performed as previously described.
The incidence of gonorrhea in Canada fell from 170.7 cases per 100,000 in 1984 to 21.1 cases per 100,000 in 1994 to 14.9 cases per 100,000 in 1997 and then rose to 20.2 per 100,000 in 2000. 4 Between 1994 and 1999, 31,676 cases of N. gonorrhoeae were reported to public health authorities in Canada (Fig. 1). Despite the drop in the incidence of gonorrhea, the prevalence of CMRNG has increased from 121 strains (2.0%) in 1994 peaking at 299 strains (9.8%) in 1997. Since 1997, the incidence has dropped slightly to 321 strains (8.1%) in 1998 but rose again to 345 strains (8.6%) in 1999. In 1994, 6167 N. gonorrhoeae isolates were reported, which declined to 4522 isolates in 1997. Since 1997, reported cases of N. gonorrhoeae have been increasing from 4868 in 1998 to 5412 in 1999 (Fig. 1). 4 Between 1994 and 1995, before the use of DNA amplification tests for diagnosing N. gonorrhoeae, the number of cases reported by the public health laboratories is used as the total number of strains tested for susceptibility. Between 1996 and 1999, an accurate figure for the number of strains tested for susceptibility is available from each province. Based on this, we estimated that 27,798 strains were tested for antimicrobial susceptibility in Canada between 1994 and 1999. Of these, 7280 N. gonorrhoeae isolates were referred to the NLSTD between 1994 and 1999, and 6727 strains were found to be resistant to at least one of the following antibiotics: penicillin, tetracycline, spectinomycin, ciprofloxacin, azithromycin, or erythromycin. A total of 553 isolates were found to be susceptible to all antibiotics.
From 1994 to 1999, there was an increasing prevalence of chromosomally mediated antimicrobial resistance, whereas the plasmid-mediated resistance strains (PPNG, PP/TRNG, and TRNG) strains all had a declining trend (unpublished data), as shown in Figure 1.
The distribution of chromosomally mediated resistance to all the antibiotics tested is summarized in Table 1. The majority of strains (1399 of 7280; 19.2%) are chromosomally resistant to penicillin, tetracycline, and erythromycin, and an additional 205 CMRNG strains (2.8%) were penicillinase-producing (PPNG). The next most common resistance identified in these strains was tetracycline and erythromycin (12.9%) at a prevalence of 3.4% of all N. gonorrhoeae strains isolated between 1994 and 1999. Strains resistant to tetracycline amounted to 4.7% (a prevalence of 1.2% among all strains) followed by ciprofloxacin resistance (2.3%), combined penicillin and tetracycline resistance (1.6%), erythromycin resistance (1.3%), azithromycin resistance (0.8%), penicillin resistance (0.12%), and combined penicillin and erythromycin resistance (0.09%).
The prevalence of CMRNG in Canada is 5.0% (1399 of 27,798) among all N. gonorrhoeae isolated during 1994 to 1999. They are not evenly distributed geographically. The geographic distribution of CMRNG strains are summarized in Table 2. The majority of the CMRNG strains were found in Ontario (429 of 1399; 30.7%), or 1.5% of all N. gonorrhoeae isolated between 1994 and 1999. British Columbia had the second most strains (364 of 1399; 26.0%), a prevalence of 1.3% of all strains isolated, followed by Quèbec (297 of 1399; 21.2%) and then Alberta (291 of 1399; 20.8%), a prevalence of 1.1% and 1.0%, respectively. The remaining 6 provinces accounted for only 1.2% of the CMRNG strains. CMRNG showed an increasing trend in some provinces as well as nationally. The CMRNG strains isolated in British Columbia have increased steadily from only 25 strains in 1994 to 133 strains in 1999. In Ontario, 46 strains were isolated in 1994, whereas in 1999, 97 strains were isolated. In Alberta, 22 strains were isolated in 1994, increasing to 105 strains in 1997 and then declining to 54 strains in 1999.
The 1054 CMRNG strains isolated between 1994 to 1998 were characterized by auxotype and serovar (A/S), all summarized in Table 3. These CMRNG strains were differentiated into a total of 97 A/S classes. The predominant auxotypes identified were nonrequiring (NR) or wild-type (724 of 1054 strains; 68.7%) or proline-requiring (P; 126 of 1054 strains; 11.9%) and then hypoxanthine-requiring (H; 23 of 1054 strains; 2.2%). The most common A/S class identified was NR/IB-01 (493 of 1054 strains; 46.8%), followed by P/IB-01 (83 of 1054 strains; 7.8%) and NR/IB-03 (72 of 1054 strains; 6.8%). Although serotyping data were not available for the 345 strains isolated in 1999, the auxotyping data are complete. The predominant auxotype identified was nonrequiring (272 strains; 78.8%), followed by proline-requiring (25 strains; 7.2%). The remaining 48 strains were classified into 12 different auxotypes.
Plasmid profiles were identified for all 1399 strains (1994–1999), and all were found to carry the Neisseria- specific cryptic 2.6-mDa plasmid. The 24.5-mDa conjugation plasmid was identified in 109 strains (7.8%).
Ciprofloxacin-resistant N. gonorrhoeae accounted for 0.6% (167 of 27,798) of all strains isolated between 1994 and 1999. The number of isolates increased from 5 strains in 1994 (0.8%) to 65 strains in 1999 (1.6%). The mode of MICs of ciprofloxacin has shifted from 0.004 μg/mL in 1990 to 0.016 μg/mL in 1999. Of all the resistant strains tested at the National Microbiology Laboratory in 1994 (n = 1448), strains exhibiting intermediate resistance (0.125–0.5 μg/mL) to ciprofloxacin accounted for 4.1%. By 1999, the proportion of intermediate to resistant strains reversed with 2.1% (n = 943) of strains exhibiting intermediate resistance, and 6.9% of all resistant strains tested exhibiting resistance to ciprofloxacin. These results indicate that the MICs of ciprofloxacin are increasing over time. Of the 167 ciprofloxacin-resistant strains identified during this period, 27.5% of these were characterized as CMRNG. However, this only represents 0.17% of all strains isolated during this period. The ciprofloxacin-resistant strains were classified into 75 A/S/P classes; the most predominant A/S/P class identified was P/IB-01/2.6 (representing 18.6% of the strains). P/IB-01/2.6 is also a common phenotype in other chromosomally mediated resistance strains. Other important phenotypes among the ciprofloxacin-resistant strains were NR/IB-01, NR/IB-03, P/IB-05, and NR/IA-06. 14
Erythromycin-resistant N. gonorrhoeae accounted for 8.8% (2440 of 27,798) of all strains isolated between 1994 and 1999. Of these, 57.3% were classified as CMRNG. Strains with higher MICs to erythromycin also have higher MICs to azithromycin. Azithromycin-resistant N. gonorrhoeae accounted for 0.2% (56 of 27,798) of all strains isolated between 1994 and 1999. The 56 azithromycin-resistant strains identified were classified into 16 A/S/P types. The predominant A/S/P class was NR/IB-01/2.6 (30 strains; 53.6%). Other important phenotypes among the azithromycin-resistant strains were NR/IB-03/2.6 and NR/IB-06/2.6. 15
Of the 27,798 strains tested within Canada between 1994 and 1999, no strains were identified with resistance to cefixime or ceftriaxone. The mode of the MICs of ceftriaxone and cefixime has shifted from 0.002 μg/mL in 1990 to 0.016 μg/mL in 1999 and 0.004 μg/mL in 1990 to 0.016 μg/mL in 1999, respectively.
Chromosomal-resistant N. gonorrhoeae is a growing problem in Canada and elsewhere. This is illustrated in a 1997 World Health Organization Western Pacific Region Gonococcal Antimicrobial Surveillance Program (WHO WPR GASP) document, which reported an incidence of CMRNG as high as 61.2% from Hong Kong and 44.0% from China. 1 During this same time period, the proportion of PPNG had declined to 5.2% in Hong Kong and 11.0% in China, no doubt reflecting the shift in therapeutic treatment for patients with gonorrhea. In the United States, the percentage of PPNG has declined from 11.0% in 1991 to 3.0% in 1998. 16 This trend of declining PPNG and increasing CMRNG seen in the United States, Hong Kong, and China is similar to the situation identified in Canada. The reverse trend has been observed in the United Kingdom. 17
Although there has been a general trend toward a decline in PPNG levels, fluoroquinolone-resistant isolates have been increasing worldwide. 10,18,19 A report from China stated that 59.3% of isolates tested in 1998 to 1999 were resistant to ciprofloxacin. 20 In neighboring Japan, the incidence of ciprofloxacin resistance increased from 3.7% 1981 to 46.8% in 1992 to 1993. 21 This trend is also observed in the United States in which the incidence of ciprofloxacin resistance increased from 0.6% in 1997 to 1.0% in 1998. 16 In Canada, ciprofloxacin resistance in N. gonorrhoeae emerged in 1994, and the incidence steadily increased from 0.3% in 1994 to 6.9% in 1999 (of all antimicrobial-resistant N. gonorrhoeae strains). In addition, the MICs of ciprofloxacin-susceptible strains have shifted from 0.004 μg/mL in 1990 to 0.016 μg/mL in 1999. Resistance to ciprofloxacin in N. gonorrhoeae strains is attributed to mutations in gyr A and par C, and the most common mutations seen in Canadian strains were amino acid substitutions of Ser→Phe at codon 91 and Asp→Gly at codon 95 of gyr A and Ser→Arg at codon 87 of par C. 14
Interestingly, resistance to azithromycin has been reported, 8,10,22 and we found cross-resistance between azithromycin and erythromycin as previously described. 22 Macrolides (erythromycin and azithromycin) are not used primarily for the treatment of gonococcal infections in Canada, but they are used in the treatment of chlamydia and in some Latin American countries as the primary treatment of gonococcal infections. 22 Strains either resistant to or less susceptible to azithromycin been reported from South America and the Caribbean. 23,24 In Canada, 56 azithromycin-resistant isolates were identified between 1997 and 1999 (0.2% of all strains tested). It has been shown that high levels of resistance to erythromycin and azithromycin require mutations in multiple loci or acquisition of rRNA methylase genes (erm). 25 Isolates found in men who have sex with men were found to be more likely to have higher erythromycin MIC values as a result of the changes within the mtr locus of the organism. The alterations to the locus, which encodes for an efflux pump, allows for the resistance of a variety of hydrophobic molecules such as fecal lipids, bile salts, and antibiotics, including erythromycin. 26 The effect of the efflux pump is more pronounced in erythromycin than azithromycin.
Although penicillin and tetracycline are no longer used in the treatment of gonococcal infections in Canada, the increase in prevalence of penicillin- and tetracycline-resistant isolates is most likely caused by selective pressure exerted in treating other bacterial infections. In the United States, the prevalence of isolates with chromosomal resistance to both penicillin and tetracycline increased from 3.0% in 1989 to 7.2% in 1998. The percentage of strains with chromosomal resistance to penicillin alone has increased from 0.5% in 1988 to 5.1% in 1998. The percentage of strains with chromosomally mediated resistance to tetracycline alone was 6.8%, which has remained relatively stable since 1989. 16
Further cause for concern was a report recently from China that found that 16.5% of isolates were resistant to ceftriaxone. 20 In Canada, although all strains are susceptible to cefixime and ceftriaxone, we have observed a shift to higher MICs for these antibiotics (from 0.002 to 0.004 μg/mL in 1990 to 0.016 μg/mL in 1999). There has also been a trend toward increased MICs for both ceftriaxone and cefixime for strains with chromosomal penicillin resistance (MICs between 1.0 μg/mL and 8.0 μg/mL).
N. gonorrhoeae antimicrobial resistance is a very important global public health concern. The ability to develop resistance to the majority of antibiotics used in the treatment of gonococcal infections shows the ability of the organisms to adapt and survive in an antibiotic-rich environment. Maintaining a surveillance program to identify the spread of antimicrobial-resistant gonococcal strains and detecting the emergence of strains with reduced susceptibility to currently recommended therapies ensures successful treatment for patients. It is a challenge to maintain national surveillance programs because more laboratories in North America are replacing cultural methods with nucleic acid-based assays to diagnose patients. In the near future, it might be necessary to set up sentinel sites to obtain cultures for surveillance purposes. However, the capture of data might not be as comprehensive as we currently have, and we might not be as sensitive and timely in the identification of outbreaks or antibiotic-resistant strains.
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