Tn3-like Transposon Research Articles

Nosocomial Transmission of Necrotizing Fasciitis: A Molecular Characterization of Group A Streptococcal DNases in Clinical Virulence

Streptococcus pyogenes, or Group A Streptococcus (GAS), is responsible for over 500,000 deaths per year. Approximately 15% of these deaths are caused by necrotizing soft-tissue infections. In 2008, we isolated an M5 GAS, named the LO1 strain, responsible for the nosocomial transmission of necrotizing fasciitis between a baby and a nurse in Belgium. To understand this unusual transmission route, the LO1 strain was sequenced. A comparison of the LO1 genome and transcriptome with the reference M5 Manfredo strain was conducted. We found that the major differences were the presence of an additional DNase and a Tn916-like transposon in the LO1 and other invasive M5 genomes. RNA-seq analysis showed that genes present on the transposon were barely expressed. In contrast, the DNases presented different expression profiles depending on the tested conditions. We generated knock-out mutants in the LO1 background and characterized their virulence phenotype. We also determined their nuclease activity on different substrates. We found that DNases are dispensable for biofilm formation and adhesion to both keratinocytes and pharyngeal cells. Three of these were found to be essential for blood survival; Spd4 and Sdn are implicated in phagocytosis resistance, and Spd1 is responsible for neutrophil extracellular trap (NET) degradation.

First Detection of High-Level Aminoglycoside-Resistant Klebsiella pneumoniae and Enterobacter cloacae Isolates Due to 16S rRNA Methyltransferases with and Without blaNDM in Uruguay

Background: The increase in antimicrobial resistance includes emerging mechanisms such as 16S ribosomal RNA methylases, which confer high-level resistance to aminoglycosides. In this regard, the most predominant genes observed worldwide are rmtB and armA, but their presence in Uruguay is unknown. Objectives: We describe the genomic characterization of isolates carrying rmtB and rmtC, together with blaNDM-5 and blaNDM-1, respectively, and rmtD in our country. Methology: Five isolates from patients admitted to three hospitals were studied. Identification and antibiotic susceptibility testing were performed using the Vitek2 System. Whole Genome Sequencing was conducted, and hybrid assembly was performed with Unicycler. In silico analysis using the Center for Genomic Epidemiology’s tools was undertaken to predict antibiotic resistance determinants, plasmid incompatibility groups, and sequence types. Results: We report three K. pneumoniae ST307 isolates with an IncR plasmid carrying blaNDM-5/blaCTX-M-15/blaTEM-1B/rmtB/dfrA14/dfrA12/sul1/qacEΔ1/ermB/mphA, one K. pneumoniae ST258 harboring an IncC plasmid containing rmtC/blaNDM-1/blaCMY-6/aac(6′)-Ib/sul1, and one E. cloacae ST88 isolate with an IncFIB/II plasmid hosting rmtD, within a novel Tn21-like transposon named Tn7825, alongside blaOXA-101/sul1/tet(G)/floR, and a new variant of blaTEM assigned as blaTEM-258. One of the strains, named UH_B2, also carried an IncM1 plasmid encoding qnrE1/blaTEM-1/blaCTX-M-8 associated with ISEcp1. Conclusions: This is the first description of plasmids harboring 16S rRNA methyltransferases in Uruguay. The association and dissemination of diverse antibiotic-resistant genes underpin the health threat they represent, highlighting the lack of available antibiotics effective against multidrug-resistant microorganisms.

Structure of TnsABCD transpososome reveals mechanisms of targeted DNA transposition

Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the transpososome and facilitate transposition. Although this pathway is well established, atomic-level structural insights of this process remain largely elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of the TnsC-TnsD-att DNA complex and the TnsABCD transpososome from the Tn7-like transposon in Peltigera membranacea cyanobiont 210A, a type I-B CRISPR-associated transposon. Our structures reveal a striking bending of the att DNA, featured by the intercalation of an arginine side chain of TnsD into a CC/GG dinucleotide step. The TnsABCD transpososome structure reveals TnsA-TnsB interactions and demonstrates that TnsC not only recruits TnsAB but also directly participates in the transpososome assembly. These findings provide mechanistic insights into targeted DNA insertion by Tn7-like transposons, with implications for improving the precision and efficiency of their genome-editing applications.

Whole genome sequencing insight into carbapenem-resistant and multidrug-resistant Acinetobacter baumannii harboring chromosome-borne blaOXA-23.

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure "ISAba1-blaADC-25." The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816.IMPORTANCEThe blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.

Natural and Engineered Guide RNA-Directed Transposition with CRISPR-Associated Tn7-Like Transposons.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.

Comparative genomics of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa.

We aimed to determine the molecular characteristics of carbapenem-resistant Pseudomonas aeruginosa strains 18081308 and 18083286, which were isolated from the urine and the sputum of two Chinese patients, respectively. Additionally, we conducted a comparative analysis between Tn6411 carrying blaIMP-1 in strain 18083286 and transposons from the same family available in GenBank. Bacterial genome sequencing was carried out on strains 18081308 and 18083286 to obtain their whole genome sequence. Average nucleotide identity (ANI) was used for their precise species identification. Serotyping and multilocus sequence typing were performed. Furthermore, the acquired drug resistance genes of these strains were identified. The carbapenem-resistant P. aeruginosa strains isolated in the present study were of sequence type ST865 and serotype O6. They all carried the same resistance genes (aacC2, tmrB, and blaIMP-1). Tn6411, a Tn7-like transposon carrying blaIMP-1, was found in strain 18083286 by single molecule real time (SMRT) sequencing. We also identified the presence of this transposon sequence in other chromosomes of P. aeruginosa and plasmids carried by Acinetobacter spp. in GenBank, indicating the necessity for heightening attention to the potential transferability of this transposon.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

VIM-type metallo-β-lactamase (MBL)-encoding genomic islands in Pseudomonas spp. in Poland: predominance of clc-like integrative and conjugative elements (ICEs).

To characterize VIM-type metallo-β-lactamase (MBL)-encoding genomic islands (GIs) in Pseudomonas aeruginosa and P. putida group isolates from Polish hospitals from 2001-2015/16. Twelve P. aeruginosa and 20 P. putida group isolates producing VIM-like MBLs were selected from a large collection of these based on epidemiological and typing data. The organisms represented all major epidemic genotypes of these species spread in Poland with chromosomally located blaVIM gene-carrying integrons. The previously determined short-read sequences were complemented by long-read sequencing in this study. The comparative structural analysis of the GIs used a variety of bioinformatic tools. Thirty different GIs with blaVIM integrons were identified in the 32 isolates, of which 24 GIs from 26 isolates were integrative and conjugative elements (ICEs) of the clc family. These in turn were dominated by 21 variants of the GI2/ICE6441 subfamily with a total of 19 VIM integrons, each inserted in the same position within the ICE's Tn21-like transposon Tn4380. The three other ICEs formed a novel ICE6705 subfamily, lacking Tn4380 and having different VIM integrons located in another site of the elements. The remaining six non-ICE GIs represented miscellaneous structures. The presence of various integrons in the same ICE sublineage, and of the same integron in different GIs, indicated circulation and recombination of the integron-carrying genetic platforms across Pseudomonas species/genotypes. Despite the general diversity of the blaVIM-carrying GIs in Pseudomonas spp. in Poland, a clear predominance of broadly spread and rapidly evolving clc-type ICEs was documented, confirming their significant role in antimicrobial resistance epidemiology.

Unexpected role of pig nostrils in the clonal and plasmidic dissemination of extended-spectrum beta-lactamase-producing Escherichia coli at farm level

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

Novel mechanisms of diversity generation in Acinetobacter baumannii resistance islands driven by Tn7-like elements.

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.

Novel resistance ICEs carrying the blaFIM-1 metallo-β-lactamase gene from an ST235 Pseudomonas aeruginosa sublineage.

FIM-1 is an acquired metallo-β-lactamase identified in a multidrug-resistant Pseudomonas aeruginosa (index strain FI-14/157) of clinical origin isolated in 2007 in Florence, Italy. Here we report on a second case of infection by FIM-1-positive P. aeruginosa (FI-17645), which occurred in 2020 in the same hospital. Both FIM-1-positive strains exhibited resistance to all anti-Pseudomonas antibiotics except colistin and cefiderocol. Comparative genomic characterization revealed that the two FIM-positive strains were closely related [core genome difference, 16 single nucleotide polymorphisms (SNPs)], suggesting a local circulation of similar strains. In the FI-14/157 index strain, the blaFIM-1 gene was associated with an ISCR19-like element that likely contributed to its capture downstream an integron platform inserted aboard a Tn21-like transposon, named Tn7703.1, which was associated with a large integrative and conjugative element (ICE) named ICE7705.1, integrated into an att site located within the 3'-end of tRNAGly CCC gene of the P. aeruginosa chromosome. In strain FI-17645, blaFIM-1 was associated with a closely related ICE, named ICE7705.2, integrated in the same chromosomal site. Similar ICE platforms, lacking the blaFIM-1-containing region, were detected in other ST235 P. aeruginosa strains from different geographic areas, suggesting a common ancestry and underscoring the role of these elements in the dissemination of resistance genes in P. aeruginosa. Sequence database mining revealed two draft P. aeruginosa genomes, one from Italy and one from the USA (both isolated in 2012), including a contig with blaFIM-1, suggesting that this resistance gene could have a broader distribution than originally anticipated.

Detection of CTX-M-15 ESBL in XDR Haemophilus parainfluenzae from a urethral swab.

Haemophilus parainfluenzae is an opportunistic pathogen causing respiratory tract infection and sexually transmitted diseases. The emergence of multidrug resistance in this species is particularly worrisome, especially since the recent description of CTX-M-15 ESBL-producing isolates in Spain. The aim of this study was to characterize a CTX-M-15-producing H. parainfluenzae clinical isolate, HP01, obtained from a urethral swab. MICs were determined with gradient strips for this isolate. Hydrolysis assays were performed with the β LACTA test. Genomic DNA from HP01 was subjected to Illumina and Oxford Nanopore sequencing to investigate the genetic environment of blaCTX-M-15. Phylogenetic analysis was performed with available H. parainfluenzae genomes from the NCBI database, including CTX-M-15 producers. HP01, an XDR isolate, was resistant to penicillin, third-generation cephalosporins, fluoroquinolones, macrolides, cyclines and co-trimoxazole and susceptible only to carbapenems and rifampicin. HP01 carried blaTEM-1, blaCTX-M-15, tet(M), catS and mef(E)/mel and harboured amino acid substitutions in PBP3, PBP5, GyrA, ParC and FolA implicated in resistance. Genomic analysis revealed that blaCTX-M-15 was carried by a Tn3-like transposon inserted into a novel integrative and conjugative element (ICE), ICEHpaSLS, present on the chromosome and belonging to the ICEHin1056 family described in Haemophilus influenzae. The tet(M)-MEGA element was also detected on the chromosome. No plasmid was found. The phylogenetic analysis showed that four H. parainfluenzae producing CTX-M-15 clustered in the same clade. Here we report the description of an XDR H. parainfluenzae producing blaCTX-M-15 isolated from a urethral swab. The blaCTX-M-15 gene was inserted into an ICE structure similar to those recently described in CTX-M-15 producers in Spain. The emergence of XDR H. parainfluenzae producing blaCTX-M-15 is a matter of great concern. Careful surveillance is required to prevent its spread.

Extensively drug-resistant Enterobacter ludwigii co-harbouring MCR-9 and a multicopy of blaIMP-1 in South Korea

In this study, we describe an Enterobacter ludwigii clinical isolate that is resistant to both carbapenems and colistin in South Korea. Antimicrobial susceptibility testing revealed that E. ludwigii CRE2104-31 was non-susceptible to all tested antibiotics except fosfomycin. Whole genome sequencing identified a 323-kbp IncHI2 plasmid, pCRE2104-31a, that was co-harbouring mobile colistin resistance (mcr)-9.1 and blaIMP-1. In comparison with other full plasmids, pCRE2104-31a exhibited the closest similarity to a plasmid from the Klebsiella pneumoniae strain CNR48 from France, with 19.9% query coverage and 99% identity. Notably, we observed five tandem repeats of blaIMP-1 and aac(6′)-Il genes, accompanied by multiple attCs within a class I integron on the Tn402-like transposon. The unit of blaIMP-1-attC-aac(6′)-Il-attC might have accumulated due to multiple convergent events. In addition to mcr-9.1 and blaIMP-1, various other antibiotic resistance-associated genes were identified in the plasmid, as follows: blaTEM-1B, aph(3′)-I, aph(3′)-Ia, aac(6′)-Il, aac(6′)-IIc, aac(6′)-IIa, aph(6)-Id, aph(3′')-Ib, aadA2b, aac(6′)-Ib3, sul, dfrA19, qnrB2, aac(6′)-Ib-cr, ere(A), and qacE. A conjugation assay showed that the mcr-9.1/blaIMP-1-co-bearing plasmid was self-transmissible to E. coli J53. However, colistin and carbapenem resistance could not be transferred to E. coli due to high incompatibility. The convergence of mcr and carbapenemase genes is thought to be host-dependent among Enterobacteriaceae. The emergence of extensively drug-resistant E. ludwigii co-harbouring MCR-9.1 and a multicopy of blaIMP-1 would pose a significant threat within the compatible Enterobacteriaceae.

Tn3-like structures co-harboring of blaCTX-M-65, blaTEM-1 and blaOXA-10 in the plasmids of two Escherichia coli ST1508 strains originating from dairy cattle in China

The purpose of this study was to determine the level of horizontal transmission of the blaCTX-M-65 gene and the role of its associated mobile genetic elements (MGEs) in the bovine-derived Escherichia coli. After PCR identification, two plasmids carrying blaCTX-M-65 were successfully transferred to the recipient E. coli J53 Azr through conjugation assays and subsequently selected for Whole-Genome sequencing (WGS) analysis. The resistance profiles of these two positive strains and their transconjugants were also determined through antimicrobial susceptibility tests. Whole genome data were acquired using both the PacBio sequencing platform and the Illumina data platform. The annotated results were then submitted to the Genbank database for accession number recording. For comparison, the genetic environment of plasmids carrying the resistance gene blaCTX-M-65 was mapped using the Easyfig software. WGS analysis revealed Tn3-like composite transposons bearing blaCTX-M-65, blaTEM-1, and blaOXA-10 in the IncHI2-type plasmids of these two E. coli ST1508 strains. A phylogenetic tree was generated from all 48 assembled E. coli isolates blaCTX-M-65, blaTEM-1, and blaOXA-10 from the NCBI Pathogen Detection database with our two isolates, showing the relationships and the contribution of SNPs to the diversity between genetic samples. This study suggests that the transmissibility of blaCTX-M-65 on Tn3-like composite transposons contributes to an increased risk of its transmission in E. coli derived from dairy cattle.

Molecular detection of Tn7-like transposons and IntI2 integron integrase in multidrug-resistant Escherichia coli and Klebsiella spp.

Abstract Background: Transposable elements are mobile DNA sequences that can move and change their location within the genome. Objectives: The aim of the study was to investigate class II integron and Tn7-like transposons and their relationship to multidrug resistance in commensal and pathogenic Klebsiella and Escherichia coli isolates. Materials and Methods: The disk-diffusion method was used to check the antibiotic susceptibility of 26 Klebsiella isolates and 76 E. coli isolates that had been isolated and preidentified using the VITEK 2 System from healthy stool, diarrhea, and urine in (urinary tract infection) specimens. The presence of the intI2 and Tn7-like transposase genes was then examined using specific primer pairs by polymerase chain reaction. Results: A total of 76% of the total isolates harbored Tn7, of which 14% of these isolates appeared to harbor intI2 in the Tn7 in three phylotypes. The other 62% of isolates had five patterns of Tn7. At the same time, the rest of the isolates (24%) were negative for the Tn7 and intI2 genes. Overall, the E. coli and Klebsiella isolates contain different Tn7-like transposons and are associated with pathogenic bacteria (P < 0.05) though some normal flora harbor the transposons. The presence of intI2 and Tn7-like transposons is associated with multidrug resistance in the studied species; intI2 in Tn7-like transposon (P < 0.029) and Tn7-like transposition genes only (P < 0.001). A significant association (P < 0.05) was detected between intI2 in the Tn7-like transposons and resistance to amoxicillin-clavulanate, piperacillin, cefotaxime, gentamicin, azithromycin, and nitrofurantoin. Conclusions: There is a correlation between multidrug resistance and the presence of Tn7-like transposition and intI2 genes.

Integrons, transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies.

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.

Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector

Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.

Modularity and diversity of target selectors in Tn7 transposons

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, whichwe show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

Complete Genome Sequence of the Extensively Drug-Resistant Extended-Spectrum β-Lactamase-Producing Proteus mirabilis Isolate HK294, Obtained from Poultry Feces in Hong Kong.

Here, we report the complete genome sequence of Proteus mirabilis isolate HK294, recovered from pooled poultry feces in Hong Kong in 2022. The chromosome contained 32 antimicrobial resistance genes, including the extended-spectrum β-lactamases blaCTX-M-65 and blaCTX-M-3. Almost all resistance genes were part of either an integrative conjugative element or a Tn7-like transposon.

Unravelling the Evolutionary Dynamics of High-Risk Klebsiella pneumoniae ST147 Clones: Insights from Comparative Pangenome Analysis.

The high prevalence and rapid emergence of antibiotic resistance in high-risk Klebsiella pneumoniae (KP) ST147 clones is a global health concern and warrants molecular surveillance. A pangenome analysis was performed using publicly available ST147 complete genomes. The characteristics and evolutionary relationships among ST147 members were investigated through a Bayesian phylogenetic analysis. The large number of accessory genes in the pangenome indicates genome plasticity and openness. Seventy-two antibiotic resistance genes were found to be linked with antibiotic inactivation, efflux, and target alteration. The exclusive detection of the blaOXA-232 gene within the ColKp3 plasmid of KP_SDL79 suggests its acquisition through horizontal gene transfer. The association of seventy-six virulence genes with the acrAB efflux pump, T6SS system and type I secretion system describes its pathogenicity. The presence of Tn6170, a putative Tn7-like transposon in KP_SDL79 with an insertion at the flanking region of the tnsB gene, establishes its transmission ability. The Bayesian phylogenetic analysis estimates ST147's initial divergence in 1951 and the most recent common ancestor for the entire KP population in 1621. Present study highlights the genetic diversity and evolutionary dynamics of high-risk clones of K. pneumoniae. Further inter-clonal diversity studies will help us understand its outbreak more precisely and pave the way for therapeutic interventions.