Background The role of plasmids in driving antimicrobial resistance (AMR) in West African Salmonella Typhi remains unexplored. This study investigates the prevalence, temporal dynamics, and genetic associations of plasmid mobility classes and replicon types in Nigerian S. typhi over nearly four decades. Results We analyzed 151 S. typhi genomes from Nigeria (1982–2018) obtained from EnteroBase. Plasmid replicon typing and mobility classification revealed that 49.7% of isolates harbored conjugative plasmids, with the IncHI1 family (IncHI1A, IncHI1B_R27, and IncFIA_HI1) being the most prevalent. Conjugative plasmids increased from 0% in the 1980s to 56% in the 2010s (logistic regression, p = 0.006). A core multidrug resistance element comprising blaTEM-1B, sul1, catA1, dfrA15, and tet(B) was identified in 49% of isolates. Strong statistical associations were observed between IncHI1 plasmids and these AMR genes, with phi coefficients reaching r = 0.95 for tet(B), r = 0.87 for blaTEM-1B, and r = 0.86 for sul1. Isolates carrying conjugative plasmids harbored nearly five times more AMR genes than non-mobilizable isolates (4.9 vs 0.9; ANOVA, p < 0.001). Temporal breakpoint analysis identified 2010 as the year AMR prevalence exceeded 50%, coinciding with the sharp rise in conjugative plasmid carriage. Conclusion Conjugative IncHI1 plasmids are the primary drivers of multidrug resistance emergence in Nigerian S. typhi, carrying a conserved core resistome that has spread rapidly since 2010. These findings underscore the critical need for plasmid surveillance in genomic epidemiology and highlight IncHI1 plasmids as key targets for monitoring AMR transmission in high-burden settings.
Conjugative plasmids in combination with translocatable elements play a key role in the spread of antibiotic resistance genes. The IncZ plasmid pIE545 is one of the group of plasmids that have been used since the 1980s to examine and compare the properties of conjugative plasmids in the I-complex and is the only member of the group for which a complete sequence was not available. pIE545 was found in a Klebsiella pneumoniae in Germany in the 1970s and was known to confer resistance to kanamycin, neomycin and sulphonamides. Here, the complete pIE545 sequence was determined and its structure examined. The plasmid is 109,697 bp, made up of an 86.7-kb backbone that includes determinants for replication, maintenance and conjugation functions, together with 23 kb of accessory regions. A 10.2-kb integrative element, IE-545, that interrupts the backbone impB gene is itself interrupted by an IS102-bounded transposon, TnaphA1-pIE545, that includes the aphA1 kanamycin/neomycin resistance gene and an ISEc52-like element. The sulphonamide resistance gene sul2 is in a fragment of GIsul2 inserted between nikB and trbC. An IS2 interrupts pilL, and a second copy of ISEc52-like is in one of the two additional alternate ends for the pilV gene found in the shufflon. The pIE545 shufflon is found in other backbones that contain a Z-type replicon but is distinct from the shufflons of I1- and I2-type plasmids. The uninterrupted version of IE-545 is found in both plasmids and chromosomes interrupting impB or umuC genes.
Carbapenem-resistant Klebsiella pneumoniae ST11 is a high-risk lineage predominantly associated with healthcare settings. Here, we report the phenotypic and genomic characterization of a KPC-producing K. pneumoniae ST11 isolate recovered from a free-living, yellow-chevroned parakeet (Brotogeris chiriri) in Brazil. Antimicrobial susceptibility testing revealed resistance to multiple drug classes, including carbapenems. Whole-genome sequencing using a hybrid short- and long-read approach resolved a 5.5 Mb chromosome and five plasmids belonging to the IncFIB/HI1B, IncFIB/FII, IncN15, IncX3, and ColRNAI groups. The blaKPC-2 gene was located within a Tn4401-like element on an IncN15 plasmid, while additional resistance determinants were embedded in mosaic Tn402-derived regions and class 1 integrons. The genome displayed a complex mobilome comprising insertion sequences, transposon derivatives, and nine intact chromosomal prophages. Multiple heavy-metal tolerance operons and efflux systems were also identified. In vivo infection assays using Galleria mellonella demonstrated increased virulence compared with a reference strain. These findings document the occurrence of a clinically relevant carbapenem-resistant K. pneumoniae ST11 isolate in a free-living bird and support the consideration of wildlife-associated samples in integrated One Health surveillance of high-risk antimicrobial-resistant clones.
T vectors are core tools for TA cloning and are widely used for the direct cloning of PCR products. In the present study, a novel versatile T vector, designated pZBT01C, was developed based on a modified lysis gene E (mE) from bacteriophage PhiX174, which allows only positive tranformants to grow. Ten unique cloning sites were introduced into the mE gene, flanked by two XcmI sites. Universal M13-48/M13-47 primer binding sites were incorporated 35 bp upstream and downstream of the TA cloning site, and the enhancer T7g10 and Shine-Dalgarno box were placed upstream to stabilize mE expression. With a compact size of 3029 bp, pZBT01C is prepared by XcmI digestion to generate 3'-T overhangs. At the induction temperature, the plasmid pZBT01C exhibited strong host-killing activity. In cloning tests, all randomly selected 20 colonies were confirmed to be positive by colony PCR. The potential of pZBT01C as an expression system was further demonstrated by temperature-inducible expression of the reporter gene GFP. Owing to its near-zero-background performance and versatile design, pZBT01C is expected to have broad application prospects in PCR product cloning and related molecular biology applications.
Antibiotic resistance genes associated with IS26-family mobile genetic elements are globally distributed in the Gram-negative plasmidome. In many cases, antibiotic resistance genes are co-located with genes of unknown function. Two particular IS26-associated gene clusters, mph(A)-mrx-mphR(A) and cysH-eamA-tet(A)-tetR(A), are prevalent on plasmids and chromosomes of common Gram-negative pathogens. While mph(A) encodes a macrolide phosphotransferase and tet(A) encodes a tetracycline efflux pump, the roles, if any, of mrx, cysH, and eamA remain poorly understood. To test the hypothesis that these genes consistently co-located with mph(A) and tet(A) act cooperatively to enhance antibiotic resistance, recombinant E. coli strains carrying various genetic combinations from each cluster were subjected to antimicrobial susceptibility testing. While the mph(A) gene alone provided minimal protection from azithromycin, inclusion of mrx increased resistance by at least 48-fold. Disruption of mrx by the insertion of a nonsense mutation or the addition of an efflux pump inhibitor abrogated this effect, indicating that the Mrx protein is a drug exporter that is required for maximal effect. In the absence of mph(A), mrx had no effect on azithromycin resistance, suggesting that Mrx only effluxes the phosphorylated form of azithromycin. Similarly, inclusion of cysH-eamA with tet(A) increased resistance to tetracycline antibiotics by at least 2-fold over tet(A) alone. Disruption of either cysH or eamA had no effect on this enhancement, but disruption of both genes eliminated it entirely. A more complete understanding of antibiotic resistance among Gram-negative bacteria requires elucidation of the roles of modulator genes co-located with primary resistance genes on mobile genetic elements.
