The origin and evolution of the gonococcal beta-lactamase plasmid, and implications for public health

The origin and evolution of the gonococcal beta-lactamase plasmid, and implications for public health | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The origin and evolution of the gonococcal beta-lactamase plasmid, and implications for public health View ORCID Profile T. A. Elsener , A. Cehovin , C. Philp , K. Fortney , S.M. Spinola , M.C.J. Maiden , C.M. Tang doi: https://doi.org/10.1101/2025.01.03.631176 T. A. Elsener 1 Sir William Dunn School of Pathology, University of Oxford , South Parks Road, Oxford OX1 3RE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for T. A. Elsener A. Cehovin 1 Sir William Dunn School of Pathology, University of Oxford , South Parks Road, Oxford OX1 3RE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site C. Philp 1 Sir William Dunn School of Pathology, University of Oxford , South Parks Road, Oxford OX1 3RE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site K. Fortney 2 Departments of Microbiology and Immunology, Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site S.M. Spinola 2 Departments of Microbiology and Immunology, Medicine 3 and Pathology and Laboratory Medicine 4 Indiana University School of Medicine , Indianapolis, IN, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site M.C.J. Maiden 5 Department of Biology, University of Oxford , 11a Mansfield Road, Oxford OX1 3SZ, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site C.M. Tang 1 Sir William Dunn School of Pathology, University of Oxford , South Parks Road, Oxford OX1 3RE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: christoph.tang{at}path.ox.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Neisseria gonorrhoeae is a leading cause of sexually transmitted infection (STI) and a priority AMR pathogen. Two narrow host range plasmids, p bla and pConj, have contributed to ending penicillin and tetracycline therapy, respectively, and undermine current prevention strategies including Doxy-PEP. Here, we investigated the origin and evolution of the beta-lactamase plasmid, p bla . We show that the interplay between p bla and pConj influences their co-occurrence and the spread of p bla in the gonococcal population. We demonstrate that p bla was acquired by the gonococcus on at least two occasions from Haemophilus ducreyi , and describe the subsequent evolutionary pathways taken by the three major p bla variants. Changes that mitigate fitness costs of p bla and the emergence of TEM beta-lactamases which confer increased resistance have contributed to the success of p bla . In particular, TEM-135, which has arisen in certain p bla variants, increases resistance to beta-lactams and only requires one amino acid change to become an extended spectrum beta-lactamase (ESBL). The evolution of p bla underscores the threat of plasmid-mediated resistance to current therapeutic and preventive strategies against gonococcal infection. Given the close relationship between p bla and pConj, widespread use of Doxy-PEP is likely to promote spread of pConj and p bla , and emergence of plasmid-mediated ESBL in the gonococcus, with dire public health consequences. INTRODUCTION Neisseria gonorrhoeae causes ∼80 million sexually transmitted infections (STIs) annually 4 and is a WHO priority pathogen due to its extensive antimicrobial resistance (AMR) 5 . N. gonorrhoeae has two resistance plasmids, pConj and p bla . These plasmids contributed to the cessation of tetracycline and penicillin therapy, and undermine doxycycline post-exposure prophylaxis (Doxy-PEP) 6 , 7 . pConj and p bla are highly prevalent in low and middle-income countries (LMICs) where syndromic treatment of STIs with doxycycline has been recommended 1 , 7 , 8 . 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 mediate tetracycline resistance 7 , 10 , and can be categorised into seven variants 8 . p bla emerged in the gonococcus in the 1970s and encodes the TEM beta-lactamase conferring penicillin resistance 11 , 12 . p bla TEM beta-lactamases require one or two amino acid changes to become an extended-spectrum beta-lactamase (ESBL) 13 , which would render third-generation cephalosporins, the current first-line treatment, ineffective 14 . p bla usually is closely associated with pConj which can mobilise p bla 8 , 15 . There are three main p bla variants, characterised by distinct gene presence/absence patterns 1 . The 7.4 kb p bla .2 (p bla Asia) has been considered the ancestral plasmid 16 . p bla .1 (5.6 kb, p bla Africa) has a deletion in the replication region, while p bla .3 (5.1 kb, p bla Rio) lacks the region implicated in p bla mobilisation 1 , 16 . Variants of p bla are associated with certain pConj variants and TEM alleles 1 . p bla .1 mostly carries TEM-1 or TEM-1 P14S , while p bla .3 is associated with TEM-135; p bla .2 carries TEM-1 or TEM-135. Importantly, the M182T substitution in TEM-135 is a ‘stepping stone’ mutation before becoming an ESBL 13 , 17 . Here, we investigated evolution and characteristics of the p bla variants. We demonstrate that the spread and distribution of p bla in gonococci results from the dynamic interplay of its intimate association with pConj, fitness costs, and resistance levels. p bla has evolved to avoid fitness costs and confer higher resistance to beta-lactams. Our results underline the threats posed by p bla and pConj, particularly through the widespread implementation of Doxy-PEP. RESULTS p bla has been acquired at least twice by the gonococcus from Haemophilus Haemophilu s spp. harbours plasmids related to p bla 18 , 19 . Therefore, we interrogated Haemophilus whole genome sequences (WGS, 4,620 isolates, 12 species, Supplementary Table 1) for p bla . We searched for Tn 2 as p bla TEM-1b is located on this transposon 20 , 21 , and confirmed the presence of p bla replicons by searching for NEIS2960, NEIS2358, and NEIS2961 1 . Tn 2 is present in 10.2% of Haemophilus influenzae (4,337 isolates), with a p bla -like plasmid only co-occurring in one H. influenzae (PubMLST id: 23482). In contrast, 22.6% of H. ducreyi (7/31 isolates) harbour TEM-1 containing p bla -like plasmids; resequencing of H. ducreyi HD183 and DMC64 22 identified distinct 9.1 and 10.9 kb plasmids, respectively. We aligned p bla .1 and p bla .2 to the H. ducreyi plasmids 22 . p bla .1 and p bla .2 are highly related to the 9.1 and 10.9 kb H. ducreyi plasmids, respectively ( Figure 1A ). Tn 2 is intact in H. ducreyi while gonococcal p bla lack tnpA and have a truncated tnpR, with distinct tnpR alleles on p bla .1 is and p bla .2/3, indicating that p bla .1 and p bla .2 were acquired independently by the gonococcus. Download figure Open in new tab Figure 1: Origin and evolution of p bla. (A) Pairwise alignments of p bla variants in H . ducreyi and N. gonorrhoeae generated with Easyfig 2 . p bla ORFs are coloured according to gene products; yellow, mobilisation proteins; orange, Tn 2 -derived genes including bla TEM; turquoise, replication initiation proteins; dark green, undefined/other gene function. Sequence identity between loci is depicted in shades of grey, as indicated. (B) Maximum likelihood tree of 414 gonococcal p bla sequences with tips coloured according to p bla variant. Circles indicate the tnpR allele and the TEM variant carried. To gain further insights into the evolutionary relationships between p bla variants, we examined a subset of p bla (414 of 2,758, Supplementary Table 2) 1 with the 10.9 kb H. ducreyi p bla as reference; plasmids were from 1979-2022 with the same proportion of variants as the whole population ( i.e. 70% p bla .1, 14% p bla .2, 16% p bla .3) 1 . Maximum likelihood phylogeny distinguished p bla variants into distinct clades, with p bla .1 split from the other variants, and p bla .3 arising from p bla .2 ( Figure 1B ). Taken together, gonococcal p bla .1 and p bla .2 arose by independent acquisition of plasmids expressing TEM-1 from H. ducreyi . These events were associated with distinct truncations of Tn 2 , with subsequent emergence of p bla .3 from p bla .2. p bla is associated with pConj variants that promote its spread To understand the association between p bla and pConj, we examined the transfer of p bla by different pConj variants. Matings were performed between isogenic strains (FA1090 or 2086_K) with Δ pilD donors and recipients to prevent transfer by transformation 23 - 25 . p bla .1 was mobilised at a frequency of ∼1% transconjugants/recipient by pConj.1 in both strains ( Supplementary Figure 1 ). p bla is commonly associated with pConj variants 1, 3, 4 and 5 ( Figure 2A ). Therefore, we evaluated p bla mobilisation by different pConj variants. The conjugation frequencies of pConj.1, 3 and 4 were >79% ( Figure 2B ) but several orders of magnitude lower for pConj.2 and pConj.7 (which are not associated with p bla , Figure 2A ). The rate of p bla mobilisation mirrored conjugation frequencies, with p bla transfer by pConj.2 undetectable ( Figure 2B ), indicating that p bla is associated with pConj variants that mobilise it efficiently, and promote its spread in the gonococcal population. Download figure Open in new tab Figure 2: p bla mobilisation by pConj. (A) Sankey plot of pConj carrying isolates (n=4,883 isolates) 1 , displaying the presence of p bla (left) and co-occurrence of p bla with individual pConj variants (right). (B) Conjugation rates of pConj variants (top) and the mobilisation rates of co-located p bla .1 (bottom). (C) Mobilisation rates of wild type and isogenic p bla variants (p bla iso ) by pConj.1. (D) The impact of single mob gene knockouts in p bla .2 on p bla 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 The restricted distribution of p bla .3 is associated with its immobility There are conflicting data about p bla .3 mobility 26 - 28 . Therefore, we assessed the mobilisation of wild-type p bla variants by pConj.1. Results demonstrate that wild-type p bla .1 and p bla .2 are mobilised efficiently, while p bla .3 mobilisation was not detected ( Figure 2C ). To assess whether p bla variant deletions are responsible for these differences, we introduced variant-specific deletions into p bla .2, generating the isogenic plasmids p bla .1 iso and p bla .3 iso . Mobilisation of the isogenic plasmids did not differ from wild-type plasmids (p=0.82, Figure 2C ), indicating the variant-specific deletions are responsible for mobilisation differences. Of note, the immobility of p bla .3 is evident from its restricted distribution in three related lineages, whilst p bla .1 and p bla .2 are found across the gonococcal population ( Supplementary Figure 2 ) 1 . Next, we examined the genes responsible for the immobility of p bla .3, and generated p bla .2 mutants lacking genes absent in p bla .3. Deletion of mobA (encoding the relaxase 29 ) abolished p bla transfer ( Figure 2D ), indicating that the pConj relaxase cannot recognise p bla oriT . Removal of NEIS2962 significantly reduced p bla mobilisation (p=0.01, Figure 2D ). NEIS2962 is related to MobC from E. coli plasmid RSF1010, which unwinds DNA at the oriT 30 . RSF1010. NEIS2962 homodimers are structurally related to MobC and predicted to recognise the p bla oriT but not a scrambled oriT sequence ( Supplementary Figure 3 ). Deletion of NEIS2964 did not impact p bla transfer. TEM-135 confers increased penicillin resistance Although p bla .3 is immobile, it is prevalent in Ng_cgc 400 s 25 and 298 ( Table 1 , Supplementary Figure 2 ) 1 . This suggests p bla .3, which carries TEM-135, confers a benefit to the gonococcus that has led to the clonal expansion of isolates with this plasmid. Therefore, we measured the penicillin MICs conferred by p bla variants. Whilst TEM-1 carrying p bla .1 and 2 conferred MICs of 8 µg/ml, p bla .3 with TEM-135 conferred a significantly higher MIC (32 µg/ml, p=0.003, Figure 3A ). To establish whether the TEM variant determines resistance levels, we changed p bla .3 TEM-135 into TEM-1 by introducing a T182M substitution. This substitution reduced the p bla .3 MIC to levels of p bla .1/p bla .2, demonstrating that TEM-135 confers elevated MICs ( Figure 3B ). We also compared resistance conferred by TEM-1, TEM-1 P14S and TEM-135 (which together account for >95% of gonococcal TEMs 1 ) expressed by p bla .2. Again, TEM-135 significantly increased MICs (128 µg/ml vs. 8 µg/ml with TEM-1, p<0.001, Figure 3C ). Download figure Open in new tab Figure 3: TEM-135 increases the MIC. (A) Penicillin G MICs of p bla variants in FA1090 isogenic strain background (one-way ANOVA on log 2 -transformed MIC values with Tukey multiple comparisons of means; *** p<0.001). (B) MICs of TEM-1 in different p bla variant backbones. (C) MICs of different TEM variants in p bla .2 backbone (one-way ANOVA on log 2 -transformed MIC values; *** p<0.001). (D) Representative Western blot of cellular enzyme levels of different TEM variants. View this table: View inline View popup Download powerpoint Table 1: pbla prevalence in different Ng_cgc400. pbla-carrying lineages with >50 isolates and >5 isolates with pbla were assessed for their % pbla carriage. To understand 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 ( Figure 3D , Supplementary Figure 4 ), consistent with increased stability of TEM-135 17 . In conclusion, the appearance of TEM-135, particularly associated with p bla .3, provides a significant benefit to the gonococcus by enhancing resistance against beta-lactams, with MICs correlating with cellular TEM levels. p bla has evolved with reduced fitness costs Plasmids often impose fitness costs, disadvantaging isolates which carry plasmids 31 . We therefore assessed the fitness costs of p bla by introducing p bla .1 into isolates from a range of lineages, and competing plasmid-carrying vs. plasmid-free strains. p bla .1 had no detectable fitness cost in any isolate ( Figure 4A ), consistent with its continued prevalence in the gonococcus ( Figure 4B ). Download figure Open in new tab Figure 4: Impact of p bla -imposed fitness cost on its prevalence in the population. (A) Fitness cost (w) of p bla .1 in clinical isolates from different p bla -free (grey) or p bla -associated lineages (turquoise, p bla .1-associated; dark green, p bla .1/p bla .2-associated; yellow, p bla .3-associated). w>1 indicates a benefit, whereas w<1 signifies a cost of plasmid carriage. (B) Proportional p bla carriage in gonococcal isolates deposited on PubMLST between 2010 and 2019 (n=12,914 isolates). Colours show p bla variant carried and numbers above bars indicate the number of samples in the respective year. (C) Fitness cost of p bla 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 p bla in FA1090 isogenic strain background was assessed by ddPCR (one-way ANOVA with Tukey multiple comparisons; *** p<0.001). (E) p bla carriage in isolates from the p bla .1/p bla .2-associated Ng_cgc 400 29 between 2011 and 2019 (n=433 isolates). Bar colours indicate p bla variant and numbers above the bars specify the number of samples in the respective year. In contrast to p bla .1 and p bla .3, p bla .2 inflicts a significant fitness cost ( Figure 4C ), and has a higher copy number (>6 vs . 1-2 plasmids/chromosome, Figure 4D ). This could explain the decreasing prevalence of p bla .2 over time seen across all available WGS ( Figure 4B ). We also examined the prevalence of p bla .1 and p bla .2 within a single lineage (Ng_cgc 400 29 which harbours both variants) to account for potential sampling bias. Between 2010 and 2020, there has been a shift from p bla .2 to p bla .1 in this lineage ( Figure 4E ). Further evidence of relative success of the p bla variants is revealed by their abundance within a lineage. p bla .1 is prevalent in lineages, whilst p bla .2 is only present at low frequency in lineages ( Table 1 ), reflecting its fitness costs and a lack of expansion of plasmid-carrying isolates within a lineage. Taken together, fitness costs imposed by p bla .2 are consistent with its low prevalence in the gonococcal population compared with p bla .1. p bla .3 with TEM-135, which evolved from p bla .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 32 . Here, we investigated the origin of the beta-lactamase plasmid p bla which is largely restricted to the gonococcus, a WHO priority pathogen. Our analysis indicates that p bla was acquired by N. gonorrhoeae at least twice from Haemophilus . Interestingly, H. ducreyi strains are divided into two clades, which diverged ∼1.9 million years ago 33 ; p bla. 1- and p bla. 2-like plasmids are in Class I and Class II isolates, respectively. Thus, the independent acquisition of p bla .1 and p bla .2 by the gonococcus likely reflects separate events involving the two clades. Both intergenic transfers were accompanied by truncation of Tn 2 , suggesting the transposase is disadvantageous in the gonococcus. 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 34 . However, ∼3.5% of men with chancroid also have urethritis 34 . Therefore, these species can occupy the same niche, providing opportunities for gene transfer. Interestingly, p bla carriage is negligible in H. influenzae and N. meningitidis which inhabit the nasopharynx. p bla was not detected in any non-invasive Neisseria spp. (41,158 isolates, 30 species), and in only three of 39,372 N. meningitidis isolates in distinct lineages (ST-5, ST-11 and ST-32 complexes). This could reflect the renal excretion of beta-lactams 35 , favouring p bla carriage for bacteria inhabiting the urogenital tract compared with other sites. Of note, the meningococcal urethritis clade (NmUC) evolved from ST-11 N. meningitidis by acquiring genetic elements (but not p bla ) from N. gonorrhoeae 36 , 37 . We found p bla in an ST-11 meningococcal isolate, suggesting p bla could appear in NmUC. Plasmids can be successful in bacterial populations by spreading into diverse lineages and/or through clonal expansion of plasmid-carrying isolates. We found that p bla is associated with pConj variants that can effectively spread p bla through the gonococcal population. Given the higher rates of pConj conjugation (>75%) compared with p bla mobilisation (∼1%), the spread of p bla into a lineage is likely to be accompanied by pConj, maintaining the close association between these plasmids. The most frequent and widespread p bla variant, p bla .1, does not impose fitness costs and is mobilised efficiently by common pConj variants such as pConj.1. p bla .2 is also mobile but imposes fitness costs. Compared with p bla .1, p bla .2 has a second replication initiation protein and additional origins of replication ( ori2 and ori3 ) 1 , 38 . This might explain differences in copy number and fitness costs of p bla .1 and p bla .2; plasmid Rep proteins can sequester host DNA replication machinery 31 , causing fitness costs. We found that p bla .2 is present in lower prevalence in lineages than p bla .1, with evidence of a shift from p bla .2 to p bla .1 in a single lineage (Ng_cgc 400 29) over time. These observations are consistent with strains carrying p bla .2 being outcompeted by plasmid-free isolates or those with other p bla variants. p bla .3-associated lineages have undergone clonal expansion indicating its successful adaptation to the gonococcus. Phylogenetic analysis indicates that TEM-135 originally arose in p bla .2. However, despite increased resistance levels conferred by TEM-135, the fitness cost of p bla .2 has likely undermined the success of TEM-135 in this p bla variant. We found that p bla .3 evolved from TEM-135 carrying p bla .2 through gene loss. This prevented the plasmid from being mobile, but with the trade-off of avoiding fitness costs. Consequently, p bla .3 has not spread in the gonococcal population but promoted the expansion of group of related lineages, likely though the increased beta-lactam resistance associated with TEM-135. In summary, since emergence of gonococcal p bla 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 1 , 8 , especially in p bla .3. The intimate relationship between p bla and pConj highlights the threat posed by increased use of tetracyclines, as already witnessed in LMICs where gonococci have remarkably high plasmid carriage 7 , 39 . Similarly, the widespread implementation of Doxy-PEP will likely increase pConj and consequently p bla carriage, and promote the appearance of ESBL-expressing p bla with minimal fitness costs. This threatens treatment of cases and their contacts with third-generation cephalosporins, undermining the use of current first-line antibiotics used to control this important human pathogen. METHODS Analysis of Haemophilus spp. and Neisseria spp. genomes Tn 2 -carrying isolates of Haemophilus spp. and Neisseria spp. were identified querying Tn 2 (GenBank accession: LC091537.1 ) against Haemophilus (accessed 24/07/2024, 4,640 isolates, 12 species) and Neisseria (accessed 24/07/2024, 41,158 isolates, 33 species) sequences in PubMLST 40 (blastN word size: 11, scoring: reward: 2; penalty: -3; gap open: 5; gap extend: 2, sequence identity >99%, alignment length >50% query). p bla -like plasmids were confirmed by the presence of NEIS2960 (sequence identity >80%; alignment length >50%) and NEIS2358, and NEIS2961 1 . Plasmid variants p bla and pConj were analysed in 15,532 gonococcal WGS (accessed 28/07/2022) 1 , 40 with isolates from 1928-2022 and 66 countries. NEIS2220 indicated the presence of pConj, and its variants defined as previously 8 . Where the Ng_cp 5 could not be assigned, plasmids were clustered with GrapeTree 41 , and variants assigned manually. p bla variants were typed using the Ng_p bla ST scheme 1 . For the population structure, isolates were grouped into core genome clusters according to the allelic profile of 1,668 core genes 42 ; isolates were grouped with a cut-off of 400 allelic differences (Ng_cgc 400 ). Phylogenetic analyses A subset of 414 p bla- carrying isolates conserving the ratio of p bla variants (70% p bla .1, 14% p bla .2, 16% p bla .3, Supplementary Table 2) 1 was selected to investigate the phylogenetic relationship of p bla variants. This included all p bla -containing isolates pre-dating 2000 (n=35). Isolates between 2000 and 2022 (n=379) were randomly selected using the r sample function 1 . Snippy v4.6.0 mapped plasmid reads to H. ducreyi DMC64 p bla (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 43 with 100 bootstrap replicates, rooted at H. ducreyi DMC64 p bla , and visualised with ape 44 and ggtree 45 , 46 . Structure predictions Analysis of NEIS2962 and RSF1010 MobC (GenBank accession: S96966.1 ) homodimers and NEIS2962 with p bla oriT 29 were performed using AlphaFold 3 47 and PyMol v2.5.4 48 . Charge distributions were visualised with the Adaptive Poisson-Boltzmann Solver (APBS) electrostatics tool 49 . Bacterial strains/growth Strains and plasmids used in this study are listed in Supplementary Tables 5 and 6, respectively. E. coli DH5α 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) 50 supplemented with 1% Vitox (Oxoid) at 37°C in 5% CO 2 . H. ducreyi was grown on chocolate agar plates supplemented with 1% IsoVitaleX at 35°C in 5% CO 2 . 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. Transformation of gonococci For electroporation, bacteria grown on GCB agar were resuspended in PBS (Sigma), adjusted to 5x10 7 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. Δ pilD :: ermC and Δ pilD :: aph constructs were transformed into N. gonorrhoeae as described previously 23 , 25 . 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 modification To generate p bla .1 iso and p bla .3 iso , p bla .2 was cut with Hin dIII-HF and Pvu II-HF (NEB), respectively. p bla .1 iso was amplified with primers TE18/19 and PrimeSTAR GXL polymerase (Takara Bio); Gibson assembly was performed with primers TE20/21. p bla .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 α . Point mutations in bla TEM were introduced using the RAIR method 51 . PCRs with primers (TE56/TE57, p bla .2 TEM-135; TE63/TE64, p bla .2 TEM-1 P14S ; TE65/TE66, p bla .3 TEM-1 ) were performed using Herculase II polymerase (Agilent), with p bla .2 or p bla .3 as template. Products were purified (Promega Wizard PCR Clean-up) and transformed into E. coli DH5 α . 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 (Δ pilD :: ermC ) and recipient (Δ pilD :: aph ) strains grown overnight were inoculated in 5 ml GCBL/1% Vitox at an OD 600 of 0.1 and grown to mid-exponential phase (OD 600 0.6 - 0.8). The bacterial density was adjusted to 10 8 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% CO 2 , 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). Competition assays Plasmids were introduced into FA1090 Δ pilD :: ermC and competed against FA1090 Δ pilD :: aph (n=4). Bacteria in PBS were adjusted to an OD 600 1, mixed 1:1, diluted to 10 5 CFU/ml and added to 200 μl Fastidious Broth 52 then grown at 37°C, 5% CO 2 , 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 p bla + vs . p bla - strains; N i and N f , p bla - strain at the beginning/end, respectively; N i , pbla and N f , pbla , same for p bla + strain). Antibiotic susceptibility testing Penicillin G MICs were assessed using the broth microdilution method 53 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 10 5 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 (Bio-Rad) as described previously 54 . ddPCR contained 1x EvaGreen super mix (Bio-Rad), and TE79/TE80 ( recA ) or TE81/TE82 ( tnpR ). After thermal cycling, data were analysed using the QX200 Droplet Reader with QuantaSoft software (Bio-Rad). Statistics and data analysis Data analysis was performed in R version 4.1.1 using base R and the tidyverse package 55 . Plots were generated with ggplot2 56 . A p value <0.05 was considered statistically significant. SUPPLEMENTARY FIGURES Download figure Open in new tab Supplementary Figure 1: p bla. 1 mobilisation in isogenic matings with the N. gonorrhoeae strains FA1090 and 2086_K (Welch two-sample t-test, p=0.66). Download figure Open in new tab Supplementary Figure 2: Minimum spanning trees of N. gonorrhoeae clustered by core genome allelic differences with distribution of p bla variants. Individual dots represent isolates that are coloured according to Ng_cgc 400 (A) or p bla variant carried (B). Download figure Open in new tab Supplementary Figure 3: (A) Alignment of MobC from E. coli plasmid RSF1010 (Genbank accession: S96966.1 ) and NEIS2962 from p bla .2 (Genbank accession: NZ_LT591911). Amino acid sequences were aligned with COBALT 3 and the alignment visualised with ESprit 9 . 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. Download figure Open in new tab Supplementary Figure 4: 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). REFERENCES ↵ Elsener , T. A. et al. There are three major Neisseria gonorrhoeae beta-lactamase plasmid variants which are associated with specific lineages and carry distinct TEM alleles . 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