Neisseria gonorrhoeae

Ngo-penA dendogram fragment
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Ng-porB
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Gonorrhea is the second most common sexually transmitted disease in the United States. Most infections are asymptomatic and hence go undetected. However, depending on muliple factors including the N. gonorrhoeae strain, infection may lead to dysuria and urogenital discharge, most commonly in males. In females, infection may progress to pelvic inflammatory disease associated with chronic pelvic pain and increased risk of ectopic pregancy and infertility. Furthermore, N. gonorrhoeae infection increases the risk of acquiring or transmitting HIV.

Although N. gonorrhoeae infection is increasingly diagnosed by nucleic-acid amplification tests, culture remains important since it is required for antimicrobial susceptibility testing. This pathogen can develop resistance to any antibiotic, facilitated by its natural competence – the ability to incorporate chromosomal or plasmid DNA from other bacteria.

For many years gonorrhea was effectively treated with penicillin, albeit increasingly high doses due to the accumulation of mutations, particularly in the penA gene encoding PBP2, a key enzyme in cell wall synthesis. Penicillin was largely abandoned, however, following the emergence in the 1970’s of strains carrying plasmid-encoded beta-lactamase. These strains remained susceptible to cefixime (oral third generation cephalosporin), which became the treatment of choice (after the rise and fall of fluoroquinolones, that is). Needless to say, cefixime resistance eventually emerged, again primarily due to penA mutations. This led to the current (and in the absence of new antibiotics, final?) recommendation from the CDC for a combination of ceftriaxone (injectable third generation cephalosporin) with either oral azithromycin or doxycycline (the latter two also active versus chlamydia). Treatment of sex partners, symptomatic or asymptomatic, is critical to controlling this disease.

Ceftriaxone-resistant N. gonorrhoeae was first detected in Japan in 2009 (strain H041), followed by France in 2010 and Spain in 2011 (strain F89). Whether or not these strains truly represent the “superbugs” they have been labelled (e.g., www.cnbc.com/id/101041141), leading to untreatable infections and a public health crisis, remains to be seen. Regardless, surveillance by susceptibility testing and strain typing are more important than ever.

As reviewed by Unemo & Dillon (1), a wide variety of methods have been developed for N. gonorrhoeae strain typing, but DNA sequence-based methods are now the preferred approach. Chief among these is NG-MAST (N. gonorrhoeae multiantigen sequence typing), a well validated system targeting variable regions within two genes, porB and tbpB, encoding highly polymorphic surface proteins. Inputting the sequences into the NG-MAST database yields porB and tbpB alleles and NG-MAST types (e.g., porB-908 and tbpB-110 = ST1407, a prevalent European clone from which ceftriaxone-resistant strain F89 arose). As emphasized by Unemo & Dillon, the sequences should also be phylogenetically analyzed (see Ng-tbpB dendrogram and Ng-porB dendrogram for examples), since it is difficult to discern relationships among strains based on allele/ST numbers alone (and, furthermore, novel alleles/STs absent in the current NG-MAST database are not uncommon). While NG-MAST is more affordable than MLST (2 genes sequenced versus 7), further cost savings and adequate strain resolution may be obtained by sequencing only porB, for which there is a database from >1000 strains facilitating phylogenetic analysis. Subsequently, tbpB can be sequenced using the same DNA sample to provide increased strain resolution if needed.

With respect to beta-lactam resistance, penA sequence typing (pubmlst.org) and phylogenetic analysis has emerged as an important adjunct to susceptibility testing (e.g., ref. 2). Importantly, penA sequence analysis can identify strains most likely to develop cefixime/ceftriaxone resistance through the acquisition of specific mutations, since these strains share a similar mosaic penA (see Ng-penA dendrogram).

In response to these needs, MicrobiType offers multiple typing services. Ng-porB and Ng-tbpB: reliable, cost-effective sequencing results are obtained using genomics-optimized primers. In addition to allele numbers, these services deliver a dendrogram (see examples above) and sequence alignment showing the relationship between your current strain, other strains submitted from your lab, and additional strains that have been extensively characterized at the genome level. Ng-MAST: combines the two services above (at discounted price), and delivers NG-MAST sequence type along with porB and tbpB allele numbers, dendrograms, and alignments. Finally, Ng-penA: delivers penA type, and dendrogram/alignments comparing your strain’s penA to >30 previously characterized penA genes from beta-lactam susceptible, penicillin-resistant, and cephalosporin resistant or reduced susceptibility strains.

(1) Unemo & Dillon, 2011, Clin. Microbiol. Rev. 24:447
(2) Unemo et al., 2012, 
Antimicrob. Agents Chemother. 56:1273

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