Genotyping of the Resistance Determinant of Neisseria Gonorrhoeae with Reduced Susceptibility to Ceftriaxone in Manaus-AM-Brazil

Gonorrhea is the second most prevalent sexually transmitted infection worldwide, with an estimated 78.3 million new cases. At the Alfredo da Matta foundation, gonorrhea as the main cause of urethral discharge with prevalence of 16.8%. Gonococci have developed resistance to all the antibiotics leaving cephalosporins as the last option for treatment. In this report, we genotype the determinants of resistance to Extended Spectrum Cephalosporins, such as penA, ponA, porB, mtrR, pilQ, of a Neisseria gonorrhoeae strain, isolated from a male patient with urethral discharge. The ST1901was identified by MLST protocol and genotyping of the penA, showed mutations on regions F505L, A511V, A517G, N542H e P522S which confirmed the presence of gonococcus with reduced susceptibility to ceftriaxone in the region.


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In last few decades, gonococci have developed resistance to all the antibiotics used as first line of treatment for gonococcal infections, leaving Extended-Spectrum Cephalosporins (ESCs) as the last remaining option for gonorrhea [1,2].With reports of reduced susceptibility or resistance to ESCs from different regions, and due to therapeutic limitations, the infection has become a serious health problem to the point that disease complications can no longer be treated in the near future, besides the possibility of the gonococcus to evolve into "superbug" [3][4][5][6][7].
Gonorrhea is the second most prevalent sexually transmitted infection worldwide, with an estimated 78.3 million new cases in 2012 [8].At the Alfredo da Matta foundation (Manaus-Brazil), gonorrhea appears as the main cause of urethral discharge, with an average of 513 cases in 20 years and prevalence of 16.8%.In this report, we describe the molecular characteristics of the N. gonorrhoeae strain NgFUAM84, isolated from the wianfe@yahoo.com.brurethral discharge of a male patient, with MIC of 0.064μg/mL for ceftriaxone in E-test (AB Biodisk, Solna, Sweden) [9].The determinants of resistance to ESCs: penA, ponA, porB, mtrR and pilQ were amplified by PCR (Proflex PCR System-Applied Biosystems) using primers previously described [10,11].The sequencing of amplicons was performed on the ABI 3130 Genetic Analyzer (Applied Biosystems).The substitutions in the residues were analyzed using the software Geneious v.10.0.10 and identified by comparison with the sequences deposited in GenBank (Figure 1).
Analysis of the gene ponA showed a single mutation at the L421P position, whereas in the gene mtrR, a single deletion of adenine (A) in the inverted position of the promoter region was identified.The NgFUAM84 also presented resistance determinant penB with substitutions at position G120K, A121D of loop 3 of PorB1b.These mutations have been associated with reduced sensitivity and resistance of gonococci to ECSs [6,14].A single substitution on position G554D was identified in PilQ (Table 1).Analysis of the penA made possible identification of substitutions at positions F505L, A511V, A517G and P552S.The PBP2 protein showed similarity of 99.3% with the XXIV allele (GenBank the MIC of 0.064μg/mL reinforces the possibility that non-mosaic penA mutations can increase the MICs of ESCs similar to those mediated by mosaic allele [3].
The mutations observed in the resistance determinants of ESCs in NgFUAM84 strain as well the identification of STs 225 and 1901, confirm the presence of gonococci with reduced susceptibility to ceftriaxone in the region and reinforces the need for monitoring of the susceptibility of gonococcus to these antibiotics and extensive research for better understanding of the resistance's mechanisms in order to maximize the effectiveness of ESCs in the treatment of gonorrhea.[4].

Table 1 :
Genotype characteristics -NgFUAM84 •Presence of the extra codon on positions 346 of NgFUAM84 PBP2