Gonorrhoea remains a major cause of reproductive tract morbidity in Africa.1,2Neisseria gonorrhoeae exhibits a remarkable ability to adapt to antibiotic pressure through generation of resistance mechanisms.3 The past 80 years have witnessed the sequential development of resistance to sulfonamides, penicillins, tetracyclines, quinolones, and most recently oral cephalosporins.4 Treatment options are now reduced, particularly in resource-poor countries where health budgets are limited and antibiotic access is poorly controlled.5
Given the rising prevalence of antibiotic resistant gonorrhoea, efforts are now underway to enhance surveillance of antibiotic resistance among N. gonorrhoeae isolates in many parts of the world, with the aim of establishing an early warning system for the emergence and possible spread of cephalosporin- resistant gonorrhea.4 To assist with this exercise, definitions for multidrug resistant and extensively drug resistant gonorrhoea have been published and the World Health Organization recently released an extended panel of N. gonorrhoeae control strains.4,6
In the last 20 years, there have been several reports from Africa documenting the prevalence of penicillinase-producing N. gonorrhoeae (PPNG), and to a lesser extent plasmid-mediated high level tetracycline resistant N. gonorrhoeae (TRNG).7–13 With the move to quinolone therapy in many African countries about 10 years ago, reports on penicillin and tetracycline resistance have become less numerous and those that exist are outdated. As a national sexually transmitted infections (STI) reference laboratory, we undertook a survey using recently isolated gonococci, collected as part of South Africa's National STI Microbiological Surveillance Program, to determine the level of phenotypic resistance to both penicillin and tetracycline. We developed a duplex polymerase chain reaction (PCR) assay for the simultaneous detction and typing of PPNG and TRNG.14,15 This duplex assay detects epidemic-associated Asia, Africa, and Toronto PPNG plasmids and both the American and Dutch variant TRNG plasmids.
MATERIALS AND METHODS
Control Neisseria gonorrhoeae Strains and Plasmids
To develop the assay, 5 control N. gonorrhoeae strains with known penicillin and tetracycline susceptibility phenotypes were used: ATCC 49226 (Penicillin minimum inhibitory concentrations [MIC] 1 mg/L; Tetracycline MIC 1 mg/L), SPL-4 (Penicillin MIC 4 mg/L; Tetracycline MIC 4 mg/L), CDC10328 (Penicillin MIC 32 mg/L, PPNG; Tetracycline MIC 0.5 mg/L), CDC10329 (Penicillin MIC 32 mg/L, PPNG; Tetracycline MIC 2 mg/L), and P681E (Penicillin MIC 8 mg/L, PPNG; Tetracycline MIC 16 mg/L, TRNG). These strains form part of the antimicrobial susceptibility testing control panel of the Gonococcal Isolate Surveillance Project (GISP).
Three gonococcal β-lactamase-encoding plasmids (pJD4, 4.4 MDa “Asia”; pJD5, 3.2 MDa “Africa”; pJD7, 3.05 MDa “Toronto”) were provided as purified DNA extracts by Dr. Jo-Anne Dillon (University of Saskatchewan, Canada).14 The pJD4, pJD5, and pJD7 plasmids produce 4911, 3083, and 2639 bp amplicons in the PCR assay, respectively. These 3 plasmids were used to transform E. cloni5-α chemically competent cells (genotype fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) (Lucigen Corporation, Middleton, WI). Supercoiled pUC19 DNA (1 ng/μL) was used as a control for transformation. Transformed cells were grown on selective Escherichia coli FastMedia LB agar Amp (Fermentas Life Sciences, Burlington, Canada) containing ampicillin (50 μg/mL). Plasmid DNA was extracted from these transformed cells to produce sufficient quantity of β-lactamase-encoding plasmid controls for the real-time PCR assay. Two control TRNG strains containing the American (TRNG-A) and Dutch (TRNG-D) Type 25.2 MDa tet(M) plasmids were used. TRNG-A and TRNG-D produced a 778 and 443 bp amplicon in the duplex assay, respectively.
The first 209 consecutive gonococcal isolates, representing 60% of the total gonococci collected in the 2008 national STI microbiologic survey, were resurrected from −70°C stock cultures. The isolates were obtained from men with urethral discharge who had signed consent forms for further research to be undertaken on their specimens subject to approval from the Human Research Ethics Committee (Medical) of the University of the Witwatersrand, Johannesburg, South Africa. Each frozen isolate suspension, previously identified as N. gonorrhoeae by in-house real-time PCR, was freshly subcultured onto selective modified New York City agar and incubated in 5% to 10% CO2 at 35°C in a humidified environment for 24 to 48 hours. Cultures were examined on both of the 2 subsequent days and colonies were subcultured onto nonselective chocolate agar to ensure purity. The identity of resurrected isolates was confirmed by Gram staining and oxidase testing.
Antibiotic Susceptibility and β-Lactamase Testing
Penicillinase production was detected using nitrocefin solution. To determine penicillin and tetracycline MICs, GC susceptibility agar plates containing GC agar base (Oxoid, Basingstoke, United Kingdom) and 1% IsoVitalex (Becton, Dickinson & Co, Sparks, MD) were inoculated with a suspension of each control or clinical gonococcal isolate (equivalent to a 0.5 McFarland standard) and E-test strips (AB Biodisk, Sweden) placed on the plates. The plates were incubated in 5% to 10% CO2 at 35°C in a humidified environment for 24 hours before reading the MIC result. The Clinical and Laboratory Standards Institute susceptibility criteria for penicillin and tetracycline were used.16 For penicillin, the breakpoints were susceptible (MIC <0.125 mg/L), reduced susceptible (MIC 0.125–1.0 mg/L), and resistant (MIC >1.0 mg/L). For tetracycline, the breakpoints were susceptible (MIC <0.5 mg/L), reduced susceptible (MIC 0.5–1.0 mg/L), and resistant (MIC >1.0 mg/L).
Development of the Duplex PCR Assay for the Detection of PPNG and TRNG
DNA was extracted from all clinical and control isolates using the X-Tractor Gene automated DNA/RNA extraction system (Corbett Life Science, Concorde, Australia). Plasmid pJD4, pJD5, and pJD7 DNA was extracted from transformed E. coli cells by using the Zippy Plasmid Miniprep Kit (Zymo Research Corp, Orange, CA). Briefly, the transformed cells were added to a kit-specific lysis buffer, neutralized, and then purified using the Fast-Spin column technology (Zymo Research Corp, Orange, CA). This method yielded plasmid DNA up to 25 μg per preparation.
The duplex PCR assay used previously published primer sequences.14,15 Primers JDA and JDB were used to amplify DNA from TEM-1 β-lactamase-containing PPNG. For TRNG detection, a universal forward primer UF that hybridizes with both TRNG plasmid variants was combined with reverse primers AR and DR specific to the American and Dutch variants, respectively. After being optimized, PCRs were carried out in a total volume of 50 μL containing 2.5 units of Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), 5 μL of 10× PCR buffer with MgCl2 (Roche Diagnostics, Mannheim, Germany), 5 μL of each 2 mmol/L dNTP (Roche Diagnostics, Mannheim, Germany), and 0.4 μL of each 100 μmol/L primer. PCR amplification was performed in the GeneAmp PCR System 9700 instrument (Applied Biosystems, Foster City, CA). After initial incubation at 94°C for 3 minutes, 30 cycles were performed with denaturing at 94°C for 20 seconds, annealing at 58°C for 1 minute, and extension at 72°C for 5 minutes. A final elongation step was performed at 72°C for 5 minutes. Amplified DNA was detected using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
The χ2 statistical test was used to assess the significance of associations. Statistical analyses were performed using Stata version 9.0 (StataCorp, TX).
Penicillin and Tetracycline Susceptibility Profiles
Penicillin MIC testing demonstrated 68 (32.5%) susceptible isolates, 87 (41.6%) isolates with reduced susceptibility, and 54 (25.8%) resistant isolates (Fig. 1). The MIC50 and MIC90 for penicillin were 0.19 and 32 mg/L, respectively. All 54 resistant isolates expressed β-lactamase. Tetracycline MIC testing demonstrated 15 (7.2%) susceptible isolates, 37 (17.7%) isolates with reduced susceptibility, and 157 (75.1%) resistant isolates (Fig. 1). The MIC50 and MIC90 for tetracycline were 6 and 16 mg/L, respectively.
Molecular Typing of PPNG Isolates
All 209 clinical isolates were tested using the duplex PCR assay (Fig. 2). Amplicons, consistent with the presence of a β-lactamase-encoding plasmid, were detected in all 54 nitrocefin-positive penicillin resistant gonococci. There were 19 isolates with a 3.1 kb amplicon product consistent with the Africa plasmid, 24 isolates with a 2.6 kb amplicon product consistent with the Toronto plasmid, and 11 isolates with a slightly smaller 2.35 kb amplicon product. The 2.35 kb product represents a novel β-lactamase-encoding plasmid that appears to be a 2560 bp deletion variant of the Asia plasmid (data not shown). None of the 54 PPNG contained the Asia plasmid and no isolate had more than one type of β-lactamase-encoding plasmid. The 3 plasmid controls pJD4, pJD5, and pJD7 produced amplicons of the correct size (4.9, 3.1, and 2.6 kb, respectively). Three of the GISP control strains (CDC 10328, CDC 10329, and p681E) contained Africa plasmids consistent with their PPNG phenotype.
Molecular Typing of TRNG Isolates
TRNG plasmid-derived amplicons were detected in 154 (73.7%) of the 209 clinical isolates (Fig. 2). There were 117 (76.0%) TRNG isolates with the American variant plasmid and 37 (24.0%) isolates with the Dutch variant plasmid. No isolates contained both plasmid variants. When used as templates for the duplex PCR assay, DNA from the 2 TRNG control strains produced amplicons of the expected size. One GISP control strain (p681E) contained a 25.2 MDa American variant plasmid.
Association of β-Lactamase-Encoding Plasmids With TRNG Isolates
Overall, 44 (81.5%) PPNG isolates and 110 (70.5%) non-PPNG isolates contained the 25.2 MDa Tet(M)-encoding plasmid, although this difference was not statistically significant (P = 0.131) (Table 1). There was a significant association between the presence of β-lactamase-encoding plasmids and the type of TRNG variant plasmid; none of the 37 TRNG Dutch variants produced penicillinase compared with 44 (37.6%) of the 117 TRNG American variants (P < 0.0001).
This study identified a new “Johannesburg” β-lactamase plasmid and documented a high prevalence of both PPNG and TRNG in Johannesburg. Our data confirm that penicillin and tetracyclines should not be used in the modern day management of gonorrhoea within South Africa. Although these antibiotics are rarely prescribed in clinic-based practice, they are still inappropriately used to treat gonorrhoea in many parts of Africa through nonclinical outlets.5 It is therefore important for African countries to have local data on PPNG and TRNG prevalence at the national reference centre level. By combining 2 previously published PCR methodologies, we developed a duplex assay to simultaneously detect PPNG and TRNG for use at our national reference centre. Our assay does not detect chromosomally mediated penicillin resistance, has yet to be validated on clinical samples, and would not be appropriate for use in more peripheral laboratories.
Penicillinase production among gonococci is mediated by a family of plasmids consisting of the Asia (4.4 MDa, 7426 bp), Africa (3.2 MDa, 5599 bp), Toronto (3.05 MDa, 5154 bp), Rio (2.9 MDa, 5154 bp), Nîmes (3.8 MDa, 6798 bp), and New Zealand (6.5 MDa, 9309 bp) types.17–19 Whilst the latter 3 have been isolated only sporadically, the Asia, Africa, and Toronto types are associated with epidemic outbreaks.17 The Asia-type plasmid (pJD4) has been sequenced in entirety and the remaining β-lactamase plasmids may be characterized as deletion derivatives of the Asia plasmid (Africa, Toronto, and Rio) or insertion derivatives of either the Asia (New Zealand) or Africa (Nîmes) plasmids.20 The PPNG assay used in the development of our duplex assay identifies the predominant 3 epidemic plasmids, that is, Asia, Africa, and Toronto types. Because of their genetic origins, the nonepidemic Rio, Nîmes, and New Zealand plasmids will produce amplicons of identical sizes to the Toronto (2.6 kb), Africa (3.1 kb), and Asia (4.9 kb) plasmids, respectively. We detected Africa and Toronto plasmids, as well as a new “Johannesburg” plasmid, among our clinical isolates.
After the emergence of high-level plasmid-mediated tetracycline resistance in the mid-1980s in both the United States and the Netherlands, TRNG have spread globally.21–23 Detection of 25.2 (40.6 kb) Tet(M)-encoding plasmids requires molecular methods.24 On the basis of restriction endonuclease mapping, the early 25.2 MDa plasmids isolated from United States- and the Netherlands-derived TRNG appeared different, and were termed “American” and “Dutch.”25 This type difference was subsequently confirmed by DNA sequencing of the tet(M) genes.26 American TRNG variants were initially reported in the United Kingdom and Africa, whereas the Dutch TRNG variants have been epidemiologically linked to Asia and South America.27 The first report of TRNG in South Africa appeared in 1994 and, even as most of the initial TRNG plasmids were of the American type, 2 new 25.2 MDa Tet(M)-encoding plasmids were described on the basis of BglI restriction site analysis, one similar to the American type plasmid and the other an American/Dutch hybrid.13 Our duplex PCR assay confirmed that the Dutch variant is now established in South Africa, although the American variant still predominates.13 Although Dutch TRNG variants isolated from other geographical locations have frequently expressed β-lactamase, none of the Dutch type TRNG identified in our study contained β-lactamase plasmids.25 The reasons underlying this observation are not clear but may reflect clonal spread of one or more β-lactamase negative Dutch TRNG strains locally.
Recent data on penicillin and tetracycline resistance among gonococci in South Africa are limited as it has not been a first- or second-line therapeutic agent to treat gonorrhoea for many years. Moodley et al reported a TRNG prevalence of 67% in rural KwaZulu Natal in 1999.10 A survey, undertaken in 2004–2005, among men presenting with urethral discharge to healthcare professionals in Pretoria reported the prevalence of plasmid-mediated and total resistance to penicillin to be 16% and 32%, respectively, whilst for tetracycline, plasmid-mediated, and total resistance was found to be 36% and 54%.11 Our study documents higher prevalence of both PPNG and TRNG in Johannesburg,
The use of molecular tools, such as the duplex assay described here, may assist national STI reference laboratories to generate key antimicrobial susceptibility data required to inform public health policy. Our study confirms that neither penicillin nor tetracycline will be of benefit to future gonorrhoea control efforts within South Africa.
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