Genomic Insights into Ceftazidime Resistance in Burkholderia pseudomallei: Discovery of A172T Mutation, and Palindromic GC-Rich Repeat Sequences Facilitating penA Duplication and Amplification.

Genomic Insights into Ceftazidime Resistance in Burkholderia pseudomallei: Discovery of A172T Mutation, and Palindromic GC-Rich Repeat Sequences Facilitating penA Duplication and Amplification | 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 Genomic Insights into Ceftazidime Resistance in Burkholderia pseudomallei : Discovery of A172T Mutation, and Palindromic GC-Rich Repeat Sequences Facilitating penA Duplication and Amplification View ORCID Profile Apichai Tuanyok , Chie Nakajima , Tiernan Noll , Md. Siddiqur Rahman Khan , Pacharapong Khrongsee , View ORCID Profile Charles A. Yowell , Yu-Ping Xiao , Vanaporn Wuthiekanun , View ORCID Profile Narisara Chantratita , Henry Heine , View ORCID Profile Kuttichantran Subramaniam , View ORCID Profile Yasuhiko Suzuki , View ORCID Profile Direk Limmathurotsakul , Ayalew Mergia doi: https://doi.org/10.1101/2025.02.11.637714 Apichai Tuanyok 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA 2 Emerging Pathogens Institute, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Apichai Tuanyok For correspondence: tuanyok{at}ufl.edu Chie Nakajima 3 Hokkaido University International Institute for Zoonosis Control , Sapporo, Hokkaido, 001-0020 Japan 4 Hokkaido University Institute for Vaccine Research and Development , Sapporo, Hokkaido, 001-0021 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tiernan Noll 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Md. Siddiqur Rahman Khan 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA 2 Emerging Pathogens Institute, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pacharapong Khrongsee 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA 2 Emerging Pathogens Institute, University of Florida , Gainesville, FL, USA 5 Faculty of Veterinary Science, Prince of Songkla University , Hatyai, Songkhla, Thailand Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charles A. Yowell 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Charles A. Yowell Yu-Ping Xiao 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vanaporn Wuthiekanun 6 Mahidol–Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University , Bangkok, Thailand Find this author on Google Scholar Find this author on PubMed Search for this author on this site Narisara Chantratita 6 Mahidol–Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University , Bangkok, Thailand 7 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University , Thailand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Narisara Chantratita Henry Heine 8 Institute for Therapeutic Innovation, Department of Medicine, College of Medicine, University of Florida , USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kuttichantran Subramaniam 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA 2 Emerging Pathogens Institute, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kuttichantran Subramaniam Yasuhiko Suzuki 3 Hokkaido University International Institute for Zoonosis Control , Sapporo, Hokkaido, 001-0020 Japan 4 Hokkaido University Institute for Vaccine Research and Development , Sapporo, Hokkaido, 001-0021 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yasuhiko Suzuki Direk Limmathurotsakul 6 Mahidol–Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University , Bangkok, Thailand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Direk Limmathurotsakul Ayalew Mergia 1 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Ceftazidime (CAZ) resistance in Burkholderia pseudomallei , the causative agent of melioidosis, complicates treatment in endemic regions. This study identified a novel A172T mutation and other known penA mutations as critical contributors to CAZ resistance in a large Thai strain collection. Frequent gene duplication and amplification (GDA) of penA , likely driven by Palindromic GC-Rich Repeat Sequences (PGCRRS), highlights the urgent need for rapid diagnostics and optimized treatment strategies to manage this life-threatening disease effectively. TEXT The growing threat of antimicrobial resistance in B. pseudomallei demands a deeper understanding of the mechanisms underlining ceftazidime (CAZ) resistance. In this study, we analyzed a collection of 58 strains, isolated from 24 patients who experienced treatment failures in Northeast Thailand over two decades, 1987 to 2007 ( 1 ). These strains were specifically chosen due to their documented Etest results indicating resistance to CAZ and amoxicillin-clavulanic acid (AMC), which developed during or after treatment ( Table 1 ). To identify the genetic factors contributing to this resistance, we employed next-generation sequencing (NGS) on the Illumina MiSeq platform as previously described ( 2 ), followed by a detailed analysis of the sequencing data using the BWA-MEM algorithm ( 3 ). Genomic alignments were conducted against the reference genome of B. pseudomallei K96243, and the data were visualized using the Artemis genome browser ( 4 ). De novo assembly of each genome was also generated using SPAdes genome assembler (Galaxy Version 3.12.0+galaxy1) or BV-BRC ( http://bv-brc.org ). Sequencing data are available through GenBank (accession no. PRJNA1196838). View this table: View inline View popup Table 1. Details of B. pseudomallei strains used in this study, MICs, and penA mutations. Our findings revealed a complex landscape of genetic alterations associated with CAZ and AMC resistance. We identified eight distinct amino acid substitutions (AAS) in the penA gene, which encodes a class A β-lactamase enzyme known to confer resistance to β-lactam antibiotics, especially CAZ and AMC ( 5 - 7 ) based on Etest results ( Table 1 ). Among these substitutions, five were known to be responsible for CAZ, AMC, or imipenem (IMP) resistance ( Fig. 1 ), while three were novel AAS mutations, I139M, P145L, and A172T. We observed that P174L, the most recently reported AAS mutation associated CAZ resistance in Hainan, China ( 8 ), was not present in our strains. Among the novel mutations, only the A172T (Alanine to Threonine) substitution near 166 ExxLN 170 , one of the Ambler’s motifs ( 9 ) in B. pseudomallei strain 490f showed a particularly strong association with increased minimal inhibitory concentrations (MICs) for CAZ. This mutation was absent in the CAZ-susceptible strain 490b, which was isolated three weeks earlier from the same patient during the hospitalization. To confirm whether this mutation was responsible for the increased CAZ MIC, allelic exchange mutagenesis was performed to introduce the A172T mutant in penA gene of B. pseudomallei Bp82, a biosafe and CAZ-susceptible strain ( 10 , 11 ); see Supplemental Text 1. The resulting mutant exhibited a 16-fold-increase in CAZ MIC, confirming the mutation’s critical role in resistance. Download figure Open in new tab Fig. 1. Amino acid substitution (AAS) mutations in PenA and in a promoter region known to be associated with amoxiclav resistance (AMC r ), imipenem resistance (IMP r ), or ceftazidime resistance (CAZ r ). A172T is a novel AAS mutation responsible for CAZ resistance identified and characterized in this study. In addition to AAS mutations, we identified a promoter-up mutation, specifically the - 78A mutation ( 12 ), of the penA gene in the CAZ-resistant B. pseudomallei strains isolated from 10 (41.6%) of the 24 patients. This mutation is known to enhance the expression of penA , thereby increasing the production of the β-lactamase enzyme and contributing to the observed resistance. Another major resistance mechanism identified was gene duplication and amplification (GDA) of the penA gene, observed in 12 CAZ-resistant strains from 10 (41.7%) of the 24 patients analyzed ( Fig. 2 ). Digital droplet PCR assays (Bio-Rad QX200™ Droplet Digital™ PCR System) using genomic DNA from B. pseudomallei K96243 as a single copy penA control confirmed that penA gene copies varied from two to nine among these strains, suggesting that penA GDA significantly increased resistance by enhancing beta-lactamase enzyme production as previously reported by us ( 2 ). In most GDA events, the junction sequences, ranging from 5 to 16 bp in length and enriched in GC content, likely resulted from homologous recombination between cruciform structures, such as stem-loop cruciform or four-way junction cruciform sequences, found at both ends of the GDA region as exemplified by strain 405a ( Fig. 3 ). We termed this genetic structure “Palindromic GC-Rich Repeat Sequences” (PGCRRS), as it appeared to mediate the homologous recombination of the GDA. We hypothesize that these cruciform structures induce replication stress in B. pseudomallei under CAZ selection, leading to double-strand breaks and complicating the DNA repair processes, thereby contributing to GDA. GDA also resulted in extra copies of variable genomic regions. For example, two copies of genes BPSS0935 – BPSS1017 were found in strain 533eii, while nine copies of genes between BPSS0935 – BPSS0949 were observed in strains 577cii and 577d ( Fig. 2 ). To confirm the GDA, initially identified in Illumina assembly contigs, we utilized the Oxford Nanopore Technologies (ONT) sequencing platform, which offers longer reads to effectively cover the GDA’s junctions. This approach confirmed the junction sequences and genomic regions affected by the GDA in three selected strains: 490f, 533a, and 942dii (BioSample accession numbers SAMN45667634, SAMN45667635, and SAMN45667647, respectively). The GDA events in all 12 strains reported in this study are described in Supplemental Table 1. The addition of ONT sequencing and the hybrid genomic assembling approach for strain 490f, as an example, are described in Supplemental Text 2. Download figure Open in new tab Fig. 2. The gene duplication and amplification (GDA) of the penA gene, highlighting affected genomic regions observed from mapping Illumina short reads of 12 CAZ-resistant B. pseudomallei strains against chromosome 2 of the reference B. pseudomallei K96243. Amplified regions, indicated by peaks in read depth, with the penA highlighted in red. Download figure Open in new tab Fig. 3. An example of gene duplication and amplification (GDA) of penA mediated by Palindromic GC-Rich Repeat Sequences (PGCRRS). Panel A : Genomic region containing penA and its neighboring genes, observed by mapping Illumina short reads from B. pseudomallei 405a against chromosome 2 of the reference strain K96243. Panel B : Hypothetical model illustrating GDA mediated by the recombination of the PGCRRS features, including: (a) a cruciform stem-loop structure, (b) a cruciform four-way junction structure, and (c) a 14-bp sequence resulting from the recombination between (a) and (b). Note: The genome coordinates shown correspond those of chromosome 2 of B. pseudomallei K96243. Additionally, we have observed significantly higher MICs for CAZ or AMC in strains that contained an AAS mutation in combination with the -78A promoter-up mutation and/or GDA. Notable, in strain 577ci, the presence of C69Y mutation occurring against the background of the S72F mutation within the Ambler’s motif 70 SxxK 73 in an earlier strain 577b, shifted the resistance phenotype from AMC to CAZ. This finding suggests that modifications to PenA’s active site may influence substrate specificity. Further investigation using the artificial intelligence - driven crystal structure analysis and molecular docking approach is warranted to elucidate the underlying mechanisms. However, in this current study, we were unable to determine the genetic or molecular basis of CAZ resistance in strain 4609e from patient #24. The penA and other CAZ resistance - associated genes, including penicillin-binding protein 3 ( BPSS1219 ) ( 13 ) in this strain were identical to those in the earlier CAZ-susceptible strain 4609a from the same patient. This observation underscores the need for additional research to identify alternative mechanisms contributing to CAZ resistance in such cases. On another note, multi-locus sequence typing (MLST) analysis revealed that in most patients, initial isolates and resistant strains shared the same sequence types (STs), except in three patients ( Table 1 : patient #14, #17, and #18), the resistant strains had different STs from those initially identified at admission. This suggests that the resistant strains arose from distinct B. pseudomallei subpopulations that were not detected in the initial samples. Given the genetic diversity of B. pseudomallei in soil as previously described ( 14 ), it is possible that these patients were exposed to multiple strain genotypes. These results highlight the genetic heterogeneity of B. pseudomallei and the potential for multiple subpopulations to contribute to treatment failures, complicating efforts to manage CAZ resistance in clinical settings. In conclusion, our findings provide critical insights into the genetic and molecular basis of CAZ resistance in B. pseudomallei . The identification of the novel A172T mutation and other previously known AAS mutations, the - 78A promoter-up mutation, and the frequent occurrence of penA GDA events significantly advance our understanding of how this pathogen adapts under antibiotic pressure. Additionally, penA -mediated CAZ resistance appears to be increasing, as reported in multiple recent studies ( 8 , 15 ). These findings from us and others emphasize the importance of developing rapid diagnostic assays that can detect these genetic alterations, guiding more effective treatment decisions in clinical practice. Implementing these insights could lead to improved management of melioidosis, ultimately reducing the morbidity and mortality associated with this challenging disease. Future research should focus on developing tools to monitor CAZ resistance in clinical settings in real-time and exploring potential therapeutic strategies that target these resistance mechanisms. Moreover, public health initiatives aimed at optimizing antibiotic use in melioidosis treatment could help mitigate the treatment failure, preserving the efficacy of CAZ and other critical antibiotics in managing this deadly disease. Supplemental Text 1 To generate a single base A172T substitution in PenA (BP1026B_II1037), we modified a technique from López CM et al., 2009 [1]. Briefly, approximately 650 base pairs upstream and 700 base pairs downstream of the target nucleotide were amplified using the following four primers in a single PCR reaction. These primers included: penA A172T 5’_fwd: 5’-GCTGAACACGACGCTGCCCGGCGACGAG-3’, penA A172T 5’_rev: 5’-CGGGCAGCGTCGTGTTCAGCTCAGGCTC-3’, penA A172T 3’_fwd: 5’-GCTGAACACGACGCTGCCCGGCGACGAG-3’, and penA A172T 3’_rev: 5’-GGGATAACAGGGTAATCCCGATACCGGCATCGTTTCGCTGCG-3’. The PCR fragments were then gel purified using the Zymoclean Gel DNA Recovery Kit. To construct the pExKm5-A172T PenA plasmid, the pExKm5 plasmid was digested using EcoRI -HF and NotI (New England Biolabs, NEB) and gel purified alongside the PCR products. Note: The plasmid pExKm5 was kindly provided by Dr. Schweizer at the University of Florida. The digested plasmid and PCR fragments were assembled using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, NEB), following the manufacturer’s protocol. The assembled plasmid was then transformed into DH5α E. coli on LB plates supplemented with 50 μg/ml of kanamycin (Km) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). White colonies were selected for plasmid amplification. B. pseudomallei Bp82 electrocompetent cells were prepared in-house by washing three times with 300 mM sucrose, as described by Choi KH et al., 2005 [2]. Two microliters of purified plasmid were electroporated into 100 μL of competent cells using a 0.2 mm cuvette (Bio-Rad) at 2500 V, 200 Ω. The transformed cells were recovered in LB broth for 2 hours with slow shaking, then plated on LB plates containing 250 μg/mL Km and 50 μg/mL X-Gluc for 48 hours. A blue Km-resistant colony was picked, and the merodiploid was resolved on 15% sucrose YT agar supplemented with 0.8 μg/mL adenine for 48 hours. White colonies were selected on LB adenine agar supplemented with 32 μg/ml ceftazidime. The resistant colonies were then screened by PCR using penA A172T 5’_fwd and penA A172T 3’_rev primers, followed by amplicon sequencing to confirm the mutation. Minimal inhibitory concentration (MIC) of ceftazidime against a PCR positive clone (CAY2) using a test strip (Liofilchem™ MTS™ Ceftazidime [CAZ] 0.016-256 μg/mL). The MIC for the mutant strain was determined as 32 ug/mL, while Bp82 had an MIC of 1.5-2 μg/mL (see below). Whole genome sequencing by Illumina was used to confirm the presence of A172T allele in penA of the mutant CAY2 (BioSample# SAMN46427263). Download figure Open in new tab Supplemental Text 2 The genome of B. pseudomallei strain 490f was sequenced using a hybrid approach combining long-read Oxford Nanopore Technologies (ONT) and short-read Illumina sequencing. Briefly, the bacteria were cultured in LB broth at 37°C with 250 rpm shaking overnight. DNA was extracted using the Promega’s Wizard® Genomic DNA Purification Kit according to the manufacturer’s instructions. Oxford Nanopore Sequencing ONT sequencing was conducted on the GridION platform using an R10.4.1 flow cell and the PCR-free ONT Ligation Sequencing Kit (SQK-NBD114.24) in combination with the NEBNext® Companion Module (E7180L). The sequencing yielded 157,168 reads with an average read length of 3,165 bp. Reads were filtered for a minimum length of 2,000 bp using SeqKit v2.4.0 [1], resulting in 69,268 reads with an average read length of 5,629 bp. Genome assembly was performed with Canu v2.2 [2], producing two circular chromosome scaffolds with an average coverage of 57.46x. Unless otherwise noted, default parameters were used for all software. Illumina Sequencing Illumina libraries were prepared using the Illumina DNA Prep Kit and NEBNext® Multiplex Oligos for Illumina (dual-indexed primers), targeting a 280-bp insert size. Paired-end sequencing (2 × 151 bp) was performed on the Illumina NextSeq platform, generating 18,124,202 reads. Quality control and adapter trimming were performed with bcl-convert1 v4.2.4. Reads were aligned to the ONT-derived genome scaffolds using Bowtie2 v2.4.2 [3]. Pilon v1.23 [4] was used to polish the draft assembly, resulting in a final average coverage of 749x. Repeat sequence correction Initial analysis of the polished sequences in CLC Genomics Workbench v20.0.4 revealed a threefold increase in read coverage of the long reads at coordinates 2.1–2.3 Mb on chromosome 2. A 150-kb region from this area was extracted and manually re-aligned to improve mapping accuracy. To resolve this, ambiguous bases (N) were added upstream and downstream of the sequence, followed by re-mapping. There was a subset of three identical repeat sequences, two of which had unique sequence endings that aligned upstream and downstream of the 150-kb region. The three repeat regions were then internally connected each other. These adjustments ensured proper assignment of the three repeat regions. ACKNOWNLEDGEMENTS This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP24wm0125008 and JP243fa627005; CRDF Global Grant OISE-9531011; and the Wellcome Trust [220211/Z/20/Z]. A.T. and C.N. received the US-Japan Cooperative Medical Sciences Programs (USJCMSP) Collaborative Award. T.N. was supported by The Lisa Conti Florida Veterinary Scholars Fund and the Linda F Hayward Florida Veterinary Scholars Program. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. REFERENCES 1. ↵ Wuthiekanun V , Amornchai P , Saiprom N , Chantratita N , Chierakul W , Koh GC , et al. Survey of antimicrobial resistance in clinical Burkholderia pseudomallei isolates over two decades in Northeast Thailand . Antimicrobial agents and chemotherapy . 2011 ; 55 ( 11 ): 5388 – 91 . OpenUrl Abstract / FREE Full Text 2. ↵ Chirakul S , Somprasong N , Norris MH , Wuthiekanun V , Chantratita N , Tuanyok A , et al. Burkholderia pseudomallei acquired ceftazidime resistance due to gene duplication and amplification . Int J Antimicrob Agents . 2019 May ; 53 ( 5 ): 582 – 8 . OpenUrl CrossRef PubMed 3. ↵ Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM . arXiv . 2013 (preprint arxiv: 1303.3997 ). 4. ↵ Carver TJ , Rutherford KM , Berriman M , Rajandream MA , Barrell BG , Parkhill J. ACT: the Artemis Comparison Tool . Bioinformatics . 2005 Aug 15; 21 ( 16 ): 3422 – 3 . OpenUrl CrossRef PubMed Web of Science 5. ↵ Tribuddharat C , Moore RA , Baker P , Woods DE . Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition . Antimicrobial agents and chemotherapy . 2003 ; 47 ( 7 ): 2082 – 7 . OpenUrl Abstract / FREE Full Text 6. Sarovich DS , Price EP , Von Schulze AT , Cook JM , Mayo M , Watson LM , et al. Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia . PLoS One . 2012 ; 7 ( 2 ): 21 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Bugrysheva JV , Sue D , Gee JE , Elrod MG , Hoffmaster AR , Randall LB , et al. Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis . Antimicrobial agents and chemotherapy . 2017 Jun ; 61 ( 6 ). 8. ↵ Yao J , Zhang Z , Tian S , Luo N , Tan J , Zhang Y , et al. Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay . Microbiology Spectrum . 2023 ; 11 ( 6 ): e01125 – 23 . OpenUrl 9. ↵ Ambler RP , Coulson AF , Frere JM , Ghuysen JM , Joris B , Forsman M , et al. A standard numbering scheme for the class A beta-lactamases . Biochemistry . 1991 ; 276 ( Pt1 ): 269 - 70 . OpenUrl CrossRef 10. ↵ Propst KL , Mima T , Choi KH , Dow SW , Schweizer HP . A Burkholderia pseudomallei delta-purM mutant is a avirulent in immune competent and immune deficient animals: candidate strain for exclusion from select agent lists . Infection and immunity . 2010 ; 78 : 3136 – 43 . OpenUrl Abstract / FREE Full Text 11. ↵ Lopez CM , Rholl DA , Trunck LA , Schweizer HP . Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei . Appl Environ Microbiol . 2009 ; 75 : 6496 – 503 . OpenUrl Abstract / FREE Full Text 12. ↵ Chirakul S , Norris MH , Pagdepanichkit S , Somprasong N , Randall LB , Shirley JF , et al. Transcriptional and post-transcriptional regulation of PenA ß-lactamase in acquired Burkholderia pseudomallei ß-lactam resistance . Sci Rep . 2018 Jul 13; 8 ( 1 ): 10652 . OpenUrl CrossRef PubMed 13. ↵ Chantratita N , Rholl DA , Sim B , Wuthiekanun V , Limmathurotsakul D , Amornchai P , et al. Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei . Proceedings of the National Academy of Sciences of the United States of America . 2011 ; 108 ( 41 ): 17165 – 70 . OpenUrl Abstract / FREE Full Text 14. ↵ Roe C , Vazquez AJ , Phillips PD , Allender CJ , Bowen RA , Nottingham RD , et al. Multiple phylogenetically-diverse, differentially-virulent Burkholderia pseudomallei isolated from a single soil sample collected in Thailand . PLoS neglected tropical diseases . 2022 Feb ; 16 ( 2 ): e0010172 . OpenUrl 15. ↵ Fen SHY , Tandhavanant S , Phunpang R , Ekchariyawat P , Saiprom N , Chewapreecha C , et al. Antibiotic susceptibility of clinical Burkholderia pseudomallei isolates in northeast Thailand during 2015-2018 and the genomic characterization of ß-lactam-resistant isolates . Antimicrobial agents and chemotherapy . 2023 May 1; 95 ( 5 ). Reference 1. López C.M. ; Rholl , D.A. ; Trunck , L.A. ; Schweizer , H.P. Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia Pseudomallei . Appl. Environ. Microbiol . 2009 , 75 , 6496 – 6503 , doi: 10.1128/AEM.01669-09 . OpenUrl Abstract / FREE Full Text 2. Choi , K.-H. ; Kumar , A. ; Schweizer , H.P. A 10-Min Method for Preparation of Highly Electrocompetent Pseudomonas Aeruginosa Cells: Application for DNA Fragment Transfer between Chromosomes and Plasmid Transformation . J. Microbiol. Methods 2006 , 64 , 391 – 397 , doi: 10.1016/j.mimet.2005.06.001 . OpenUrl CrossRef PubMed Web of Science References 1. Shen W , Le S , Li Y , Hu F. 2016 . SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation . PLoS One 11 : e0163962 . OpenUrl CrossRef PubMed 2. Koren S , Walenz BP , Berlin K , Miller JR , Bergman NH , Phillippy AM . 2017 . Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation . Genome Res 27 : 722 – 736 . OpenUrl Abstract / FREE Full Text 3. Langmead B , Salzberg SL . 2012 . Fast gapped-read alignment with Bowtie 2 . Nat Methods 9 : 357 – 359 . OpenUrl CrossRef PubMed Web of Science 4. Walker BJ , Abeel T , Shea T , Priest M , Abouelliel A , Sakthikumar S , Cuomo CA , Zeng Q , Wortman J , Young SK , Earl AM . 2014 . Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement . PLoS One 9 : 1 – 14 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 11, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Genomic Insights into Ceftazidime Resistance in Burkholderia pseudomallei: Discovery of A172T Mutation, and Palindromic GC-Rich Repeat Sequences Facilitating penA Duplication and Amplification Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Genomic Insights into Ceftazidime Resistance in Burkholderia pseudomallei : Discovery of A172T Mutation, and Palindromic GC-Rich Repeat Sequences Facilitating penA Duplication and Amplification Apichai Tuanyok , Chie Nakajima , Tiernan Noll , Md. Siddiqur Rahman Khan , Pacharapong Khrongsee , Charles A. Yowell , Yu-Ping Xiao , Vanaporn Wuthiekanun , Narisara Chantratita , Henry Heine , Kuttichantran Subramaniam , Yasuhiko Suzuki , Direk Limmathurotsakul , Ayalew Mergia bioRxiv 2025.02.11.637714; doi: https://doi.org/10.1101/2025.02.11.637714 Share This Article: Copy Citation Tools Genomic Insights into Ceftazidime Resistance in Burkholderia pseudomallei : Discovery of A172T Mutation, and Palindromic GC-Rich Repeat Sequences Facilitating penA Duplication and Amplification Apichai Tuanyok , Chie Nakajima , Tiernan Noll , Md. Siddiqur Rahman Khan , Pacharapong Khrongsee , Charles A. Yowell , Yu-Ping Xiao , Vanaporn Wuthiekanun , Narisara Chantratita , Henry Heine , Kuttichantran Subramaniam , Yasuhiko Suzuki , Direk Limmathurotsakul , Ayalew Mergia bioRxiv 2025.02.11.637714; doi: https://doi.org/10.1101/2025.02.11.637714 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17651) Bioengineering (13873) Bioinformatics (41887) Biophysics (21424) Cancer Biology (18566) Cell Biology (25465) Clinical Trials (138) Developmental Biology (13365) Ecology (19871) Epidemiology (2067) Evolutionary Biology (24293) Genetics (15591) Genomics (22478) Immunology (17715) Microbiology (40331) Molecular Biology (17150) Neuroscience (88492) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9817) Zoology (2268)