Rising quinolone resistance in Neisseria gonorrhoeae isolates from New Delhi

Rising quinolone resistance in Neisseria gonorrhoeae isolates from New Delhi

Bhalla, P

Background & objectives: Treatment for gonorrhoea with fluoroquinolones is recommended. However, reduced susceptibility and treatment failure with fluoroquinolones has recently been reported. We undertook to study the antibiotic susceptibility pattern and the incidence of quinolone resistance in 36 consecutive isolates of Neisseria gonorrhoeae from April to November 2000.

Method: Antibiotic susceptibility testing was performed by the Kirby Bauer disc diffusion technique and minimum inhibitory concentration (MIC) of ciprofloxacin was determined by the agar dilution method. Penicillinase producing N. gonorrhoeae (PPNG) were identified by using the nitrocefin disc method.

Results: Thirty six strains of N. gonorrhoeae obtained from 44 consecutive male patients (81.9%) were studied. By the disc diffusion method, only 3 (8.3%) of these isolates were found to be sensitive to ciprofloxacin. All isolates were sensitive to ceftriaxone while 23 (63.9%) were sensitive to tetracycline and 12 (33.3%) to penicillin. Four (11.1%) of the N. gonorrhoeae isolates were PPNG. Twenty seven (75%) isolates were found to be resistant to ciprofloxacin by MIC determination.

Interpretation & conclusion: Incidence of ciprofloxacin resistance amongst N. gonorrhoeae isolates is on the rise in New Delhi. Periodic monitoring of antimicrobial susceptibility pattern of N. gonorrhoeae to antimicrobials other than quinolones is essential to prevent treatment failure in patients with gonorrhoea.

Key words Antimicrobial susceptibility – Neisseria gonorrhoeae – quinolone

Antimicrobial resistance of Neisseria gonorrhoeae to penicillin and tetracycline is well known and has been increasing over the years. The emergence of resistant strains has led to the increased use of broad spectrum cephalosporins and fluoroquinolones for the treatment of uncomplicated gonorrhoea1. However, since 1993 resistance to fluoroquinolones has been reported from Hong Kong, Japan and Philippines with sporadic reports from the US, UK, Australia, Canada and Rwanda2. The resistance to fluoroquinolones in N. gonorrhoeae is due to changes at a number of sites in genes gyrA and parC at chromosomal level which encode for DNA gyrase and topoisomerase IV enzymes on which quinolones act. Once chromosomal resistance develops to one fluoroquinolone, cross resistance is seen to other drugs of the same class but at variable MICs2. Results of a study3 suggest that the low accumulation of quinolone in the cell is one of the mechanisms of quinolone resistance in N. gonorrhoeae isolated in Japan. Further investigations are necessary in the mechanisms of fluoroquinolone resistance in N. gonorrhoeae, such as changes in porin protein and the lipopolysaccharide structure associated with outer membrane permeability, an active efflux system and alteration in DNA gyrase3. The development and spread of resistance is encouraged by the use of suboptimal doses of antibiotics and also contributed to by the extensive use of fluoroquinolones in treating many other infections.

In India, penicillin and tetracycline resistance has been reported but there have been few reports on fluoroquinolone resistance4. A study conducted in New Delhi in 1994 reported an incidence of 12 per cent quinolone resistant N. gonorrhoeae (QRNG)4 while another study5 conducted between 1995-99 reported an overall incidence of 21.5 per cent QRNG (ciprofloxacin MIC > 1 (mu)g/ml). Surveillance for antimicrobial resistance should be an integral part of a routine STD laboratory programme but if resources are limited a sample of approximately 20-50 consecutive isolates may be tested periodically6. Thus, the present study was conducted to examine the antimicrobial susceptibility of consecutive gonococcal isolates and to compare the same with the antimicrobial -susceptibility of the isolates studied in 1994, to detect change if any, in the susceptibility of N. gonorrhoeae to the commonly used antimicrobial agents.

Material & Methods

Patient group: Forty four consecutive male patients clinically suspected to have acute gonococcal urethritis presenting to the STD clinic of the Lok Nayak Hospital, New Delhi between April and November 2000 were included in the study. Urethral swabs were taken from all these patients for direct microscopy and culture for N. gonorrhoeae.

Direct microscopy and culture of N. gonorrhoeae: Gram stained smear was prepared from the urethral swab to look for pus cells and intracellular Gram negative diplococci. Immediately after collection the urethral specimen was inoculated on to modified Thayer Martin Medium7 [Chocolate agar + vancomycin colistin nystatin trimethoprim (VCNT) (HiMedia, India)] in the STD clinic and transported in a candle jar to the STD laboratory where it was incubated at 37 deg C in 5-10 per cent CO^sub 2^ for 48-72 h.

Growth of N. gonorrhoeae was identified by colony morphology, Gram staining, oxidase test and carbohydrate utilization tests7. All isolates were maintained by daily subcultures on chocolate agar [Gonococcus agar (HiMedia, India) + 2% haemoglobin (Difco, USA) + GC growth supplement (HiMedia, India)] or stored in trypticase soy broth (Difco, USA) with 20 per cent glycerol (HiMedia, India) at -70 deg C for further testing.

Disc diffusion technique: Antibiotic sensitivity testing of gonococcal isolates was carried out using the Kirby Bauer disc-diffusion method8 on chocolate agar. Antibiotic discs (HiMedia, India) used were ciprofloxacin (5 (mu)g); ceftriaxone (30 (mu)g), penicillin (10 IU) and tetracycline (30 (mu)g). The inhibition zone diameter was measured in millimeters and interpreted as sensitive, less sensitive and resistant based on NCCLS criteria9. beta lactamase production was detected by chromogenic cephalosporin method using nitrocefin disc10 (Difco, USA).

MIC determination: Minimum inhibitory concentration of ciprofloxacin in the range of 0.002 to 32 (mu)g/ml for all N. gonorrhoeae isolates was determined by agar dilution method11 on chocolate agar. MIC was read as the lowest concentration of ciprofloxacin that allowed no visible growth. Isolates with MIC values = 1 (mu)g/ml were considered resistant12.

Control strains: WHO reference gonococcal strains A-E and provisional reference cultures (SEARO/ WHO) H, I, J obtained from Regional STD Reference Laboratory, Safdarjung Hospital, New Delhi were used for contr6l of disc diffusion and MIC testing11.

Chi-square test was used to analyse the data statistically

Results

Urethral specimens from 44 male patients with symptoms and signs of acute urethritis were subjected to direct microscopy and culture for N. gonorrhoeae and 36 were found to be positive by direct microscopy and culture while four were positive only by direct microscopy.

Antibiotic susceptibility testing of these 36 isolates by disc diffusion revealed that only 3 (8.3%) isolates were fully sensitive to ciprofloxacin. All isolates were sensitive to ceftriaxone. Penicillin sensitivity was observed in 12 (33.3%) isolates while 23 (63.9%) were sensitive to tetracycline (Table). Four (11.1%) N. gonorrhoeae isolates were found to be pencillinase producing N. gonorrhoeae (PPNG) by chromogenic cephalosporin technique. All these PPNG strains had an inhibition zone diameter of = 1 (mu)g/ml). Only one of the nonPPNG (3.1%) strains was suspected to be a TRNG on the basis of disc diffusion tests. On the basis of MIC of ciprofloxacin, 27 (75%) of the gonococcal isolates were found to be resistant (MIC >= 1 (mu)g/ml), with 22 (81.4%) of these having a MIC of >= 4 (mu)g/ml. Only 2 (5.6%) of the isolates were sensitive (MIC

Thirty five patients of gonorrhoea were treated with ciprofloxacin (500 mg) and were followed up for clinical and microbiological cure. Treatment failure was detected in 25 patients (71.4%) on clinical grounds. The gonococcal isolates from all these patients had been reported as being resistant to ciprofloxacin.by disc diffusion and MIC.

Comparison between the disc diffusion and MIC values of ciprofloxacin for N. gonorrhoeae isolates showed that all isolates with zone diameter = 4 (mu)g/ml. Also all isolates with zone diameter = 1 (mu)g/ml. However, only 50 per cent of strains with a zone diameter of 27-35 mm had MIC of >= 1 (mu)g/ml. Among the 3 isolates with zone diameter >= 36 mm (sensitive by disc diffusion), 2 had MIC value

A comparison between the MIC values of quinolones observed in the present study and a similar study conducted in 1994(4) in the same hospital revealed a rapid rise in percentage of isolates showing quinolone MIC >= 1 (mu)g/ml.

In 1994(4) only 12 per cent of gonococcal isolates had MIC value >= 1 (mu)g/ml in comparison to 75 per cent in 2000. This difference was highly significant (P

Discussion

In the present study, ciprofloxacin resistance (MIC >= 1 (mu)g/ml) was seen in 75 per cent of N. gonorrhoeae isolates. This was much higher than that observed earlier in the same institution (12%)4. A study5 conducted between 1995-1999 in a tertiary care centre in the same city (Delhi) reported an increasing incidence of 3.5, 13.6 and 22 per cent QRNG (MIC > 1 (mu)g/ml) in 1996, 1997 and 1999 respectively, This suggests that fluoroquinolone resistance in N. gonorrhoeae has emerged in India also and is on a rapid rise over the past few years. Another study conducted in Philippines has reported an incidence of 63 per cent QRNG13.

The percentage of PPNG isolates in our study was 11.1 per cent. The incidence of PPNG reported in other studies from New Delhi varies from 0-8 per cent4,5, while another study14 in Mumbai reported an incidence of 13.6 per cent. While incidence as high as 59 per cent has been reported from Central Africa15. All PPNG and TRNG strains were found to be resistant to ciprofloxacin (MIC >= 1 (mu)g/ml). .

Out of the 27 QRNG (MIC >= 1 (mu)g/ml), 7 were resistant to penicillin while 1 strain was resistant to tetracycline.

The increase in the percentage of QRNG in the present study (75% vs 12% in 1994) suggests a need to switch over to other oral formulations such as cefixime as the first line treatment for gonorrhoeae. Because of the prevalence of QRNG in parts of Asia, treatment with a non quinolone regimen is suggested1. The recommended regimens for uncomplicated gonococcal urethritis include in order of preference cefixime 400 mg orally in a single dose or ceftriaxone 125 mg I/M in a single dose16.

Comparison of MIC and disc diffusion gave good agreement for sensitive strains by disc diffusion (zone diameter >= 36 mm) and MIC value (

Thus, we conclude that there has been a rapid rise of quinolone resistant N. gonorrhoeae isolates from 12 per cent in 1994(4) to 75 per cent in 2000 in a major STI) centre in New Delhi. Susceptibility testing by diffusion method can be used for preliminary detection of ciprofloxacin resistance and should be subsequently confirmed by determination of MIC. Continued use of fluoroquinolones for the treatment of uncomplicated gonorrhoea in the presence of resistant strains can lead to selective pressure and further spread of resistant strains. Hence, it is suggested that periodic monitoring of susceptibility of consecutive N. gonorrhoeae isolates should be a routine activity at all regions of India.

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P. Bhalla, S. Vidhani, B.S.N. Reddy*, S. Chowdhry* & M.D. Mathur

Departments of Microbiology & *Skin & STD, Maulana Azad Medical College & Associated Lok Nayak Hospital, New Delhi, India

Received July 19, 2001

Reprint requests : Dr Preena Bhalla, Professor, Department of Microbiology, Maulana Azad Medical College & Associated Lok Nayak Hospital, New Delhi 110002, India

Copyright Indian Council of Medical Research Mar 2002

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