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Original Study

Trends in Antimicrobial Resistance in Neisseria gonorrhoeae Isolated in Canada: 2000–2009

Martin, Irene BSc*; Jayaraman, Gayatri PhD, MPH; Wong, Tom MD, MPH; Liu, Gary MD*; Gilmour, Matthew PhD*on Behalf of the Canadian Public Health Laboratory Network

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doi: 10.1097/OLQ.0b013e31822c664f
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Neisseria gonorrhoeae infection is the second most commonly reported bacterial sexually transmitted infection in the Canada with over 11,000 cases reported in 2009.1 Reported rates of gonorrhea declined from 217.3 cases per 100,000 population in 1980 to 14.9 cases per 100,000 population in 1997, but since this time, reported gonorrhea rates have more than doubled to 33.1 cases per 100,000 population in 2009.1 Untreated infections can lead to complications in both sexes, with more severe consequences for women. Serious complications of N. gonorrhoeae infections include pelvic inflammatory disease, infertility, and ectopic pregnancy in women; epididymitis in men; and disseminated bloodstream infections in both the sexes.2 Like other nonulcerative sexually transmitted infections (STIs), gonorrhea has been associated with an increase risk of HIV infection and transmission.3 The prevention and control of gonorrhea remain a very important public health issue.4 Treatment of gonorrhea has relied on highly effective single-dose therapies for rapid cure of the infection and prevention of transmission to sexual partners. However, the development of antimicrobial resistance in gonorrhea poses on-going challenges both for individual case management and population-based disease control. Over the years, gonococcal resistance to penicillin, tetracycline, and erythromycin have precluded their use as inexpensive therapies. In light of more recent emergence of significant resistance to quinolones,5–8 the Canadian Guidelines on Sexually Transmitted Infections removed quinolones as the first-line treatment for gonorrhea and recommended oral cefixime as the first-line treatment option.9 Furthermore, the guidelines recommend that any antibiotic not be prescribed in situations where resistance rates reach a level greater than 3% to 5%.9 In this report, we present trends in antimicrobial susceptibilities between 2000 and 2009, results from a pan-Canadian, laboratory-based antimicrobial susceptibility program coordinated by the Public Health Agency of Canada (PHAC).

MATERIALS AND METHODS

N. gonorrhoeae Isolates

Since 1985, the National Microbiology Laboratory at the PHAC has conducted ongoing monitoring of antimicrobial susceptibilities in N. gonorrhoeae isolates as part of the National N. gonorrhoeae Surveillance Program. Between 2000 and 2009, a total of 10,993 N. gonorrhoeae strains were submitted from provincial public health laboratories to PHAC for antimicrobial susceptibility testing. Since isolates are submitted to PHAC only when the provincial laboratories identify resistance to at least one antibiotic or if the provincial laboratories do not conduct any antimicrobial susceptibility testing, the total number of isolates retrieved from cultures in each jurisdiction was used as the overall denominator to calculate resistance proportion (n = 40,875). When information was available, duplicate strains from the same patient were excluded from the analyses. To standardize the susceptibility testing among laboratories, proficiency surveys are conducted 2 times a year. Isolates were subcultured on gonococcal (GC) medium base (Difco Laboratories, Detroit, MI) containing 0.2% BioX10 (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 (BHI) broth containing 20% glycerol and stored at −80°C.

The results presented in this report represent N. gonorrhoeae isolates received by PHAC from all provincial public health laboratories in Canada. In addition to the isolates, information on age and sex of the patient, province of residence, date of isolation, and anatomical site of infection were also submitted to PHAC. In this study, isolates obtained from both males and females of all ages were included.

Strain Characterization and Antimicrobial Susceptibility Testing

Antimicrobial susceptibilities of N. gonorrhoeae to azithromycin (compliments of Pfizer, Pointe-Claire/Dorval, Québec); cefixime (compliments of Wyeth-Ayerst Laboratories, Mason, MI); ciprofloxacin (compliments of Bayer, Etobicoke, Ontario); spectinomycin (compliments of Pharmacia & Upjohn, Kalamazoo, MI); ceftriaxone, erythromycin, penicillin, and tetracycline (Sigma-Aldrich Canada Ltd., Oakville, Ontario) were determined using the agar dilution method with a GC medium base containing 1% Kelloggs defined supplement11 and 2-fold dilutions of antibiotic. N. gonorrhoeae American Type Culture Collection ATCC49226 and the World Health Organization strains WHO B, WHO C, WHO D were used as controls. Minimum inhibitory concentration (MIC) (the minimum concentration of antibiotic which will inhibit the growth of the organism) interpretations were based on the criteria of the Clinical Laboratory Standards Institute: penicillin resistance MIC ≥2.0 μg/mL (testing range, 0.004–128.0 μg/mL); tetracycline resistance MIC ≥2.0 μg/mL (testing range, 0.064–64.0 μg/mL); ciprofloxacin resistance MIC ≥1.0 μg/mL (testing range, 0.001–64.0); spectinomycin resistance MIC ≥128.0 μg/mL (testing range, 4.0–256.0 μg/mL); ceftriaxone susceptibility ≤0.25 μg/mL (testing range, 0.000125–0.5 μg/mL); cefixime susceptibility ≤0.25 μg/mL (testing range, 0.00025–0.5 μg/mL).12 The breakpoints for erythromycin and azithromycin resistance were both MIC ≥2.0 μg/mL (testing ranges include 0.032–32.0 μg/mL and 0.016 – 32.0 μg/mL, respectively).13–15 Plasmid profiles of the N. gonorrhoeae isolates were performed as previously described.16

A 2 × 2 χ2 test was used to compare 2 proportions to detect significant differences between groups of isolates (P values were calculated with a 95% confidence interval). Statistical analysis was carried out using EpiCalc 2000 version 1.02 software.

RESULTS

N. gonorrhoeae Isolates and Demographic Characteristics

Between 2000 and 2009, a total of 40,875 isolates were tested at provincial laboratories for susceptibility to antimicrobial agents. PHAC received 10,993 (26.9%) of these isolates. A median of 1163 isolates were submitted each year (a range between 800 and 1532 isolates per year) (Table 1). The majority of N. gonorrhoeae strains was submitted from the central provinces (73.1%) followed by the Western provinces (18.2%), Prairie provinces (5.0%), and the Eastern provinces (3.6%).

TABLE 1
TABLE 1:
Number of Neisseria gonorrhoeae Isolates Tested at the National Microbiology Laboratory, Public Health Agency of Canada (PHAC) Each Year, 2000–2009

Demographic data were available on those isolates submitted to PHAC for susceptibility testing. Of those isolates with information on gender (92.4%, 10,162/10,993), 81.3% were collected from males. A majority of isolates from males with known site of collection were urethral (88.0%, 3341/3797 isolates), followed by rectal (6.5%) and pharyngeal (4.5%) sites. Among females with known site of specimen collection, the majority of sources was cervical (87.1%, 1281/1470 isolates), followed by vaginal (8.0%) and pharyngeal (1.5%). Over time, the proportion of cervical isolates collected dropped significantly from 89.5% in 2000 to 59.5% in 2009 (P < 0.001), whereas the rate of vaginal isolates increased significantly over the same period from 8.0% to 32.4%, respectively (P < 0.001). The rate of male urethral isolates dropped from 90.4% of isolates in 2000 to 64.6% in 2009 (P < 0.001), whereas the rate of rectal isolates increased over the same period from 5.3% to 22.9% (P < 0.001). Age was available for 92.0% of the isolates (10,114/10,993 isolates). The mean age was 31.5 years (range, 1–86 years). A higher proportion of isolates submitted for testing are from males (81.3%) compared to the number of male cases reported via routine surveillance of diagnosed cases (55.7%). In addition, the mean age of males submitting isolates was 33.3 years compared to a mean age of 27.7 years among reported male cases during this time period. As this is a laboratory-based surveillance system based on information provided on the laboratory requisition form, no data were systematically collected on sexual orientation, ethnicity, clinical presentation, or treatment offered to permit analyses of these factors.

Antimicrobial Susceptibility Trends

In 2000, 24.5% of all isolates (1092/4458 isolates) tested by PHAC were found to be resistant to at least 1 antibiotic. By 2006, the proportion of isolates showing any antibiotic resistance had significantly increased to 35.0% (1472/4201, P < 0.001). However, there has been a significant increase in the modal MICs of certain antibiotics over time, to be described in greater detail later in the text.

Penicillin, Tetracycline, and Erythromycin

Overall, 11.8% (4812/40,875), 21.3% (8690/40,875), and 14.2% (5787/40,875) of isolates were resistant to penicillin, tetracycline, and erythromycin, respectively. Penicillin resistance decreased between 2000 and 2003 from 14.2% (633/4458 isolates) to 5.6% (240/4235 isolates, P < 0.001), and then increased to 18.7% by 2009 (580/3106, P < 0.001), as shown in Figure 1. Tetracycline followed this same pattern with a decrease in resistance levels between 2000 and 2003 from 23.3% (1040/4458 isolates) to 14.3% (608/4235 isolates, P < 0.001) and then significantly increased again to 24.7% by 2009 (767/3106 isolates, P < 0.001, Fig. 1). In 2000, 12.5% (559/4459 isolates) were found to be erythromycin resistant and by 2009, this proportion had significantly increased to 21.3% (660/3106 isolates, P < 0.001), Figure 1.

Figure 1.
Figure 1.:
Antimicrobial susceptibility of Neisseria gonorrhoeae isolates tested in Canada between 2000 and 2009.

From 2000 to 2009, although there was an increasing prevalence of isolates that were chromosomally resistant N. gonorrhoeae (CMRNG) defined as resistance to penicillin (MIC ≥2.0 μg/mL, tetracycline (≥2.0 μg/mL and ≤8.0 μg/mL), and erythromycin (MIC ≥2.0 μg/mL),17 the plasmid-mediated resistance strains (penicillinase-producing N. gonorrhoeae [PPNG], tetracycline-resistant N. gonorrhoeae [TRNG] strain, and PP/TRNG strain), all had a declining trend (Fig. 2). The rate of CMRNG increased from 3.9% in 2000 (177/4458 isolates) to 15.2% by 2009 (472/3106 isolates, P < 0.001) and 4.7% of isolates were characterized as probable CMRNG (one of the MIC values of either penicillin, tetracycline, or erythromycin = 1 μg/mL, the other 2 ≥2.0 μg/mL). During the same period, the PPNG isolates decreased from 3.0% (135/4458 isolates) to 0.9% (28/3106 isolates, P < 0.001). The TRNG isolates decreased from 3.4% (151/4458 isolates) in 2000 to 1.6% (51/3106 isolates, P < 0.001) in 2009.

Figure 2.
Figure 2.:
Trends of plasmid mediated penicillinase-producing Neisseria gonorrhoeae (PPNG), tetracycline-resistant (TRNG), chromosomal mediated resistant (CMRNG), and probable CMRNG in Canada from 2000 to 2009.

Plasmid profiles were determined for all PPNG, TRNG, and PPNG/TRNG isolates. The β-lactamase gene was encoded in 3 different types of plasmids of sizes 3.05 Mda (Toronto type), 3.2 Mda (Africa type), and 4.5 Mda (Asia type). The 3.2-Mda (Africa type) plasmid was the most common type among the 933 PPNG strains isolated between 2000 and 2009 at 78.8%, followed by the 3.05 Mda (Toronto type) plasmid at 13.8%, and then the 4.5 Mda (Asia type) plasmid at 6.1%. These plasmids coexisted with the 2.6-Mda cryptic plasmid and sometimes with the 24.5-Mda conjugal plasmid. The 3.2-Mda (Africa type) plasmid is also the most common β-lactamase encoding plasmid in 455 PPNG/TRNG strains at 59.9%. The 25.2-Mda plasmid that encodes tetracycline resistance (Tet M) coexisted with the cryptic plasmid in the TRNG and PPNG/TRNG strains. Among the TRNG isolates tested between 2000 and 2009, 99.6% had the 2.6 and 25.2 Mda plasmids. Of all the isolates tested, 0.12% carried only the 24.5-Mda conjugal plasmid, whereas 10.0% of isolates carried the 24.5-Mda conjugal plasmid along with other plasmids.

Ciprofloxacin

Resistance to ciprofloxacin was identified in 13.2% of all N. gonorrhoeae strains isolated between 2000 and 2009 (5377/40,875 isolates, Fig. 1). The percentage of isolates resistant to ciprofloxacin significantly increased from 1.3% in 2000 (59/4539 isolates) to 25.5% (791/3106, P < 0.001), with a peak during 2007 when 30.2% of isolates were resistant to ciprofloxacin. The modal MICs of ciprofloxacin shifted from 0.016 μg/mL in 2000 to 16.0 μg/mL in 2009. Of the 791 ciprofloxacin-resistant isolates identified during 2009, all were also resistant to at least one other antibiotic; 475 (60.0%) were characterized as chromosomally resistant to penicillin, tetracycline, and erythromycin (CMRNG).

Azithromycin

Overall, 0.17% of isolates (69/40,875) were azithromycin resistant. The modal MIC of azithromycin shifted from 0.25 μg/mL in 2001 to 0.5 μg/mL by 2007 through to 2009. Each of the 69 azithromycin-resistant isolates were associated with resistance to at least one other antibiotic, 39.1% (27/69) were CMRNG and azithromycin resistant and 31.9% (22/69) were CMRNG and resistant to both ciprofloxacin and azithromycin. Twenty-six azithromycin-resistant isolates were identified with higher-level resistance (MIC = 8–64 μg/mL), whereas the remaining 43 isolates were identified with lower-level resistance (MIC = 2–4 μg/mL).

Spectinomycin, Cefixime, Ceftriaxone

None of the 10,993 isolates tested at PHAC between 2000 and 2009, were resistant to spectinomycin, cefixime, or ceftriaxone. However, there has been a shift in the modal MICs of ceftriaxone from 0.016 μg/mL in 2000 to 0.063 μg/mL in 2009. There was also a shift in the MICs of cefixime, although a modal MIC can not be determined since isolates were evenly distributed over 3 MICs (0.016 μg/mL, 0.032 μg/mL, 0.125 μg/mL). A total of 208 N. gonorrhoeae isolates with decreased susceptibility MICs to ceftriaxone and cefixime were identified between 2001 and 2009 (Fig. 3). Fifty isolates (2001, n = 3; 2006, n = 3; 2007, n = 1; 2008, n = 15; 2009, n = 28) were identified with MICs of 0.125 μg/mL and 0.25 μg/mL to ceftriaxone and cefixime, respectively. An additional 12 isolates (2003, n = 1; 2004, n = 2; 2007, n = 2; 2008, n = 4; 2009, n = 3) were identified with a cefixime MIC of 0.25 μg/mL. Six isolates (2007, n = 1; 2008, n = 1; 2009, n = 4) were identified with a ceftriaxone MIC of 0.25 μg/mL. Three isolates (one each in 2004, 2007, and 2008) were identified with a cefixime MIC of 0.5 μg/mL, classified as nonsusceptible to cefixime by CLSI guidelines. These isolates with decreased susceptibility MICs to cephalosporins have been identified from several provinces around the country. Also, more isolates with reduced-susceptibility to cefixime than for ceftriaxone have been identified. Preliminary data suggest the trend toward a “right” shift in MICs continued during 2010 as well as an increase in the incidence of isolates with reduced susceptibility to cephalosporins (data not shown). These results indicate that the MICs of these third-generation cephalosporins are likely increasing over time.

Figure 3.
Figure 3.:
Neisseria gonorrhoeae strains (208) with decreased susceptibility to cefixime (0.25 and 0.5 μg/mL) and ceftriaxone (0.125 μg/mL and 0.25 μg/mL) MICs isolated between 2001 and 2009.

DISCUSSION

Although there may be jurisdictional variations, overall, high levels of resistance to ciprofloxacin are well established in Canada. There has been a noticeable increase in modal MICs of N. gonorrhoeae to third-generation cephalosporins, specifically ceftriaxone and cefixime. There is also an increasing prevalence of N. gonorrhoeae isolates that are chromosomally resistant to penicillin, tetracycline, and erythromycin.

Although there are regional differences in ciprofloxacin resistance in Canada (unpublished data), overall resistance to fluoroquinolones has steadily increased from 0.3% in 199417 to 1.3% in 2000 and 25.5% by 2009. The emergence of ciprofloxacin resistance in Canada is not unexpected and has been reported worldwide.18,19 In 2007, CDC recommended that fluoroquinolones no longer be used to treat gonococcal infections20 and Canada followed suit in 2008.9

The increased MICs for the third-generation cephalosporins seen in this study add to the rising concern worldwide regarding multidrug-resistant N. gonorrhoeae. Between 2000 and 2009, there was a shift in the modal MICs of ceftriaxone from 0.016 μg/mL to 0.063 μg/mL in 2009. Before that, in 1990, the mode of the MICs of ceftriaxone was 0.002 μg/mL and cefixime 0.004 μg/mL.17 Increased MICs for cefixime and ceftriaxone in N. gonorrhoeae isolates have also been reported from additional Southeast Asian countries, Europe, and Australia.21–24 In Japan, cefixime was removed from the national treatment guidelines due to the reports of treatment failure.25 To date, no treatment failure with injectable ceftriaxone has been reported for urogenital gonorrhea; however, there has been a recent report of a treatment failure of pharyngeal gonorrhea infection in Sweden.26 Recommended doses of ceftriaxone differ among countries: in Japan, an intravenous 1 g dose is used; in other Asian countries, 250 mg of intramuscular ceftriaxone is used; the United States updated treatment to 250 mg of intramuscular ceftriaxone in December 2010, whereas Canada recommends a 125-mg intramuscular dose (currently under review in light of the recent surveillance information).9,25,27

Increases in MICs for third-generation cephalosporins have been linked to chromosomally mediated penicillin resistance, and it is possible that they are related to mosaic and nonmosaic penA and penB (porB1b) alleles.23,28,29 Two penA gene alterations are possible: the first is the acquisition of a penA mosaic allele which encodes an altered penicillin-binding protein 2; the second is alterations in amino acids (A501, G542, P551) of penicillin-binding protein 2 in nonmosaic penA alleles.30–34 Mutations in the promoter and/or coding sequence of the repressor gene mtrR, which cause overexpression of the MtrCDE efflux pump system have also been associated with a decrease in cephalosporin susceptibility.30,32,35,36 Finally, porB1b gene mutations (the penB resistance determinant) that alter amino acid G101 and A102 in the outer membrane PorB1b porin result in decreased permeability, and thus further decreased susceptibility to cephalosporins.30,32,35–38 Other not yet identified resistance determinants may also exist.30,36

Alternative options in the Canadian STI Guidelines for the treatment of gonococcal infections include a single dose of 2 g of oral azithromycin. Continued monitoring of azithromycin MICs is also essential as it is the recommended treatment for Chlamydia trachomatis infection, therefore commonly exposing circulating N. gonorrhoeae coinfection to it. The modal MICs for azithromycin have shifted from 0.25 μg/mL in 2000 to 0.5 μg/mL in 2007 through to 2009, 56.4% of isolates tested in 2009 had an MIC of 0.5 μg/mL, a difference of only 2 dilutions from the resistance breakpoint of 2 μg/mL used in Canada and the United States,15 and only one dilution from the European EUCAST resistance breakpoint of 1 μg/mL.39 Resistance to azithromycin and erythromycin has been associated with mutations in the mtr efflux pump or with the acquisition of rRNA methylase genes (erm).17 There have been reports of the emergence of high-level azithromycin-resistant (>256 μg/mL) N. gonorrhoeae first detected in 2004 in Scotland40 and since then reported from England and Wales,23 Italy,41 and Argentina.42 MICs of azithromycin of that level have not yet been identified in Canada.

The final alternative therapy in the Canadian STI Guidelines is a single dose of 2 g of intramuscular spectinomycin. No resistance to spectinomycin was observed during this study period and although the Guidelines recommend it as a treatment alternative, access to this drug is complicated as, in Canada, it requires ordering through a Special Access Program. Continued monitoring of spectinomycin susceptibilities is essential as during its use in the mid-1980s isolates with high levels of resistance were reported43,44 and have more recently been reported in Russia.45

The threat of untreatable gonococcal infection is a serious global public health concern.46 It is difficult to estimate the frequency of gonococcal disease, especially in the resource-limited countries, with the greatest burden of disease. These countries also often have limited prevention activities and are likely to be the source of new patterns of gonococcal resistance.4 The WHO recently reported that approximately 82 million people yearly are affected by gonococcal infections worldwide.47 Gonococcal infections are on the rise in Canada1 and around the world particularly among those who are economically and socially disadvantaged.48 Circulating antimicrobial-resistant isolates have the additional threat of contributing shared resistances with other Neisseria spp. or even other bacterial species, as is possible when the resistance genes are plasmid mediated.

Possible solutions suggested to control antimicrobial resistance in N. gonorrhoeae include multidose cephalosporin regimens, higher cephalosporin doses, and drug cycling.23 Additional gonococcal control measures include the screening of populations at risk for infection, prevention counseling, and partner referral.4

One of the major challenges faced by the laboratories that perform surveillance of antimicrobial resistance of N. gonorrhoeae is the shift from the use of cultures (required for antimicrobial susceptibility testing) to the nucleic acid amplification test for the diagnosis of gonorrhea, explaining the decrease in the number of isolates available for testing as gonorrhea rates increase in the population. Obtaining cultures of N. gonorrhoeae is of foremost importance, and this must be orchestrated with diagnostic laboratories and sexually transmitted infection clinics. A network of sentinel laboratories could be established to ensure cultures are performed to retrieve isolates for surveillance purposes. Susceptibility testing is required for both patient management and surveillance purposes and control as increased susceptibilities to third-generation cephalosporins are emerging. The current passive Canadian surveillance system for N. gonorrhoeae, limited by the collection of only resistant isolates which may introduce a bias and cause a lack of representativeness is being reviewed with the objective of creating an enhanced sentinel laboratory surveillance system capable of integrating epidemiologic and laboratory information for N. gonorrhoeae. The objectives of surveillance would be to determine the incidence and trends of antimicrobial resistance in N. gonorrhoeae and the notification of treatment failures. This is required to develop population-level evidence-based public health interventions, to ensure timely response to emerging resistances, to characterize both antimicrobial susceptible and resistant strains of gonorrhea in order to understand the spread of strains in Canada, and to inform Canadian STI Guidelines.

ACKNOWLEDGEMENTS

Canadian Public Health Laboratory Network members include Vanessa Allen, Public Health Laboratories, Public Health Ontario, Toronto, Ontario; Anne-Marie Bourgault and Brigitte Lefebvre, Laboratoire de Santé Publique du Québec, Ste-Anne-de-Bellevue, Québec; Linda Hoang, BCCDC Public Health Microbiology & Reference Laboratory, Vancouver, British Columbia; Marguerite Lovgren, Provincial Laboratory for Public Health, Edmonton, Alberta; Paul Van Caeseele, Cadham Provincial Laboratory, Manitoba Health, Winnipeg, Manitoba; Greg Horsman, Saskatchewan Provincial Public Health Laboratory, Regina, Saskatchewan; Richard Garceau, Hôpital Dr G.L. Dumont, 330 University Ave, Moncton, New Brunswick; David Haldane, Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia; Lei Ang, Queen Elizabeth Hospital, Charlottetown, Prince Edward Island; Sam Ratnam, Newfoundland Public Health Laboratory, St. John's, Newfoundland. The authors thank Pam Sawatzky, from the National Microbiology Laboratory, for technical assistance.

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