Carbapenem-resistant Enterobacter aerogenes strains are a major clinical problem because of the lack of effective alternative antibiotics. However, viruses that lyze bacteria, called bacteriophages, have potential therapeutic applications in the control of antibiotic-resistant bacteria. In the present study, a lytic bacteriophage specific for E. aerogenes isolates, designated vB_EaeM_φEap-3, was characterized. Based on transmission electron microscopy analysis, phage vB_EaeM_φEap-3 was classified as a member of the family Myoviridae (order, Caudovirales). Host range determination revealed that vB_EaeM_φEap-3 lyzed 18 of the 28 E. aerogenes strains tested, while a one-step growth curve showed a short latent period and a moderate burst size. The stability of vB_EaeM_φEap-3 at various temperatures and pH levels was also examined. Genomic sequencing and bioinformatics analysis revealed that vB_EaeM_φEap-3 has a 175,814-bp double-stranded DNA genome that does not contain any genes considered undesirable for the development of therapeutics (e.g., antibiotic resistance genes, toxin-encoding genes, integrase). The phage genome contained 278 putative protein-coding genes and one tRNA gene, tRNA-Met (AUG). Phylogenetic analysis based on large terminase subunit and major capsid protein sequences suggested that vB_EaeM_φEap-3 belongs to novel genus “Kp15 virus” within the T4-like virus subfamily. Based on host range, genomic, and physiological parameters, we propose that phage vB_EaeM_φEap-3 is a suitable candidate for phage therapy applications.

A carbapenem-nonsusceptible Enterobacter aerogenes strain named 3-SP was isolated from a human case of pneumonia in a Chinese teaching hospital. NDM-1 carbapenemase is produced by a pNDM-BJ01-like conjugative plasmid designated p3SP-NDM to account for carbapenem resistance of 3-SP. p3SP-NDM was fully sequenced and compared with all publically available pNDM-BJ01-like plasmids. The genetic differences between p3SP-NDM and pNDM-BJ01 include only 18 single nucleotide polymorphisms, a 1 bp deletion and a 706 bp deletion. p3SP-NDM and pNDM-BJ01 harbor an identical Tn125 element organized as ISAba125, blaNDM-1, bleMBL, ∆trpF, dsbC, cutA, ∆groES, groEL, ISCR27 and ISAba125. The blaNDM-1 surrounding regions in these pNDM-BJ01-like plasmids have a conserved linear organization ISAba14-aphA6-Tn125-unknown IS, with considerable genetic differences identified within or immediately downstream of Tn125. All reported pNDM-BJ01-like plasmids are exclusively found in Acinetobacter, whereas this is the first report of identification of a pNDM-BJ01-like plasmid in Enterobacteriaceae.

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.

Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

Cronobacter sakazakii 505108 was isolated from a sputum specimen of a neonate with severe pneumonia. C. sakazakii 505108 co-harbors 3 resistance plasmids of the IncHI2, IncX3, and IncFIB incomparability groups, respectively. These 3 plasmids have acquired several accessory modules, which carry an extremely large number of resistance genes, especially including those involved in resistance to carbapenems, aminoglycoside, tetracyclines, and phenicols and sulphonamide/trimethoprim. These plasmid-borne antibiotic resistance genes were associated with insertion sequences, integrons, and transposons, indicating that the assembly and mobilization of the corresponding accessory modules with complex chimera structures are facilitated by transposition and/or homologous recombination. This is the first report of fully sequence plasmids in clinical Cronobacter, which provides a deeper insight into plasmid-mediated multi-drug resistance in Cronobacter from hospital settings.