Molecular analyses of TEM genes and their corresponding penicillinase-producing Neisseria gonorrhoeae isolates in Bangkok, Thailand

Abstract

Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant, bla(TEM-135), that may be a precursor in the transitional stage of a traditional bla(TEM-1) gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the bla(TEM-135) gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the bla(TEM-135) allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a bla(TEM) variant (bla(TEM-135)) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

Fig 1

MAMA-PCR for bla_TEM-135 detection. (A) The TEM PCR primer set (TEM-F and TEM-R), which can amplify a 231-bp amplicon from bla_TEM-1 and bla_TEM-135, and the MAMA-PCR primer set, specific for bla_TEM-135 (MAMA-F and MAMA-R), are shown schematically with arrows. The sequence of primer MAMA-R (middle) and the corresponding regions from bla_tem135 (top) and bla_tem1 (bottom) are also shown. (B) The PCR results for the Japanese penicillinase-producing N. gonorrhoeae (PPNG) TEM-135 and PPNG TEM-1 isolates, which were used as controls in all PCRs, are presented.

Fig 2

Minimum spanning tree analysis of multilocus sequence typing (MLST) STs observed in penicillinase-producing N. gonorrhoeae (PPNG) isolates cultured in Thailand in 2005 to 2007. Numbers beside the circles denote ST. Solid lines, dashed lines, and dotted lines show the interrelationship of “single-locus variant,” “double-locus variant,” and “triple-locus variant,” respectively. The types directly or indirectly connected through single- or double-locus-variant relationships were judged to form one cluster. Each cluster is shaded gray. Sizes of circles reflect the numbers of isolates belonging to each type (for details, see text and tables). The only PPNG TEM-135 isolate belonging to cluster A (ST7822) is marked with an asterisk, and its plasmid type is given.

Fig 3

Molecular characterization of penicillinase-producing N. gonorrhoeae PPNG TEM-135 isolates. (A) Sequence types revealed by multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) are shown, along with por and tbpB alleles. (B and C) Neighbor-joining clustering showing similarity of por alleles (B) and tbpB alleles (C) from PPNG TEM-135 isolates.