Identification of TEM-135 β-Lactamase in Neisseria gonorrhoeae Strains Carrying African and Toronto Plasmids in Argentina

TEXT

Antibiotic resistance in Neisseria gonorrhoeae is a global public health problem (1). Through the years, N. gonorrhoeae has developed resistance to the first-line antibiotics used for treatment (2). Penicillinase-producing N. gonorrhoeae (PPNG) isolates with plasmid-mediated high-level resistance to penicillin were first reported in 1976 and have spread since then (3). These first PPNG isolates contained TEM-1-type β-lactamase plasmids encoded by transposon TnA (Tn2), which are responsible for the transference and dissemination of resistance (4). To date, eight plasmid types have been described in N. gonorrhoeae and named after their epidemiological origin as African, Toronto/Rio, Asian, Nîmes, New Zealand, Johannesburg, and Australian plasmids (5–7). The PPNG isolates carrying a bla_TEM-135 gene were found in Thailand, Japan, and China and recently reported in Australia and 15 other countries (8–12). TEM-135 differs from TEM-1 by a single nucleotide substitution at position 539, resulting in a single amino acid substitution, M182T. This substitution is also found in TEM-type extended-spectrum β-lactamase (ESBL) but has little effect on enzyme activity, and it has been proposed to stabilize substitutions near the active site, which collaboratively results in the emergence of a stable ESBL (13–15). The first PPNG isolate in Argentina was reported in 1980 and has since been disseminated in our country (16). The prevalence of PPNG isolates has been increasing through the years, with the highest level of 40% in the 1990's. The aim of this study was to detect bla_TEM-135 and investigate plasmid types carrying β-lactamase in the PPNG strains in 2008 and 2012 in Argentina.

We studied 49 and 94 PPNG isolates collected in 2008 and 2012, respectively, from GASSP-AR, which includes 70 laboratories distributed all around the country. The MIC_S (μg/ml) of penicillin, ciprofloxacin, ceftriaxone, and azithromycin for the isolates were determined by the agar dilution method (17, 18). The N. gonorrhoeae ATCC 49226 and World Health Organization (WHO) reference strains were used for quality control in antimicrobial susceptibility testing (19). Mismatch amplification mutation assay (MAMA) PCR was performed to identify the bla_TEM-135 allele, and TEM PCR was used to recognize both the bla_TEM-1 and bla_TEM-135 alleles (20). The whole bla_TEM, porB, and tbpB genes of all isolates that were MAMA PCR positive were amplified and sequenced to confirm the bla_TEM-135 allele, and genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed (21, 22). For assignment of porB and tbpB allele numbers and NG-MAST sequence types (STs), the NG-MAST website (http://www.ng-mast.net) was used. A neighbor-joining tree with the partial porB nucleotide sequence (490 bp) was generated by using Mega4 software. Multiple-sequence alignments of the nucleotide and amino acid sequences of bla_TEM were performed by using the BioEdit (version 7.2.5) software. The scheme proposed by Ambler et al. was used to number the amino acid positions (23). The amino acid sequences identified in the present study were compared to sequences at the β-Lactamase Classification and Amino Acid Sequences for TEM, SHV, and OXA Extended-Spectrum and Inhibitor-Resistant Enzymes website (http://www.lahey.org/studies). The plasmid profile was studied by using a boiling plasmid extraction method, and the relationship between the former and both bla_TEM-1 and bla_TEM-135 was investigated (24, 25).

In this study, two plasmid types were found in our isolates from 2008 and 2012. Isolates carrying the African plasmid were the most common (30/49 [61.2%] and 69/94 [73.4%], respectively), and those harboring the Toronto/Rio plasmid were the least common (19/49 [38.8%] and 25/94 [26.6%], respectively). Eighteen of the 49 PPNG from 2008 and 26 of the 94 PPNG from 2012 were MAMA PCR positive. All MAMA PCR-negative isolates were TEM PCR positive, suggesting the presence of the bla_TEM-1 allele in these isolates. Sequencing analysis of 44 MAMA PCR-positive isolates revealed a T→C substitution at position 539, confirming the bla_TEM-135 allele. However, two isolates showed an additional G→A substitution at position 547. The nucleotide sequence revealed a bla_TEM allele encoding a variant that, compared to TEM-1, carried the amino acid substitutions M182T and A185T. This combination of point mutations was original, and the new variant was assigned the name TEM-220 (http://www.lahey.org/studies) (Fig. 1). Studies based on high-resolution structures of TEM variants containing M182T have shown that Thr182 acts as an N-cap residue for the 183-to-195 helix, forming an additional hydrogen bond with the NH of Ala185 (26, 27). The plasmid profile and its association with bla_TEM revealed in the Toronto/Rio plasmid the presence of bla_TEM-135 in 16/19 (84.2%) isolates from 2008 and 24/25 (96%) isolates from 2012, while in the African plasmid, bla_TEM-1 was present in 30/30 (100%) isolates from 2008 and 67/69 (97.1%) from 2012. Only 2/19 isolates from 2008 harboring the Toronto/Rio plasmid carried bla_TEM-220 and 2/69 isolates from 2012 with the African plasmid were observed to carry the bla_TEM-135 allele. All isolates were resistant to penicillin and susceptible to ceftriaxone. The penicillin MICs for 50% of the isolates tested (MIC₅₀) for TEM-135- and TEM-1-producing isolates were 16 and 32 μg/ml, respectively. The ceftriaxone MIC₅₀ for both TEM-135- and TEM-1-producing isolates was 0.008 μg/ml. All TEM-135-producing isolates, except one from 2008 that was resistant to ciprofloxacin (MIC: 4 μg/ml), were susceptible to ciprofloxacin and azithromycin. The two isolates containing the bla_TEM-220 allele were susceptible to ceftriaxone (MIC: 0.004 μg/ml). NG-MAST was used to investigate the diversity and relatedness of 44 MAMA PCR-positive isolates. The 44 isolates were divided into 16 different NG-MAST STs, and the most frequent STs were 10979 (29.5%) and 10972 (22.7%), three STs, 10974, 10975, and 10977, were represented by three isolates, and 10 isolates were associated with the single ST 4432, 10973, 10976, 10978, 10980, 10981, 10982, 10983, 10984, or 10985. The two isolates carrying African plasmids and containing the bla_TEM-135 allele were associated with one ST, 4990. The phylogenetic analysis of isolates carrying the bla_TEM-135 allele divided the isolates into two main clades, which represent N. gonorrhoeae serogroups PorB1b (WII/III) and PorB1a (WI). The comparison of isolates in our study carrying the bla_TEM-135 allele with isolates previously described from Japan, Thailand, and China revealed that ST211 from Thailand and STs 568, 641, 2313, 2833, and 8801 from China had a porB gene sequence identical to that of some of the Argentinian isolates and were associated with clade PorB1a (WI), (Fig. 2).

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FIG 1

Alignment of multiple TEM amino acid sequences identified in 44 N. gonorrhoeae isolates that were MAMA PCR positive.

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FIG 2

Phylogenetic analysis of partial porB gene sequences in PorB1a (WI) PPNG isolates from Argentina that contained bla_TEM-135 and previously identified bla_TEM-135-carrying isolates from Thailand, Japan, and China.

According to our data from GASSP-AR, 295 and 405 N. gonorrhoeae isolates were reported in Argentina in 2008 and 2012. Even though an increase was observed during the period, it is believed that there was underreporting. In our country, gonorrhea diagnosis depends on syndromic management; this has resulted in a lack of specimens and cultures (28). It is a big problem that not only compromises the surveillance program but increases selective pressure and facilitates the development of drug resistance (29). Prevalence data on and plasmid profiles of PPNG isolates from South America are limited (30). In our study, the prevalence of PPNG isolates was 16.6% from 2008 and 23.2% from 2012 and the plasmid profile revealed two types of circulating plasmids, with the African-type plasmid being the most abundant. Of note, 40/42 (95.2%) isolates possessing the bla_TEM-135 allele carried the Toronto/Rio-type plasmid and two isolates had the African-type plasmid. Interestingly, two isolates from 2008 carrying Toronto/Rio plasmids showed two substitutions at position 539 (T→C) and 547 (G→A), resulting in the amino acid substitutions M182T and A185T. The presence of bla_TEM-135 and the novel bla_TEM-220 allele in the Toronto/Rio and African plasmids circulating in our country and the association of bla_TEM-135 as a possible direct precursor of ESBL suggest a need for surveillance studies to monitor the presence of PPNG isolates and β-lactamase type and epidemiological data to assess the distribution of these isolates in the population. No differences in the MICs of penicillin, ceftriaxone, and other antimicrobials were observed in isolates producing TEM-1, TEM-135, and TEM-220. A phylogenetic tree of partial porB gene sequences showed that some of the previously identified bla_TEM-135 isolates from Thailand and China contained an porB gene identical to that of some of the bla_TEM-135-carrying isolates from Argentina, indicating a common evolutionary origin.

This study shows the first evidence of bla_TEM-135 in N. gonorrhoeae isolates in Argentina. About 30% of the PPNG isolates studied contained the bla_TEM-135 allele, and two isolates had a new bla_TEM-220 allele. It thus seems important for surveillance programs to investigate not only the plasmid type in PPNG isolates and the associated bla_TEM allele but also the phenotypic characteristics and geographic distribution of isolates.