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Background Klebsiella pneumoniae is a leading cause of intractable hospital-acquired multidrug-resistant infections and carbapenemase-producing K. pneumoniae (CP Kp ) are particularly feared. Most of the clinical isolates produce capsule as a major virulence factor. Recombination events at the capsule locus are frequent and responsible for capsule diversity within Klebsiella spp . Capsule diversity may also occur within clonal bacterial populations generating differences in colony aspect. However, little is known about this phenomenon of phenotypic variation in CP Kp and its consequences. Results Here, we explored the genetic causes of in vitro switching from capsulated, mucoid to non-mucoid, non-capsulated phenotype in eight clinical CP Kp isolates. We compared capsulated, mucoid colony variants with one of their non-capsulated, non-mucoid isogenic variant. The two colony variants were distinguished by their appearance on solid medium. Whole genome comparison was used to infer mutations causing phenotypic differences. The frequency of phenotypic switch was strain-dependent and increased along with colony development on plate. We observed, for 72 non-capsulated variants that the loss of the mucoid phenotype correlates with capsule deficiency and diverse genetic events, including transposition of insertion sequences or point mutations, affecting genes belonging to the capsule operon. Reduced or loss of capsular production was associated with various in vitro phenotypic changes, affecting susceptibility to carbapenem but not to colistin, in vitro biofilm formation and autoaggregation. Conclusions The different impact of the phenotypic variation among the eight isolates in terms of capsule content, biofilm production and carbapenem susceptibility suggested heterogeneous selective advantage for capsular loss according to the strain and the mutation. Based on our results, we believe that attention should be paid in the phenotypic characterization of CP Kp clinical isolates, particularly of traits related to virulence and carbapenem resistance.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA , while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA -carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS 6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS 6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS 6 family genes in disseminating plasmid-borne optrA . Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS 6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. Key points • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids. Graphical Abstract

The ability of bacteria to undergo rapid evolution and adapt to changing environmental circumstances drives the public health crisis of multiple antibiotic resistance, as well as outbreaks of disease in economically important agricultural crops and animal husbandry. Prokaryotic transposable elements (TE) play a critical role in this. We describe here the structure and organization of TnCentral ( https://tncentral.proteininformationresource.org/ [or the mirror link at https://tncentral.ncc.unesp.br/ ]), a web resource for prokaryotic transposable elements (TE). TnCentral currently contains ∼400 carefully annotated TE, including transposons from the Tn 3 , Tn 7 , Tn 402 , and Tn 554 families; compound transposons; integrons; and associated insertion sequences (IS). These TE carry passenger genes, including genes conferring resistance to over 25 classes of antibiotics and nine types of heavy metal, as well as genes responsible for pathogenesis in plants, toxin/antitoxin gene pairs, transcription factors, and genes involved in metabolism. Each TE has its own entry page, providing details about its transposition genes, passenger genes, and other sequence features required for transposition, as well as a graphical map of all features. TnCentral content can be browsed and queried through text- and sequence-based searches with a graphic output. We describe three use cases, which illustrate how the search interface, results tables, and entry pages can be used to explore and compare TE. TnCentral also includes downloadable software to facilitate user-driven identification, with manual annotation, of certain types of TE in genomic sequences. Through the TnCentral homepage, users can also access TnPedia, which provides comprehensive reviews of the major TE families, including an extensive general section and specialized sections with descriptions of insertion sequence and transposon families. TnCentral and TnPedia are intuitive resources that can be used by clinicians and scientists to assess TE diversity in clinical, veterinary, and environmental samples. IMPORTANCE The ability of bacteria to undergo rapid evolution and adapt to changing environmental circumstances drives the public health crisis of multiple antibiotic resistance, as well as outbreaks of disease in economically important agricultural crops and animal husbandry. Prokaryotic transposable elements (TE) play a critical role in this. Many carry “passenger genes” (not required for the transposition process) conferring resistance to antibiotics or heavy metals or causing disease in plants and animals. Passenger genes are spread by normal TE transposition activities and by insertion into plasmids, which then spread via conjugation within and across bacterial populations. Thus, an understanding of TE composition and transposition mechanisms is key to developing strategies to combat bacterial pathogenesis. Toward this end, we have developed TnCentral, a bioinformatics resource dedicated to describing and exploring the structural and functional features of prokaryotic TE whose use is intuitive and accessible to users with or without bioinformatics expertise.

Insertion sequences are the most common mobile genetic elements found in bacterial genomes, and hence they significantly impact bacterial evolution. We observe insertion sequence movement at the center of large-scale deletions and duplications that occurred during laboratory evolution experiments with Escherichia coli and Pseudomonas fluorescens , involving three distinct types of transposase. We raise the possibility that the transposase does not mediate DNA cleavage but instead inserts into existing DNA breaks. Our research highlights the importance of insertion sequences for the generation of large-scale genomic rearrangements and raises questions concerning the mechanistic basis of these mutations.

Colistin is a last resort antibiotic for the treatment of carbapenemase producing Klebsiella pneumoniae. The disruption of the mgrB gene by insertion sequences (ISs) is a mechanism mediating colistin resistance. Plasmids encode mobilizable IS elements which integrate into the mgrB gene in K. pneumoniae causing gene inactivation and colistin resistance. The species prevalence of mgrB‐gene disrupting insertion elements ISL3 (ISKpn25), IS5 (ISKpn26), ISKpn14, and IS903B present on plasmids were assessed. IS containing plasmids were also scanned for antimicrobial resistance genes, including carbapenem resistant genes. Plasmids encoding ISs are abundant in K. pneumoniae. IS903B was found in 28 unique Inc groups, while ISKpn25 was largely carried by IncFIB(pQil) plasmids. ISKpn26 and ISKpn14 were most often found associated with IncFII(pHN7A8) plasmids. Of the 34 unique countries which contained any of the IS elements, ISKpn25 was identified from 26. ISKpn26, ISKpn14, and IS903B ISs were identified from 89.3%, 44.9%, and 23.9% plasmid samples from China. Plasmids carrying ISKpn25, ISKpn14, and ISKpn26 IS have a 4.6‐, 6.0‐, and 6.6‐fold higher carbapenemase gene count, respectively, relative to IS903B‐carrying plasmids. IS903B bearing plasmids have a 20‐, 5‐, and 5‐fold higher environmental source isolation count relative to ISKpn25, ISKpn14, and ISKpn26 bearing plasmids. ISKpn25 present on IncFIB(pQil) sourced from clinical settings is established across multiple countries, while ISKpn26, ISKpn14, and IS903B appear most often in China. Carbapenemase presence in tandem with IS elements may help promote an extensively drug resistant profile in K. pneumoniae limiting already narrow treatment options. Klebsiella pneumoniae is the dominant species carrying colistin‐resistance inducing insertion sequence elements ISKpn25, ISKpn26, ISKpn14, and IS903B on plasmids. ISKpn25, ISKpn26, ISKpn14 bearing plasmids have a high carriage of carbapenemase genes and are associated with clinical source isolation relative to IS903B. Clinically derived plasmids that harbor both carbapenemases and IS elements may engender an extensively drug‐resistant phenotype during colistin therapy in hospitals, limiting already narrow treatment options.

The interplay between insertion sequences (ISs) and antibiotic resistance genes (ARGs) in Acinetobacter baumannii contributes to resistance against specific antibiotics. Conventionally, genetic variations and ARGs have been utilized for predicting resistance phenotypes, with the potential pivotal role of IS elements largely overlooked. Our study advances this approach by integrating both rule-based and machine learning models to predict AMR in A. baumannii . This significantly enhances the accuracy of AMR prediction, emphasizing the pivotal function of IS elements in antibiotic resistance. Notably, we uncover a series of conserved sequence patterns linking IS elements and ARGs, which outperform ARGs alone in phenotypic prediction. Our findings are crucial for bioinformatics strategies aimed at studying and tracking AMR, offering novel insights into combating the escalating AMR challenge.

Background The convergence of resistance and hypervirulence in Klebsiella pneumoniae represents a significant public health threat, driven by the horizontal transfer of plasmids. Understanding factors affecting plasmid transfer efficiency is essential to elucidate mechanisms behind emergence of these formidable pathogens. Methods Hypermucoviscous K. pneumoniae strains were serially passaged in LB medium to investigate capsule-deficient phenotypes. Capsule-deficient mutants were analyzed using genetic sequencing to identify the types and insertion sites of insertion sequences (IS). Bioinformatics and statistical analyses based on the NCBI and National Microbiology Data Center (NMDC) database were used to map the origins and locations of IS elements. Conjugation assays were performed to assess plasmid transfer efficiency between encapsulated and capsule-deficient strains. A murine intestinal colonization model was employed to evaluate virulence levels and IS excision-mediated capsule restoration. Results Our research revealed that a hypervirulent K. pneumoniae (hvKP) strain acquired a bla NDM-1 -bearing IncX3 plasmid with IS 5 and IS Kox3 elements. These IS elements are capable of inserting into capsular polysaccharide synthesis genes, causing a notably high frequency of capsule loss in vitro. The IS-mediated capsular phase variation, whether occurring in the donor or recipient strain, significantly increased the conjugation frequency of both the resistance plasmid and the virulence plasmid. Additionally, capsular phase variation enhanced bacterial adaptability in vitro. Experiments in mouse models demonstrated that capsule-deficient mutants exhibited reduced virulence and colonization capacity. However, during long-term intestinal colonization, IS element excision restored capsule expression, leading to the recovery of hypervirulence and enhanced colonization efficiency. Conclusions Our findings reveal that IS elements mediate capsular phase variation by toggling gene activity, accelerating the genomic convergence of multidrug resistance and hypervirulence in K. pneumoniae , as well as facilitating adaptive transitions in different environments.

Multidrug-resistant Gram-negative carriers of Klebsiella pneumoniae carbapenemases (KPCs) often subvert antibiotic therapy due to inadequate sensitivity in laboratory detection. Although unstable gene amplification has been recognized to crucially contribute to underestimation or misestimation of antimicrobial resistance in clinical isolates, the precise mechanisms underlying carbapenem resistance driven by amplification of blaKPC-2 remain obscure. Here, we reported that IS26-mediated amplification of blaKPC-2 rapidly and robustly gave rise to carbapenem hyperresistant phenotypes in an Escherichia coli clinical strain following sublethal meropenem or tobramycin preexposure. Intriguingly, IS26 also underpinned amplification of a 47 kb multiple drug resistance (MDR) region encompassing nine antibiotic resistance genes and six IS26 insertion sequences. Tandem-repeat analysis and experimental validation demonstrated that blaKPC-2 amplification was indeed mediated by IS26, which was further experimentally shown to involve intricate genetic rearrangement. Such gene amplification arose dynamically under antibiotic stress and subsided upon antibiotic withdrawal. Instead of reducing the amplification of the IS26-flanked MDR region, drug combinations in vitro exacerbated it. Our study, thus, provides valuable insights into how dynamic gene amplification processes can precipitously transform resistance status and complicate diagnosis. IMPORTANCE Klebsiella pneumoniae carbapenemases (KPCs) are powerful β-lactamases that enable Gram-negative pathogens to destroy clinically important carbapenems in antibiotic therapies. In particular, KPC-2 is difficult to detect due to a lack of instrument sensitivity in regular laboratory screens, which leads to misdiagnosis and poor treatment outcomes. It remains unclear how blaKPC-2 rapidly induces exceedingly high-level resistance against carbapenems following the challenges of sublethal antibiotics. Here, we demonstrated that, under sublethal doses of antibiotics, insertion sequence IS26 mediated rapid amplification of multiple resistance determinants, including blaKPC-2 and a multiple drug resistance (MDR) region, which was accompanied by intricate genetic rearrangement.

Summary Genome phasing is a recently developed assembly method that separates heterozygous eukaryotic genomic regions and builds haplotype‐resolved assemblies. Because differences between haplotypes are ignored in most published de novo genomes, assemblies are available as consensus genomes consisting of haplotype mixtures, thus increasing the need for genome phasing. Here, we review the operating principles and characteristics of several freely available and widely used phasing tools (TrioCanu, FALCON‐Phase, and ALLHiC). An examination of downstream analyses using haplotype‐resolved genome assemblies in plants indicated significant differences among haplotypes regarding chromosomal rearrangements, sequence insertions, and expression of specific alleles that contribute to the acquisition of the biological characteristics of plant species. Finally, we suggest directions to solve addressing limitations of current genome‐phasing methods. This review provides insights into the current progress, limitations, and future directions of de novo genome phasing, which will enable researchers to easily access and utilize genome‐phasing in studies involving highly heterozygous complex plant genomes.

Background Insertion sequences (ISs) are mobile repeat sequences and most of them can copy themselves to new host genome locations, leading to genome plasticity and gene regulation in prokaryotes. In this study, we present functional and evolutionary relationships between IS and neighboring genes in a large-scale comparative genomic analysis. Results IS families were located in all prokaryotic phyla, with preferential occurrence of IS 3 , IS 4 , IS 481 , and IS 5 families in Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria and Firmicutes as well as in eukaryote host-associated organisms and autotrophic opportunistic pathogens. We defined the concept of the IS-Gene couple (IG), which allowed to highlight the functional and regulatory impacts of an IS on the closest gene. Genes involved in transcriptional regulation and transport activities were found overrepresented in IG. In particular, major facilitator superfamily (MFS) transporters, ATP-binding proteins and transposases raised as favorite neighboring gene functions of IS hotspots. Then, evolutionary conserved IS-Gene sets across taxonomic lineages enabled the classification of IS-gene couples into phylum, class-to-genus, and species syntenic IS-Gene couples. The IS 5 , IS 21 , IS 4 , IS 607 , IS 91 , IS L3 and IS 200 families displayed two to four times more ISs in the phylum and/or class-to-genus syntenic IGs compared to other IS families. This indicates that those families were probably inserted earlier than others and then subjected to horizontal transfer, transposition and deletion events over time. In phylum syntenic IG category, Betaproteobacteria, Crenarchaeota, Calditrichae, Planctomycetes, Acidithiobacillia and Cyanobacteria phyla act as IS reservoirs for other phyla, and neighboring gene functions are mostly related to transcriptional regulators. Comparison of IS occurrences with predicted regulatory motifs led to ~ 26.5% of motif-containing ISs with 2 motifs per IS in average. These results, concomitantly with short IS-Gene distances, suggest that those ISs would interfere with the expression of neighboring genes and thus form strong candidates for an adaptive pairing. Conclusions All together, our large-scale study provide new insights into the IS genetic context and strongly suggest their regulatory roles.