Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 3, 2007, pp. 225-229

Indian Journal of Medical Microbiology, Vol. 25, No. 3, July-September, 2007, pp. 225-229

Original Article

Epidemilogical analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping

Khaki P, Bhalla P, Sharma P, Chawla R, Bhalla K

Department of Microbiology, Mualana Azad Medical College, New Delhi - 110 002
Correspondence Address: Department of Microbiology, Mualana Azad Medical College, New Delhi - 110 002, preenadr@gmail.com

Date of Submission: 25-Aug-2006
Date of Acceptance: 10-Feb-2007

Code Number: mb07064

Abstract

Keywords: A/S typing, antimicrobial susceptibility testing, auxotyping, Neisseria gonorrhoeae, serotyping

Gonorrhea remains one of the most common sexually transmitted disease (STD) in developing countries. Two important factors, which are responsible for the high rate of prevalence of gonorrhea, are increasing antimicrobial resistance of N. gonorrhoeae and presence of a large reservoir of asymptomatic carriers that unknowingly transmit the disease to their sexual contacts.

The study of relationship between isolates of N. gonorrhoeae can contribute to the identification of outbreaks in sexual networks, re-infection, temporal and geographic changes, sexual abuse and monitoring of antibiotic resistance. ^[1],[2] Ideally, a system for classification of N. gonorrhoeae should be typable, reproducible and easy to perform, economical, rapid and give a high discriminatory index. Epidemiological studies can be carried on the basis of phenotypic and genotypic methods; however, the latter are more costly in terms of equipment, reagents and analyzing software and need more expertise. Various phenotypic techniques that have been used for epidemiological analysis of N. gonorrhoeae isolates include, antimicrobial susceptibility, auxotyping and serotyping. In India, only a few studies exploring the epidemiological aspects of N. gonorrhoeae have been carried out. ^[3],[4] Therefore, the aim of the present study was to analyze the epidemiological relationship between gonococcal strains isolated from patients attending sexually transmitted disease clinic and their sexual partners, on the basis of antimicrobial susceptibility testing, auxotyping and serotyping.

Materials and Methods

Gonococcal strains
Sixty consecutive N. gonorrhoeae strains isolated from 62 males with urethritis, 22 females with endocervicitis and 10 sexual contacts of these patients attending the STD clinic of Lok Nayak Hospital, New Delhi from January 2004 to June 2005, were studied.

The samples were inoculated directly onto the modified Thayer Martin medium and incubated at 35-36.5°C in moist air containing 5% CO ₂ for 48h. The colonies suspected to be N. gonorrhoeae were presumptively identified by Gram stain, oxidase test and superoxol test. Confirmation of identity after subculture on chocolate agar was based on rapid carbohydrate utilization test. Gonococcal isolates were stored at -70°C in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) containing 20% glycerol. ^[5]

Antimicrobial susceptibility testing
All the isolates were examined for susceptibility to penicillin (10 IU), ciprofloxacin (5 mg), tetracycline (30 mg), ceftriaxone (30 mg), spectinomycin (100 mg), cefixime (5 mg) and azithromycin (15 mg) by the agar disc diffusion method. In addition, the minimum inhibitory concentrations (MICs) were determined by E-test. The E-test was performed as specified in the manufacturer′s (AB Biodisk, Sweden) product package insert. N. gonorrhoeae ATCC 49226 was included for quality control of antimicrobial susceptibility testing. The antimicrobial susceptibility was interpreted according to Clinical and Laboratory Standards Institute (CLSI) and Centers for Disease Control (CDC) guidelines. ^[6],[7] β-lactamase production was assayed using nitrocefin discs (BBL Cefinase, Becton Dickinson and Company, Sparks, MD, USA). ^[5]

Auxotyping
Auxotyping was performed using the method described by La Scolea and Young. ^[8] The strains were tested for their requirements for arginine, proline, uracil, hypoxanthine, serine, isolocine, cysteine and cystine. Isolates with no special nutritional requirements were classified as non-requiring (NR).

Serotyping
The isolates of N. gonorrhoeae were serotyped using the co-agglutination technique, as described by Coghill and Young. ^[9] Serotyping was performed using the Phadebact Monoclonal GC kit and the Phadebact GC serovar test (Boule Diagnostic AB, Hudding, Sweden), which consisted of five WI reagents (Ao, Ar, As, At and Av) and nine WII/III reagents (Bo, Bp, Br, Bs, Bt, Bu, Bv, Bx, By). All isolates were classified into serogroups WI and WII/III and further subdivided into serovars.

Discriminatory index
The discriminatory index for the various typing methods was calculated as described previously. ^[10]

Results

A total of 60 gonococcal strains were isolated from 52 (83.87%) out of 62 men with urethritis, 4 (18.18%) out of 22 women with endocervicitis and 4 (40%) out of 10 sexual contacts of these cases.

Antimicrobial susceptibility testing
All isolates were found to be susceptible to ceftriaxone, spectinomycin, cefixime and azithromycin. 59(98.3%), 20(33.3%) and 14(20%) strains were resistant to ciprofloxacin (quinolone resistant N. gonorrhoeae , QRNG), penicillin and tetracycline respectively. MIC ₉₀ for ciprofloxacin, penicillin, tetracycline, ceftriaxone and spectinomycin was 8, 3, 24, 0.016 and 12 µg/mL respectively [Table - 1]. Twelve (20%) strains were found to be penicillinase producing N. gonorrhoeae (PPNG) and 11(18.3%) were tetracycline resistant N. gonorrhoeae (TRNG). We observed nine antibiotic resistance patterns with antimicrobial susceptibility testing [Table - 2]. The discriminatory index for antimicrobial susceptibility testing was 0.58.

Auxotyping
The strains belonged to eight different auxotypes. The most common auxotypes were non-requiring (40 %), proline requiring (31.7%) and arginine requiring (11.7%) auxotypes [Table - 3]. All PPNG and TRNG strains were either NR or Pro auxotype. Auxotyping had a discriminatory index of 0.61.

Serotyping
Out of the total 60 strains studied, 28 (46.7%) isolates belonged to WI (IA) serogroup and 31 (51.7%) were of WII/III (IB) serogroup and one strain was WI and WII/WIII serogroup combination. Among the serogroup WI isolates, serovar Aost (50 %) was the most prevalent, followed by Arost (25%) and Ast (14.3%). The serogroup WII/WIII isolates were differentiated into 12 serovars, of which the most prevalent serovar was Boprt (48.4%) followed by Btuvy (13%), Bopty (9.6%). Thus 71 % of the serogroup WII/III strains included only three serovars [Table - 3]. One strain gave a positive agglutination reaction with both WI and WII/III serogroup reagents and was found to be of Av/Bx serovar. Serotyping had a discriminatory index of 0.81.

Auxotype/serovar typing
Sixty isolates were subdivided in to 29 auxotype/serovar (A/S) classes. The main A/S classes [Table - 3] were NR/Aost (10%), NR/Arost (10%), Pro/Aost (10%), Pro/Boprt (10%) and NR/Boprt (10%). A/S typing gave a highest discriminatory index (0.94).

Discriminatory index of typing methods
Amongst all the typing methods, A/S typing was found to have the highest discriminatory index [Table - 4]. However, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.

Discussion

The ability to characterize the strains of infectious agents that cause disease is central to epidemiological surveillance and public health decisions. Apart from being highly discriminatory an ideal bacterial typing system for epidemiological studies should be easy to perform and inexpensive. Typing systems used to characterize individual strains of N. gonorrhoeae include antimicrobial susceptibility testing, serotyping, auxotyping and molecular typing. Although molecular techniques give a higher discriminatory index, the main limitation of these methods are that they are expensive in terms of equipment, reagents and analyzing software, need more expertise, being laborious and the results from different laboratories are also difficult to compare. In India, a few epidemiological studies on N. gonorrhoeae, based on antimicrobial susceptibility testing, serotyping and auxotyping have been carried out. ^{[3],[4],[11],[12],[13],[14]}

As regards antimicrobial resistance pattern as an epidemiological marker, the incidence of PPNG reported from various parts of the India varies from 8-46.7%. ^{[3],[12],[14]} In a previous study from New Delhi in 1998, 28% TRNG was detected. ^[12] The majority of these studies categorized N. gonorrhoeae only into PPNG and non-PPNG or into TRNG and non-TRNG.

In the present study, we observed nine antibiotic resistance patterns. Fifty-nine (98%) isolates were resistant to ciprofloxacin (QRNG), while 12(20%) isolates were PPNG and 11(18.3%) isolates were TRNG. PPNG strains could be further subdivided into four categories and TRNG could be categorized into three categories as the basis of resistance to other antimicrobial agents tested. Thirty-five percent of strains were resistant to at least two antibiotics. The discriminatory index for antimicrobial susceptibility testing was 0.58.

The number of different auxotypes that have been reported by other studies is between 4-16. Although NR and proline requiring auxotypes often predominate, several other auxotypes have been detected only in certain parts of the world. ^{[1],[15],[16],[17],[18]} NR and proline requiring auxotypes were also most common auxotypes in Mumbai and Delhi. ^[3],[4] In our study also, the most prevalent auxotype was NR (40%), followed by proline requiring (31.7%) and arginine requiring (11.7%) auxotypes. Restriction of certain auxotypes to different geographical areas can help in global epidemiological studies. We found some new auxotypes that have not been previously reported before in Delhi, which indicates changes in the pool of circulating strains. Auxotyping gave a discriminatory index of 0.61.

In our study, 28 (46.7%) isolates belonged to WI (IA) serogroup and 31 (51.7%) were of WII/III (IB) serogroup and one strain was a combination of WI and WII/WIII serogroups. The data shows that serogroups WII/III are most prevalent in Delhi. This finding is in contrast to the results from a previous study, in which the serogroup WI was found to be the most prevalent serogroup in Mumbai. ^[3] However, this study did not identify the isolates up to serovar level.

From our data it could be deduced that there is a wide variety of serovars of N. gonorrhoeae circulating in New Delhi, but the serovars Aost (50%), Arost (25%) and Ast (14.3%) in the serogroup WI and those of Boprt (48.4%) and Btuvy (13%) in the serogroup WII/III, predominate. Similar studies carried out in other parts of the world showed that serovars Arost and Arst belonging to serogroup WI and Boprt and Bropyt belonging to serogroup WII/III were most common serovars. ^[1],[9] However, Dillon et al . reported that serovars Arost and Arot in the serogroup WI and serovars Boprt and Brpyust in the serogroup WII/III were most prevalent in Jamaica. ^[18] In the present study, one strain that cross-reacted with both Av and Bx reagents (Av/Bx) was detected. This result is consistent with previous studies in which the same Av/Bx strain has been reported. ^[9],[16] Serotyping had a discriminatory index of 0.81.

The results of our study showed that serotyping in combination with auxotyping (A/S typing) provided greater discrimination between isolates than the use of only one of these techniques. We observed 29 A/S classes, the most prevalent A/S classes being NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. In a study by Dillon et al ., the most common A/S classes were NR/Arot, Pro/Arost, NR/Bropt and Pro/Bropt. ^[18] A/S typing provided a discriminatory index of 0.94, which is an acceptable level of discrimination in a typing method. ^[10]

Our findings showed WI serogroup to be the most prevalent serogroup among the PPNG and TRNG isolates. This finding is in accordance with previous reports that the most prevalent serogroup among PPNG strains was WI. ^[3] The diversity of the auxotypes and serovars of PPNG and TRNG isolates suggests that these isolates in Delhi had multiple sources and did not represent the introduction and spread of a single strain. All isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.

Our results illustrate that the discrimination is enhanced when auxotyping and serotyping are used in combination to produce auxotype/serovar (A/S) classes. Although antimicrobial susceptibility testing had less discriminatory index, it could be performed as a routine test for ongoing monitoring of antimicrobial susceptibility of N. gonorrhoeae strains.

References

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