Molecular Characterization of Penicillinase–Producing Neisseria gonorrhoeae Isolated in Two Time Periods,2003–2004 and 2014

Abstract

The emergence of antibiotic resistant strains poses a great concern for gonorrhea treatment. The aim of this study was to characterize penicillinase–producing Neisseria gonorrhoeae (PPNG) isolates collected in Italy in two time frames, 2003–2004 and 2014–2015. A total of 80 PPNG were characterized for the bla_TEM gene variant and the plasmid type. Furthermore, gonococci were typed using Neisseria gonorrhoeae multiantigen sequence typing. Antibiotic susceptibility assay was performed for penicillin, ciprofloxacin, ceftriaxone, and spectinomycin by Etest and minimum inhibitory concentration (MIC) test strip methods. The β-lactamase production was detected using nitrocefin test. Among PPNG isolates, four bla_TEM alleles were identified as follows: bla_TEM-1, bla_TEM-228, bla_TEMP14S, and bla_TEM-135. The African plasmid possessed the bla_TEM-1, bla_TEM-228, and bla_TEMP14S, whereas bla_TEM-135 was identified in Toronto/Rio and Asian plasmids. The percentage of isolates with the bla_TEM-1-carrying African plasmid increased from 42.5% in 2003–2004 to 55% in 2014–2015; conversely, the isolates with bla_TEM-135-carrying Toronto/Rio plasmid decreased from 57.5% to 35%. Among the isolates carrying the Toronto/Rio plasmids possessing bla_TEM-135, sequence type (ST)661 and ST5624 were found to be the predominant STs in both periods 2003–2004 and 2014–2015, respectively. More than half of the PPNG isolates were resistant to ciprofloxacin. Increase in the isolates carrying the African plasmid possessing bla_TEM-1 and a parallel decrease of the bla_TEM-135-carrying Toronto/Rio plasmid was observed. Moreover, PPNG isolate harbored Toronto/Rio plasmid with bla_TEM-135 belonged mainly to two major STs (ST661 and ST5624). Given the possible role of a mutated bla_TEM gene as an additional mechanism to extended spectrum β-lactamase resistance, it is crucial to monitor gonococci carrying these resistance genes.

Introduction

N eisseria gonorrhoeae infection represents an important public health threat due to widespread multiple antimicrobial resistance.¹ Over time, N. gonorrhoeae has developed resistance to a wide range of antibiotics and, more recently, to third-generation cephalosporins.^1–3 For this reason, public health national and international initiatives aim to monitor the spread and the main molecular characteristics of resistant gonococci.^2,4

Due to its attitude to alter genetic material, N. gonorrhoeae has developed several mechanisms of resistance through gene transfer or specific mutations, which result in the increase in the number of resistant gonococci.¹

The worldwide dissemination of antimicrobial resistant (AMR) N. gonorrhoeae and the resistance plasmids carrying β-lactamase–encoding gene (bla_TEM) has caused an increased interest in penicillinase–producing Neisseria gonorrhoeae (PPNG) isolates. PPNG results in high-level resistance to penicillin and, although no longer used for gonorrhea treatment, a significant proportion of strains have retained their resistance to this antibiotic.^1,5

Several studies have described the prevalence and the molecular characterization of PPNG and the bla_TEM gene variants.^5–11

Among the eight plasmid types carrying the bla_TEM gene, Asian, African, and Toronto/Rio plasmids were the most frequently described and widespread globally.^1,5 The bla_TEM gene encodes for β-lactamase able to hydrolyze the cyclic amide bond in the β-lactam ring inactivating the molecular activity.^1,12

The bla_TEM-1 and bla_TEM-135, which only differ by one single nucleotide polymorphism, were the most commonly identified gene variants among PPNG isolates.^1,5,6 Recently, a new bla_TEM-220 carried by Toronto/Rio plasmid was identified in PPNG isolated in Argentina.^8,9

Molecular typing by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) has improved the molecular epidemiology of gonococci¹³; furthermore, an association between specific sequence types (STs) and bla_TEM alleles was observed.^9,11

In Italy, the proportion of PPNG isolates decreased significantly from 77% in 2003 to 7% in 2012.¹⁴ In our prior studies no further information about bla_TEM variants was available. This prompted us to perform a retrospective analysis of plasmid and bla_TEM variants responsible for penicillin resistance in 80 gonococci collected in Italy in two time frames, 2003–2004 and 2014–2015. Molecular typing by NG-MAST was evaluated to identify the most representative ST. Finally, the minimum inhibitory concentration (MIC) determination for the susceptibility to penicillin, ceftriaxone, ciprofloxacin, and spectinomycin was also determined.

Materials and Methods

PPNG isolate collection

N. gonorrhoeae isolates were collected by 14 laboratories collaborating with the Department Infectious Diseases, Istituto Superiore di Sanità in Rome, as Reference Center for the antimicrobial susceptibility study on gonorrhea and as part of European Gonococcal Antimicrobial Surveillance Program (EURO-GASP).¹⁵

The isolates were initially determined to be PPNG on the basis of nitrocefin disc testing (Oxoid Ltd.) after growth on Thayer-Martin agar (Oxoid Ltd.) with 1% of IsoVitalex (Oxoid Ltd.) at 37°C in a 5% CO₂ atmosphere. For each isolate, information on the type of clinical specimen and of patient was anonymously provided and recorded using Epi Info software (version 3.3.2). Eighty N. gonorrhoeae isolates resulted in penicillin resistant and producing β-lactamase; they were randomly selected from two time frames 2003–2004 and 2014–2015, 40 isolates for each period.

Ethical approval was not required since the isolates were collected and stored as part of routine clinical care by the laboratories participating in the study.

Molecular characterization of bla_TEM gene, plasmid type, and NG-MAST

Plasmid DNA was extracted from a fresh subculture using a boiling extraction method.⁸

Plasmid type and bla_TEM gene were identified as already described.^16,17 Multiple-sequence alignments of nucleotide and amino acid sequence were performed using ClustalW (http://ebi.ac.uk/Tools/msa/clustalo/). The amino acid sequences of β-lactamase were compared to the sequences in the Lactamase Engineering Database (http://laced.uni-stuttgart.de/) and numbering according to the scheme proposed by Ambler et al.¹⁸

Genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen), according to the manufacturer's instructions. Sequencing of porB and tbpB was achieved using primers and amplification parameters, as previously described.¹³ The porB and tbpB alleles, as well as the STs, were assigned according to NG-MAST website (http://ng-mast.net).

Antimicrobial susceptibility testing

All isolates were examined to define the MIC for penicillin, ciprofloxacin, ceftriaxone, and spectinomycin. Penicillin susceptibility was evaluated using Etest method (bioMerieux, Sweden) on isolate collected before 2009. Ciprofloxacin, ceftriaxone, and spectinomycin were all tested using MIC test strip method (Liofilchem Diagnostici, Italy) from 2003 up to now. The World Health Organization (WHO) N. gonorrhoeae O, M, and N reference strains were used for quality control.¹⁹ The MIC values were interpreted according to the EUCAST clinical breakpoint criteria (v. 7.1, 2017) (http://eucast.org/clinical_breakpoints/).

Antimicrobial susceptibility data for the PPNG belonging to the first time period (2003–2004) were previously described.^14,20

Results

Patient characteristics

Of all the isolates selected for this study between 2003 and 2004, the 87.5% (35/40) were isolated from the urethra of males and the remaining endocervix. The median age was 35 years for both sexes. Among males who have declared their sexual orientation, 26 were men who have sex with women (MSW), whereas 3 were men who have sex with men (MSM). All the females declared to be heterosexual (data not shown).

In 2014–2015, the majority of isolates (38/40; 95%) were retrieved from male patients with a median age of 30.5 years. For two cases gender information was not available. Among the patients who declared their sexual orientation, 16 were MSW and 21 were MSM (data not shown).

Characterization of PPNG isolated in 2003–2004

bla_TEM-135, bla_TEM-1, and a new bla_TEM, the bla_TEM-228 (accession no.: KY851075), have been identified. The bla_TEM-228 showed one nucleotide substitution at position 547 of the gene (G → A) resulting in a single amino acid substitution, A185T.

The bla_TEM-135 in Toronto/Rio plasmid was the most frequent (23/40; 57.5%), followed by bla_TEM-1 (16/40; 40%) and bla_TEM-228 (1/40; 2.5%), both carried by African plasmid types (Table 1).

Table 1.

Plasmid Types and bla_TEM Gene Variants in Eighty Penicillinase–Producing Neisseria gonorrhoeae Isolates Collected in Two Time Periods, 2003–2004 and 2014–2015, in Italy

		bla_TEM-variant
		N (%)
	Plasmid type	bla_TEM-1	bla_TEM-135	bla_TEM-228	bla_TEMP14S	Total
2003–2004	African	16 (40.0%)	0 (0.0%)	1 (2.5%)	0 (0.0%)	17
	Toronto/Rio	0 (0.0%)	23 (57.5%)	0 (0.0%)	0 (0.0%)	23
2014–2015	African	21 (52.5%)	0 (0.0%)	0 (0.0%)	1 (2.5%)	22
	Asian	0 (0.0%)	4 (10.0%)	0 (0.0%)	0 (0.0%)	4
	Toronto/Rio	0 (0.0%)	14 (35.0%)	0 (0.0%)	0 (0.0%)	14
	Total	37	41	1	1	80

Forty strains were assigned to 18 STs, with ST661 (19/40; 47.5%) being the predominant, followed by ST660 (3/40; 7.5%), ST655, and ST656 with two isolates each, and 14 STs (ST69, ST331, ST657, ST658, ST659, ST662, ST663, ST665, ST666, ST667, ST669, ST1249, ST1253, and ST1479) with single isolates (Fig. 1).

FIG. 1.

Distribution of bla_TEM allele/plasmid type and ST in 80 Neisseria gonorrhoeae isolates collected in Italy in 2003–2004 and 2014–2015. ST, sequence type.

Penicillin MIC values ranged from 4 to >32 mg/L; 80% (32/40) were resistant to ciprofloxacin (MIC >0.06 mg/L) (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr).

Characterization of PPNG isolated in 2014–2015

As shown in Table 1, bla_TEM-1, bla_TEM-135, and bla_TEMP14S have been detected. The bla_TEM-1 possessed by the African plasmid was the predominant type (21/40; 52.5%). The bla_TEM-135 allele (18/40; 45%) was located on Toronto/Rio (14/40; 35%) and Asian plasmids (4/40; 10%), whereas one isolate harbored bla_TEMP14S on the African plasmid.

Forty strains were assigned to 21 STs, with ST5624 as the most frequent (12/40; 30%), followed by ST1582 (3/40; 7.5%). There were 2 isolates each of ST5793, ST9918, and ST13231, whereas 16 STs (ST661, ST995, ST1461, ST2886, ST3104, ST4995, ST5364, ST9541, ST11657, ST11658, ST13071, ST13228, ST13229, ST13230, ST13232, and ST13239) were all identified in only 1 isolate (Fig. 1). Five STs (ST13229, ST13230, ST13231, ST1232, and ST13239) were novel to the NG-MAST database.

In the period 2014–2015, the penicillin MIC values ranged from 6 to >32 mg/L; 75% (30/40) were resistant to ciprofloxacin (MIC >0.06 mg/L) (Supplementary Table S2).

Overall, none of the samples were resistant to ceftriaxone (MIC values ranging between 0.002 and 0.012 mg/L) or spectinomycin (MIC >64 mg/L) (Supplementary Tables S1 and S2).

Association among bla_TEM alleles, plasmid types, and NG-MAST

In 2003–2004, bla_TEM-135/Toronto/Rio was mainly associated with ST661 (19/40; 47.5%), as well as ST655, ST657, and ST1479_. Conversely, isolates harboring bla_TEM-1/African belonged to 13 STs (ST69, ST331, ST656, ST658, ST659, ST660, ST662, ST663, ST665, ST666, ST669, ST1249, and ST1253). One isolate, related to ST667, harbored the bla_TEM-228/African (Fig. 1).

In 2014–2015, ST5624 was the most frequent (12/40; 30%), of which nine harbored bla_TEM-135/Toronto/Rio; two were bla_TEM-135/Asian; and one isolate possessed bla_TEM-1/African (Fig. 1).

A total of four STs (ST661, ST9541, ST9918, and ST13232) were also associated to bla_TEM-135/Toronto/Rio. Two PPNG isolates belonging to ST4995 and ST11658 harbored bla_TEM-135/Asian.

Conversely, the broadest diversity of STs (ST995, ST1461, ST1582, ST2886, ST3104, ST5793, ST11657, ST13071, ST13228, ST13229, ST13230, ST13231, and ST13239) was associated with isolates that harbored bla_TEM-1/African. The bla_TEMP14S/African was identified in only one isolate belonging to ST5364 (Fig. 1).

Discussion

Although penicillin is no longer used in clinical practice for the treatment of gonorrhea, the plasmid–mediated penicillin resistant gonococci (PPNG) have been widely described.^1,5–9,11

According to previous studies,^5,6 few mutations in bla_TEM-1 and a single mutation in bla_TEM-135 have been described to be more likely associated with extended-spectrum β-lactamase (ESBL) in N. gonorrhoeae. This is very relevant considering that ceftriaxone combined with azithromycin is the currently recommended treatment for gonorrhea worldwide.^21–24

For this reason, a key component of a successful response plan to gonococcal AMR is to conduct timely surveillance of resistance and to characterize the genetic elements of resistant strains since resistance frequencies can vary by country.^4,6–8,11 Certainly, the majority of PPNG isolates were currently recorded in Western Pacific and South East Asia.^25,26 In 2013, the WHO GASP reported the highest percentage in the Western Pacific Region, such as in the Philippines (85%), Brunei (66%), Singapore (61%), and Hong Kong (54%).²⁵

According to the European Centre for Disease Control (ECDC) surveillance report 2014,⁴ there was a different distribution of PPNG also in Europe, from 0% in Cyprus, Estonia, Latvia, and Poland to 29.1% in Norway. Overall, the proportion of PPNG remained fairly constant from 8% to 14.1% in Europe.⁴

This study provides an updated picture of the prevalence, the diversity of the bla_TEM alleles, identifying their plasmid vectors, and the genotype of a subsample of PPNG isolates circulating in Italy in the time frame 2003–2004 and 2014–2015.

In accordance with the ECDC report,⁴ we observed an association between gonococcal infections due to PPNG and heterosexual patients, for the cases reported in 2003–2004. Conversely, in 2014–2015, PPNG was mostly associated with MSM. This may be, in part, due to the increase of gonorrhea cases among MSM (data not shown).

bla_TEM-1 and bla_TEM-135 were prevalent in both periods, as well as a higher prevalence of African and Toronto/Rio was observed in overall study periods with the exception of Asian plasmid of more recent detection (2014–2015). The bla_TEM-1 was carried by African plasmid, whereas the bla_TEM-135 was mostly found in Toronto/Rio and rarely in Asian plasmids. Compared to our results, the spread of bla_TEM-135 was mainly attributed to Asian and to a lesser extent by Toronto/Rio plasmids, as reported in other European countries.^6,10

A different trend of isolates carrying plasmid type and bla_TEM gene was observed. In particular, Toronto/Rio harboring bla_TEM-135 decreased from 57.5% in 2003–2004 to 35% in 2014–2015. On the contrary, African plasmid possessing bla_TEM-1 increased from 42.5% in 2003–2004 to 55% in 2014–2015.

The decrease of isolates carried by Toronto/Rio plasmid that possessed bla_TEM-135 allele and the parallel increase of those with African plasmid carrying bla_TEM-1 suggest a change in the molecular plasmid profile of PPNG circulating in the country. Furthermore, compared to Toronto/Rio and Asian plasmids, the African plasmid exhibits a broad diversity of bla_TEM alleles.

ST661 and ST5624 were prevalent in the two periods, respectively, even though it observed a higher variability among PPNG of more recent isolation. As previously described,^14,20 gonococci belonging to ST661 have been recovered up to 2007 and were characterized by ciprofloxacin, penicillin, and tetracycline resistance pattern. Conversely, ST5624 was associated with gonococci of recent isolation and mainly characterized by ciprofloxacin resistance.

Similar to what was reported in England, Wales, and France,^6,10 ST5624 was more likely to be associated with Toronto/Rio plasmid harboring bla_TEM-135.

In this scenario, overall the susceptibility for penicillin, ciprofloxacin, ceftriaxone, and spectinomycin was retained rather stable over the time by the PPNG isolates circulating in Italy. Moreover, in accordance with Cole et al.,⁶ most of the PPNG isolates harboring bla_TEM-135 allele were ciprofloxacin resistant.

Of note, all the PPNG isolated in 2014–2015 were included in the study, whereas the PPNG of the first period, 2003–2004, represented around 50% of the total. The different percentages of PPNG between the two periods (53% vs. 9.2%) could be, in part, explained by a change in the molecular characteristics of the gonococcal populations over time in terms of plasmid–mediated resistance capability.

The risk of emergence and spread of ESBL–producing N. gonorrhoeae caused by mutations in the bla_TEM requires careful epidemiological and molecular monitoring.

Footnotes

Acknowledgments

Neisseria gonorrhoeae Italian Study Group: I. Dal Conte: STI Clinic, Department of Infectious Disease, Amedeo di Savoia Hospital, Turin, Italy; S. Del Re, G. Gregori: Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Turin, Italy; M.L. Stella: STI Clinic, Amedeo di Savoia Hospital, Turin, Italy; A. Di Carlo, A. Cristaudo, M. Giuliani, A. Latini, A. De Santis: San Gallicano Dermatologic Institute, IRCCS, Rome, Italy; L. Corti, S. Ramoni, D. Fanoni: Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Milan, Italy; A. Matteelli: Clinic of Infectious and Tropical Diseases, University of Brescia, Italy; M.A. De Francesco: “Dipartimento di Medicina Molecolare e Traslazionale, Sezione di Microbiologia,” University of Brescia, Italy; M. Gulletta: Clinic of Infectious and Tropical Diseases, University of Brescia, Italy; M.P. Landini: Unit of Clinical Microbiology, University Hospital S. Orsola-Malpighi, Rizzoli Orthopedic Institute, Bologna, Italy; A. D'Antuono: Unit of Dermatology, St. Orsola-Malpighi, University of Bologna, Bologna, Italy; R. Antonetti, A. Di Taranto, R. De Nittis: Department of Clinical Pathology, “Azienda Ospedaliera- Universitaria OOR,” Foggia, Italy; M. Gaino, P. Ober, R. Predazzer: Microbiology and Virology Laboratory, “Santa Chiara Hospital, Trento,” Trento, Italy; M. Busetti: Microbiology Unit, University Hospital ASUITs, Trieste, Italy; S. Del Monte: STI Clinic of San Lazzaro Dermatologic Hospital, A. O. Città della Salute e della Scienza di Torino, Turin, Italy; S. Brossa: Microbiology and Virology Laboratory, Molinette Hospital, Turin, Italy; E. Stroppiana: Dermatologic Clinic, ‘A.O.U. Città della Salute e della Scienza’ Turin, Italy; L. Levorato: Microbiology Section, Department of Experimental Medicine, University of Perugia; M.A. Latino, E. Gaido: Department of “Medicina di Laboratorio,” P.O. Sant'Anna, Città della Salute e della Scienza di Torino, Turin, Italy; M.C. Cava: Microbiology and Virology Laboratory, Sandro Pertini Hospital, Rome, Italy; M. Rotondi: Marilab s.r.l., Ostia, Italy; F. Poletti: Dept. of Infectious Diseases, “Ospedale Castelli ASL VCO,” Verbania, Italy. This work was supported by the Ministry of Health-CCM Project 2014 “Sorveglianza di laboratorio della farmaco resistenza di Neisseria gonorrhoeae come malattia emergente.”

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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Molecular Characterization of Penicillinase–Producing Neisseria gonorrhoeae Isolated in Two Time Periods,2003–2004 and 2014–2015,in Italy

Abstract

Introduction

Materials and Methods

PPNG isolate collection

Molecular characterization of blaTEM gene, plasmid type, and NG-MAST

Antimicrobial susceptibility testing

Results

Patient characteristics

Characterization of PPNG isolated in 2003–2004

Characterization of PPNG isolated in 2014–2015

Association among blaTEM alleles, plasmid types, and NG-MAST

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

Molecular characterization of bla_TEM gene, plasmid type, and NG-MAST

Association among bla_TEM alleles, plasmid types, and NG-MAST