Origin, evolution, and success of pbla, the gonococcal beta-lactamase plasmid, and implications for public health

Tabea A. Elsener; Ana Cehovin; Connor Philp; Kate Fortney; Stanely M. Spinola; Martin C. J. Maiden; Christoph M. Tang

doi:10.1371/journal.ppat.1013151

Abstract

Neisseria gonorrhoeae is a leading cause of sexually transmitted infection (STI) and a priority AMR pathogen. Two narrow host range plasmids, pbla and pConj, have contributed to ending penicillin and tetracycline therapy, respectively, and undermine current prevention strategies including doxycycline post-exposure prophylaxis (Doxy-PEP). Here, we investigated the origin and evolution of the beta-lactamase plasmid, pbla. We demonstrate that pbla was likely acquired by the gonococcus from Haemophilus ducreyi, and describe the subsequent evolutionary pathways taken by the three major pbla variants. We show that the ability of pConj to spread pbla promotes their co-occurrence in the gonococcal population and the spread of pbla. Changes that mitigate fitness costs of pbla and the emergence of TEM beta-lactamases that confer increased resistance have contributed to the success of pbla. In particular, TEM-135, which has arisen in certain pbla variants, increases resistance to beta-lactams and only requires one amino acid change to become an extended spectrum beta-lactamase (ESBL). The evolution of pbla underscores the threat of plasmid-mediated resistance to current therapeutic and preventive strategies against gonococcal infection. Given the close relationship between pbla and pConj, widespread use of Doxy-PEP is likely to promote spread of both plasmids, strains which carry pConj and are resistant against third generation cephalosporins, and the emergence of plasmid-mediated ESBL in the gonococcus, with significant public health consequences.

Author summary

Neisseria gonorrhoeae is a threat to sexual and reproductive health. With no available vaccine against gonococcal disease, control of infection depends on having effective antimicrobials for the treatment of cases and their contacts. However, strains of N. gonorrhoeae have acquired resistance to all classes of antibiotics, including third generation cephalosporins, the mainstay of treatment for gonococcal disease. The gonococcus can carry two resistance plasmids, the conjugative plasmid pConj and beta-lactamase plasmid pbla, which have rendered treatment with tetracycline/doxycycline and penicillin ineffective, respectively. Here, we investigated the origin and evolution of pbla, and the molecular basis of its success in the gonococcal population. We show that pbla was likely acquired from Haemophilus ducreyi and has adapted to the gonococcus both through gene loss that reduces fitness costs, and through mutations that increase resistance and favour the development of an extended spectrum beta-lactamase. Our findings show that the ability of pConj to mobilise pbla promotes their co-occurrence, highlighting the potential to co-select for pbla by favouring pConj-containing strains through doxycycline use. Our results highlight the continuing adaptation of pbla to its host, which could undermine treatment options in the future.

Citation: Elsener TA, Cehovin A, Philp C, Fortney K, Spinola SM, Maiden MCJ, et al. (2025) Origin, evolution, and success of pbla, the gonococcal beta-lactamase plasmid, and implications for public health. PLoS Pathog 21(5): e1013151. https://doi.org/10.1371/journal.ppat.1013151

Editor: H. Steven Seifert, Northwestern University Feinberg School of Medicine, UNITED STATES OF AMERICA

Received: February 11, 2025; Accepted: April 23, 2025; Published: May 6, 2025

Copyright: © 2025 Elsener et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: o TAE is funded by the Oxford-Hoffman Graduate Scholarship, Jesus College with the Sir William Dunn School of Pathology. Work in CMT’s laboratory is supported by the Wellcome Trust (https://wellcome.org/, grant codes 214374/Z/18/Z and 221924/Z/20/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Neisseria gonorrhoeae causes ~80 million cases of sexually transmitted infection (STIs) annually [1] and is a WHO priority pathogen due to its extensive antimicrobial resistance (AMR) [2]. N. gonorrhoeae has two resistance plasmids, pConj and pbla, which contributed to the cessation of tetracycline and penicillin therapy, respectively. Furthermore, pConj carrying tetM confers resistance to doxycycline as well as tetracycline, so undermines the ability of doxycycline post-exposure prophylaxis (Doxy-PEP) to prevent gonococcal infection [3,4]. pConj and pbla are highly prevalent in low and middle-income countries (LMICs) where syndromic treatment of STIs with doxycycline has been recommended [5,4,6]. Therefore, it is important to understand the factors driving the success of these plasmids in gonococcal populations.

pConj is a 39–42 kb conjugative plasmid, that can confer high-level tetracycline/doxycycline resistance [4,7], and can be categorised into seven variants [6]. pbla emerged in the gonococcus in the 1970s and encodes a TEM beta-lactamase conferring penicillin resistance [8,9]. pbla TEM beta-lactamases require a few amino acid changes to become an extended-spectrum beta-lactamase (ESBL) [10], which would render third-generation cephalosporins, the current first-line treatment, ineffective [11]. pbla is usually closely associated with pConj which can mobilise pbla [6,12]; around 85% of strains that harbour pbla also contain pConj.

There are three main pbla variants, characterised by the presence/absence of certain genes [5]. The 7.4 kb pbla.2 (also referred to as pbla Asia) has been considered the ancestral plasmid [13]. pbla.1 (5.6 kb, pbla Africa) is the commonest variant and has a deletion in the replication region, while pbla.3 (5.1 kb, pbla Rio/Toronto) lacks the region implicated in pbla mobilisation [5,13]. Variants of pbla are associated with certain pConj variants and TEM alleles [5]. pbla.1 mostly carries TEM-1 or TEM-1_P14S, while pbla.3 is associated with TEM-135; pbla.2 carries TEM-1 or TEM-135. Importantly, the M182T substitution in TEM-135 is a ‘stepping stone’ mutation before the enzyme becomes an ESBL [10,14].

Here, we investigated the origin, evolution and characteristics of the three pbla variants. We demonstrate that the spread and distribution of pbla in gonococci results from the dynamics of its association with pConj, fitness costs, and resistance levels. pbla is found with mobile pConj variants and has evolved to avoid fitness costs and confer higher resistance to beta-lactams. Our results underline the threats posed by pbla and pConj, particularly if their spread and prevalence in the gonococcal population is promoted by the widespread implementation of Doxy-PEP.

Results

pbla has been acquired by the gonococcus from Haemophilus

Haemophilus spp. are known to harbour plasmids related to pbla [15–20]; however, without nucleotide sequence data, these early studies could not characterise the precise relationship between Haemophilus and Neisseria beta-lactamase plasmids. Therefore, we interrogated Haemophilus whole genome sequences (WGS) in the PubMLST database [21] (6,896 isolates, 12 species, S1 Table) for pbla. We searched for Tn2 as pbla TEM-1b is located on this transposon [22,23], and confirmed the presence of pbla replicons by searching for NEIS2960, NEIS2358 (repA), and NEIS2961 (mobA) [5]. Tn2 is present in 12.5% of Haemophilus influenzae (6,403 isolates) and 19.3% Haemphilus parainfluenzae (269 isolates), while pbla-like replicons were detected in 0.3% of H. influenzae and 1.5% H. parainfluenzae isolates. However, Tn2 and pbla sequences only co-occurred in two isolates of H. influenzae (PubMLST ids: 23482 and 33361) and H. parainfluenzae (PubMLST ids: 16289 and 34872).

In contrast, seven of 31 (22.6%) H. ducreyi isolates (S2 Table) harbour TEM-1 containing pbla-like plasmids. H. ducreyi strains can be divided into two clades/classes [24], and a pbla.1-like plasmid in Class I isolate and a pbla.2-like plasmid in a Class II isolates. The sequences of plasmids from H. ducreyi HD183 (Class I, 9.1 kb) and DMC64 (Class II, 10.9 kb) [25] were aligned to gonococcal pbla.1 and pbla.2, and were found to be highly similar (> 83% nucleotide identity, Fig 1A). pbla.1 and the 9.1 kb H. ducreyi plasmid carry repB and one copy of NEIS2964. Both the 10.9 kb H. ducreyi plasmid and gonococcal pbla.2 carry two rep alleles, repA and repB, as well as two copies of NEIS2964. The major difference is that Tn2 is intact in the H. ducreyi plasmids while gonococcal pbla lack tnpA and have a truncated tnpR. These findings are consistent with pbla transfer from H. ducreyi into the gonococcus with the event associated with truncation of Tn2.

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Fig 1. Evolutionary relationships between pbla variants.

(A) Schematic representation of alignment of gonococcal and H. ducreyi pbla variants to H. ducreyi pbla.2. Aligned regions are represented as black bars, with deletion regions and nucleotide polymorphisms indicated in white within. Insertions are shown as black lines above the bars. ORFs on H. ducreyi pbla.2 are coloured according to gene function; yellow, mobilisation proteins; orange, Tn2-derived genes including blaTEM; light blue, replication initiation proteins; purple, other gene function. (B) Maximum likelihood tree of 414 gonococcal pbla sequences with tips coloured according to pbla variant; the tree was rooted at H. ducreyi pbla.2. Circles indicate the tnpR allele and the TEM variant carried.

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Two alternative scenarios can explain the occurrence of two distinct pbla variants in both H. ducreyi and N. gonorrhoeae: i) independent introductions of pbla.1 and pbla.2, with both introductions associated with a truncation of Tn2, or ii) introduction of pbla.2 into the gonococcus with pbla.1 emerging independently in H. ducreyi and N. gonorrhoeae through the deletion of repB and one copy of NEIS2964. Comparison of the Tn2 deletion site in gonococcal pbla variants demonstrates that the plasmids carry distinct tnpR alleles (allele 3 and 2 for pbla.1 and pbla.2, respectively), as their truncations differ by a single nucleotide (S1A Fig), supporting the hypothesis of two independent Tn2 truncations/introductions. We also examined the pbla.1 deletion site (spanning repB and one copy of NEIS2964). H. ducreyi pbla.2 carries two copies of NEIS2964 (alleles 2 and 3) which differ by three nucleotides (S1B Fig). Gonococcal pbla.2 carries alleles 1 and 2. H. ducreyi pbla.1 carries allele 3, whereas gonococcal pbla.1 is associated with allele 2, suggesting pbla.1 arose independently in H. ducreyi and N. gonorrhoeae. Therefore, analysis of Tn2 truncations and pbla deletions is inconclusive about whether a single or multiple introductions of pbla occurred into the gonococcus.

To investigate the evolutionary relationships between gonococcal pbla variants further, we examined a subset of pbla (414 of 2,758, S3 Table) [5] with the 10.9 kb H. ducreyi pbla as reference; plasmids were from 1979-2022 with the same proportion of variants as the whole population (i.e., 70% pbla.1, 14% pbla.2, 16% pbla.3) [5]. A maximum likelihood phylogeny placed pbla variants into distinct clades, with pbla.1 split from the other variants. While H. ducreyi pbla carry TEM-1, TEM-1_P14S arose in pbla.1 while TEM-135 arose in pbla.2, with pbla.3 emerging from TEM-135 carrying pbla.2 (Fig 1B).

pbla is associated with pConj variants that promote its spread

To understand the association between pbla and pConj, we examined the ability of different pConj variants to transfer pbla. Initially, matings were performed between isogenic strains (FA1090 or 2086_K) to eliminate any barriers to horizontal gene transfer between unrelated strains [26]. Additionally, ΔpilD donors and recipients were constructed to block transformation [27–29], and the transfer pbla.1 by pConj.1, the commonest combination of these plasmids [5], was measured. pbla.1 was mobilised at a frequency of ~1% transconjugants/recipient for FA1090 and 2086_K (S1 Fig), while no pbla transfer was detected in the absence of pConj.

Conjugative plasmids can block the acquisition of other plasmids by expressing entry exclusion proteins [30]. Entry exclusion could impede pbla mobilisation, as the initial transfer of pConj during conjugation could block subsequent acquisition of pbla. pConj encodes a predicted lipoprotein annotated as TrbK entry exclusion protein [31], with 21% amino acid similarity with Agrobacterium fabrum Ti plasmid TrbK (locus tag: ATU_RS23180, Genbank: NC_003065.3). To examine whether pConj in a recipient can impair the transfer of pbla, we compared the mobilisation rates of pbla into pConj-free and pConj-harbouring recipients. There was no difference in pbla transfer into pConj-free (1.1%) and pConj-containing recipients (0.9%, Welch two-sample t-test, p = 0.87, S1 Fig), indicating that surface exclusion is unlikely to limit pbla spread.

We next evaluated pbla mobilisation by pConj variants that are frequently associated with pbla (i.e. pConj 1, 3, and 4, Fig 2A) and by variants that are infrequently associated with pbla (i.e. pConj.2 and 7). The conjugation frequencies of pConj.1, 3 and 4 were >79% (Fig 2B) but several orders of magnitude lower for pConj.2 and pConj.7 which are not associated with pbla (Fig 2A). The rate of pbla mobilisation correlated with conjugation frequencies (Fig 2B), with pbla transfer by pConj.2 undetectable in our assays (limit of detection, L.O.D. = 0.001%). Taken together, results indicate that pbla is associated with pConj variants that mobilise it efficiently and promote its spread in the gonococcal population.

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Fig 2. pbla mobilisation by pConj.

(A) Sankey plot of pConj carrying isolates (n = 4,883 isolates) [5], displaying the presence of pbla (left) and co-occurrence of pbla with individual pConj variants (right). (B) Conjugation rates of pConj variants (top) and the mobilisation rates of co-located pbla.1 (bottom). The limit of detection (L.O.D.) is indicated as a dashed line. (C) Mobilisation rates of wild type and isogenic pbla variants (pbla^iso) by pConj.1. (D) The impact of single mob gene knockouts in pbla.2 on pbla mobilisation frequencies. All assays consist of three individual repeats and were analysed by one-way ANOVA with Tukey multiple comparisons; n.s. p > 0.05, *** p < 0.001.

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The immobility of pbla.3 is reflected in its restricted distribution

There are conflicting reports about the mobility of pbla.3 [32–34]. Therefore, we assessed the mobilisation of wild-type pbla variants by pConj.1. Results indicate that wild-type pbla.1 and pbla.2 were mobilised efficiently (transfer rate, ~ 1%), while pbla.3 mobilisation was not detected (Fig 2C). To assess whether pbla variant deletions are responsible for these differences, we introduced variant-specific deletions into pbla.2, generating the isogenic plasmids pbla.1^iso and pbla.3^iso. Mobilisation of the isogenic plasmids did not differ from wild-type plasmids (p = 0.82, Fig 2C), indicating the 2.3 kb deletion in pbla.3 compared with pbla.2 is responsible for the lack of pbla.3 transfer. The immobility of pbla.3 is evident from its restricted distribution in three related lineages, whilst pbla.1 and pbla.2 are found across the gonococcal population (S3 Fig) [5].

Mobilisable plasmids deploy diverse strategies to exploit the conjugative machinery of co-existing conjugative plasmids [35]. Some mobilisable plasmids encode their own relaxase which recognises and nicks their origin of transfer (oriT) and then guides the plasmid DNA through the Type 4 secretion system encoded by the conjugative plasmid. Alternatively, the oriT of mobilizable plasmids can be recognised by the relaxase encoded by a conjugative plasmid. Therefore, we next examined the genes responsible for the immobility of pbla.3 by generating isogenic pbla.2 mutants lacking genes absent in pbla.3, and assessing their ability to be mobilised between pilD mutants of FA1090 by pConj1. The pbla.3-characteristic deletion spans mobA (NEIS2961, encoding the relaxase [36]), mobC (NEIS2962) and NEIS2964. Deletion of mobA abolished pbla transfer (Fig 2D), indicating that pbla mobilisation depends on its own relaxase and not the pConj relaxase. Removal of NEIS2962 significantly reduced pbla mobilisation (p = 0.01, Fig 2D). NEIS2962 is related to MobC from E. coli plasmid RSF1010, which unwinds DNA at the oriT [37]. NEIS2962 homodimers are structurally related to MobC and predicted to recognise the pbla oriT but not a scrambled oriT sequence (S4 Fig). Deletion of both copies of NEIS2964 did not impact pbla.2 transfer.

TEM-135 confers increased penicillin resistance

If a plasmid is beneficial to its host, one would expect that plasmid carrying isolates to be highly prevalent in a lineage, due to their competitive advantage of isolates without the plasmid. Therefore, to assess the prevalence of pbla in certain lineages, we resolved the gonococcal population structure by clustering isolates according to allelic differences in loci that are core to the gonococcus [38], and defined N. gonorrhoeae core genome clusters (Ng_cgc₄₀₀) with a cut-off of 400 allelic differences. Although pbla.3 is immobile, it is highly prevalent in Ng_cgc₄₀₀s 25 and 298 with 56.8 and 39.6% of isolates in these lineages carrying pbla, respectively, Table 1 and S3 Fig) [5]. This suggests pbla.3, which carries TEM-135, confers a benefit to the gonococcus that has led to the clonal expansion of isolates containing this pbla variant. To test this, we measured the penicillin MICs conferred by pbla variants. Whilst TEM-1 carrying pbla.1 and 2 conferred MICs of 8 µg/ml, pbla.3 with TEM-135 conferred a significantly higher MIC (32 µg/ml, p = 0.003, Fig 3A). To establish whether the TEM variant determines resistance levels, we changed pbla.3 TEM-135 into TEM-1 by introducing a T182M substitution. This substitution reduced the MIC conferred by pbla.3 to levels of pbla.1 and pbla.2, demonstrating that TEM-135 confers elevated MICs (Fig 3B). We also compared resistance conferred by TEM-1, TEM-1_P14S and TEM-135 (which together account for >95% of gonococcal TEMs [5]) expressed by pbla.2. Again, TEM-135 significantly increased MICs (128 µg/ml vs. 8 µg/ml with TEM-1, p < 0.001, Fig 3C).

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Table 1. The percentage of isolates carrying pbla in three largest Ng_cgc400 that carry each variant.

https://doi.org/10.1371/journal.ppat.1013151.t001

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Fig 3. TEM-135 increases the MIC.

(A) Penicillin G MICs of pbla variants in FA1090 isogenic strain background (one-way ANOVA on log₂-transformed MIC values with Tukey multiple comparisons of means; *** p < 0.001). (B) MICs of TEM-1 in different pbla variant backbones. (C) MICs of different TEM variants in pbla.2 backbone (one-way ANOVA on log₂-transformed MIC values; *** p < 0.001). (D) Cellular levels of TEM variants were assessed by Western blot analysis. The image is representative of three biological repeats.

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To explore the basis for the different MICs, we assessed cellular TEM levels. Levels of mature TEM-135 (29 kDa) were significantly higher than TEM-1 or TEM-1_P14S (Figs 3D and S5), consistent with increased stability of TEM-135 [14]. In conclusion, the appearance of TEM-135, particularly associated with pbla.3, provides a significant benefit to the gonococcus by enhancing resistance against beta-lactams, with MICs correlating with cellular TEM levels.

Successful pbla variants have evolved with reduced fitness costs

Plasmids often impose fitness costs, disadvantaging isolates that carry plasmids [39]. We therefore assessed the fitness costs of pbla by introducing pbla.1 into isolates from a range of lineages (S3 Fig) and competing plasmid-carrying vs. plasmid-free strains over 24 hrs. pbla.1 had no detectable fitness cost in any isolate (Fig 4A), consistent with its continued prevalence in the gonococcus (Fig 4B). We also compared the fitness costs of the three pbla variants in wild-type FA1090. In contrast to pbla.1 and pbla.3 which impose no fitness cost, pbla.2 inflicts a significant fitness cost which was evident within 24 hrs (Fig 4C). This is associated with a higher copy number for pbla.2 (>6 copies/chromosome) than the other variants (1–2 copies/chromosome, Fig 4D).

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Fig 4. Impact of pbla-imposed fitness cost on its prevalence in the population.

(A) Fitness cost (w) of pbla.1 in clinical isolates from different pbla-free (grey) or pbla-associated lineages (light blue, pbla.1-associated; purple, pbla.1/pbla.2-associated; yellow, pbla.3-associated). w > 1 indicates a benefit, whereas w < 1 signifies a cost of plasmid carriage. (B) Proportional pbla carriage in gonococcal isolates deposited on PubMLST between 2010 and 2019 (n = 12,914 isolates). Colours show pbla variant carried and numbers above bars indicate the number of samples in the respective year. (C) Fitness cost of pbla variants in FA1090 isogenic strain background were assessed in four independent replicates (one-way ANOVA with Tukey multiple comparisons, n.s. p > 0.05; * p < 0.05). (D) Copy number of pbla in FA1090 isogenic strain background was assessed by ddPCR (one-way ANOVA with Tukey multiple comparisons; *** p < 0.001). (E) pbla carriage in isolates from the pbla.1/pbla.2-associated Ng_cgc₄₀₀ 29 between 2011 and 2019 (n = 433 isolates). Bar colours indicate pbla variant and numbers above the bars specify the number of samples in the respective year.

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The fitness costs of pbla.2 could explain its decreasing prevalence seen when analysing all available WGS in the PubMLST database from 2010 onwards (Fig 4B). To account for any bias in sampling, we also examined the prevalence of pbla.1 and pbla.2 within a lineage (Ng_cgc₄₀₀ 29) which harbours both these variants. Between 2010 and 2020, there has been a shift from pbla.2 to pbla.1 in this lineage. In 2011, 55.6% of pbla sequences were pbla.2 and 33.3% pbla.1, while only pbla.1 was recovered from isolates of Ng_cgc₄₀₀ 29 after 2018 (Fig 4E).

As the observed changes in pbla variant prevalence could result from uneven sampling, we further assessed the relative success of pbla variants by their abundance within lineages; if a strain acquires a plasmid which offers an advantage it will undergo clonal expansion and outcompete other strains belonging to the same lineage but lacking the plasmid. Therefore, we examined the percentage of strains with pbla in the three largest lineages carrying each pbla variant. pbla.1 is highly prevalent in lineages (50–60% of pbla carriage in major pbla.1 lineages, Table 1). In contrast, pbla.2 is only present at low frequency in lineages (<20%, Table 1), or is found in lineages with other variants. pbla.3 which expresses TEM-135 with no obvious fitness cost is found in a high percentage of strains in a lineage (39–84%).

Taken together, fitness costs imposed by pbla.2 are consistent with its low prevalence in the gonococcal population compared with pbla.1. pbla.3 with TEM-135, which arose from pbla.2, confers elevated penicillin resistance without fitness costs, and is associated with the success of a small group of related lineages.

Discussion

Plasmids are important vehicles for AMR, with resistance plasmids amongst the most diverse and mobile [40]. Here, we investigated the origin of the relatively conserved beta-lactamase plasmid pbla which is largely found in the gonococcus, a WHO priority pathogen. Our analysis indicates that pbla was likely acquired by N. gonorrhoeae from another cause of STI, H. ducreyi. We describe the evolution of pbla since its emergence in N. gonorrhoeae, and its association with pConj. Gene loss and the appearance of novel TEM alleles influence the benefits and costs of pbla to the gonococcus. These traits are associated with the success and distribution of pbla variants within the gonococcal population.

In women, N. gonorrhoeae primarily causes cervicitis, while H. ducreyi causes ulcers at the vaginal entrance and cervix. In men, N. gonorrhoeae primarily causes urethritis and H. ducreyi mainly causes penile ulcers [41]. Therefore, these species can occupy the same niche, providing ample opportunities for gene transfer. The presence of pbla.1- and pbla.2-like plasmids in H. ducreyi could indicate independent introductions of pbla.1 and pbla.2 into the gonococcus or independent emergence of pbla.1 in H. ducreyi and N. gonorrhoeae. Whilst we cannot reject either scenario, the independent emergence of pbla.1 in H. ducreyi through the deletion of repB/NEIS2964 allele 2 and repB/NEIS2964 allele 3, respectively, is the most parsimonious explanation.

pbla is found relatively frequently in N. gonorrhoeae and H. ducreyi, which inhabit the genitourinary tract, but is seldom present in pathogens which inhabit the nasopharynx (e.g., H. influenzae and N. meningitidis); pbla was not detected in any non-invasive Neisseria spp. (41,158 isolates, 30 species). This could reflect the renal excretion of beta-lactams [42], favouring pbla carriage among bacteria inhabiting the urogenital tract compared with other sites. The meningococcal urethritis clade (NmUC) evolved from ST-11 N. meningitidis by acquiring genetic elements from N. gonorrhoeae [43,44]. So far, pbla has not been reported in NmUC. However, we previously found an ST-11 N. meningitidis isolate harbouring pbla [5]. This raises the possibility that NmUC might acquire pbla, which could provide an entry point of this plasmid into the meningococcal population, and the emergence of beta-lactamase producing N. meningitidis.

Plasmids can be successful in bacterial populations either by horizontal transmission and their ability to spread into diverse lineages, or through vertical transmission through their stable inheritance while being beneficial to their host. Our data indicates that the success of pbla depends both on its own mobility and its association with pConj variants that can effectively mediate its spread. Given the higher rates of pConj conjugation (>75%) compared with pbla mobilisation (~1%), the spread of pbla into a lineage is likely to be accompanied by pConj, maintaining the close association between these plasmids. Interesting, unlike many other conjugative plasmids [45], pConj lacks entry exclusion, so pbla can enter bacteria already containing pConj, allowing proliferation of bacteria with successful pbla/pConj pairs, such as pbla.1/pConj.1, across the gonococcal population.

The most frequent and widespread pbla variant, pbla.1, is mobilised efficiently by common pConj variants and does not impose fitness costs. pbla.2 is also mobile but imposes fitness costs. Compared with pbla.1, pbla.2 has a second replication initiation protein and additional origins of replication (i.e., ori2 and ori3) [5,46] which might increase copy number and/or impose the fitness costs of pbla.2; plasmid Rep proteins can sequester host DNA replication machinery [39], causing fitness costs. We found that pbla.2 is present in lower prevalence in lineages than pbla.1, with evidence of a shift from pbla.2 to pbla.1 in a single lineage (Ng_cgc₄₀₀ 29) over time. Whilst the observed shift in pbla variants could reflect sampling bias, it is consistent with strains carrying pbla.2 being outcompeted by plasmid-free isolates or those with other pbla variants. Further evidence is provided by a longitudinal 10 year study of over 1,700 gonococcal isolates from a single city in China (Guangzhou), which reported a marked increase in pbla.1 (pbla Africa, from 18.4 to > 90% of isolates) with a concurrent fall in pbla.2-containing strains (from 81.6 to 7.6%) due to expansion of successful clones harbouring pbla.1 [47].

pbla.3-associated lineages have undergone clonal expansion indicating its successful adaptation to the gonococcus. Phylogenetic analysis indicates that TEM-135 originally arose in pbla.2. However, despite increased resistance levels conferred by TEM-135, the fitness cost of pbla.2 has likely undermined the success of TEM-135 in this pbla variant. We found that pbla.3 evolved from TEM-135 carrying pbla.2 through gene loss. This prevented the plasmid from being mobile, but with the trade-off of avoiding fitness costs. Consequently, the prevalence of pbla.3 in gonococci is not due to transfer between isolates, but through the expansion of pbla.3-carrying isolates, potentially through the increased beta-lactam resistance associated with TEM-135 and/or its association with an otherwise successful lineage. Further work is required to distinguish between these and other possibilities.

Since the emergence of gonococcal pbla in the 1970s, the evolutionary trajectory of this plasmid has been marked by its association with pConj variants that enable its spread through the population, the appearance of plasmid variants with minimal costs, and emergence of TEMs promoting higher resistance (e.g., TEM-135). A major concern is that the ESBL-permissive M182T substitution in TEM-135 is already widespread in gonococci [5,6], especially in pbla.3.

The intimate relationship between pbla and pConj also highlights the threat posed by increased use of tetracyclines, as already witnessed in LMICs where gonococci have remarkably high plasmid carriage [4,48]. Similarly, the implementation of Doxy-PEP will increase selection for the carriage of pConj in gonococci and will thence select the isolates/lineages harbouring the plasmid. A significant threat is posed by isolates carrying pConj as well as chromosomal mutations conferring cefotaxime resistance [49]. The spread of these isolates, as well as pbla (which itself could become an ESBL-plasmid) have the potential to undermine the successful treatment of cases and their contacts with third-generation cephalosporins, the first-line antibiotics currently used to control gonococcal infection.

Methods

Analysis of Haemophilus spp. and Neisseria spp. genomes

Tn2-carrying isolates of Haemophilus spp. and Neisseria spp. were identified querying Tn2 (GenBank accession: LC091537.1) against Haemophilus (accessed 8/4/2025, 6,403 isolates, 12 species) and Neisseria (accessed 24/07/2024, 41,158 isolates, 33 species) sequences in PubMLST [21] (blastN word size: 11, scoring: reward: 2; penalty: -3; gap open: 5; gap extend: 2, sequence identity >99%, alignment length >50% query). pbla-like plasmids were confirmed by the presence of NEIS2960 (sequence identity >80%; alignment length >50%) and NEIS2358, and NEIS2961[5].

Plasmid variants

pbla and pConj were analysed in 15,529 gonococcal WGS on PubMLST (accessed 28/07/2022, S4 Table) [5,21] with isolates from 1928-2022 and 66 countries. NEIS2220 indicates the presence of pConj, and variants were defined according to gene presence/absence and specific alleles of plasmid genes [6]. pbla variants were typed using the Ng_pblaST scheme [5]. For the population structure, isolates were grouped into core genome clusters according to the allelic profile of 1,668 core genes [38]; isolates were grouped with a cut-off of 400 allelic differences (Ng_cgc₄₀₀).

Phylogenetic analyses

A subset of 414 pbla-carrying isolates conserving the ratio of pbla variants (70% pbla.1, 14% pbla.2, 16% pbla.3, S3 Table) [5] was selected to investigate the phylogenetic relationship of pbla variants. This included all pbla-containing isolates pre-dating 2000 (n = 35). Isolates between 2000 and 2022 (n = 379) were randomly selected using the r sample function [5]. Snippy v4.6.0 mapped plasmid reads to H. ducreyi DMC64 pbla (minimum coverage, 4 and base quality, 25). Multiple sequence alignments were generated with snippy-core v4.6.0/snippy-clean v4.6.0. Maximum likelihood trees were generated using RaxML-ng v1.2.2 [50] with 100 bootstrap replicates, rooted at H. ducreyi DMC64 pbla, and visualised with ape [51] and ggtree [52,53].

Structure predictions

Analysis of NEIS2962 and RSF1010 MobC (GenBank accession: S96966.1) homodimers and NEIS2962 with pbla oriT [36] were performed using AlphaFold 3 [54] and PyMol v2.5.4 [55]. Charge distributions were visualised with the Adaptive Poisson-Boltzmann Solver (APBS) electrostatics tool [56].

Bacterial strains/growth

Strains and plasmids used in this study are listed in S5 and S6 Tables, respectively. E. coli DH5 was grown on Luria-Bertani (LB) agar or in liquid LB shaking at 180 rpm. N. gonorrhoeae was grown on Gonococcal Base Media (GCB) agar plates or liquid media (GCBL) [57] supplemented with 1% Vitox (Oxoid) at 37°C in 5% CO₂. H. ducreyi was grown on chocolate agar plates supplemented with 1% IsoVitaleX at 35°C in 5% CO₂. Antibiotics were added as follows: for E. coli, carbenicillin 100 μg/ml; for N. gonorrhoeae, carbenicillin 2.5 μg/ml; erythromycin 1 μg/ml; kanamycin 50 μg/ml, and tetracycline 2 μg/ml.

Characterisation of H. ducreyi plasmids

Genomic DNA was isolated from H. ducreyi by harvesting bacteria from plates and the DNeasy Blood/Tissue Kit (Qiagen) with the modifications that cells were incubated in lysis buffer with 20 mg/ml lysozyme at 37°C for 2 hours and then proteinase K overnight at 56°C. Plasmids were analysed by Sanger sequencing using primers listed in S7 Table. Sequence similarity of plasmid sequences was assessed with ClustalW with default parameters [58] and sequences were mapped onto H. ducreyi DMC64 pbla using Snapgene v6.1.1 (Insightful Science; available from snapgene.com) to investigate deletion sites.

Transformation of gonococci

For electroporation, bacteria grown on GCB agar were resuspended in PBS (Sigma), adjusted to 5x10⁷ CFU/ml then washed three times with 20% glycerol/ 1% MOPS (Sigma); electroporation was performed with 2.5 kV, 200 Ω, 25 mF. Cells were recovered in 1 ml of GCBL with 2% Vitox and plated on GCB agar. Plates were incubated for 3 hours, cells collected, and then transferred to selective media.

∆pilD::ermC and ∆pilD::aph constructs were transformed into N. gonorrhoeae as described previously [27,29]. In brief, 1 μg of DNA was spotted onto plates, allowed to dry, and bacteria streaked over the spots. Plates were incubated for 8 hours, then bacteria were transferred onto selective agar. Transformants confirmed by PCR/Sanger sequencing.

Plasmid modifications

All primers are listed in S8 Table. To generate pbla.1^iso and pbla.3^iso, pbla.2 was cut with HindIII-HF and PvuII-HF (NEB), respectively. pbla.1^iso was amplified with primers TE18/19 and PrimeSTAR GXL polymerase (Takara Bio); Gibson assembly was performed with primers TE20/21. pbla.3^iso was amplified in two fragments with TE7/TE17 and TE9/TE16. Plasmids were assembled using Gibson Hifi (NEB) and transformed into E. coli DH5.

Point mutations in blaTEM were introduced using the RAIR method [59]. PCRs with primers (TE56/TE57, pbla.2 TEM-135; TE63/TE64, pbla.2^{TEM-1 P14S}; TE65/TE66, pbla.3^TEM-1) were performed using Herculase II polymerase (Agilent), with pbla.2 or pbla.3 as template. Products were purified (Promega Wizard PCR Clean-up) and transformed into E. coli DH5.

tetM⁺ pConj.7 was constructed by amplifying tetM from N. gonorrhoeae WHO N using primers TE34/TE35. Flanking regions were amplified with primers TE36/37 and TE38/39, then joined by Gibson assembly; the product was amplified with TE36/39, then introduced into N. gonorrhoeae NG028 by transformation. All constructs were confirmed by sequencing.

Conjugation and mobilisation assays

Donor (∆pilD::ermC) and recipient (∆pilD::aph) strains grown overnight were inoculated in 5 ml GCBL/1% Vitox at an OD₆₀₀ of 0.1 and grown to mid-exponential phase (OD₆₀₀ 0.6 - 0.8). The bacterial density was adjusted to 10⁸ CFU/ml and donor and recipient strains mixed in a 10:1 ratio. Bacteria (5 µl) were spotted onto GCB agar and incubated for 6 hours at 37°C, 5% CO₂, harvested in 200 μl GCBL, then plated to GCB agar with antibiotics. Conjugation and mobilisation frequencies were defined as the number of transconjugants/recipients (n = 3). Plasmids used in the different assays are listed in S6 Table together with the WGS of the corresponding isolates available on PubMLST.

Competition assays to determine fitness costs

Plasmids were introduced into FA1090 ∆pilD::ermC and competed against FA1090 ∆pilD::aph (n = 4). Bacteria in PBS were adjusted to an OD₆₀₀ 1, mixed 1:1, diluted to 10⁵ CFU/ml and added to 200 μl Fastidious Broth [60] then grown at 37°C, 5% CO₂, shaking at 180 rpm. After 24 hours, strains were enumerated by spotting on selective media. Fitness costs (w) were calculated by:

(w, relative fitness of pbla⁺ vs. pbla^- strains; and , pbla^- strain at the beginning/end, respectively; and , same for pbla⁺ strain).

Antibiotic susceptibility testing

Penicillin G MICs were assessed using the broth microdilution method [61] in 96-well plates with 2-fold Penicillin dilutions in water (50 μl); strains grown overnight on GCB agar were resuspended in PBS (Sigma), then diluted in 2x FB/2% Vitox to 10⁵ CFU/ml. Bacteria (50 μl) were transferred into each well and incubated for 24 hours.

SDS page and Western blot analysis

Bacteria were grown to mid-exponential phase, added to an equal volume of 2x SDS-PAGE buffer, run on 12% SDS-polyacrylamide gels, and transferred to Protan nitrocellulose membranes (GE Healthcare) using the Trans-Blot Turbo System (Bio-Rad). Membranes were blocked in PBS/0.5% Tween-20/5% milk, washed thrice and incubated with the primary antibodies (Rabbit anti-RecA, Abcam, ab63797, 1:5,000; Mouse anti-TEM, Abcam, 8A5.A10, 1:1,000) for 2 hours. After washing, membranes were incubated with secondary antibodies (LI-COR Biosciences, 925–68071 IRDye 680RD Goat anti-Rabbit IgG and 925–32210 IRDye 800CW Goat anti-Mouse IgG) at a final dilution of 1:10,000 for 1 hour, washed, then imaged using LI-COR Biosciences.

Plasmid copy number

Copy number of recA and plasmid tnpR were quantified using the QX200 Droplet Digital PCR system (ddPCR, Bio-Rad) as described previously [62]. ddPCR contained 1x EvaGreen super mix (Bio-Rad), and TE79/TE80 (recA, S8 Table) or TE81/TE82 (tnpR, S8 Table). After thermal cycling, data were analysed using the QX200 Droplet Reader with QuantaSoft software (Bio-Rad).

TEM mRNA expression

pbla.1^TEM-1 carrying isolates were sub-cultured to mid-exponential phase in 5 ml GCBL/1% Vitox. Cells were harvested from 1 ml of culture and RNA was extracted using the Qiagen RNeasy Mini Kit together with the RNA protect Reagent (Qiagen, #76506) and on column RNase-free DNase I (Qiagen, #79254) treatment according to the manufacturer’s instruction (protocol RY28). RNA was subsequently reverse transcribed to cDNA using the LunaScript RT SuperMix Kit (NEB, #E3010). The cDNA was used in ddPCRs with primers TE79/80 (recA) and TE97/98 (blaTEM, S8 Table). The no-RT and no template reactions served as negative controls.

Statistics and data analysis

Data analysis was performed in R version 4.1.1 using base R and the tidyverse package [63]. Plots were generated with ggplot2 [64]. A p value <0.05 was considered statistically significant.

Supporting information

S1 Fig. (A) Analysis of the Tn2 deletion region: the truncation affecting tnpR in gonococcal pbla.1 and pbla.2 differ by a single nucleotide.

Schematic representation of tnpR sequences of gonococcal and H. ducreyi pblas is shown on the bottom with the SNP displayed as a white line and the site of the Tn2 truncation in relation to H. ducreyi pbla indicated with a triangle. Displayed above is the nucleotide alignment of gonococcal pbla to H. ducreyi DMC64 pbla.2. (B) Sequence alignment of NEIS2964 alleles -1 to -3) with SNPs highlighted in white.

https://doi.org/10.1371/journal.ppat.1013151.s001

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S2 Fig. pbla.1 mobilisation by pConj.1 in isogenic matings.

(A) pbla.1 mobilisation in the N. gonorrhoeae strains FA1090 and 2086_K (Welch two-sample t-test, p = 0.66). (B) Mobilisation rates of pbla.1 into FA1090 with or without pConj (Welch two-sample t-test, p = 0.87).

https://doi.org/10.1371/journal.ppat.1013151.s002

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S3 Fig. Minimum spanning trees of N. gonorrhoeae clustered by core genome allelic differences using Grapetree [65] with distribution of pbla variants.

Individual dots represent isolates that are coloured according to Ng_cgc₄₀₀ (A) or pbla variant carried (B). Strains used in this study are indicated in panel A.

https://doi.org/10.1371/journal.ppat.1013151.s003

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S4 Fig. (A) Alignment of MobC from E. coli plasmid RSF1010 (Genbank accession: S96966.1) and NEIS2962 from pbla.2 (Genbank accession: NZ_LT591911).

Amino acid sequences were aligned with COBALT [66] and the alignment visualised with Esprit [67]. Identical residues are shown in white on red background, residues with a similarity score >0.7 are framed in blue and the remaining residues are shown in black. (B) Superimposed AlphaFold structure prediction of MobC from the E. coli plasmid RSF1010 (salmon, Genbank accession: S96966.1) and NEIS2962 (blue) dimers (Match Align: 677.7, RMSD: 0.775Å) (C) Electrostatics prediction of NEIS2962 homodimer with oriT sequence using the Adaptive Poisson Boltzman Solver Electrostatics Plugin. Negatively and positively charged regions are shown in red and blue, respectively.

https://doi.org/10.1371/journal.ppat.1013151.s004

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S5 Fig. Cellular levels of different TEM variants in an isogenic FA1090 background.

TEM/RecA ratios of whole cell lysates were visualised by Western blot analysis and quantified with the LI-COR system (one-way ANOVA with Tukey multiple comparisons, n.s. p > 0.05; *** p < 0.001).

https://doi.org/10.1371/journal.ppat.1013151.s005

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S1 Table. Genes in Haemophilus spp. related to pbla.

https://doi.org/10.1371/journal.ppat.1013151.s006

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S2 Table. Genes in H. ducreyi isolates related to pbla.

https://doi.org/10.1371/journal.ppat.1013151.s007

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S3 Table. Examples of gonococcal pbla used for phylogeny.

https://doi.org/10.1371/journal.ppat.1013151.s008

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S4 Table. N. gonorrhoeae isolates used for phylogeny.

https://doi.org/10.1371/journal.ppat.1013151.s009

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S5 Table. Bacterial strains used in this study.

https://doi.org/10.1371/journal.ppat.1013151.s010

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S6 Table. Plasmids used in this study.

https://doi.org/10.1371/journal.ppat.1013151.s011

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S7 Table. Primers used for sequencing.

https://doi.org/10.1371/journal.ppat.1013151.s012

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S8 Table. Primers used for strain construction.

https://doi.org/10.1371/journal.ppat.1013151.s013

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Figures

Abstract

Author summary

Introduction

Results

pbla has been acquired by the gonococcus from Haemophilus

pbla is associated with pConj variants that promote its spread

The immobility of pbla.3 is reflected in its restricted distribution

TEM-135 confers increased penicillin resistance

Successful pbla variants have evolved with reduced fitness costs

Discussion

Methods

Analysis of Haemophilus spp. and Neisseria spp. genomes

Plasmid variants

Phylogenetic analyses

Structure predictions

Bacterial strains/growth

Characterisation of H. ducreyi plasmids

Transformation of gonococci

Plasmid modifications

Conjugation and mobilisation assays

Competition assays to determine fitness costs

Antibiotic susceptibility testing

SDS page and Western blot analysis

Plasmid copy number

TEM mRNA expression

Statistics and data analysis

Supporting information

S1 Fig. (A) Analysis of the Tn2 deletion region: the truncation affecting tnpR in gonococcal pbla.1 and pbla.2 differ by a single nucleotide.

S2 Fig. pbla.1 mobilisation by pConj.1 in isogenic matings.

S3 Fig. Minimum spanning trees of N. gonorrhoeae clustered by core genome allelic differences using Grapetree [65] with distribution of pbla variants.

S4 Fig. (A) Alignment of MobC from E. coli plasmid RSF1010 (Genbank accession: S96966.1) and NEIS2962 from pbla.2 (Genbank accession: NZ_LT591911).

S5 Fig. Cellular levels of different TEM variants in an isogenic FA1090 background.

S1 Table. Genes in Haemophilus spp. related to pbla.

S2 Table. Genes in H. ducreyi isolates related to pbla.

S3 Table. Examples of gonococcal pbla used for phylogeny.

S4 Table. N. gonorrhoeae isolates used for phylogeny.

S5 Table. Bacterial strains used in this study.

S6 Table. Plasmids used in this study.

S7 Table. Primers used for sequencing.

S8 Table. Primers used for strain construction.

References