Evolution in the trends of antimicrobial resistance in Neisseria gonorrhoeae isolated in Durban over a 5 year period: impact of the introduction of syndromic management

Prashini Moodley, Chinsamy Pillay, Rabia Goga, Ayesha B. M. Kharsany and A. Willem Sturm,*

Department of Medical Microbiology and Africa Centre for Population Studies and Reproductive Health, School of Infection, Nelson R. Mandela School of Medicine, University of Natal, Private Bag 7, Congella, 4013 Durban, South Africa


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial susceptibility testing was performed on isolates of Neisseria gonorrhoeae obtained from patients attending the City Health STD clinic in Durban, KwaZuluNatal, using the following drugs: penicillin, tetracycline, ciprofloxacin, ofloxacin, ceftriaxone, spectinomycin, erythromycin and azithromycin. These isolates were collected over a 6 year period from 1995 to 2000. Four hundred and fifteen strains were tested: 61 in 1995, 198 in 1997, 98 in 1998/99 and 58 in 1999/2000. A shift to the right is observed in the susceptibilities of N. gonorrhoeae to the currently recommended drugs in the syndromic management guidelines viz. penicillin, tetracycline, ceftriaxone, ciprofloxacin, spectinomycin and erythromycin. The prevalence of penicillinase-producing N. gonorrhoeae is currently c. 30%, whereas that of plasmid-mediated tetracycline-resistant N. gonorrhoeae is c. 50%. There is a definite association between the MICs of strains falling within the penicillin and tetracycline chromosomally resistant group, and strains exhibiting a decreased susceptibility to ciprofloxacin and ceftriaxone. The MICs of azithromycin showed a similar distribution when compared with erythromycin for 1999/2000 isolates. We postulate that the presence of efflux pumps might play a role in the increasing MICs that we observe among structurally unrelated groups of drugs. Furthermore, widespread use of these antimicrobials in the community may offer a selective advantage to the development of resistance. The implications of this are far reaching and the local susceptibility trends of N. gonorrhoeae need to be monitored constantly to direct therapy.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Decreasing the prevalence of sexually transmitted diseases (STDs) is a priority in developing countries, especially in large parts of Africa where the prevalence of HIV and STDs has reached astronomical levels.1,2 In resource-poor settings, such as most of KwaZuluNatal, South Africa, the diagnosis and control of STDs has been hampered by the lack of appropriate laboratory support and trained health care personnel. For the want of an appropriate point-of-care test with good sensitivity and reasonable specificity to detect the common STD pathogens, the World Health Organization (WHO) introduced treatment guidelines for the syndromic management of symptomatic patients.3

The syndromic approach uses clinical algorithms based on the patient's symptoms and clinical signs to determine antimicrobial therapy. However, the aetiology of the different syndromes and the antimicrobial susceptibility patterns of the microbes involved may vary significantly in different areas or even within the same area. This highlights the need for area-specific research when addressing control programmes. Periodic monitoring of the prevalence of organisms and their susceptibility profiles provides essential clues towards the adjustment of local syndromic management guidelines.

In 1995, the Department of Health for KwaZuluNatal adopted and modified the WHO guidelines for the treatment of STDs in the region. The discharge syndrome is treated with ciprofloxacin 250 mg stat., with doxycycline 200 mg daily for 7 days in male, supplemented with metronidazole 2 g stat. in the non-pregnant female. During pregnancy, these drugs are replaced by ceftriaxone, 125 mg im stat. and erythromycin 500 mg qds for 7 days. Spectinomycin is used in case of ß-lactam hypersensitivity. Although these guidelines have been in place for 5 years, STD control has been perceived as being far from optimal with the area being ill-famed as having one of the highest HIV and STD prevalence rates in the world.

Up until 1993, Neisseria gonorrhoeae strains were susceptible to most antimicrobial agents listed in the current syndromic management protocols.4 We report on trends in susceptibility to these drugs over the last 5 years and relate these findings to the introduction of syndromic management for STDs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates of N. gonorrhoeae were collected from patients attending the City Health STD Clinic in Durban, South Africa in 1995, 1997, end of 1998/beginning of 1999 and end of 1999/beginning of 2000. Surveillance studies were not conducted in 1996. Isolates were grown from specimens obtained from patients presenting to the clinic with genital discharge syndrome during surveillance studies. Such studies are performed at regular intervals at this clinic. Data on antibiotic consumption and population size were obtained from the KwaZuluNatal Provincial Administration and Department of Tourism, respectively.

Urethral and cervical specimens were obtained and New York City plates [GC agar base supplemented with yeast autolysate, lincomycin, colistin, amphotericin B, trimethoprim (Oxoid Ltd, Basingstoke, UK) and lysed horse blood] were inoculated for the isolation of N. gonorrhoeae. Plates were incubated at 37°C in 5% CO2 for 48 h. Suspected colonies of N. gonorrhoeae were identified by means of Gram's stain, oxidase test, ß-galactosidase, hydroxyprolylaminopeptidase, {gamma}-glutamylaminopeptidase and acid production from glucose, lactose and maltose.

MICs of penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, ofloxacin, erythromycin and azithromycin were determined by means of the agar dilution method. Two-fold serial dilutions of antibiotics were added to molten GC agar base (Oxoid) supplemented with 1% isovitalex at a temperature of 45°C. After solidification, these plates were seeded with 104 cfu/spot of bacteria by means of a multipoint inoculator and incubated at 37°C in CO2 for 24 h. N. gonorrhoeae ATCC 49226, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 10418 were used as controls. Erythromycin and spectinomycin were not tested for isolates obtained in 1995. Plates for the MICs for N. gonorrhoeae of erythromycin from the 1997, 1998/99 batches were incubated in CO2. Plates for MICs of erythromycin and azithromycin in the 1999/00 batch were incubated at 37°C in CO2 as well as at 37°C in air. The CO2-incubated plates demonstrated two-fold higher MICs than those incubated in air. MICs of previous batches of the macrolides were therefore adjusted accordingly.5,6 The antimicrobial susceptibility was judged using breakpoint criteria defined by the NCCLS7 for all but erythromycin and azithromycin, for which interpretive criteria for N. gonorrhoeae are not available. Susceptibility to penicillin and tetracycline were defined as follows: tetracyclinesusceptible strains (TET-S), MIC <= 0.25 mg/L; plasmid-mediated tetracycline-resistant N. gonorrhoeae (TRNG), MIC >= 16 mg/L; chromosomally mediated tetracycline-resistant N. gonorrhoeae (CMRNGT), MIC 1–8 mg/L; penicillin-susceptible strains (PEN-S), MIC <= 0.06 mg/L; decreased penicillin-susceptible N. gonorrhoeae (DPNG), 0.06–4 mg/L in the absence of ß-lactamase. Strains testing positive for ß-lactamase production by the chromogenic cephalosporin method are referred to as penicillinase-producing N. gonorrhoeae (PPNG).

Calculations for the comparison of data to illustrate trends were made using the {chi}2 test for linear trends. Fisher's exact test was used for comparisons between different years.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 415 N. gonorrhoeae strains obtained from different patients were tested: 61 in 1995, 198 in 1997, 98 in 1998/99 and 58 in 1999/2000.

The prevalence of PPNGs doubled from 1995 to 1997 (P = 0.02) and has remained at c. 30% since then (Figure 1Go). The doubling of the PPNG prevalence in 1997 was associated with a decrease in the DPNGs (P < 0.001) and an increase in PEN-S in that year. In 1998/99 and 1999/00, a significant increase in the DPNGs ({chi}2trend = 49.9, P < 0.001) was seen and this was accompanied by a decrease in PEN-S strains ({chi}2trend = 49.2, P < 0.001). There was a dramatic increase in the number of strains exhibiting high-level resistance to tetracycline (TRNG) between 1997 (3%) and 1998/99 (51%) (P < 0.001). This increase was counteracted by a reduction in CRMNGT in 1998/99. A doubling of the prevalence of CMRNGT was accompanied by a marked decrease in the number of strains fully susceptible to tetracycline in 1999/00 (7%).



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Figure 1. Trends in susceptibility to (a) penicillin ({blacksquare}, PPNG; , DPNG; {square}, PEN-S) and (b) tetracycline ({blacksquare}, TRNG; {blacksquare}, CRNGT; {square}, TET-S) of N. gonorrhoeae isolates over a 5 year period.

 
Table 1Go shows the trends in susceptibility patterns for the remaining drugs. All 1995 strains had MICs of ciprofloxacin <=0.007 mg/L, but only 71% fell in this category in 1999/00. In addition, over 10% of strains demonstrating MICs of >=0.03 mg/L were seen in 1998/99 and 1999/00. The same trend was observed with ofloxacin but at higher MIC levels. The susceptibility to erythromycin also changed over this period. The MICs in 1997 ranged from <=0.007 to 0.5 mg/L, but these values changed to 0.03–4 mg/L for the 1999/00 strains. The data for azithromycin (available for 1999/00 only) showed a similar distribution of MICs when compared with erythromycin for the same period (Figure 2Go). No resistance to ceftriaxone was detected. However, an increase in MICs was observed with c. 50% of strains in 1999/00. A similar pattern was observed for spectinomycin, as evidenced by the increase in MICs with 28% of strains exhibiting MICs >= 16 mg/L in 1997, 52% in 1998/99 and 100% in 1999/00. Two strains showed a marked decrease in susceptibility to the drug with an MIC of 64 mg/L.


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Table 1. Activity of five antimicrobial agents on Neisseria gonorrhoeae isolates from 1995 (n = 61), 1997 (n = 198), 1998/99 (n = 98) and 1999/2000 (n = 58)
 


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Figure 2. Susceptibility range for erythromicin ({blacksquare}) and azithromycin ({square}) of N. gonorrhoeae isolates from the 1999/2000 survey (n = 58).

 
A large proportion of DPNG and CRMNGT isolates in 1999/2000 exhibited increased MICs of ciprofloxacin, ceftriaxone, erythromycin, azithromycin and spectinomycin (Table 2Go).


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Table 2. Percentage of DPNG and CRMNGT isolates in 1999/2000 with increased MICs of ciprofloxacin, erythromycin, azithromycin, ceftriaxone and spectinomycin
 
The amount of drug, prescribed for any indication, of the classes of antimicrobial drugs that are advised for treatment of STDs is given in Table 3Go. The consumption of penicillins per potential user of the provincial health care system increased significantly between 1996 and 1999. The use of cephalosporins remained constant. Macrolides, mainly erythromycin, were the most frequently prescribed antimicrobial agents. The use of quinolones rose sharply from 1996 to 1997 and levelled off thereafter.


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Table 3. Total consumption of antimicrobial agents used for syndromic management of STDs in the provincial healthcare system of KwaZuluNatal
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To establish the aetiological diagnosis of sexually transmitted infections is one of the most challenging issues in human medicine. Most of the 333 million estimated new infections per year8 occur in developing countries, where clinical and laboratory diagnosis is hampered by the lack of medical personnel and laboratory facilities. However, even in more affluent countries where these are available, aetiological diagnosis proves to be difficult because of the low sensitivity of both the clinical diagnosis and even the most sophisticated of tests. Furthermore, a positive test for one organism does not exclude the presence of others, because mixed infections occur frequently.9 These difficulties in reaching a diagnosis have resulted in the implementation of syndromic management for STDs.3 However, the standardization of antimicrobial treatment creates a selective advantage for organisms resistant to the drugs used.10 Hence, sexually transmitted infections caused by antibiotic-resistant organisms are becoming a major therapeutic problem in many parts of the world with resistance among strains of N. gonorrhoeae posing a particular challenge.11–17

Syndromic management was introduced on a large scale in KwaZuluNatal in 1995. From then until 2000, the distribution of MICs for N. gonorrhoeae of all antimicrobials recommended, has shifted to the right, indicating increased resistance. These shifts have resulted in clinically significant resistance to penicillins and tetracyclines. For the other drugs the increase in MICs has had no clinical impact as yet. Others have reported similar trends in resistance.13,14,16–19 Mechanisms that cause increase in MICs among unrelated groups of antibiotics are well recognized. Events leading to the expression or upregulation of efflux pumps produce a phenotype with resistance to several structurally unrelated antibiotics.20 Integrons provide bacteria with a tool to accumulate resistance genes.21 Although such genetic elements have not been reported in N. gonorrhoeae, these are found with increasing frequency in Gram-negative bacteria. In addition, a site-specific recombinase has recently been identified in N. gonorrhoeae.22 Such enzymes play a role in the acquisition of resistance gene cassettes.

The gradual increase in MICs as observed with the quinolones, the macrolides/azalides, ceftriaxone and spectinomycin might be the result of a series of independent events. The emergence of plasmid-mediated high-level resistance to penicillin and tetracycline indicates either the acquisition of genes from other organisms, or the introduction of an already resistant gonococcal strain that takes advantage of the selective environment created by the syndromic management. Both these events may occur simultaneously in gonococci, as has been demonstrated in the past. The initial low level chromosomally mediated resistance to penicillin (MIC >= 2 mg/L)23 was followed by the emergence of plasmid-mediated high-level resistance (MIC >= 8 mg/L) through the production of ß-lactamase.18 The emergence of these PPNG strains in Durban, KwaZuluNatal, occurred in 1977.24 By 1985, the prevalence of PPNG among strains from Durban was 29%.25 In 1988, the use of penicillin as first-line therapy for N. gonorrhoeae was curtailed and patients were treated with a cephalosporin or spectinomycin. This was changed to ciprofloxacin with the introduction of the syndromic management guidelines in 1995.

We recently reported on quinolone resistance in N. gonorrhoeae in South Africa.26 Molecular characterization of clinical strains from other areas of the world has shown that resistance occurs in a stepwise fashion, which can be detected by increasing MICs to the drug. The number of organisms with decreased susceptibility to the quinolones in KwaZuluNatal has increased since 1995 (Table 1Go). This South African province uses a 250 mg stat. dose of ciprofloxacin instead of the WHO- and CDCrecommended 500 mg. Reports on susceptibility from other parts of South Africa where the 500 mg stat. dose is used are lacking. The question as to whether the use of this low dose has fast-tracked this emergence of decreased susceptibility is therefore difficult to answer. Selection of resistance in bacteria is thought to be related to the duration of treatment in individual patients as well as to the total amount of drug used within a population.10,27 The stat. dose of ciprofloxacin reflects the shortest course possible. Therefore, the vast amounts of antimicrobial agents used in the population is the most likely explanation for our observations. However, if mechanisms conferring resistance to more than one drug are involved, then the selective pressure does not have to come from ciprofloxacin usage. The extremely high consumption of macrolides (Table 3Go) provides a selective advantage for these strains with decreased susceptibility to both erythromycin and ciprofloxacin (29% in 1999/2000). Monitoring the emergence of these pre-resistant strains will assist in the prediction of future evolution of quinolone resistance in N. gonorrhoeae.

Although tetracycline derivatives are not the treatment of choice for gonorrhoea, these are the recommended drugs for the treatment of Chlamydia trachomatis.3 However, since differentiation between gonorrhoea and chlamydial infection is not made when syndromic management is applied, all patients with gonorrhoea are exposed to this group of drugs. Such exposure also occurs when asymptomatic patients or those with unrecognized symptoms28 are treated with tetracyclines for indications other then STDs. Plasmid-mediated high-level resistance to tetracycline in N. gonorrhoeae (>=16 mg/L) was first reported in 1983 in the USA. Resistance in Africa was first described in Zaire.29 In 1994, the first high-level resistant strains in South Africa were reported from Bloemfontein.30 Plasmid-mediated high-level resistance to tetracycline emerged in KwaZuluNatal in 1998 and spread rapidly. Within a period of 18–24 months the prevalence of these strains highly resistant to tetracycline jumped from 3% to >50%. This resistance is plasmid-mediated and has been shown to be caused by the presence of the tetM gene.31

Erythromycin is widely used for the treatment of genital ulcer disease, vaginal discharge in pregnancy and the treatment of respiratory tract infections among children. Mutations leading to resistance to erythromycin may confer resistance to azithromycin.32,33 Our data support this (Figure 2Go). Azithromycin has been recommended as an alternative in the treatment of uncomplicated N. gonorrhoeae and C. trachomatis infections. Recent failure of azithromycin therapy in patients with uncomplicated urethritis is alarming,15,34 but might have been predicted since it is structurally similar to erythromycin. In South Africa, this drug is not recommended for uncomplicated urethritis/cervicitis. However, it is commonly used in the private sector for the treatment of respiratory tract infections and sexually transmitted infections. The somewhat rapid increase in MICs of N. gonorrhoeae in our area to azithromycin should not be taken lightly, since this heralds the ineffectiveness of this antibiotic for the treatment of gonorrhoea.

Ceftriaxone is currently the drug of choice for the treatment of N. gonorrhoeae in pregnancy.35 Recently, the use of this drug has increased in both the public and private sectors as first-line therapy for bacterial meningitis. In addition the drug is widely used in the private sector for the treatment of other childhood infections.

Spectinomycin is a recommended alternative to ciprofloxacin for the treatment of N. gonorrhoeae in pregnancy.35 Our data reveal an increase in the MICs of this drug. This is interesting, because the main mechanism of resistance to this drug is a one-step chromosomal mutation that results in full resistance.36 Our observation suggests the presence of another mechanism, most likely a shared efflux pump.

The main question is whether we need to respond to the increase in MICs of the different drugs in the absence of documented resistance-related clinical failure. Experience with Streptococcus pneumoniae, another community-acquired pathogen, suggests that gradual increase in MICs will eventually lead to clinically significant resistance.37 The high rates of resistance to penicillin, and the decrease in susceptibility to azithromycin and erythromycin, occurred despite the fact that none of these agents is recommended in the treatment guidelines for genital discharge. In addition, a decrease in susceptibility to ceftriaxone and spectinomycin is seen, although these drugs are infrequently used for the treatment of vaginal discharge in pregnancy and are administered as a stat. dose. The shift to the right in the distribution of MICs of these antimicrobials may therefore be consistent with the widespread use of these agents in the community for other indications. It is therefore doubtful whether changes in syndromic management guidelines at this stage, or a decrease of the total consumption of antibiotics by reverting to aetiology-based management of STDs, will make any difference in the absence of a more rational use of the same drugs for other indications.


    Notes
 
* Corresponding author. Tel: +27-31-2604395; Fax: +27-31-2604431; E-mail: sturm{at}med.und.ac.za Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Gerbase, A. C., Rowley, J. T. & Mertens, T. E. (1998). Global epidemiology of sexually transmitted diseases. Lancet 351, Suppl. 3, S1112–4.

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4 . Chenia, H. Y., Pillay, B., Hoosen, A. A. & Pillay, D. (1997). Antibiotic susceptibility patterns and plasmid profiles of penicillinase-producing Neisseria gonorrhoeae strains in Durban, South Africa, 1990-1993. Sexually Transmitted Diseases 1, 18–22.

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Received 4 December 2000; returned 26 February 2001; revised 1 May 2001; accepted 14 June 2001