Molecular typing of Neisseria gonorrhoeae byrestriction fragment length polymorphisms

C L Poh, H P Khng, C K Lim, G K Loh

AbstractObjective-To characterise Neisseriagonorrhoeae isolates by restriction frag-ment length polymorphisms (RFLPs) inribosomal RNA genes.Design-Generation of RFLP patternsby HincII restriction of rRNA genes fol-lowed by hybridisation with a non-

radioactive labelled broad spectrum 16+ 23S rRNA gene probe. This typingmethod was developed and comparedwith MAb based serotyping.Specimens-Forty three randomlycollected isolates from Bangkok (27isolates) and Singapore (16 isolates) werestudied.Results-The RFLP patterns generatedwere reproducible and highly discrimin-atory between strains. Analysis ofRFLPsproduced by HincII restriction of rRNAgenes established 9 patterns amongst the43 isolates examined. Strains presentwithin a common serovar could be fur-ther subdivided by RFLP typing. Iden-tical RFLP patterns were found in somestrains that belonged to variousserovars.Conclusion-RFLP typing based on

heterogeneities of rRNA gene restrictionpatterns could be advantageously used tocomplement monoclonal antibody basedserotyping for further subdivision ofserovars. Higher sensitivity of this com-bined approach would enable better dif-ferentiation of strains in epidemiologicalstudies.

Department ofMicrobiology,National University ofSingapore, LowerKent Ridge Road,Singapore 0511C L PohH P KhngC K LimG K LohAddress correspondence to:Dr C L PohAccepted for publication28 November 1991

IntroductionAuxotyping and serovar determination havebeen accepted as the two major phenotypicsystems for typing of Neisseria gonorrhoeae.Currently, the most widely employed methodto differentiate N gonorrhoeae isolates is one

based on a combination of both methods,known as the auxotype-serovar (A/S) clas-sification system.' However, it has severallimitations. Large numbers of isolates in Asiaare either Prototrophic or of Pro- auxotype2.These isolates may in fact be very differentstrains despite sharing common auxotypes.Similarly, strains belonging to a particularserovar may not be identical as suggested bycomparisons of other phenotypic characteris-tics such as antimicrobial susceptibility pat-terns, plasmid profiles and auxotypes. Otherproblems include the occurrence of non-typa-ble strains, the need to develop new mono-

Genitourin Med 1992;68:106-1 10

clonals in anticipation of possible genetic driftin Protein I (PI), the continual production ofmonoclonals, batch to batch variations ofmonoclonals and the technical reproducibilityof coagglutination reactions especially whenusing monoclonal antibodies such as 2D6,2G2 and 6D93.Molecular typing methods such as re-

striction endonuclease analysis (REA) of gen-omic DNA has been shown to be a sensitivetechnique for differentiation ofN gonorrhoeaeserovars.45 However, owing to the complexityof DNA bands generated in either agarose or

polyacrylamide gels, meaningful interpreta-tions of DNA fingerprints are best made withquantitative comparisons of dendograms gen-erated.

Specific DNA probes such as ribosomalribonucleic acid (rRNA) based probes havebeen found to generate fewer bands in gels,making both interpretations and comparisonsmuch easier. This approach has been found toprovide excellent discrimination andreproducibility in the epidemiological typingof diverse groups of bacteria, for example, inthe differentiation of Legionella pneumophilaserogroup 16, coagulase-negative staphy-lococci7, Helicobacter pylori8 and meningococci9.The aim of this study was to investigate the

application ofrRNA gene probe for the detec-tion of restriction fragment length polymor-phisms (RFLPs) in N gonorrhoeae strains andto compare the rRNA gene patterns withserological characterisation.

Materials and methodsGonococcal strainsA group of 27 random isolates collected in 1984from Bangkok which had been serotyped withMAbs was kindly provided by SM Bygdeman.The remaining 16 strains used in this studywere randomly isolated over 6 months in 1984from male patients with urethritis from theMiddle Road Hospital, Singapore. Serologicalcharacteristics of this latter set of strains havebeen described previously.5

Preparation ofDNAGenomic DNA was prepared using the in situextraction method as described previously.5

Restriction enzyme digestion and electrophoresisRestriction endonucleases EcoRI, CfoI, ClaIand HincII were purchased from BethesdaResearch Laboratories, Inc, Gaithersburg,Md, USA. DNA entrapped in agarose pelletswas digested with restriction enzymes using

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Molecular typing of Neisseria gonorrhoeae by restriction fragment length polymorphisms

buffers and reaction conditions as describedpreviously.5 The digested DNA was electro-phoresed at 25 V for 16 h in horizontal 0.8%(wt/vol) agarose (type II medium electroen-domosis grade, Sigma, USA) gel in a TAEbuffer system (0 04 M Tris, 0.04 M acetic acid,0-002 M EDTA [pH 8.0]). After electro-phoresis, the gel was stained with ethidiumbromide, examined by UV transilluminationand photographed. Southern blotting ofDNAfragments to a nylon membrane (Hybond-N,RNN 303 N, Amersham) was performed in avacuum pump unit (Vacugen; Pharmacia-LKB) and the membranes were baked for 2 h at80°C.

Preparation of non-radioactive labelled rRNAprobeThe rRNA gene probe derived from plasmidpHF 1 1, which encodes the 16S and 23SrRNA genes of Pseudomonas aeruginosa was agift from K H Schleifer.`0 The plasmid wasdigested with BstEII (New England Biolab,USA). The digested DNA fragments wereseparated by gel electrophoresis on 0.8%agarose gels. The 2.2 kb fragment containingthe rRNA genes were isolated from agarose andlabelled with horseradish peroxidase (ECLGene detection system RPN 2101, Amersham)following the manufacturer's instructions.Briefly, the DNA at a final concentration of 10-20 ng/pl was heated at 55°C for 10 min, thencooled on ice for 5 min. An equivalent volumeof labelling reagent (horseradish peroxidasecomplexed with a positively charged polymer)was added. Cross-linking was obtained withglutaraldehyde. The reaction tube was spun at12,000 x g for 5 s to settle the reaction mixturein the bottom of the tube and incubated for 10min at 37C.

A B

lybridisation with proberhe membrane filters were soaked in pre-ybridisation solution (Amersham Inter-Lational, UK) containing 0.5 M NaCl for 30nin at 42°C. After prehybridisation, theabelledDNA probe was added to the hybridis-tion solution and the contents were mixed.'he blots were then incubated with agitation at2°C overnight. Washing of the blots wasarried out as instructed by the manufacturer.*or the computation of DNA fragment sizes,unbda DNA cleaved by HindIII (BoehringerAannheim, Germany) was used as a MWttandard.

)etection of the non-radioactive label7wo detection reagents provided by thenanufacturer were mixed in equal volumes'efore use. Detection reagent 1 contained.ydrogen peroxide, the substrate for peroxi-lase. Detection reagent 2 contained luminolvhich produces blue light upon oxidation. Forwo membranes, 25 ml of each reagent werenixed, poured on the membranes, andncubated at room temperature for one minute.rhe excess detection buffer was drained offandhe blots were wrapped in Saran Wrap avoidingir bubble formation. The blots were placed)NA-side up in a film cassette with a sheet ofutoradiography film (Hyperfilm MP, Amer-ham) and exposed for one min, after which itvas replaced with a second sheet of film. Theime of exposure of the second film wasstimated from the appearance of the first filmfter it was developed. Generally, 20 minxposure time was used.

tesultsRNA gene restriction patterns ofgonococci_xperiments with digestion of total genomic

Kb

..:.?.__E-v-_L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... 13.9

11.88.0

4.6

_ _ .... - 36L~~~~~~~~~~~~~.

_4 1.4

Fig 1 Restriction endonuclease patterns of totalgenomic DNA (A) and ribosomal RNA gene restriction patterns (B)ofN. gonorrhoeae strains digested with HincII. Lane 1, strain All; lane 2, strain A14; lane 3, strain A15; lane 4,strain A16; lane 5, strain A17; lane 6, strain A18; lane 7, strain A19; lane 8, strain A20.

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1 2 3 4 5 6 7 8 9 kb

- 1-13.9

-11-8- 8-0

F- 36

_- 23

Fig 2 Schematic representation ofRFLPs of 43 N.gonorrhoeae strains obtained after digestion of total DNAwith HincII and hybridisation with rRNA gene probefrom P. aeruginosa.

for each strain remained identical when DNAprepared from randomly selected strains grownon separate occasions during the course of thestudy were compared. Similarly, no differencesin RFLP pattems were observed for strainssubjected to serial subcultures.

Comparison ofRFLP with serologicalcharacterisationAnalysis ofthe hybridisation patterns ofHincI Idigests showed that there were several strainsbelonging to the same serovar displaying thesame RFLP pattem. For example, four strainsof serogroup WI/serovar Aedih belonged toRFLP type 7 (table 2). However, strains be-longing to the same serogroup/serovar could befurther subdivided based on different RFLPpattems generated. Eleven strains of serogroupWI/serovar Aedih were subdivided into fourRFLP types whilst two groups comprising sixstrains of WII: Back and five strains of WIII:Beghjk were each further resolved into threeRFLP types. RFLP type 2 was the predomin-ant group observed among the 9 HincII gen-erated RFLP types. It was represented by 18isolates which were distributed amongst dif-ferent serogroups/serovars.

DNA employing a variety of restriction endo-nucleases showed that HincII restrictionproduced rRNA gene restriction patterns thatwere most disriminatory as judged by thenumber and distribution of band positionsafter hybridisation with P aeruginosa16 + 23 SrRNA genes (fig 1). Enzymes such as

EcoRI and CfoI which generated one or twohybridising bands were not chosen as no

heterogeneity was observed in the hybridisa-tion patterns of various strains examined.

Digestion of genomic DNA by HincII was

found to produce between 8 to 12 hybridisingfragments ranging in size from 1-4 to 13-9 kb.Nine RFLP patterns comprising differentmolecular sized DNA fragments were revealedby HincII digestion of the 43 isolates (fig 2).The nine patterns possessed four common

rRNA gene fragments which corresponded tosizes of approximately 4-6, 5 0, 5-4 and 8.0 kb,respectively. RFLP type 1 (isolates All, A18and A20, fig 1) differed from RFLP type 2(isolates A14 and A15, fig 1) by the presence ofasingle rRNA-containing fragment of 14 kb.RFLP types 3 and 8 had hybridisation patternsthat were significantly different from the rest.The remaining five RFLP patterns were quiteclosely related and each differed from the otherby the presence of one to two rRNA gene

fragments.The distribution of gonococci into RFLP

types was not uniform. Some of the patternswere shared by numerous strains, for example,RFLP type 2 was represented by 18 strainswhile others were characterised by one to twostrains (table 1). Only one isolate each was

assigned to EFLP types 3, 6 and 8. This couldbe due to the small number ofisolates examinedrather than a very low prevalence of thesepatterns.

Reproducibility and stability ofRFLP patterns

The characteristic patterns of bands observed

DiscussionRestriction site heterogeneity within rRNAgenes has been found to generate RFLP pat-tems suitable for differentiation of geneticallydiverse groups of bacteria. The RFLP methodis based on the detection ofminor differences ina genetic locus such as that encoding rRNAgenes. For this study, we have chosen a broadspectrum probe encoding rRNA genes clonedfrom P aeruginosa. An earlier study by Stull etal' showed that identical rRNA gene patternscould be produced by either a homologous or a

Table 1 Restriction fragment length polymorphismpatterns ofNgonorrhoeae strains

RFLP type Strain RFLP type Strain

1 Al 3 42380AIOAllA18A20 4 A17B3 D34B6B8D14D35 5 A19

D37D47

2 A4 31487A5A9A14 6 D25A15B4 7 B2B5 B7Bll B9B15 B14D5 D18DIOD12 8 45469D15D21D28 9 A16D30 69844D32D42

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Table 2 Relationship between RFLP type and serological characteristics of the43 Ngonorrhoeae strains

Serogroup and RFLP Serogroup and RFLPserovar Strain type serovar Strain type

WI:Aed D28 2 WI:Bajk A14 1D12 2

WI:Aedih A15 2 D14 1A18 1 D15 2B2 7 D37 5B3 1B4 2 WII: Bak D18 7B6 1B7 7B9 7BR1 2 WII:Bcegjk All 1B14 7 D5 245469 8 31487 5

WII: Bacejk A5 2 WII:Bcgk A16 942380 3

WII:Bcgjk A4 2D32 2

WII: Bacjk A19 5B15 2D25 6D42 2 WII:Bck D30 2D47 5

WII: Back AIO 1 WIlI: Beghjk Al 1A17 4 A9 2A20 1 B5 2D21 2 D8 1D34 4 69844 9D35 1

heterologous probe. Many investigators haveused a broad spectrum 16 + 23 S rRNA fromE coli that is available commercially. However,the need to make a cDNA copy employingreverse transcriptase would make theprocedure unnecessarily complicated andcostly.'2 The 2.2 kb BstEII fragment encodingthe 16 + 23 S genes served as a good broadspectrum probe in this study as hybridisingbands were seen with every strain. No strainwas non-typable with this probe. RFLP pat-terns generated using this probe were alsofound to be both stable and reproducible.The results of this study showed that RFLP

typing based on rRNA gene heterogeneity wasmuch simpler to interpret than the complexrestriction fragment patterns of total genomicDNA digests. The rRNA gene patternsobtained using a non-radioactive probe inhybridisation reactions was found to be bothsharp and highly discriminatory. A non-radioactive labelled probe was chosen as resultsfrom Koblavi et all" demonstrated that therRNA gene patterns obtained with the ECLgene detection system was as sensitive as 32pautoradiography.The choice of restriction endonucleases used

was critical to the divergence of rRNA genehybridisation patterns obtained. More pro-nounced heterogeneity of rRNA hybridisationpatterns could be generated by restrictionendonucleases that have different restrictionsites within the rRNA cistrons. If a single copyrRNA gene is highly conserved and an enzymeused for restriction recognised a single site andgenerates only a single DNA fragment ofidentical molecular weight in all the strainsexamined, then this enzyme should not beselected. In our study, EcoRI was found not tohave any restriction site in the DNA fragmentencoding the rRNA genes and a singlehybridising band was observed in all the strains

examined. Both ClaI and HincII generatedgood restriction endonuclease (RE) digestionpatterns of total genomic DNA. However, theHincII rRNA gene hybridisation patterns werefound to be more useful for differentiation ofstrains as greater heterogeneity was observed inthe number of hybridising bands in HincIIrRNA gene restriction patterns than thosegenerated by ClaI.The HincII rRNA gene restriction patterns

observed were very much related as there weresignificant numbers of common DNA bandspresent in each ribotype. The highly conservednature of rRNA genes was reflected by thepresence ofthese commonDNA bands. Never-theless, heterogeneities in the rRNA genesexisted and these were marked by the presenceof uniqueDNA bands. These formed the basisof the present typing system. For example,RFLP types 1 and 2 were differentiated fromeach other in the presence or absence of aDNAband at 1.4 Kb.Based on RFLPs generated with HincII

enzyme, we found no absolute correlation be-tween rRNA gene restriction patterns andserotyping using monoclonal antibodies. Manystrains with the same RFLP patterns werefound distributed amongst several serovars (forexample RFLP types 1, 2 and 5). Strains withidentical and different RFLP types were alsofound within each serovar. The present datademonstrate that both typing methods could beused advantageously to complement each otheras isolates were subdivided by the alternativetechnique. RFLP typing is observed to offer anincreased sensitivity over serotyping for dif-ferentiating serologically identical strains andnon-typable strains. A combined typing systembased on monoclonal antibody serotyping andRFLP subtyping will be very useful for dif-ferentiating N gonorrhoeae strains inepidemiological studies. The option of usingthe RFLP technique on a non-radioactive basismakes this approach especially promising foruse in non-reference labaratories and indeveloping countries.

We acknowledge the support of a National University ResearchGrant No RP 900369. We are grateful to Professor S MBydgeman (Sweden) for the MAb serotyped N gonorrhoeaestrains and the kind gift of the pHF 1 1 probe from Professor KH Schleifer (Germany).

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