Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring blaCMY-2 and blaSHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries.

The objective of this study was to elucidate the genetic and evolutionary relatedness of blaCMY-2- and blaSHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring blaCMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, blaSHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.

Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam.

Acquired ß-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all ß-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of ß-lactamase gene blaCMY-2, encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of blaCMY-2R were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of blaCMY-2R on the IncP1 plasmid, but possessed anther blaCMY-2R on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of blaCMY-2R significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.

Emergence of a Salmonella Rissen ST469 clinical isolate carrying bla NDM-13 in China.

New Delhi metallo-ß-lactamase-13 (NDM-13) is an NDM variant that was first identified in 2015 and has not been detected in Salmonella species prior to this study. Here we describe the first identification of a Salmonella Rissen strain SR33 carrying bla NDM-13. The aim of this study was to molecularly characterize SR33's antimicrobial resistance and virulence features as well as investigate the genetic environment of bla NDM-13. The Salmonella Rissen SR33 strain was isolated from a patient with fever and diarrhea. SR33 belonged to ST469, and it was found to be multidrug-resistant (MDR) and to carry many virulence genes. Phylogenetic analysis showed that SR33 shared a close relationship with most of the Chinese S. Rissen ST469 strains. bla NDM-13 was located in a transmissible IncI1 plasmid pNDM13-SR33. Sequence analysis of bla NDM-13-positive genomes downloaded from GenBank revealed that a genetic context (ΔISAba125-bla NDM-13-ble MBL-trpF) and a hybrid promoter (consisting of -35 sequences provided by ISAba125 and -10 sequences) were conserved. ISAba125 was truncated by IS1294 in three plasmids carrying bla NDM-13, including pNDM13-SR33. To our knowledge, this is the first report of bla NDM-13 carried by Salmonella. The emergence of bla NDM-13 in a clinical MDR S. Rissen ST469 strain highlights the critical need for monitoring and controlling the dissemination of bla NDM-13. bla NDM-13 carried by a transmissible IncI1 plasmid may result in an increased risk of bla NDM-13 transmission. IS1294 may be involved in the movement of bla NDM-13.

IS1294 Reorganizes Plasmids in a Multidrug-Resistant Escherichia coli Strain.

The aims of this study were to elucidate the role of IS1294 in plasmid reorganization and to analyze biological characteristics of cointegrates derived from different daughter plasmids. The genetic profiles of plasmids in Escherichia coli strain C21 and its transconjugants were characterized by conjugation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern hybridization, whole-genome sequencing (WGS) analysis, and PCR. The traits of cointegrates were characterized by conjugation and stability assays. blaCTX-M-55-bearing IncI2 pC21-1 and nonresistant IncI1 pC21-3, as conjugative helper plasmids, were fused with nonconjugative rmtB-bearing IncN-X1 pC21-2, generating cointegrates pC21-F1 and pC21-F2. Similarly, pC21-1 and pC21-3 were fused with nonconjugative IncF33:A-:B- pHB37-2 from another E. coli strain to generate cointegrates pC21-F3 and pC21-F4 under experimental conditions. Four cointegrates were further conjugated into the E. coli strain J53 recipient at high conjugation frequencies, ranging from 2.8 × 10-3 to 3.2 × 10-2. The formation of pC21-F1 and pC21-F4 was the result of host- and IS1294-mediated reactions and occurred at high fusion frequencies of 9.9 × 10-4 and 2.1 × 10-4, respectively. Knockout of RecA resulted in a 100-fold decrease in the frequency of plasmid reorganization. The phenomenon of cointegrate pC21-F2 and its daughter plasmids coexisting in transconjugants was detected for the first time in plasmid stability experiments. IS26-orf-oqxAB was excised from cointegrate pC21-F2 through a circular intermediate at a very low frequency, which was experimentally observed. To the best of our knowledge, this is the first report of IS1294-mediated fusion between plasmids with different replicons. This study provides insight into the formation and evolution of cointegrate plasmids under different drug selection pressures, which can promote the dissemination of MDR plasmids. IMPORTANCE The increasing resistance to ß-lactams and aminoglycoside antibiotics, mainly due to extended-spectrum ß-lactamases (ESBLs) and 16S rRNA methylase genes, is becoming a serious problem in Gram-negative bacteria. Plasmids, as the vehicles for resistance gene capture and horizontal gene transfer, serve a key role in terms of antibiotic resistance emergence and transmission. IS26, present in many antibiotic-resistant plasmids from Gram-negative bacteria, plays a critical role in the spread, clustering, and reorganization of resistance determinant-encoding plasmids and in plasmid reorganization through replicative transposition mechanisms and homologous recombination. However, the role of IS1294, present in many MDR plasmids, in the formation of cointegrates remains unclear. Here, we investigated experimentally the intermolecular recombination of IS1294, which occurred with high frequencies and led to the formation of conjugative MDR cointegrates and facilitated the cotransfer of blaCTX-M-55 and rmtB, and we further uncovered the significance of IS1294 in the formation of cointegrates and the common features of IS1294-driven cointegration of plasmids.

Silent transmission of an IS1294b-deactivated mcr-1 gene with inducible colistin resistance.

Global dissemination of the mobile colistin resistance mcr-1 is of particular concern as colistin is one of the last-resort antibiotics for the treatment of severe infections caused by carbapenem-resistant Gram-negative bacteria. In this study, an inactive form of mcr-1 in a fluoroquinolone-resistant and colistin-susceptible uropathogenic Escherichia coli isolate (ECO3347) was characterised. The mcr-1 gene was deactivated by insertion of a 1.7-kb IS1294b element flanked by two tetramers (GTTC) and located on a 62-kb pHNSHP45-like plasmid (p3347-mcr-1). Single-step and multistep selections were used to induce colistin resistance in vitro in ECO3347. ECO3347 acquired colistin resistance (MIC = 16-32 mg/L) only after a serial passage selection with increasing concentrations of colistin (2-8 mg/L). Deactivated mcr-1 was re-activated by loss of IS1294b without any remnants in most colistin-resistant mutants. In addition, a novel amino acid variant (Leu105Pro) in the CheY homologous receiver domain of PmrA was detected in one colistin-resistant mutant. Plasmid p3347-mcr-1+ carrying the re-activated mcr-1 gene is transferrable to E. coli J53 recipient with a high conjugation rate (ca. 10-1 cells per recipient cell). Transconjugants showed an identical growth status to J53, suggesting lack of a fitness cost after acquiring p3347-mcr-1+. These results highlight that the disrupted mcr-1 gene has the potential for wide silent dissemination with the help of pHNSHP45-like epidemic plasmids. Inducible colistin resistance may likely compromise the success of clinical treatment and infection control. Continuous monitoring of mcr-1 is imperative for understanding and tackling its dissemination in different forms.