The increase in reports of Neisseria gonorrhoeae resistance to antibiotics has been a factor of concern to the public health system in many countries.1 The “Fundação de Dermatologia Tropical e Venereologia Alfredo da Matta” in Manaus, Brazil, registered 3167 sexually transmitted disease cases from which 288 were gonococcal infections showing a presence of β-lactamase–producing strains. These findings stimulated us to study the genotypes of these gonococci.2 Currently, nucleotides methods such as Multi-Antigen Sequencing Typing (NG-MAST) and Mutilocus Sequence Typing (MLST) are widely used in various studies that seek a better understanding of the molecular epidemiology of gonococci.3 The control of this pathogen relies on important factors such as prevention, early diagnosis, rapid therapeutic intervention, monitoring of resistance, and the knowledge of genotypic characteristics of circulating strains and their possible temporal changes that may hinder efforts of prevention and control.4
One hundred twenty gonococci were isolated from sexually transmitted disease clinic attendees between November 2008 and June 2009. From this total, 14 β-lactamase–producing isolates were identified by cefinase (BD BBLTM92) and were subjected to resistance testing using E-test (AB Biodisk) to the antibiotics—azithromycin, ceftriaxone, ciprofloxacin, chloramphenicol, penicillin, and tetracycline—and also classified for the serogroup A (WI) or B (WII/WIII) by manufacturer’s recommendations (Bactus AB). Reference values and all procedures including phenotypic characterization such as tetracycline-resistant N. gonorrhoeae were performed on minimal inhibitory concentration (MIC) values of 16 μg/mL or greater, as described previously.5–7 For genotyping, the DNA were purified with PureLink Genomic DNA Mini Kit (Invitrogen), amplified by polymerase chain reaction, and sequenced with BigDye Terminator v3.1 in a ABI 3130 (Applied Biosystems), using primers to por and tbpB genes (http://www.ng-mast.net/).8 Another technique used to characterize the gonococci was MLST (http://pubmlst.org/neisseria/), comprising the following genes: abcZ, adK, aroE, fumC, gdH, pdhC, and pgM.9 Subsequently, the sequences were compared with respective database sites to identify alleles or sequence types (STs). The ATCC 19424, 9826, and 49226 strains were used in all quality control steps. This study is part of a doctoral thesis research project approved by the Ethical Committee of the Fundação Alfredo da Matta.
Among the clinic attendees, 10 were male and 4 were female aged between 21 and 48 years (mean, 28.3 years). Review of medical records revealed that each of the male attendees complained of urethral discharge and dysuria. One female patient was asymptomatic, whereas the others had symptoms including yellowish vaginal discharge, foul-smelling discharge, dysuria, lower abdominal pain, and hypogastric pain. After the collection of biological samples, all patients were treated with 500 mg of ciprofloxacin orally and presented with clinical cure upon return to the clinic.
Laboratory tests confirmed the diagnosis of N. gonorrhoeae infection, and all isolates presented positivity in the β-lactamase assay and therefore were penicillinase-producing N. gonorrhoeae, with MICs between 12 and 32 μg/mL. Four of these isolates presented MICs between 24 and 32 μg/mL to tetracycline, being classified phenotypically as penicillinase-producing N. gonorrhoeae/ tetracycline-resistant N. gonorrhoeae. The other antimicrobial tests showed that the strains were sensitive to azithromycin (0.047–0.380 μg/mL), ceftriaxone (0.002–0.006), ciprofloxacin (0.002–0.125), and chloramphenicol (0.125–0.380). The serotyping showed that 12 isolates belonged to serogroup B (WII/III), whereas 2 were A (WI) (Table 1). Genotyping by NG-MAST allowed the identification of 6 new alleles for the por and 1 unknown combination of por and tbpB alleles that resulted in 7 new STs. All sequences were signed with NG-MAST numbers, and the respective nucleotide position mutations are described in Table 1. Four strains were identified as ST6218; 2 as ST6219, ST6220, ST6221, ST6222; and 1 as ST6223 and ST6841. No new alleles were observed in the genotyping of tbpB. For this gene, the most frequently identified allele was 32, present in 10 of the isolates. Two other strains presented allele 1149 allele, and 2 presented allele 22 for the tbpB gene (Table 1).
The MLST genotyping allowed the identification of a new allele for the gene aroE, named as 653. This new allele was observed in 2 isolates resulting in new STs identified: ST9322 and ST9324. The sequence analysis of the other strains also enabled the identification of 7 new STs, which were registered in the MLST database (Table 1). Two other strains showed sequences compatible with ST1902 and ST1599.
The 14 strains described in this study were also submitted to a comparative analysis against a data set containing all the known MLST sequences deposited in the Neisseria spp. database with the eBURSTv3 tool. The set analyzed contained 6717 isolates, from which 5265 were divided into 310 groups, whereas the other 1452 strains, including 4 samples identified in this study (STs 9322, 9324, 9329, 9349), were not grouped, being considered singletons. All of the other 10 strains in our study were placed in a single group. The eBURST analysis also showed that the most probable ancestral of the group in which the samples were classified is the ST1590. Two other genotypes found in this study, ST1599 and ST1902, were already part of the data set. The new ST9304 has the highest probability of being the founder of a subgroup formed by 2 STs previously described (ST1898 and ST1902) and 2 others identified in this study, ST9347 and ST9360. The new ST9325 is part of a subgroup formed by STs: 1579 (the most probable founder), 1896, and 1901 (Fig. 1). The sequences of new alleles, STs, and the epidemiological information have now been registered in the respective databases of NG-MAST and MLST.
The typing of isolates of N. gonorrhoeae by coagglutination or by molecular techniques has been widely used in epidemiological studies to study the development of gonococcal infection, antibiotics resistance, and genotypic identification.10–12 The knowledge of the molecular structure of these isolates circulating in the community may contribute significantly to initiatives focused on the control and prevention of gonorrhea by tracking disease transmission.4
The results of this study allowed the identification of the serogroups and serotypes usually observed in other regions outside of Amazonas, where the frequency of serotype B (WII/III) group is higher than serotype A (WI).13–16 The resistance to penicillin and tetracycline suggests that the high rates observed in previous studies are trends that remain.17
Regarding genotyping, the identification of new STs reflects an important contribution of studying gonococci with a molecular epidemiology approaches. The new STs described here place the Amazon region in a global epidemiological context, as the findings contribute significant information on the genetic characteristics of gonococci in this region.
Another important contribution was demonstrated by the eBurst analysis, which showed that ST1590 is the common ancestor for most strains in this study. This ST has already been described as sensitive to quinolones in Greece. However, it is remarkable that it has also been identified in Japan as a strain with reduced sensitivity to cefixime.18,19 For the new ST9304, the eBURST analysis demonstrated that it forms a new subgroup (ST1898, ST9360, and ST9347). We also note that both ST1599 and ST1902 have been previously detected in the United Kingdom and South Africa.9 In contrast, it was not possible to associate the new STs identified in our study with the ones from other regions because of the lack of information related to the antimicrobial resistance, serotyping, or demographic data from other settings. Regarding the STs identified by the NG-MAST, we observed that allele 1149 of the tbpB present in the new ST6222 has also been described in the ST5521 (Nerteley Quaye, personal communication). However, this gonococcus presented some phenotypic similarity to 2 other isolates having the same tbpB allele, sensitivity to erythromycin, ceftriaxone, and ciprofloxacin, but being different by not producing β-lactamase.
The association between these STs demonstrates that most of the gonococci present a particular genetic profile, distinctive when compared with strains from other regions. The mutation in the allele aroE reinforces the aspect of these genes, which is their low mutation capacity, but when all of our data were combined, 8 new STs not previously described were identified.20 Regarding the alleles of the NG-MAST, several mutations were observed, which is in agreement with the high substitution rate of this gene.10 Both phenotype and genotype characteristics of the gonococci have been widely used in studies with epidemiological objectives in regions where there are already reports of emerging gonococci resistance to ciprofloxacin and cephalosporin, highlighting the continued need for research on different therapeutic options.12,15,21
In conclusion, the frequencies of the serogroups identified in our study are similar to those observed in other countries, and all strains are susceptible to some antibiotics. The results also focus attention on the need for more research on gonococci in Brazil because studies covering the molecular epidemiology of these pathogens are scarce.11,22 The new STs indicate that most of the isolates have their own molecular characteristics not previously described and that it is important to continue this type of research to better understand the relationships between this pathogen, patients, and infection transmission to other contacts.
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