Indian July-September Journal of 2007 Medical Microbiology, (2007) 25 (3):225-9 Original Article 225 EPIDEMILOGICAL ANALYSIS OF NEISSERIA GONORRHOEAE ISOLATES BY ANTIMICROBIAL SUSCEPTIBILITY TESTING, AUXOTYPING AND SEROTYPING P Khaki, *P Bhalla, P Sharma, R Chawla, K Bhalla Abstract Purpose: This study was carried out to analyze the epidemiology of gonorrhea based on antimicrobial susceptibility testing, auxotyping and serotyping in New Delhi, India. Methods: Sixty gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts were studied. The isolates were subjected to antimicrobial susceptibility testing, auxotyping and serotyping for epidemiological characterization. Results: We observed nine antibiotic resistance patterns. Ninety-eight percent of isolates were resistant to ciproßoxacin, while 20% isolates were penicillinase producing N. gonorrhoeae (PPNG) and 18.3% isolates were tetracycline resistant N. gonorrhoeae (TRNG). Eight auxotypes were observed, of which the NR (non-requiring), proline requiring and arginine requiring were most common auxotypes. On the basis of serotyping alone, the gonococcal isolates could be differentiated into three serogroups and 18 serovars. Serogroup WI represented 46.7% and WII/III represented 51.7% of isolates and one strain was WI and WII/WIII serogroup combination. When results of auxotyping and serotyping were combined (A/S) 29 A/S classes could be identiþed. The most prevalent A/S classes were NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. Conclusions: Although A/S typing had the highest discriminatory index, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods. Key words: 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 classiþcation 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 *Corresponding author (email: <preenadr@gmail.com>) Department of Microbiology (PK, PB, RC, KB), Mualana Azad Medical College, New Delhi - 110 002, and International Centre for Genetic Engineering and Biotechnology (PS), New Delhi - 110 067, India Received: 25-08-06 Accepted: 10-02-07 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 modiþed Thayer Martin medium and incubated at 35-36.5ºC in moist air containing 5% CO 2 for 48h. The colonies suspected to be N. gonorrhoeae were presumptively identiþed by Gram stain, oxidase test and superoxol test. ConÞrmation of identity after subculture on chocolate agar was based on rapid carbohydrate utilization test. Gonococcal isolates were stored at -70ºC 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), ciproßoxacin (5 µg), tetracycline (30 µg),
226 Indian Journal of Medical Microbiology vol. 25, No. 3 ceftriaxone (30 µg), spectinomycin (100 µg), ceþxime (5 µg) and azithromycin (15 µg) by the agar disc diffusion method. In addition, the minimum inhibitory concentrations (MICs) were determined by E-test. The E-test was performed as speciþed 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 nitroceþn discs (BBL CeÞnase, 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 Þve 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 classiþed 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 ciproßoxacin (quinolone resistant N. gonorrhoeae, QRNG), penicillin and tetracycline respectively. MIC 90 for ciproßoxacin, 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 Table 1: Antimicrobial susceptibility of N. gonorrhoeae isolates by disc diffusion method Antibiotic n=60, No. (%) MIC 90 S I R Penicillin 1 (1.7) 39 (65) 20 (33.3)* 3 Tetracycline 34 (56.7) 12 (20) 14 (23.3)** 24 Ciproßoxacin 0 1 (1.7) 59 (98.3) 8 Ceftriaxone 60 (100) 0 0 0.016 Spectinomycin 60 (100) 0 0 12 CeÞxime 60 (100) 0 0 ND Azithromycin 60 (100) 0 0 0.125 *Penicillinase producing N. gonorrhoeae: 12(20%), **Tetracycline resistant N. gonorrhoeae: 11 (18.3%), MIC - Minimum inhibitory concentrations, S - Sensitive, I - Intermediate, R - Resistant, Figures in parentheses are in percentage Table 2: Antibiotic resistance patterns of gonococcal isolates Antibiotic resistance pattern No. (%) of isolates QRNG* 30 (50) QRNG+PPNG # 8 (13) PPNG 1 (1.7) QRNG+CMPRNG ## 7 (11.67) QRNG+TRNG 8 (13.33) QRNG+CMTRNG 2 (3.33) QRNG+PPNG+ TRNG 2 (3.33) QRNG+PPNG+CMTRNG 1 (1.67) QRNG+CMPRNG+TRNG 1 (1.67) Total 60 *Quinolone resistant N. gonorrhoeae, # Penicillinase producing N. gonorrhoeae, ## Chromosomally mediated penicillin resistant N. gonorrhoeae, Tetracycline resistant N. gonorrhoeae, Chromosomally mediated tetracycline resistant N. gonorrhoeae 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
July-September 2007 Khaki et al - Epidemiological Typing of Neisseria gonorrhoeae 227 Table 3: Distribution of gonococcal strains into 29 auxotype/serovars Serogroup Serovar No. (%) Auxotyping NR Pro Arg AUH AH PAUH H Islu 24(40) 19(31.7) 7(11.7) 4(6.6) 3(5) 1(1.6) 1(1.6) 1(1.6) WI Aost 14 (50) 6 6-1 1 - - - (n=28) Arost 7 (25) 6-1 - - - - - Ast 4 (14.3) - 3-1 - - - - Arst 2 (7.1) - - 2 - - - - - At 1 (3.6) - 1 - - - - - - WII/III Boprt 15 (48.4) 4 6 3 1 - - 1 - (n=31) Btuvy 4 (13) 2 1-1 - - - - Bopty 3 (9.6) 3 - - - - - - - Bpr 1 (3.2) - - - - 1 - - - Bsy 1 (3.2) 1 - - - - - - - Bsuy 1 (3.2) - - - - 1 - - - Bpty 1 (3.2) - 1 - - - - - - Bptuvx 1 (3.2) - - - - - 1 - - Bost 1 (3.2) - 1 - - - - - - Bst 1 (3.2) 1 - - - - - - - Bpt 1 (3.2) - - 1 - - - - - Bsty 1 (3.2) 1 - - - - - - - Wl/Wll/lll (n=1) Av/Bx 1 (1.6) - - - - - - - 1 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 Table 4: Discriminatory indices of typing methods for gonococcal isolates Typing method No. of types Discriminatory index Antimicrobial 9 0.58 susceptibility testing Auxotyping 8 0.61 Serotyping 18 0.81 A/S typing 29 0.94 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 difþcult to compare. In India, a few epidemiological studies on N. gonorrhoeae, based on antimicrobial susceptibility testing, serotyping and auxotyping have been carried out. 3,4,11-14 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 ciproßoxacin (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-Þve percent of strains were resistant to at least two antibiotics. The discriminatory index for antimicrobial susceptibility testing was 0.58.
228 Indian Journal of Medical Microbiology vol. 25, No. 3 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. 1,15-18 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 Þnding 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 Þnding 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. 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