Prevalence of bla TEM-220 gene in Penicillinase-producing Neisseria gonorrhoeae strains carrying Toronto/Rio plasmid in Argentina, 2002 - 2011

Abstract

Background: Penicillinase-producing Neisseria gonorroheae (PPNG) was first isolated in 1976. PPNG strains carrying bla TEM-1 and bla TEM-135 gene have been described in different countries. Recently, a novel bla TEM-220 allele was detected in PPNG isolates carrying Toronto/Rio plasmid. The prevalence and characteristics of TEM-220 strains worldwide are unknown, and therefore, it needs to be studied. The purpose of this study was to detect bla TEM-220 gene in PPNG strains possessing Toronto/Rio plasmid over a period of ten years in Argentina, and to evaluate the proportion of isolates producing non-TEM-220 containing the T539C substitution in the bla TEM allele.

Methods: One hundred and fifty one PPNG isolates carrying Toronto/Rio plasmid were studied between 2002 and 2011. A mismatch amplification mutation assay (MAMA) PCR was used to identify the T539C substitution in the bla TEM allele and a MAMA-PCR protocol was developed to detect the G547A substitution in the bla TEM-220. The reference agar dilution method of the Clinical and Laboratory Standard Institute (CLSI) was used for susceptibility testing to five β-lactams antibiotics, ciprofloxacin, tetracycline and azithromycin. In all TEM-220-producing isolates, the whole bla TEM gene was sequenced and the isolates were typed using N. gonorroheae multiantigen sequence typing (NG-MAST).

Results: MAMA PCR successfully identified the G547A substitution in the bla TEM-220 allele. The proportion of isolates that possessed the bla TEM-220 allele was 2.6 %, and 93.2 % MAMA TEM-220 PCR-negative isolates showed the T539C substitution in the bla TEM gene. No differences in the susceptibility to five beta-lactam antibiotics tested were observed in PPNG isolates TEM-220-producing and PPNG isolates carrying the T539C substitution in the bla TEM gene. All TEM-220 isolates were indistinguishable by NG-MAST.

Conclusion: This is the first study which shows the prevalence of bla TEM-220 in N. gonorrhoeae isolates carrying Toronto/Rio plasmid in Argentina. Although the bla TEM-220 allele does not appear to be associated with an extended spectrum beta-lactamase (ESBL) phenotype of resistance, a single nucleotide polymorphism added to the bla TEM-220 or bla TEM containing the T539C substitution could lead to the emergence of ESBL. Thus, it is imperative to investigate in surveillance programs, not only the plasmid type in PPNG isolates and the bla TEM allele associated, but phenotypical characteristics and geographical distribution of isolates.

Fig. 1

Alignment and difference of DNA sequence between the bla _TEM-1, bla _TEM-135, and bla _TEM-220 gene. The nucleotide substitutions found in the different types of bla _TEM alleles are in bold type. The forward primer MAMA TEM-220 used in this study is shown schematically with arrows. Underlined letter indicates the nucleotide alteration near the 3′ end of the primer to enhance the 3′ mismatch effect

Fig. 2

Discrimination of PPNG isolates with the G547A substitution in bla _TEM allele by using T220-f/T220-r primers. Lanes 1 and 2, indicate the 204 bp PCR products from strains IM4519 and IM4520 with the G547A mutation in bla _TEM allele; lanes 3, 4, 5 and 6 indicate PPNG control strains containing bla _TEM-1 and bla _TEM-135, respectively, without amplification; lane M, molecular size marker