This work aimed to characterize a 29-kb bla_KPC-2-carrying plasmid, pR31-KPC, from a multidrug resistant strain of Pseudomonas aeruginosa isolated from the sputum of an elderly patient with multiple chronic conditions in China. The backbone of pR31-KPC is closely related to four other bla_KPC-2-carrying plasmids, YLH6_p3, p1011-KPC2, p14057A, and pP23-KPC, none of which have been assigned to any of the known incompatibility groups. Two accessory modules, the IS26-bla_KPC-2-IS26 unit and IS26-ΔTn6376-IS26 region, separated by a 5.9-kb backbone region, were identified in pR31-KPC, which was also shown to carry the unique resistance marker bla_KPC-2. A comparative study of the above five plasmids showed that p1011-KPC2 may be the most complete plasmid of this group to be reported, while pR31-KPC is the smallest plasmid having lost most of its conjugative region. Regions between the iterons and orf207 in the backbone may be hot spots for the acquisition of exogenous resistance entities. The accessory regions of these plasmids have all undergone several biological events when compared with Tn6296. The further transfer of bla_KPC-2 in these plasmids may be initiated by either the Tn3 family or IS26-associated transposition or homologous recombination. The data presented here will contribute to a deeper understanding of bla_KPC-2 carrying plasmids in Pseudomonas.

The need for novel antibiotics has become imperative with the increasing prevalence of antibiotic resistance in Gram-negative bacteria in clinics. Acting as a permeability barrier, lipopolysaccharide (LPS) protects Gram-negative bacteria against drugs. LPS is synthesized in cells and transported to the outer membrane (OM) via seven lipopolysaccharide transport (Lpt) proteins (LptA–LptG). Of these seven Lpt proteins, LptC interacts with LptA to transfer LPS from the inner membrane (IM) to the OM, and assembly is aided by LptD/LptE. This interaction among the Lpt proteins is important for the biosynthesis of LPS; therefore, the Lpt proteins, which are significant in the assembly process of LPS, can be a potential target for new antibiotics. In this study, a yeast two-hybrid (Y2H) system was used to screen compounds that could block LPS transport by inhibiting LptA/LptC interaction, which finally disrupts the biosynthesis of the OM. We selected the compound IMB-0042 for this study. Our results suggest that IMB-0042 disrupts LptA/LptC interaction by binding to both LptA and LptC. Escherichia coli cells, when treated with IMB-0042, showed filament morphology, impaired OM integrity, and an accumulation of LPS in the periplasm. IMB-0042 inhibited the growth of Gram-negative bacteria and showed synergistic sensitization to other antibiotics, with low cytotoxicity. Thus, we successfully identified a potential antibacterial agent by using a Y2H system, which blocks the transport of LPS by targeting LptA/LptC interaction in Escherichia coli.

Background: The spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) among humans and food-producing animals has been widely reported. However, the transmission routes and associated risk factors remain incompletely understood. Methods: Here, we used commensal Escherichia coli bacteria strains from faeces of pigs and local citizens [HEG: high exposure group (pig breeders, butchers or restaurant chefs) and LEG: low exposure group (other occupations)] to explore the dynamics of ARB and ARG transmission between animals and humans. Results: Most ARGs (96%) present in pigs were shared with humans. Carriage rates of the shared ARGs suggest two transmission patterns among pigs, the HEG and LEG: one pattern was highest in pigs, gradually decreasing in the HEG and LEG (e.g. floR and cmlA1); the other pattern was increasing from pigs to the HEG but then decreasing in the LEG (e.g. mcr-1.1). Carriage rates of the HEG were higher than in the LEG in both patterns, implicating the HEG as a crucial medium in transmitting ARB and ARGs between food-producing animals and humans. Moreover, frequent inter/intragroup transmission via strains, plasmids and/or mobile elements was evident. Carriage of mcr-1.1 on human-gut-prevalent plasmids possibly promoted its enrichment in the HEG. Conclusions: The HEG is a crucial factor in transmitting ARB and ARGs between food-producing animals and humans. Rational measures to contain the risks of occupational exposure are urgently needed to keep dissemination of antibiotic resistance in check and safeguard public health.

An increase in the carbapenem-hydrolyzing capacity of class D β-lactamase has been observed in strains of multiple species, posing a significant challenge to the control of antibiotic resistance. In this study, we aimed to investigate the genetic diversity and phylogenetic characteristics of new bla_OXA-48-like variants derived from Shewanella xiamenensis. Three ertapenem-non-susceptible S. xiamenensis strains were identified, one isolated from the blood sample of an inpatient, the other two isolated from the aquatic environment. Phenotypic characterization confirmed that the strains were carbapenemase producers and exhibited antimicrobial resistance patterns to ertapenem, with some showing lower susceptibility to imipenem, chloramphenicol, ciprofloxacin, and tetracycline. No significant resistance to cephalosporins was observed. Sequence analysis revealed that one strain harbored bla_OXA-181 and the other two strains harbored bla_OXA-48-like genes, with open reading frame (ORF) similarities with bla_OXA-48 ranging from 98.49% to 99.62%. The two novel bla_OXA-48-like genes, named bla_OXA-1038 and bla_OXA-1039, respectively, were cloned and expressed in E. coli. The three OXA-48-like enzymes demonstrated significant hydrolysis activity against meropenem, and the classical β-lactamase inhibitor had no significant inhibitory effect. In conclusion, this study demonstrated the diversity of the bla_OXA gene and highlighted the emergence of novel OXA carbapenemases in S. xiamenensis. Further attention to S. xiamenensis and OXA carbapenemases is recommended for the effective prevention and control of antibiotic-resistant bacteria.

ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5′-GXXT-3′ sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase.