Identification of TEM-135 ␤-Lactamase in Neisseria gonorrhoeae

Strains Carrying African and Toronto Plasmids in Argentina

R. Gianecini,a C. Oviedo,a A. Littvik,b E. Mendez,b L. Piccoli,b S. Montibello,b P. Galarzaa
Servicio de Enfermedades de Transmisión Sexual, Instituto Nacional de Enfermedades Infecciosas (INEI)-ANLIS Dr. Carlos G. Malbrán, Ciudad Autónoma de Buenos Aires,
Argentinaa; Gonococcal Antimicrobial Susceptibility Surveillance Program-Argentina (GASSP-AR), Ciudad Autónoma de Buenos Aires, Argentinab

One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and
2012 were examined to detect blaTEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two
PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The blaTEM-135
allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two blaTEM-135 isolates were

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related to previously described isolates from Thailand and China, indicating a common evolutionary origin.

A ntibiotic 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 re-
ported in 1976 and have spread since then (3). These first PPNG
isolates contained TEM-1-type ␤-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 epi-
demiological origin as African, Toronto/Rio, Asian, Nîmes, New
Zealand, Johannesburg, and Australian plasmids (5–7). The
PPNG isolates carrying a blaTEM-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 nucle-
otide substitution at position 539, resulting in a single amino acid
substitution, M182T. This substitution is also found in TEM-type
extended-spectrum ␤-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 blaTEM-135 and investigate plasmid
types carrying ␤-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 laborato-
ries distributed all around the country. The MICS (␮g/ml) of pen-
icillin, ciprofloxacin, ceftriaxone, and azithromycin for the iso-
lates 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 antimi-
crobial susceptibility testing (19). Mismatch amplification muta-
tion assay (MAMA) PCR was performed to identify the blaTEM-135
allele, and TEM PCR was used to recognize both the blaTEM-1 and
blaTEM-135 alleles (20). The whole blaTEM, porB, and tbpB genes of
all isolates that were MAMA PCR positive were amplified and
sequenced to confirm the blaTEM-135 allele, and genotyping by N.
gonorrhoeae multiantigen sequence typing (NG-MAST) was per-
formed (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 par-
tial porB nucleotide sequence (490 bp) was generated by using Mega4
software. Multiple-sequence alignments of the nucleotide and amino
acid sequences of blaTEM were performed by using the BioEdit (ver-
sion 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
␤-Lactamase Classification and Amino Acid Sequences for TEM,
SHV, and OXA Extended-Spectrum and Inhibitor-Resistant En-
zymes 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 blaTEM-1 and blaTEM-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%], respec-
tively), 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 iso-
lates were TEM PCR positive, suggesting the presence of the
blaTEM-1 allele in these isolates. Sequencing analysis of 44 MAMA
PCR-positive isolates revealed a T¡C substitution at position
539, confirming the blaTEM-135 allele. However, two isolates
showed an additional G¡A substitution at position 547. The nu-
cleotide sequence revealed a blaTEM allele encoding a variant that,
compared to TEM-1, carried the amino acid substitutions M182T
and A185T. This combination of point mutations was original,
Received 8 July 2014 Returned for modification 4 August 2014
Accepted 24 October 2014
Accepted manuscript posted online 3 November 2014
Citation Gianecini R, Oviedo C, Littvik A, Mendez E, Piccoli L, Montibello S,
P Galarza. 2015. Identification of TEM-135 ␤-lactamase in Neisseria gonorrhoeae
strains carrying African and Toronto plasmids in Argentina. Antimicrob Agents
Chemother 59:717–720. doi:10.1128/AAC.03838-14.
Address correspondence to P. Galarza, pgalarza@anlis.gov.ar.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.03838-14

January 2015 Volume 59 Number 1 Antimicrobial Agents and Chemotherapy aac.asm.org 717
Gianecini et al.

FIG 1 Alignment of multiple TEM amino acid sequences identified in 44 N. gonorrhoeae isolates that were MAMA PCR positive.

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and the new variant was assigned the name TEM-220 (http://www only compromises the surveillance program but increases selec-
.lahey.org/studies) (Fig. 1). Studies based on high-resolution tive pressure and facilitates the development of drug resistance
structures of TEM variants containing M182T have shown that (29). Prevalence data on and plasmid profiles of PPNG isolates
Thr182 acts as an N-cap residue for the 183-to-195 helix, forming from South America are limited (30). In our study, the prevalence
an additional hydrogen bond with the NH of Ala185 (26, 27). The
plasmid profile and its association with blaTEM revealed in the
Toronto/Rio plasmid the presence of blaTEM-135 in 16/19 (84.2%)
isolates from 2008 and 24/25 (96%) isolates from 2012, while in
the African plasmid, blaTEM-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 blaTEM-220 and
2/69 isolates from 2012 with the African plasmid were observed to
carry the blaTEM-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 iso-
lates were 16 and 32 ␮g/ml, respectively. The ceftriaxone MIC50
for both TEM-135- and TEM-1-producing isolates was 0.008 ␮g/
ml. All TEM-135-producing isolates, except one from 2008 that
was resistant to ciprofloxacin (MIC: 4 ␮g/ml), were susceptible to
ciprofloxacin and azithromycin. The two isolates containing the
blaTEM-220 allele were susceptible to ceftriaxone (MIC: 0.004 ␮g/
ml). NG-MAST was used to investigate the diversity and related-
ness of 44 MAMA PCR-positive isolates. The 44 isolates were di-
vided 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 blaTEM-135 allele were associ-
ated with one ST, 4990. The phylogenetic analysis of isolates car-
rying the blaTEM-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 blaTEM-135 allele with isolates previously described
from Japan, Thailand, and China revealed that ST211 from Thai-
land 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).
According to our data from GASSP-AR, 295 and 405 N. gon-
orrhoeae 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 FIG 2 Phylogenetic analysis of partial porB gene sequences in PorB1a (WI)
diagnosis depends on syndromic management; this has resulted in PPNG isolates from Argentina that contained blaTEM-135 and previously iden-
a lack of specimens and cultures (28). It is a big problem that not tified blaTEM-135-carrying isolates from Thailand, Japan, and China.

718 aac.asm.org Antimicrobial Agents and Chemotherapy January 2015 Volume 59 Number 1

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 blaTEM-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 blaTEM-135 and the novel blaTEM-220
allele in the Toronto/Rio and African plasmids circulating in
our country and the association of blaTEM-135 as a possible di-
rect precursor of ESBL suggest a need for surveillance studies to
monitor the presence of PPNG isolates and ␤-lactamase type
and epidemiological data to assess the distribution of these
isolates in the population. No differences in the MICs of pen-
icillin, ceftriaxone, and other antimicrobials were observed in
isolates producing TEM-1, TEM-135, and TEM-220. A phylo-
genetic tree of partial porB gene sequences showed that some of
the previously identified blaTEM-135 isolates from Thailand and
China contained an porB gene identical to that of some of the
blaTEM-135-carrying isolates from Argentina, indicating a com-
mon evolutionary origin.
This study shows the first evidence of blaTEM-135 in N. gonor-
rhoeae isolates in Argentina. About 30% of the PPNG isolates
studied contained the blaTEM-135 allele, and two isolates had a new
blaTEM-220 allele. It thus seems important for surveillance pro-
grams to investigate not only the plasmid type in PPNG isolates
and the associated blaTEM allele but also the phenotypic character-
istics and geographic distribution of isolates.
Nucleotide sequence accession number. The sequence data
for the blaTEM-220 gene have been deposited in the GenBank nu-
cleotide database under accession number KM998962.
ACKNOWLEDGMENTS
We are in debt to Martin Vacchino from the National Reference Lab-
oratory, INEI-ANLIS Dr. Carlos G. Malbrán, for his valuable contri-
bution.
This study was conducted as part of the reference work of the National
Reference Laboratory, Argentina, and the Gonococcal Antimicrobial Sus-
ceptibility Surveillance Program-Argentina, which is funded by the Na-
tional Administration of Laboratories and Institute of Health (ANLIS)
Dr. Carlos G. Malbrán—Ministry of Health.
REFERENCES
1. World Health Organization. 2012. Global incidence and prevalence of
selected curable sexually transmitted infections—2008. World Health Or-
ganization, Geneva, Switzerland.
2. Tapsall JW, Ndowa F, Lewis DA, Unemo M. 2009. Meeting the public
health challenge of multidrug- and extensively drug-resistant Neisseria
gonorrhoeae. Expert Rev Anti Infect Ther 7:821– 834. http://dx.doi.org/10
.1586/eri.09.63.
3. Ashford WA, Golash RG, Henning VG. 1976. Penicillinase-producing
Neisseria gonorrhoeae. Lancet ii:657– 658.
4. Dillon JA, Yeung KH. 1989. Beta-lactamase plasmids and chromo-
somally mediated antibiotic resistance in pathogenic Neisseria species.
Clin Microbiol Rev 2(Suppl):S125–S133.
5. Pagotto F, Aman AT, Ng LK, Yeung KH, Brett M, Dillon JA. 2000.
Sequence analysis of the family of penicillinase-producing plasmids of
Neisseria gonorrhoeae. Plasmid 43:24 –34. http://dx.doi.org/10.1006/plas
.1999.1431.
6. Müller EE, Fayemiwo SA, Lewis DA. 2011. Characterization of a novel
␤-lactamase-producing plasmid in Neisseria gonorrhoeae: sequence anal-
ysis and molecular typing of host gonococci. J Antimicrob. Chemother
66:1514 –1517. http://dx.doi.org/10.1093/jac/dkr162.
January 2015 Volume 59 Number 1 Antimicrobial Agents and Chemotherapy
TEM-135 ␤-Lactamase in Neisseria gonorrhoeae Strains
7. Trembizki E, Buckley C, Lawrence A, Lahra M, Whiley D. 2014.
Characterization of a novel Neisseria gonorrhoeae penicillinase-producing
plasmid, Australia 2012. Antimicrob Agents Chemother 58:4984 – 4985.
http://dx.doi.org/10.1128/AAC.02993-14.
8. Srifeungfung S, Roongpisuthipong A, Asavapiriyanont S, Lolekha R,
Tribuddharat C, Lokpichart S, Sungthong P, Tongtep P. 2009. Preva-
lence of Chlamydia trachomatis and Neisseria gonorrhoeae in HIV-
seropositive and gonococcal antimicrobial susceptibility: an update in
Thailand. Jpn J Infect Dis 62:467– 470.
9. Ohnishi M, Ono E, Shimuta K, Watanabe H, Okamura N. 2010.
Identification of TEM-135 ␤-lactamase in penicillinase-producing Neis-
seria gonorrhoeae strains in Japan. Antimicrob Agents Chemother 54:
3021–3023. http://dx.doi.org/10.1128/AAC.00245-10.
10. Chen SC, Yin YP, Dai XQ, Yu RX, Han Y, Sun HH, Ohnishi M, Unemo
M, Chen XS. 2013. Prevalence and molecular epidemiological typing of
penicillinase-producing Neisseria gonorrhoeae and their blaTEM-135 gene
variants in Nanjing, China. Sex Transm Dis 40:872– 876. http://dx.doi.org
/10.1097/OLQ.0000000000000037.
Downloaded from http://aac.asm.org/ on June 13, 2020 by guest
11. Muhammad I, Golparian D, Dillon JA, Johansson A, Ohnishi M, Sethi
S, Chen SC, Nakayama SI, Sundqvist M, Bala M, Unemo M. 2014.
Characterisation of blaTEM-135 genes and types of ␤-lactamase plasmids in
Neisseria gonorrhoeae—the prevalent and conserved blaTEM-135 has not
recently evolved and existed in the Toronto plasmid from the origin. BMC
Infect Dis 14:454 – 460. http://dx.doi.org/10.1186/1471-2334-14-454.
12. Whiley D, Trembizki E, Buckley C, Freeman K, Lawrence A, Limnios A,
Pearson J, Smith H, Stevens K, Lahra MM. 29 September 2014. Penicil-
linase-producing plasmid types of Neisseria gonorrhoeae clinical isolates
from Australia. Antimicrob Agents Chemother http://dx.doi.org/10.1128
/AAC.04120-14.
13. Sideraki V, Huang W, Palzkill T, Gilbert HF. 2001. A secondary drug
resistance mutation of TEM-1 ␤-lactamase that suppresses misfolding and
aggregation. Proc Natl Acad Sci U S A 98:283–288. http://dx.doi.org/10
.1073/pnas.011454198.
14. Wang X, Minasov G, Shoichet BK. 2002. Evolution of an antibiotic
resistance enzyme constrained by stability and activity trade-offs. J Mol
Biol 320:85–95. http://dx.doi.org/10.1016/S0022-2836(02)00400-X.
15. Jacquier H, Birgy A, Le Nagard H, Mechulam Y, Schmitt E, Glodt J,
Bercot B, Petit E, Poulain J, Barnaud G, Gros PA, Tenaillon O. 2013.
Capturing the mutational landscape of the beta-lactamase TEM-1. Proc
Natl Acad Sci U S A 110:13067–13072. http://dx.doi.org/10.1073/pnas
.1215206110.
16. Fiorito S, Fernandez Cabo M, Granados P, Galarza P. 1993. Primer
informe en la República Argentina de resistencia a penicilina en Neisseria
gonorrhoeae mediada por el plásmido de 3,2 MDa (africano). Infect Mi-
crobiol Clin 5:78 – 84.
17. Clinical and Laboratory Standards Institute. 2012. Methods for dilution
antimicrobial susceptibility test for bacteria that grow aerobically; ap-
proved standard—ninth edition M07A9. Clinical and Laboratory Stan-
dards Institute, Wayne, PA.
18. Clinical and Laboratory Standards Institute. 2014. Performance stan-
dards for antimicrobial susceptibility testing; twenty-fourth informa-
tional supplement M100-S24. Clinical and Laboratory Standards Insti-
tute, Wayne, PA.
19. Unemo M, Fasth O, Fredlund H, Limnios A, Tapsall JW. 2009. Pheno-
typic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae
reference strain panel intended for global quality assurance and quality
control of gonococcal antimicrobial resistance surveillance for public
health purposes. J Antimicrob. Chemother 63:1142–1151. http://dx.doi
.org/10.1093/jac/dkp098.
20. Nakayama S, Tribuddharat C, Prombhul S, Shimuta K, Srifuengfung S,
Unemo M, Ohnishi M. 2012. Molecular analyses of TEM genes and their
corresponding penicillinase-producing Neisseria gonorrhoeae isolates in
Bangkok, Thailand. Antimicrob Agents Chemother 56:916 –920. http://dx
.doi.org/10.1128/AAC.05665-11.
21. Martin IM, Ison CA, Aanensen DM, Fenton KA, Spratt BG. 2004. Rapid
sequence-based identification of gonococcal transmission clusters in a
large metropolitan area. J Infect Dis 189:1497–1505. http://dx.doi.org/10
.1086/383047.
22. Speldooren V, Heym B, Labia R, Nicolas-Chanoine MH. 1998. Discrim-
inatory detection of inhibitor-resistant ␤-lactamases in Escherichia coli by
single-strand conformation polymorphism-PCR. Antimicrob Agents
Chemother 42:879 – 884.
23. Ambler RP, Coulson AF, Frère JM, Ghuysen JM, Joris B, Forsman M,
aac.asm.org 719
Gianecini et al.
Levesque RC, Tiraby G, Waley SG. 1991. A standard numbering scheme
for the class A ␤-lactamases. Biochem J 276:269 –270.
24. Dillon JA, Carballo M. 1990. Molecular epidemiology and novel combi-
nations of auxotype, serovar, and plasmid content in tetracycline-resistant
Neisseria gonorrhoeae isolated in Canada. Can J Microbiol 36:64 – 67. http:
//dx.doi.org/10.1139/m90-013.
25. Dillon JR, Nasim A, Nestmann ER. 1985. Recombinant DNA method-
ology, p 1–126. In Dillon JR, Bezanson GS, Yeung K-H (ed), Basic tech-
niques. John Wiley & Sons, Inc., New York, NY.
26. Kather I, Jakob RP, Dobbek H, Schmid FX. 2008. Increase folding
stability of TEM-1 ␤-lactamase by in vitro selection. J Mol Biol 383:238 –
251. http://dx.doi.org/10.1016/j.jmb.2008.07.082.
27. Salverda M, GM de Visser J, Barlow M. 2010. Natural evolution of
TEM-1 ␤-lactamase: experimental reconstruction and clinical relevance.
FEMS Microbiol Rev 34:1015–1036. http://dx.doi.org/10.1111/j.1574-6976
.2010.00222.x.
720 aac.asm.org Antimicrobial Agents and Chemotherapy
28. Ministerio de Salud y Ambiente de la República Argentina. 2004.
Guía de manejo de las infecciones de transmisión sexual, Argentina.
Ministerio de Salud y Ambiente de la República Argentina, Buenos
Aires, Argentina.
29. Patel AL, Chaudhry U, Sachdev D, Nagpal Sachdeva P, Bala M,
Saluja D. 2011. An insight into the drug resistance profile & mecha-
nism of drug resistance in Neisseria gonorrhoeae. Indian J Med Res 134:
419 – 431.
30. Starnino S, GASP-LAC Working Group, Galarza P, Carvallo ME, Ben-
zaken AS, Ballesteros AM, Cruz OM, Hernandez AL, Carbajal JL,
Borthagaray G, Payares D, Dillon JA. 2012. Retrospective analysis of
antimicrobial susceptibility trends (2000-2009) in Neisseria gonorrhoeae
isolates from countries in Latin America and the Caribbean shows evolv-
ing resistance to ciprofloxacin, azithromycin and decreased susceptibility
to ceftriaxone. Sex Transm Dis 39:813– 821. http://dx.doi.org/10.1097
/OLQ.0b013e3182631c9f.
Downloaded from http://aac.asm.org/ on June 13, 2020 by guest
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