neisseria gonorrhoeae is the causative agent of gonorrhea , which continues to be one the most common sexually transmitted infections worldwide [ 1 , 2 ] . control and preventive measures for infection are based on epidemiological analyses as well as antibiotic susceptibility of the n.   gonorrhoeae isolates . the process of typing of neisseria gonorrhoeae isolates can be used to identify the population of strains that circulate in different communities , temporal , and geographic changes among the strains , transmission patterns , and antibiotic resistance outbreaks [ 3 , 4 ] . the widely used phenotypic methods are based on auxotyping , serotyping , and antimicrobial susceptibility testing . however , these methods have some limitations such as reproducibility and discriminatory power [ 59 ] . therefore , different molecular genetic methods have been developed for the characterization of gonococcal strains include ribotyping [ 10 , 11 ] , pulsed - field gel electrophoresis ( pfge ) , arbitrarily primed pcr , amplified fragment length polymorphism ( aflp ) , opa - typing [ 15 , 16 ] , and sequence typing based on one or more genes [ 4 , 13 , 16 , 17 ] . ribotyping refers to the use of southern blot analysis for detecting polymorphisms that are associated with the ribosomal dna regions . the method has appeared to be good reproducibility , stability , and typeability for characterization of gonococcal isolates . however , it provides less discrimination between isolates of n.   gonorrhoeae [ 10 , 11 ] . opa - typing is a pcr - based rflp technique , which relies on amplifying the 11 opa genes and subsequent digestion with restriction enzymes , to generate restriction fragment length polymorphism . the method has shown to be highly discriminatory enough to be applied to large collections of gonococcal isolates . the opa - typing is also useful to confirm self - reported sexual contacts and to identify sex partners and short chains of transmission [ 15 , 16 , 18 ] . there are hardly any data about molecular epidemiological analysis of n.   gonorrhoeae isolates by opa - typing and ribotyping in india . epidemiological analysis of n.   gonorrhoeae isolates by genotypic methods has not been carried out in india so far . in this study , we determined the potential utility of opa - typing and ribotyping for differentiation of gonococcal strains . sixty consecutive n.   gonorrhoeae strains isolated from among 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 , 2006 to june , 2007 were studied . the samples were inoculated directly onto the selective modified thayer - martin medium ( mtm ) medium and incubated at 3536.5c containing 5%co2 for 48 hours . the colonies suspected to be n.   gonorrhoeae were identified by gram stain , oxidase , superoxol tests , and rapid carbohydrate utilization test ( rcut ) . genomic dna was isolated from confluent ( 1824 hours ) growth of gonococcal cells by using a wizard genomic dna purification kit ( promega corp . , briefly , the bacterial cell was suspended in 500 l of tris - edta buffer ( ph 7.8 ) . the suspensions were pelleted and resuspended in 500 l of nuclei lysis solution ( promega ) and incubated for 5 minutes at 80c . however , 4 l of rnase ( 10 mg / ml ) ( sigma chemical co. , usa ) was added to cell lysate and incubated for 1 hour at 37c . the lysate was adjusted to 0.7 m nacl and 1% cetyltrimethylammonium bromide ( ctab ) , mixed thoroughly , and incubated for 10 minutes at 65c in order to precipitate cell wall debris , denatured proteins , and polysaccharides . the supernatant was extracted twice with phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) and twice with chloroform / isoamyl alcohol ( 24 : 1 ) . chromosomal dna was precipitated with isopropanol , washed with 70% ethanol , resuspended in 50 l of tris - edta buffer ( ph 7.8 ) , and stored at   20c . chromosomal dna was digested with avaii and hincii , and the restricted fragments were separated by electrophoresis on 0.8% agarose gels in tris - borate ( tb ) buffer at 40 v for 16 hours . dna fragments were transferred to positively charged nylon membrane ( roche diagnostic gmbh , mannheim , germany ) from agarose gels by the method of southern . the blot was hybridized with the addition of digoxigenin - dutp labeled cdna probe at 68c for 1824 hours . however , 16s and 23s rrna from e. coli ( roche diagnostic gmbh , mannheim , germany ) was reverse transcribed into cdna with superscript ii reverse transcriptase ( invitrogen , calif , usa ) . the cdna was then labeled by random priming with digoxigenin - dutp according to the manufacturer 's directions ( roche diagnostic gmbh , mannheim , germany ) . the sizes of the hybridized fragments from at least four blots were averaged after measuring distances of band migration in comparison with the digoxigenin labeled dna molecular weight marker iii ( roche diagnostic gmbh , mannheim , germany ) . amplification was carried out in a 50 l volume containing 0.5 g of each opa forward and reverse primer ( opa-01 , 5-atgtcaggcggatttagcc-3 , and opa-04 , 5-aatgaggcttcgtgggttttg-3 ) , 100 ng of chromosomal dna , 0.2 mm of each dntp , 2.5 u of taq dna polymerase , and the buffer provided by the manufacturer with 75 mm tris hcl ( ph 9.0 ) , 2 mm mgcl2 , 50 mm kcl , 20 mm ( nh4)2so4 ( biotools , b&m labs , s.a . , cycle conditions were set at 94c for 5 minutes ( hot - start ) ; 30 cycles of 94c for 1 minute , 64c for 1 minute , and 72c for 1 minute ; a final extension reaction at 72c for 10 minutes . the pcr products ( 670 bp ) were precipitated with ethanol , washed with 70% ethanol , and resuspended in 50 l of te buffer . the opa gene fragments ( 1.5 g ) were digested overnight at 65c with 10 u of taqi in a volume of 30 l using the buffer recommended by manufacturer . the resulting restriction fragments was fractionated on a precast 12% polyacrylamide gel and visualized by ethidium bromide staining . the reproducibility of ribotyping and opa - typing was examined by analysis of the same bacterial strain on three separate occasions . the stability of the techniques was examined by daily subcultures of n.   gonorrhoeae   isolates . the relatedness values were calculated as the proportion of unweighted mismatches of fragments between the different patterns . computed similarity cluster analysis of the patterns was done by the unwieghted pair - group average algorithm , using the dice coefficient to analyze the similarities of the banding patterns . statistical methods like chi - square test and fisher 's exact test were performed to determine the significant association of various demographic data using the spss version 13.0 for windows . 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 . hybridization of avaii fragments with labeled rrna probes yielded 6 ribotype patterns ( table 1 ) . twenty - three of the 60 isolates demonstrated pattern 1 ; 16 isolates had patterns 2 ; 12 isolates demonstrated pattern 3 ; 4 isolates had pattern 4 ; 3 isolates pattern 5 ; 2 isolates pattern 6 . analysis with hincii produced highly resolved fragments suitable for analysis , and fragment sizes ranged from 0.46 to 18.36 kb ( figure 1 ) . twelve rflp patterns were observed with hincii , from which 16 of the 60 isolates demonstrated pattern 1 ; 10 isolates had pattern 2 ; 8 isolates had pattern 3 ; patterns 46 comprised 4 isolates each ; patterns 79 contained 3 isolates each ; patterns 10 and 11 had 2 isolates each . all isolates recovered from index case and their sexual contacts were found to be identical by ribotyping . the characteristic isolate banding patterns with hincii remained unchanged when dna was prepared from randomly selected strains grown on separate occasions , and the ribotypes did not change after three individual strains were subcultured 1 , 10 , and 20 times . for opa - typing , the 11 opa genes were amplified and the pcr products were digested with restriction enzymes taqi and hpaii . cluster analysis of hpaii opa - type patterns revealed a total 54 opa - types , from which 48 ( 88.9% ) were unique and 6 ( 11.1% ) formed clusters ( table 1 and figure 2 ) . of the 48 isolates with unique opa - types , none were known to be from sexual contacts . opa - typing with taqi was slightly less discriminatory than typing with hpaii ( table 1 ) . the opa - types from isolates that were epidemiologically unrelated ( unconnected ) were distinct , whereas those from the sexual contacts were identical . pcr amplifications of the opa genes were performed from three isolates of n.   gonorrhoeae grown on separate occasions , from which the opa - types were identical . the stability of the opa - typing during subculture in vitro was examined by obtaining opa - types from dna prepared from daily subcultures of three isolates of n.   gonorrhoeae . no differences in the opa - types of the isolates were seen after 1 , 10 , and 20 subcultures . the ability to identify accurately the strains of infectious agents that cause disease is central to epidemiological surveillance and public health decisions . the widely available typing systems have the potential to provide information useful to the control and prevention of n.   gonorrhoeae infections and to guide health interventions . all typing systems for epidemiological studies should have established reproducibility , and be highly discriminating in order to distinguish epidemiologically unrelated infections . the shortcomings of phenotypic typing methods have led to development of molecular genetic methods , which minimize problems with reproducibility or discriminatory power [ 59 , 12 , 15 , 16 ] . there is hardly any data about epidemiological studies of n.   gonorrhoeae isolates based on molecular typing in india . therefore , our aim was to assess molecular typing of n.   gonorrhoeae isolates using ribotyping and opa - typing . ribotyping has the advantage over restriction endonuclease analysis in that the rflp patterns contain fewer bands for comparison because only fragments with rrna sequences are visualized . more pronounced heterogeneity of rrna hybridization patterns could be generated by restriction endonuclease that have different restriction sites within the rrna genes . in our study , rflp patterns generated with hincii ( 12 ribotype patterns ) showed greater heterogeneity than those generated by avaii ( 6 ribotype patterns ) . on the basis of combined hincii and avaii - generated rflp patterns , 60 isolates of n.   gonorrhoeae the results of this study showed that ribotyping was much simpler to interpret than the complex restriction endonuclease patterns of total chromosomal dna . the results suggested that there may be sufficient heterogeneity of rrna patterns within n.   gonorrhoeae isolates and the method may to be useful for differentiation of strains in epidemiological studies . a similar study has shown that rflp patterns generated by hincii could be used to differentiate 43 isolates of n.   gonorrhoeae into nine patterns . a previous study of ribotype patterns generated by combination of smai and avaii digests of n.   gonorrhoeae differentiated 23 proline - citrulline - uracil requiring ( pcu ) auxotypes into four groups . a canadian study indicated that the ribotyping by smai could be classified 27 ornithine - uracil - hypoxanthine ( ouh ) or citrulline - uracil - hypoxanthine ( cuh ) or ornithine - hypoxanthine ( oh)/ia-2 auxotypes of n.   gonorrhoeae into five ribotype patterns . the present study showed that common dna bands were present in the rflp patterns for all the n.   gonorrhoeae isolates in accordance with previous studies [ 10 , 11 , 22 ] . opa - typing is a technique based on diversity ( polymorphism ) of opa genes of n.   gonorrhoeae . a single gonococcus possesses 11 different opa genes , which encodes the outer membrane proteins . the extensive variation and rapid evolution of the opa gene repertoire make it likely that gonococci with identical opa - types are very closely related genetically and epidemiologically . major potential use of opa - typing is the identification of groups of individuals in sexual networks by the isolation of gonococci with the same       opa - type from gonococcal populations recovered in a restricted geographic area within a limited time period . in this study , opa - typing of the 60 isolates revealed a total of 54 opa - types , from which 48 ( 88.9% ) were represented by single isolates . the opa - types from isolates that were epidemiologically unrelated were distinct , while those from the sexual contacts were identical . as the gonococci within these were indistinguishable by ribotyping , and were from patients living in the same part of delhi attending the std clinic within a short time period , it is possible that they were from individuals who were connected by a short chain of infection transmission or were form common core group ( sexual contacts ) . this result indicates that     opa - typing is highly useful for characterizing gonococcal strains from sexual contacts in full agreement with previous studies [ 15 , 16 , 18 ] . we found opa - typing to be more discriminatory than ribotyping and could differentiate between gonococci of the same ribotype . the technique even with a single restriction enzyme ( hpaii ) has a high level of discrimination ( 99.7% ) between epidemiologically unrelated isolates in concordance with results in previous studies [ 15 , 16 , 18 , 23 ] . the results of both methods indicate that gonococcal infection among the patients studied was not derived from common core groups . in conclusion , the molecular methods such as opa - typing and ribotyping can be used for epidemiological characterization of gonococcal strains .
control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of   n. gonorrhoeae isolates . in the present study the potential utility of opa - typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined . 
 sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes , avaii and hincii , and opa - typing with taqi and hpaii for epidemiological characterization of gonococcal population . 
 ribotyping with avaii yielded 6 ribotype patterns while twelve rflp patterns were observed with hincii . 
 opa - typing of the 60 isolates revealed a total 54 opa - types , which 48 were unique and 6 formed clusters . 
 fifty - two opa - types were observed with taqi - digested pcr product while opa - typing with hpaii demonstrated 54 opa - types . 
 the opa - types from isolates that were epidemiologically unrelated were distinct , whereas those from the sexual contacts were identical . 
 the results showed that opa - typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype . 
 the technique even with a single restriction enzyme has a high level of discrimination ( 99.9% ) between epidemiologically unrelated isolates . in conclusion 
 , the molecular methods such as opa - typing and ribotyping can be used for epidemiological characterization of gonococcal strains .