A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital. Strain 628 produces KPC-2 and CTX-M-55 encoded by two different conjugative plasmids, i.e., the IncFIIK plasmid p628-KPC and the IncI1 plasmid p628-CTXM, respectively. blaKPC-2 is captured by a Tn1721-based unit transposon with a linear structure ΔTn3-ISKpn27-blaKPC-2-ΔISKpn6-ΔTn1721, and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. blaKPC-2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region, respectively), which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core -35/-10 elements TAATCC/TTACAT and TTGACA/AATAAT, respectively. blaCTX-M-55 is mobilized in an ISEcp1-blaCTX-M-55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. blaCTX-M-55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region), corresponding to the ISEcp1-provided P1 promoter with the core -35/-10 element TTGAAA/TACAAT. All the above detected promoters display a characteristic of constitutive expression. Coexistence of blaKPC and blaCTX-M in K. pneumoniae has been reported many times, but this is the first report to gain deep insights into genetic platforms, promoters, and expression of the two coexisted bla genes with determination of entire nucleotide sequences of the two corresponding plasmids.

As the overuse of antibiotics has not yet been strictly limited in urban areas, drug-resistant Escherichia coli has become a fatal pressure for bacteremia treatment. Considering the outstanding performance of bacteriophages in vitro , bacteriophages may serve as an alternative to heal chronic refractory infections. In this study, a 49,890 bp double-stranded circular DNA phage, Henu8, was isolated and was able to lyse the group of E. coli strains tested in this study. Prominent biological characterization revealed that the highly adsorbed bacteriophage Henu8 could form a fully transparent plaque with a narrow translucent halo. The optimal multiplicity of infection of the bacteriophage Henu8 was 0.01, with a burst size of 275 PFU/cell. Genomic analysis revealed a G + C content of 44.17% Henu8, in which 65 open reading frames were located, which could be assigned as a new species in the genus Hanrivervirus of the subfamily Tempevirinae . The effective antibacterial ability and the obvious biofilm destruction and inhibition capability of phage Henu8 were observed. The time-killing assay demonstrated the synergetic potential of Henu8 with antibiotics in vitro for E. coli eradication. Henu8 has profound medicinal potential in a mouse bacteremia model. These studies indicate that Henu8 is a novel bacteriophage with therapeutic potential alone or in combination with antibiotics for clinical treatment. IMPORTANCE: The findings described in this study constitute concrete evidence that it is possible to significantly synergize the antimicrobial activity of bacteriophages and antibiotics. We showed that the newly isolated potent bacteriophage Henu8 lyses Escherichia coli rapidly but tends to produce resistant bacteria. The bacteriophage Henu8 has synergistic antimicrobial effects with several antibiotics and is not susceptible to developing resistance. These results provide further evidence that bacterial resistance to phages arises, possibly at an adaptive cost to sensitivity to antibiotics. Therefore, the findings of this study are important for increasing the potential of phages for clinical applications and developing new approaches to improve their therapeutic efficacy against bacterial drug resistance.

Extended-spectrum beta-lactamase-producing Gram-negative bacteria are common in the community and hospitals. To monitor ESBLs mediated by the CTX-M genotype, we collected clinical ESBL pathogenic strains from a hospital in central China and observed a strain of Escherichia coli, namely Ec15103 carrying bla_CTX–M–14, bla_CTX–M–64 and bla_TEM–1, isolated from the blood of a 7-day-old infant in 2015. Strain Ec15103 contains two drug resistance plasmids: pEc15103A, an IncFI-type plasmid that cannot be conjugatively transferred and carries the drug resistance genes bla_TEM–1, aacC2, aadA5, sul1, mph(A), sul2, strAB, and tetA(A); and pEc15103B, an IncK2/Z-type plasmid that carries the conjugation transfer gene and bla_CTX–M–14. In addition, bla_CTX–M–64 is located on the chromosome of Ec15103, and it is the first report of pathogen with bla_CTX–M–64 located on its chromosome (the search terms used “blaCTX-M-64” and “chromosome”). bla_CTX–M–14 and bla_CTX–M–64 are carried by ISEcp1-mediated transposon Tn6503a and Tn6502, respectively. The conjugation transfer ability of pEc15103B was significantly inhibited by zidovudine (AZT) and linoleic acid (LA) and that expression of bla_CTX–M–14, bla_CTX–M–64 and bla_TEM–1 at the mRNA level did not change based on the concentration of cefotaxime or ampicillin. Co-occurrence of bla_CTX–M–14 and bla_CTX–M–64 in a single isolate will enhance the drug resistance of bacteria, and the presence of bla_CTX–M–64 in the chromosome may make the resistance more maintain. This fact will facilitate its dissemination and persistence under different antimicrobial selection pressures. It is essential to prevent these strains from further spreading in a hospital environment.