Skip Navigation


JAC Advance Access originally published online on January 27, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):478-487; doi:10.1093/jac/dkm544
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
61/3/478    most recent
dkm544v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Liao, M.
Right arrow Articles by Dillon, J.-A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liao, M.
Right arrow Articles by Dillon, J.-A. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Clusters of circulating Neisseria gonorrhoeae strains and association with antimicrobial resistance in Shanghai

Mingmin Liao1,2, Kelli Bell1, Wei-Ming Gu3, Yang Yang3, Nelson F. Eng1, Wenkai Fu1, Lei Wu3, Chu-Guang Zhang3, Yue Chen4, Ann M. Jolly4 and Jo-Anne R. Dillon1,2,*

1 Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3 2 Department of Microbiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5 3 Shanghai Skin Disease and STD Hospital, 196 Wu Yi Road, Shanghai 200050, China 4 Department of Epidemiology and Community Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5

* Correspondence address. College of Arts and Science, University of Saskatchewan, Room 226, Arts Building, 9 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A5. Tel: +1-306-966-4232; Fax: +1-306-966-8839; E-mail: j.dillon{at}usask.ca

Received 14 August 2007; returned 12 December 2007; revised 19 October 2007; accepted 18 December 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 Supplementary data
 References
 
Objectives: (i) To distinguish Neisseria gonorrhoeae isolates in Shanghai by porB typing; (ii) to ascertain the congruence of porB DNA sequence typing with cases linked epidemiologically; (iii) to determine the association of specific PorB mutations with antimicrobial resistance to penicillin or tetracycline.

Methods: porB DNA sequences of 174 N. gonorrhoeae isolates, collected from 143 male patients and 31 female sexual partners in Shanghai were determined. Phylogenetic analysis was used to determine sequence associations and concordance with epidemiologically linked cases. PorB protein sequences were compared with the wild-type sequence to identify mutations associated with antimicrobial resistance to penicillin and tetracycline.

Results: porB1a genotypes comprised 27.0% of the isolates and included 15 distinct DNA sequences, while 73.0% of the isolates carried porB1b genotypes with 63 distinct DNA sequences. porB DNA sequence typing was congruent with patient-reported sexual contacts. In addition, porB DNA sequence analysis revealed a number of strains with identical DNA sequences not identified through traditional epidemiological methods. The porB1b isolates had a significantly higher percentage of chromosomally mediated resistance to tetracycline and higher MIC50s to penicillin and ciprofloxacin. G120K/A121D mutations were observed in 71.1% of PIB isolates and were associated with resistance to penicillin and/or tetracycline. The majority of the PIA isolates (82.1%) also carried G120D/A121G double mutations. The index of discrimination for porB DNA sequence analysis was 95%.

Conclusions: The porB1b genotype was found to be predominant in Shanghai. porB DNA sequence typing was sufficiently discriminatory for differentiating N. gonorrhoeae isolates and was congruent with epidemiological linkages. Novel porB sequences of N. gonorrhoeae and novel mutations of PorB proteins were identified.

Keywords: N. gonorrhoeae , molecular epidemiology , molecular typing , sexually transmitted diseases


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 Supplementary data
 References
 
Neisseria gonorrhoeae is highly variable in its genotype and phenotype.1–3 This genetic variability has been exploited to characterize gonococcal strains circulating in a given community, thereby identifying patterns of specific strain transmission and guiding control strategies.4–8 DNA sequence analysis of gonococcal porB genes can provide a high index of discrimination for isolates of N. gonorrhoeae7,9 and has been used to identify circulating strains/clusters,4,10–13 to track strain transmission in sexual contacts4,7,8,11,14 and to investigate antibiotic resistance.15–17

PorB proteins have been associated with cell viability, pathogenesis18 and antimicrobial susceptibility.16,17 The two porB alleles, porB1a and porB1b, are mutually exclusive in N. gonorrhoeae isolates, although some hybrid genes have been identified.19,20 These alleles encode one of the two PorB proteins, either PIA or PIB.21 Gonococcal PorB is a transmembrane protein comprising eight highly variable loops separated by nine conserved regions.6,22 The loops, which are surface exposed, can elicit an immune reaction during infection6,23 and were targeted for differentiating isolates when serovar determination with monoclonal antibodies was widely available.24–30

Gonorrhoea is a significant public health problem worldwide.31 The rate of sexually transmitted infections (STIs) in Shanghai, the trade capital of China and a city with a population of more than 17 million, has increased over the past two decades and is currently the highest in China; between 2001 and 2005 (STI Reports, Shanghai), gonorrhoea prevalence in Shanghai was 75–107 per 100 000 when compared with rates of 13–19 per 100 000 in China overall.32–34 The genotypic characterization and transmission patterns of N. gonorrhoeae in Shanghai have not been studied previously. This report summarizes a pilot study undertaken to understand STI strain transmission in patients accessing the Shanghai Skin Disease and STD Hospital over a 7 month period in 2004–05. We determined the porB DNA sequences of isolates, ascertained what strains comprised circulating clusters and associated porB types with antimicrobial susceptibility. The congruence of porB sequence typing and epidemiological sexual links was ascertained, and clusters identified by porB sequence analysis were compared with those revealed by patients' self-reported connections.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 Transparency declarations
 Supplementary data
 References
 
Patient recruitment, N. gonorrhoeae isolate collection and identification

Male patients accessing the STD Clinic of the Shanghai Skin Disease and STD Hospital (the Shanghai Hospital), China, with gonorrhoea, were referred to trained interviewers by attending physicians. A consent form and an anonymous questionnaire were administered to each patient to gather demographic information, STI history and information related to sexual practices. Male patients with gonorrhoea (n = 143) were consecutively recruited as index patients between November 2004 and May 2005. Sexual partners were identified by index patient nomination and were invited into the study, of whom, 31 females were N. gonorrhoeae positive. Subsequently, a total of 174 N. gonorrhoeae isolates were included in this study comprising 143 isolates from male index patients and 31 isolates from their female sexual partners. Alternatively, these isolates comprised 113 isolates from individual male patients, 58 isolates from 29 sexual pairs and 3 isolates from 1 index patient and 2 female sexual partners, respectively. The primary isolation and identification of gonococcal isolates was performed at the Diagnostic Centre of the Shanghai Skin Disease and STD Hospital, as reported previously.35 After primary isolation, isolates were subcultured on GC agar base (Oxoid; GuangZhou LOSO Science Ltd) supplemented with 1% IsoVitalex (Oxoid) prior to antimicrobial susceptibility testing and chromosomal DNA extraction procedures. Isolates were stored at –80°C in Brain Heart Infusion broth (Difco; distributed by Shanghai Chemical Reagent Co., China National Medicine Group, Shanghai, China) containing 20% glycerol. Ethical approval for this study was obtained from the Ottawa Hospital Research Ethics Board and the Ethics Committee of the Shanghai Municipal Bureau of Public Health.

Determination of antimicrobial susceptibility

The MICs for all isolates to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone were determined using an agar dilution method36 and results were reported previously.35 β-Lactamase production was determined for all isolates using a chromogenic cephalosporin test (Oxoid). Interpretative MIC criteria36 and antimicrobial resistance phenotypes included the following classifications: CIPR (ciprofloxacin resistance MICs ≥1 mg/L), PPNG (β-lactamase positive), TRNG (isolates having tetracycline MICs ≥16 mg/L), PP/TRNG (β-lactamase positive and MICs to tetracycline ≥16 mg/L), CMPR (chromosomal penicillin resistance; non-PPNG isolates with penicillin MICs ≥2 mg/L), CMTR (chromosomal tetracycline resistance; non-TRNG isolates with tetracycline MICs ≥2–8 mg/L) and CMRNG (non-PPNG and non-TRNG isolates with penicillin MICs ≥2 mg/L and tetracycline MICs ≥2 to ≤8 mg/L).

Preparation of chromosomal DNA

N. gonorrhoeae isolates were retrieved from storage and incubated for 18–24 h at 35°C in a humid environment supplied with 5% CO2. Bacterial suspensions were prepared in 0.9% saline to a turbidity equivalent to that of a 0.5 McFarland standard (Remel, Lenexa KS, USA; ~108 cfu/mL). One millilitre of the cell suspension was used for chromosomal DNA extraction. Gonococcal DNA was extracted using the Genomic DNA Purification Kit following the manufacturer’s manual (Shanghai Promega Biological Products Ltd, Shanghai, China). DNA was eluted with 100 µL of ddH2O. The quality of DNA was verified by agarose electrophoresis.

Amplification and DNA sequence analysis of porB, pilA and abcZ

porB was amplified by PCR using porB-F and porB-R primers (purchased from Invitrogen Canada, Burlington, Ontario, Canada) as described previously (Table 1).11 The PCR mixture (50 µL) contained 5 µL of genomic DNA, 2.5 U of Taq DNA polymerase (Amersham Bioscience), 1x PCR buffer with 1.5 mM MgCl2, 0.25 mM dNTPs, 0.5 µM of each primer. Amplification (Perkin Elmer 9600 Thermo Cycler, Wellesley, MA, USA) was performed as follows: an initial denaturing step at 94°C for 4 min, followed by 30 sequential cycles of 94°C for 1 min, 50°C for 45 s and 72°C for 1 min, and a final extension phase at 72°C for 10 min. Following the purification of PCR products (PCR Purification Kit, Qiagen, Mississauga, Ontario, Canada), DNA sequences of both strands were determined using the Applied Biosystems 3730x1 DNA Analyzer (Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada) with primers porB-F or porB-R, respectively (Table 1). The porB DNA sequences determined in our study comprise seven polymorphic areas for encoding the surface-exposed loops (I–VII) and six conserved interspace regions (II–VII), which accounts for 85% of the entire porB gene.21,37 The sequences of these regions were determined and used for analysis because they can be ascertained in a single DNA sequencing reaction of upper- and lower-strand of DNA and because the discriminatory power is high enough for sufficient molecular epidemiology studies.38–40


View this table:
[in this window]
[in a new window]

  		Table 1. Primers used in this study

 
An initial experiment was conducted to determine the discriminatory power of porB, pilA or abcZ DNA sequence analysis either alone or in combination.8 Thus, 33 clinical isolates were randomly selected from a clinical collection in Shanghai in 2004 prior to this study. The identification of N. gonorrhoeae for these isolates was confirmed by the oxidase test, Gram's stain and glucose utilization tests, as reported previously.35 porB (primers indicated above), pilA (primers pilA-F/pilA-R) and abcZ (primers abcZ-F/abcZ-R) were PCR-amplified (Table 1).8 PCR parameters were those described above except for an extension phase of 2 min for abcZ. Each strand of the amplicons was sequenced with the primers listed in Table 1.

Verification and editing of DNA sequences

DNA sequences were verified by the alignment of the complementary strands using Multialin version 5.4.1 (http://prodes.toulouse.inra.fr, date last accessed 20 September 2006)41 and occasionally by manual examination.4,7,9 Sequences were aligned using ClustalW42 and edited using Jalview Alignment Editor (http://www.ebi.ac.uk/clustalw, date last accessed 28 September 2006).43 porB sequences (e.g. porB1a and porB1b) from N. gonorrhoeae MS11 (porB1a, GenBank #J03029) or FA1090 (porB1b, GenBank #J03017) were used as prototypes. porB genotypes were assigned to porB1a or porB1b based on the presence or absence of two nucleotide sequences in the loop V coding region, as described previously;6,9,21 porB1a was characterized by the absence of ~50 nucleotides (nt) corresponding to the positions nt681–705 and nt718–736 of porB1b. DNA sequences of pilA (GenBank #AF520319) and abcZ (GenBank #AF520352) were previously described by Viscidi and Demma.8

Discriminatory powers

Simpson’s index of diversity (ID) was used to determine the abilities of either single gene sequence analysis (porB, pilA or abcZ) or multilocus sequencing typing (MLST) methods to discriminate between isolates.38,39 For MLST, each unique sequence of porB, pilA and abcZ was assigned a distinct allele number. Each isolate was given a 3-integer allelic profile and each unique profile was given a number representing a sequence type.

Phylogenetic analysis

Edited DNA sequences were separated into two data sets (e.g. porB1a and porB1b). Phylogenetic analyses were conducted with PAUP version 4.0b10.44 Alignments were not stripped of gaps before phylogenic analysis.4,9 Maximum parsimony trees were found with 1000 heuristic search including parsimony-informative characters in stepwise (random) addition, and tree bisection and reconstruction as branch swapping algorithm. MAXTREES were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Branch support for all parsimony analyses was estimated by performing 1000 bootstrap replicates45 with a heuristic search consisting of 10 random-addition replicates for each bootstrap replicate. Trees were constructed using Treeview.46

To determine clusters of N. gonorrhoeae isolates based on porB sequences, an average distance tree (using BLOSUM62) for the porB1a or porB1b data set, respectively, was constructed using ClustalW.42 The program allows for selection of continuous cut-off values of alignment distances. Upon selection of a cut-off value, we defined two clusters for porB1a isolates and three clusters for porB1b isolates.

To determine the congruence of porB sequence typing and epidemiological linkages, a phylogram of porB sequences from 30 sexual relationships comprising 30 male index patients and 31 correspondent sexual partners were constructed using the ClustalX program.42

Analysis of porB types/mutations and antimicrobial susceptibility

In order to avoid analysis bias due to clonal effects, only gonococcal isolates from the male index patients (n = 143) were used in this analysis. They comprised the majority of the isolates (90%, 143/159) tested, as reported previously.35 To determine the co-relationship between amino acid mutations of PorB proteins and the antimicrobial susceptibilities of relevant isolates, each porB sequence from 143 male index patients was translated into an amino acid sequence using Proteomics and Sequence Tools of ExPasy Proteomics Server (http://ca.expasy.org, date last accessed 20 October 2006). Alignments of the deduced protein sequences were carried out using ClustalW. porB sequences from N. gonorrhoeae MS11 (porB1a, GenBank #J03029) or FA1090 (porB1b, GenBank #J03017) were used as prototypes. Chi-squared tests were performed on the tetracycline antimicrobial susceptibility profiles for porB1a and porB1b isolates to determine the significance of the percentage differences among data sets. Statistical tests for percentages of isolates resistant to penicillin, ciprofloxacin, spectinomycin or ceftriaxone are not indicated since most of the isolates were resistant to penicillin and ciprofloxacin, and were susceptible to spectinomycin and ceftriaxone.

Gonococcal porB sequence deposit

porB DNA sequences identified in this study were blasted against the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST, date last accessed 3 April 2007) and were deposited in GenBank with deposit numbers of EF540591 [GenBank] –EF540669 [GenBank] .


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 Transparency declarations
 Supplementary data
 References
 
Discrimination of N. gonorrhoeae isolates by porB, pilA and abcZ DNA sequence analysis

The ID values for porB, pilA and abcZ DNA sequence typing or MLST by combining these three genes were determined. In 33 isolates randomly selected from a clinical collection in Shanghai in 2004, the ID values were 95%, 74% and 68% for single locus typing of porB, pilA or abcZ, respectively, and 99% for the combination of the three loci (data not shown). Thus, we determined that porB DNA sequence typing alone would be sufficiently discriminatory to use in subsequent analyses.

Identification of porB clusters of N. gonorrhoeae

The 174 N. gonorrhoeae isolates tested included 47 isolates (27.0%) typed as porB1a and 127 isolates (73.0%) typed as porB1b [Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. The porB1a isolates comprised two distinct clusters, porB1a-1 and porB1a-2 (Figure 1), with a total of 15 different DNA sequence groups. Cluster porB1a-1 included 37 isolates displaying nine DNA sequence groups. The majority (29/37) of porB1a-1 isolates comprised two different sequence groups with either 14 isolates (all having sequence #166) or 15 isolates (all having sequence #26), respectively; the former contained two epidemiologically linked pairs and an epidemiologically linked triplet, and the latter had two epidemiologically linked pairs. One porB1a-1 sequence (#63F) comprised two isolates from a sexual pair. Each of the other six sequences comprised single isolates (Table S1). Cluster porB1a-2 contained 10 isolates having a total of six unique DNA sequences; one porB1a-2 sequence group contained five isolates (representative sequence #115), which included one sexual pair. Each of the other five sequences comprised a single isolate (Table S1).


Figure 1
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Phylogenetic tree of N. gonorrhoeae isolates with porB1a DNA sequences. Phylogenetic analysis was conducted with the PAUP program. The tree was constructed using Treeview.46 The porB1a isolates are grouped into clusters porB1a-1 and porB1a-2. Branch lengths are shown proportional to the amount of change along the branches. The scale distance bar indicates the number of nucleotide substitutions per site. Bootstrap values >50% are shown in circles over the branches. A representative isolate number for porB sequence is shown at the tips of branches; and the number of isolates with identical sequences is shown adjacent to the representative isolate (in parentheses). The black stars indicate that the sequences are identical to previously reported porB1a sequences in GenBank.

 
The porB1b isolates were divided into three clusters porB1b-1, porB1b-2 and porB1b-3 comprising 63 individual DNA sequences (Figure 2). Cluster porB1b-1 contained 72 isolates with 27 distinct porB1b sequence groups: one sequence contained a large group of 33 isolates (representative sequence #104, Figure 2, black arrow) comprising 12 isolates from six sexual pairs and 21 individual isolates. Seven sequences each comprised two isolates from epidemiologically linked pairs; six sequences each contained two isolates with no confirmed sexual linkages (Table S1). Each of the other 13 DNA sequence groups of the porB1b-1 cluster comprised an individual isolate (Table S1). Cluster porB1b-2 had 15 unique DNA sequence groups comprising a total of 23 isolates; one group (representative sequence #29) comprised five isolates including one pair; another sequence group (representative sequence #118) had four isolates including one sexual pair; and another sequence group was from a single sexual pair. Each of the other 12 sequence groups contained a single isolate (Table S1). Cluster porB1b-3 had 21 sequence groups comprising 32 isolates; one sequence group (representative sequence #16) comprised five isolates with no sexual linkages; each of the other seven sequence groups contained two isolates and five of them were sexual pairs. Each of the other 13 sequence groups in the porB1b-3 cluster contained a single isolate (Table S1).


Figure 2
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Phylogenetic tree of N. gonorrhoeae isolates with porB1b DNA sequences. Tree construction and annotation are described in Figure 1. porB1b isolates are grouped into clusters porB1b-1, porB1b-2 and porB1b-3. The black arrow indicates a group of 33 isolates having an identical porB1b sequence, and this sequence is identical to a previously reported porB1b sequence in GenBank.

 
Congruence of porB sequence typing and epidemiological linkages

The porB sequences of N. gonorrhoeae isolates (n = 61) from 29 epidemiologically identified pairs as well as an additional male patient having two female partners were analysed for congruence (Figure 3a). Fifteen of these isolates exhibited porB1a genotypes which were distributed into four sequences: one sequence comprised seven isolates including two pairs (56/56F and 61/61F) and the epidemiologically linked triplet (75/75F1/75F2); a second sequence comprised two pairs (26/26F and 162/162F); each of the other porB1a sequences comprised one pair (63/63F and 164/164F, respectively). Forty-six isolates comprising 23 pairs were typed as porB1b representing 20 DNA sequences (Figure 3a): one sequence comprised 12 isolates from six pairs (34/34F, 64/64F, 89/89F, 101/101F, 134/134F and 135/135F); each of other 15 sequences comprised one pair (Figure 3a). Two epidemiologically linked pairs (41/41F and 141/141F) exhibited different porB1b sequences (Figure 3a, asterisks).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Congruence of porB types and sexual contacts. (a) Average distance tree of 61 porB sequences from 30 sexual partnerships. Branch lengths shown are proportional to the amount of genetic change. The identity of each sequence is shown at the tips of the branches; for example, #63 represents sequence number 63 from a male patient and 63F for the sequence from the female partner of #63. Stars indicate that sequences of the two epidemiologically linked pairs were different. Vertical lines indicate isolates that carry an identical sequence. (b and c) Congruence of porB sequence analysis and epidemiological data. The diagrams are drawn based on the phylogenetic tree in (a). Open rectangles, isolates from male index patients; filled circles, isolates from females; solid line, isolates with an identical porB sequence. Epidemiological linkages depicted by open circles. Open dashed rectangles represent isolates from several pairs sharing an identical porB sequence. Dashed circles are isolates from sexual pairs having different porB sequences (c). The black arrow represents the trios.

 
In order to better illustrate the correlation between porB sequence and epidemiologically linked isolates, DNA sequence and epidemiological data were plotted (Figure 3b and c). All linked porB1a isolates exhibited identical porB sequences (Figure 3b); seven isolates comprising two sexual pairs and the linked trio had an identical sequence, and two pairs shared a porB1a sequence (Figure 3b, open dashed rectangles). Of 23 sexually linked porB1b pairs, 21 pairs had identical sequences (Figure 3c), while 2 pairs showed different porB sequences between isolates (Figure 3c, dashed circle); 12 isolates from six sexual pairs shared an identical porB1b sequence (Figure 3c, open dashed rectangles).

Antimicrobial susceptibility and porB types

The antimicrobial susceptibilities of 143 clinical gonococcal isolates (porB1a, n = 39; porB1b, n = 104) from male patients have been reported previously.35 Most isolates were resistant to penicillin (porB1a 89.8% and porB1b 95.3%), ciprofloxacin (porB1a 91.5% and porB1b 99.2%) and tetracycline (porB1a 38.4% and porB1b 62.5%). More than 95% of the isolates were susceptible to spectinomycin, and all isolates were susceptible to ceftriaxone, although decreasing levels of susceptibility were noted (Figure 4). The percentage of tetracycline-resistant isolates was significantly higher in porB1b isolates than in porB1a isolates (P < 0.025). Nevertheless, porB1b isolates had higher MIC50s to penicillin (≥64.0 mg/L) or ciprofloxacin (8.0 mg/L) than porB1a isolates (8.0 mg/L for penicillin and 3.0 mg/L for ciprofloxacin). Plasmid-mediated resistance to penicillin and tetracycline accounted for 59.0% (porB1a) and 57.7% (porB1b) of the isolates, and there was no significant difference between the two genotypes. The percentage of porB1a or porB1b isolates with chromosomally mediated resistance to penicillin and tetracycline was 48.7% and 65.4%, respectively, and the latter (porB1b) was significantly higher (Table 2). The percentages of CMPR, CMTR or CMRNG for porB1b isolates were 21.2%, 24.0% and 20.2%, respectively, whereas those of porB1a isolates were 38.5%, 5.1% and 5.1%, respectively (data not shown). A few isolates had MICs to penicillin at intermediate levels, but there was no significant difference of percentages between porB1a (10.3%, n = 4) and porB1b (4.8%, n = 5) phenotypes. The percentages of tetracycline susceptible isolates (S) and isolates with intermediate levels of MICs (I) were significantly higher in porB1a (61.5%) than porB1b (37.5%) isolates (Table 2). There was no difference in the MIC50s to spectinomycin (16.0 mg/L) or ceftriaxone (0.03 mg/L) between porB1a and porB1b isolates (Figure 4). The MIC90 to ciprofloxacin for porB1b isolates (32.0 mg/L) was significantly higher than that for porB1a isolates (8.0 mg/L), while the MIC90s to penicillin, tetracycline, spectinomycin and ceftriaxone were not significantly different between porB1a and porB1b isolates (data not shown).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Antimicrobial susceptibility and porB types of 143 N. gonorrhoeae isolates from male patients. Bars indicate percentages of isolates that were classified as susceptible (white), intermediate (hatched) and resistant (black). PEN, penicillin; TET, tetracycline; CIP, ciprofloxacin; SPT, spectinomycin; CRO, ceftriaxone. Stars indicate statistically significant difference of TET resistance between the two porB types.

 


View this table:
[in this window]
[in a new window]

  		Table 2. Antimicrobial resistant profiles of porB1a and porB1b N. gonorrhoeae isolates

 
Mutations at residues G120 and A121 of gonococcal PorB proteins (i.e. PIB and PIA) are associated with decreased susceptibility to antibiotics.16,17 Of 104 PIB isolates in Shanghai (Table 3), 98.1% (102/104) carried mutations at residue G120: 84.6% (88/104) with a G120K mutation, 11.5% (12/104) with a G120D mutation, 1% (1/104) with a G120R and 1% (1/104) with a G120N mutation. The majority of the PIB isolates (87.5%, 91/104) had mutations at residue A121: A121D, A121G, A121N or A121H mutations accounted for 73.1% (76/104), 6.7% (7/104), 6.7% (7/104) or 1% (1/104) of the sequences, respectively. Double mutations of PIB G120/A121 were observed in 86.5% (90/104) of the isolates, including G120K/A121D (71.1%, 74/104), G120K/A121G (6.7%, 7/104), G120K/A121N (5.7%, 6/104), G120K/A121H (1%, 1/104), G120R/A121D (1%, 1/104) and G120N/A121N (1%, 1/104). A single G120 mutation (G120D) was noted in 11.5% (12/104) of the sequences, while a single A121 mutation (A121D) was observed in 1% (1/104). Two PIB isolates (2/104) did not carry any mutations at these two residues (Table 3). The mutations G120R or A121N have not been reported previously. Of 39 PIA isolates, the G120 mutation, G120D, was observed in 82.1% (32/39) of the sequences, while the rest (7/39) did not have a G120 mutation. All PIA isolates had a mutation at residue A121 (A121G). Double mutations of G120D/A121G were observed in 82.1% of PIA isolates, while 17.9% of PIA isolates had a single A121G mutation (Table 3).


View this table:
[in this window]
[in a new window]

  		Table 3. Mutations at the residues G120 and A121 of porB in N. gonorrhoeae isolates

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 Transparency declarations
 Supplementary data
 References
 
porB sequence analysis has been a useful method for studying the molecular epidemiology of N. gonorrhoeae isolates including identifying circulating clusters,4,10–13 tracking strain transmission networks8,9,11,14,15,47 and investigating mechanisms of antibiotic resistance.15,16 This method has a high discriminatory index similar to NG-MAST.13 Furthermore, sequence analysis of six variable regions (VRs) of porB has the same discriminatory power as sequencing the entire gene.6,22,48,49 This study aimed to distinguish circulating strain types of N. gonorrhoeae in Shanghai and to determine the discriminatory powers of various gene sequence analysis methods. We demonstrated that porB sequence typing alone has ID of 95%, very similar to multiple locus analysis of porB, pilA and abcZ (ID = 99%).

We identified 78 porB sequences (15 porB1a and 63 porB1b) from 174 clinical isolates in Shanghai. The majority of these sequences (75/78) had not been previously reported to the GenBank database except for two porB1a sequences and one porB1b sequence. The porB1a sequence (GenBank accession #540667) from our study was identical to a porB1a sequence (GenBank accession #AF09018)37 reported from Nairobi, Kenya. Another of our porB1a sequences (GenBank accession EF540668 [GenBank] ) was identical to a sequence previously reported (GenBank accession #AF304403)7 for an isolate from Baltimore, MD, USA. One of our porB1b sequences (GenBank accession #EF540666) was identical to a previously reported porB1b sequence (GenBank accession #AF304396)7 from Baltimore, MD, USA.

Our results demonstrate that the porB1b genotype of N. gonorrhoeae is predominant in Shanghai. The geographical distribution of N. gonorrhoeae isolates with porB1a and porB1b genotypes has been studied in several countries/regions. For example, the porB1b type is more prevalent than the porB1a type in N. gonorrhoeae isolates in Germany,29 Russia,50 Sweden51 and the USA.48 Prior to DNA sequence analysis to establish the prevalence of porB types, serological analysis was used to indicate temporal and geographic differences between PIA and PIB phenotypes. For example, the PIB type (serogroups WII/III) was predominant in non-PPNG strains in Jamaica25 and Greece,52 while PIA was prevalent in the Philippines and Singapore.29 However, these studies used highly selected isolates and may not be fully representative of the broader N. gonorrhoeae population. The reason for type differences in different regions is not well understood, although host physiological factors, behavioural characteristics and control programs such as antibiotic use may play roles in evolutionary selection.53,54

Our results confirm that porB sequence analysis was congruent with identified sexual contacts, confirming epidemiological information. However, a small number of patient self-identified sexual contacts exhibited different porB sequences, suggesting that the corresponding male and/or female patients may have had multiple sexual partners infected with different N. gonorrhoeae strains or that the patients may have had a mixed strain infection; mixed strain infections are common in sexually active groups.12,55 To detect multiple strains from cultures, porB-sequence typing of multiple individual colonies from the primary isolation plate is required. Alternatively, opa-typing of multiple individual colonies2,55 or porB VR typing from clinical non-cultured samples12,49 can be used; however, these methods were not included in the present study.

As reported in previous studies,4,7 several clusters with an identical porB sequence but not associated through epidemiological connections were present in Shanghai. For example, three groups in the porB1a cluster were identified comprising 14, 15 and 5 isolates, respectively. In porB1b isolates, there were four groups each comprising 33, 4, 5 and 4 isolates, respectively. These groups were not identified through epidemiological methods. Furthermore, seven groups with two isolates each were classified by porB sequence analysis while there were no identified epidemiological connections between the two isolates (Figure 2 and Table S1). Because discussions about sex are not well accepted in China, the patient self-reported sexual linkages are likely underestimated and thus sexual networks created from this information cannot be readily observed. This may result in discordance between porB typing and sexual linkage data. It should also be noted that isolates with identical porB sequences could possibly be further differentiated using different typing methods, such as opa-typing and MLST methods.2,7,8 Therefore, in-depth analysis of demographic, social and geographical data may reveal better concordance and, in combination with genotypic data, will better define specific transmission clusters.

China has experienced an increasing burden of antimicrobial resistance of N. gonorrhoeae isolates.56 This has provoked the ongoing review and modification of guidelines so that recommended antibiotics for the treatment of gonorrhoea are effective.35 The current recommended treatment of choice for uncomplicated gonococcal infections in Shanghai, China, includes ceftriaxone or spectinomycin, whereas penicillin, tetracycline and quinolones are no longer recommended.57 Antimicrobial resistance in N. gonorrhoeae isolates is mediated through chromosomal mutation of various genes, plasmid-mediated mechanisms or both.58 Different levels of antimicrobial susceptibility in N. gonorrhoeae isolates have been associated with various mutations in porB.16,17,59,60 Our results demonstrate that porB1b isolates are less susceptible, supporting previous findings as determined by serotyping before DNA sequence typing became feasible.24–27,29 Gonococcal strains harbouring protein 1A (serogroup WI strains) were reported as more sensitive to antibiotics such as penicillin, tetracycline, erythromycin, cefoxitin, cefuroxime or cefotaxime.24,52 Furthermore, 97.3% of 115 CMRNG isolates were reported as PIB serotypes, while 88% of isolates with PIA serotypes were penicillin or tetracycline susceptible.61 It would be useful to compare porB gene sequences with previously reported serovars so that comparison can be made for temporal trends and continuity of typing data.

Specific mutations in porB1b (spontaneously or experimentally) have been associated with decreased susceptibility to hydrophilic antibiotics such as β-lactams and tetracyclines.16,59 Mutations causing this decreased susceptibility include amino acid replacements of the hydrophobic amino acids glycine G120 and alanine A121 by lysine (K) and aspartate (D), respectively, i.e. G120K or A121D.17,59 Between 40% and 47% of porB1b isolates have been reported to have these double mutations.16,59 In our study, the majority of porB1b isolates (71.1%) exhibited G120K/A121D double mutations, remarkably higher than previously reported,4,17,59 suggesting that these mutations may play a significant role in the high percentages of penicillin and tetracycline resistance in N. gonorrhoeae isolates from Shanghai. We discovered that porB1b G120 or A121 can be replaced by amino acids other than lysine and aspartate such as arginine (R), histidine (H) or asparagine (N). The novel replacements at these two residues include G120R, G120N, A121G, A121N or A121H, accounting for 14.5% of the porB1b isolates. It is presently unknown whether these mutations confer gonococcal antibiotic resistance as well and this hypothesis should be confirmed experimentally. The porB1a isolates exhibited different mutations at G120 and A121 residues than porB1b. The majority of our porB1a isolates (82.1%) had G120D/A121G double mutations, similar to porB1a isolates from Baltimore, MD, USA, which had G120D/A121D double mutations.60 However, A121 was replaced by a hydrophobic amino acid (G) in our isolates, while in Baltimore isolates it was replaced by a hydrophilic amino acid (D). The impact on antimicrobial susceptibility of these differences needs to be further investigated as there was no susceptibility data shown for the Baltimore strains.60

In summary, this study illustrates the porB-typing based molecular epidemiology of N. gonorrhoeae from Shanghai. porB sequence analysis is a useful tool to track strain transmission4,7,8,11,14 and to provide additional information on sexual networks to the client's self-reporting methods.48,62,63 porB sequence analysis can provide more information on sexual networks when compared with conventionally obtained epidemiological data, revealing hidden epidemiological linkages. The G120K/A121D mutations in porB1b were common in N. gonorrhoeae isolates from Shanghai and were associated with chromosomally mediated penicillin and tetracycline resistance. Novel replacements at the residues G120 and A121 were identified, and the association with antimicrobial resistance in N. gonorrhoeae warrants further investigation.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
Transparency declarations
 Supplementary data
 References
 
This work was supported by the Canadian Institutes of Health Research (CIHR, Grant #OPC-67017). N. F. E. was supported by a CIHR Doctoral Research Award.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
Supplementary data
 References
 
None to declare.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 Supplementary data
References
 
Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
This report was presented, in part, at the Fifteenth International Pathogenic Neisseria Conference, Cairns, Australia, 2006. We are grateful to Ms Yan Li (University of Saskatchewan) for assistance with phylogenetic tree construction using PAUP software.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 Supplementary data
 References
 
1 Fredlund H, Falks L, Jurstrand M, et al. Molecular genetic methods for diagnosis and characterization of Chlamydia trachomatis and Neisseria gonorrhoeae: impact on epidemiological surveillance and interventions. APMIS (2004) 112:771–84.[CrossRef][Web of Science][Medline]

2 O’Rourke M, Ison CA, Renton AM, et al. Opa-typing: a high-resolution tool for studying the epidemiology of gonorrhoea. Mol Microbiol (1995) 17:865–75.[CrossRef][Web of Science][Medline]

3 Spratt BG, Bowler LD, Zhang QY, et al. Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species. J Mol Evol (1992) 34:115–25.[Web of Science][Medline]

4 Unemo M, Olcen P, Berglund T, et al. Molecular epidemiology of Neisseria gonorrhoeae: sequence analysis of the porB gene confirms presence of two circulating strains. J Clin Microbiol (2002) 40:3741–9.[Abstract/Free Full Text]

5 Unemo M, Olcen P, Jonasson J, et al. Molecular typing of Neisseria gonorrhoeae isolates by pyrosequencing of highly polymorphic segments of the porB gene. J Clin Microbiol (2004) 42:2926–34.[Abstract/Free Full Text]

6 van der Ley P, Heckels JE, Virji M, et al. Topology of outer membrane porins in pathogenic Neisseria spp. Infect Immun (1991) 59:2963–71.[Abstract/Free Full Text]

7 Viscidi RP, Demma JC, Gu J, et al. Comparison of sequencing of the por gene and typing of the opa gene for discrimination of Neisseria gonorrhoeae strains from sexual contacts. J Clin Microbiol (2000) 38:4430–8.[Abstract/Free Full Text]

8 Viscidi RP, Demma JC. Genetic diversity of Neisseria gonorrhoeae housekeeping genes. J Clin Microbiol (2003) 41:197–204.[Abstract/Free Full Text]

9 Unemo M, Olcen P, Albert J, et al. Comparison of serologic and genetic porB-based typing of Neisseria gonorrhoeae: consequences for future characterization. J Clin Microbiol (2003) 41:4141–7.[Abstract/Free Full Text]

10 Bash MC, Zhu P, Gulati S, et al. por variable-region typing by DNA probe hybridization is broadly applicable to epidemiologic studies of Neisseria gonorrhoeae. J Clin Microbiol (2005) 43:1522–30.[Abstract/Free Full Text]

11 Hobbs MM, Alcorn TM, Davis RH, et al. Molecular typing of Neisseria gonorrhoeae causing repeated infections: evolution of porin during passage within a community. J Infect Dis (1999) 179:371–81.[CrossRef][Web of Science][Medline]

12 Lynn F, Hobbs MM, Zenilman JM, et al. Genetic typing of the porin protein of Neisseria gonorrhoeae from clinical noncultured samples for strain characterization and identification of mixed gonococcal infections. J Clin Microbiol (2005) 43:368–75.[Abstract/Free Full Text]

13 Martin IM, Ison CA, Aanensen DM, et al. Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area. J Infect Dis (2004) 189:1497–505.[CrossRef][Web of Science][Medline]

14 Cooke SJ, de la Paz H, Poh CL, et al. Variation within serovars of Neisseria gonorrhoeae detected by structural analysis of outer-membrane protein PIB and by pulse-field gel electrophoresis. Microbiology (1997) 143:1415–22.[Abstract/Free Full Text]

15 Lundback D, Fredlund H, Berglund T, et al. Molecular epidemiology of Neisseria gonorrhoeae-identification of the first presumed Swedish transmission chain of an azithromycin-resistant strain. APMIS (2006) 114:67–71.[CrossRef][Web of Science][Medline]

16 Olesky M, Hobbs M, Nicholas RA. Identification and analysis of amino acid mutations in porin IB that mediate intermediate-level resistance to penicillin and tetracycline in Neisseria gonorrhoeae. Antimicrob Agents Chemother (2002) 46:2811–20.[Abstract/Free Full Text]

17 Olesky M, Zhao S, Rosenberg RL, et al. Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations. J Bacteriol (2006) 188:2300–8.[Abstract/Free Full Text]

18 Hook EW, Handsfield HH. Sexually Transmitted Diseases—Holmes KK, Sparling PF, Mardh PA, et al, eds. (1999) New York: McGraw-Hill. 451–66.

19 Cooke SJ, Jolley K, Ison CA, et al. Naturally occurring isolates of Neisseria gonorrhoeae, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules. FEMS Microbiol Lett (1998) 162:75–82.[CrossRef][Web of Science][Medline]

20 Gill M, Jayamohan J, Lessing MPA, et al. Naturally occurring PIA/PIB hybrids of Neisseria gonorrhoeae. FEMS Microbiol Lett (1994) 119:161–6.[CrossRef][Web of Science][Medline]

21 Gotschlich EC, Seiff ME, Blake MS, et al. Porin protein of Neisseria gonorrhoeae: cloning and gene structure. Proc Natl Acad Sci USA (1987) 84:8135–9.[Abstract/Free Full Text]

22 Carbonetti NH, Sparling PF. Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae. Gene (1987) 84:9084–8.

23 Ison CA, Tanna A, Easmo CS. Evaluation of a fluorescent monoclonal antibody reagent for identification of cultured Neisseria gonorrhoeae. J Med Microbiol (1988) 26:121–3.[Abstract/Free Full Text]

24 Dillon JR, Bygdeman SM, Sandström EG. Serological ecology of Neisseria gonorrhoeae (PPNG and non-PPNG) strains: Canadian perspective. Genitourin Med (1987) 63:160–8.[Web of Science][Medline]

25 Dillon JR, Carballo M, King SD, et al. Auxotypes, plasmid contents, and serovars of gonococcal strains (PPNG and non-PPNG) from Jamaica. Genitourin Med (1987) 63:233–8.[Web of Science][Medline]

26 Dillon JA, Carballo M. Molecular epidemiology and novel combinations of auxotype, serovar, and plasmid content in tetracycline-resistant Neisseria gonorrhoeae isolated in Canada. Can J Microbiol (1990) 36:64–7.[Web of Science][Medline]

27 Knapp JS, Tam MR, Nowinski RC, et al. Serological classification of Neisseria gonorrhoeae with use of monoclonal antibodies to gonococcal outer membrane protein I. J Infect Dis (1984) 150:44–8.[Web of Science][Medline]

28 Ng LK, Dillon JR. Typing by serovar, antibiogram, plasmid content, riboprobing, and isoenzyme typing to determine whether Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil for growth are clonal. J Clin Microbiol (1993) 31:1555–61.[Abstract/Free Full Text]

29 Sandström EG, Chen KCS, Buchanan TM. Serology of Neisseria gonorrhoeae: coagglutination serogroups WI and WII/WIII correspond to different outer membrane protein I molecules. Infect Immun (1982) 38:462–70.[Abstract/Free Full Text]

30 Tam MR, Buchanan TM, Sandström EG, et al. Serological classification of Neisseria gonorrhoeae with monoclonal antibodies. Infect Immun (1982) 36:1042–53.[Abstract/Free Full Text]

31 Tapsall JW. Current concepts in the management of gonorrhoea. Expert Opin Pharmacother (2002) 3:147–57.[CrossRef][Medline]

32 Chen XS, Gong XD, Liang GJ, et al. Epidemiologic trends of sexually transmitted diseases in China. Sex Transm Dis (2000) 27:138–42.[Web of Science][Medline]

33 Cohen MS, Ping G, Fox K, et al. Sexually transmitted diseases in the People’s Republic of China in Y2K: back to the future. Sex Transm Dis (2000) 27:143–5.[Web of Science][Medline]

34 Parish WL, Laumann EQ, Cohen MS, et al. Population-based study of chlamydial infection in China: a hidden epidemic. JAMA (2003) 289:1265–73.[Abstract/Free Full Text]

35 Yang Y, Liao M, Gu W, et al. Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai. J Antimicrob Chemother (2006) 58:868–72.[Abstract/Free Full Text]

36 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Seventh Edition: Approved Standard M7-A7 (2006) Wayne, PA, USA: CLSI.

37 Fudyk TC, Maclean IW, Simonsen JN, et al. Genetic diversity and mosaicism at the por locus of Neisseria gonorrhoeae. J Bacteriol (1999) 181:5591–9.[Abstract/Free Full Text]

38 Dillon JA, Rahman M, Yeung KH. Discriminatory power of typing schemes based on Simpson’s index of diversity for Neisseria gonorrhoeae. J Clin Microbiol (1993) 31:2831–3.[Abstract/Free Full Text]

39 Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol (1988) 26:2465–6.[Abstract/Free Full Text]

40 van Looveren M, Ison CA, Vandamme MIP, et al. Evaluation of the discriminatory power of typing methods for Neisseria gonorrhoeae. J Clin Microbiol (1999) 37:2183–8.[Abstract/Free Full Text]

41 Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res (1988) 16:10881–90.[Abstract/Free Full Text]

42 Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res (1997) 25:4876–82.[Abstract/Free Full Text]

43 Clamp M, Cuff J, Searle SM, et al. The Jalview Java alignment editor. Bioinformatics (2004) 20:426–7.[Abstract/Free Full Text]

44 Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods) (2002) Sunderland, Massachusetts: Sinauer Associates. Version 4.0b10.

45 Felsenstein J. Confidence intervals on phylogenetics: an approach using bootstrap. Evolution (1985) 39:783–91.[CrossRef][Web of Science]

46 Page RDM. Treeview: an application to display phylogenetic trees on personal computers. Comput Appl Biosci (1996) 12:357–8.[Free Full Text]

47 Ward H, Ison CA, Day SE, et al. A prospective social and molecular investigation of gonococcal transmission. Lancet (2000) 356:1812–7.[CrossRef][Web of Science][Medline]

48 McKnew DL, Lynn F, Zenilman JM, et al. Porin variation among clinical isolates of Neisseria gonorrhoeae over a 10-year period, as determined by por variable region typing. J Infect Dis (2003) 187:1213–22.[CrossRef][Web of Science][Medline]

49 Thompson DK, Deal CD, Ison CA, et al. A typing system for Neisseria gonorrhoeae based on biotinylated oligonucleotide probes to PIB gene variable regions. J Infect Dis (2000) 181:1652–60.[CrossRef][Web of Science][Medline]

50 Il’ina EN, Malakhova MV, Vereshchagin VA, et al. Molecular typing of N. gonorrhoeae strains prevalent in the Russian Federation. Bull Exp Biol Med (2003) 136:179–82.[CrossRef][Web of Science][Medline]

51 Berglund T, Fredlund H, Giesecke J. Epidemiology of the reemergence of gonorrhoea in Sweden. Sex Transm Dis (2001) 28:111–4.[CrossRef][Web of Science][Medline]

52 Tzanakaki G, Mavrommati L, Tzelepi E, et al. Serological classification in relation to auxotypes, plasmid contents, and susceptibilities to antimicrobials of PPNG and non-PPNG strains isolated in Greece. Genitourin Med (1989) 65:171–6.[Web of Science][Medline]

53 Derrick JP, Urwin R, Suker J, et al. Structural and evolutionary inference from molecular variation in Neisseria porins. Infect Immun (1999) 67:2406–13.[Abstract/Free Full Text]

54 Posada D, Crandall KA, Holmes EC. Recombination in evolutionary genomics. Annu Rev Genet (2002) 36:75–97.[CrossRef][Web of Science][Medline]

55 Martin IM, Ison CA. Detection of mixed infection of Neisseria gonorrhoeae. Sex Transm Dis (2003) 79:56–8.

56 Ye S, Su X, Wang Q, et al. Fifteen year monitoring of drug resistance of gonococci. Chin J Hum Sex (2004) 13:1–3.

57 Wang Q, Zhang G. Guidelines for Diagnosis and Treatment of Sexually Transmitted Diseases 2007. Shanghai, china: Shanghai Sciences and Technology Publishing. Center for Control of Sexually Transmitted Diseases, Center for Disease Control, China (China CDC).

58 Dillon JR, Pagotto F. Importance of drug resistance in gonococci: from mechanisms to monitoring. Curr Opin Infect Dis (1999) 12:35–40.[Web of Science][Medline]

59 Morse SA, Lysko PG, McFarland L, et al. Gonococcal strains from homosexual men have outer membranes with reduced permeability to hydrophobic molecules. Infect Immun (1982) 37:432–8.[Abstract/Free Full Text]

60 Pérez-Losada M, Viscidi RP, Demma JC, et al. Population genetics of Neisseria gonorrhoeae in a high-prevalence community using a hepervariable outer membrane porB and 13 slowly evolving housekeeping genes. Mol Biol Evol (2005) 22:1887–902.[Abstract/Free Full Text]

61 Dillon JR. Epidemiologie moleculaire de la resistance de Neisseria gonorrhoeae aux antibiotiques. Nales De L’Institut Pasteur (1994) 5:148–56.

62 Choudhury B, Risley CL, Ghani AC, et al. Identification of individuals with gonorrhoea within sexual networks: a population-based study. Lancet (2006) 368:139–46.[CrossRef][Web of Science][Medline]

63 Day S, Ward H, Ison C, et al. Sexual networks: the integration of social and genetic data. Soc Sci Med (1998) 47:1981–92.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Clin. Microbiol.Home page
M. Liao, S. Helgeson, W.-M. Gu, Y. Yang, A. M. Jolly, and J.-A. R. Dillon
Comparison of Neisseria gonorrhoeae Multiantigen Sequence Typing and porB Sequence Analysis for Identification of Clusters of N. gonorrhoeae Isolates
J. Clin. Microbiol., February 1, 2009; 47(2): 489 - 491.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
61/3/478    most recent
dkm544v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Liao, M.
Right arrow Articles by Dillon, J.-A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liao, M.
Right arrow Articles by Dillon, J.-A. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?