Seven mobile oxazolidinone resistance genes, including cfr , cfr (B), cfr (C), cfr (D), cfr (E), optrA , and poxtA , have been identified to date. The cfr genes code for 23S rRNA methylases, which confer a multiresistance phenotype that includes resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A compounds. The optrA and poxtA genes code for ABC-F proteins that protect the bacterial ribosomes from the inhibitory effects of oxazolidinones.

Antibiotic resistance and the ability to form biofilms of Enterococcus faecalis have compromised the choice of therapeutic options, which triggered the search for new therapeutic strategies, such as the use of phage endolysins and antimicrobial peptides. However, few studies have addressed the synergistic relationship between these two promising options. Here, we investigated the combination of the phage endolysin Ply2660 and the antimicrobial peptide LL-37 to target drug-resistant biofilm-producing E. faecalis. In vitro bactericidal assays were used to demonstrate the efficacy of the Ply2660–LL-37 combination against E. faecalis. Larger reductions in viable cell counts were observed when Ply2660 and LL-37 were applied together than after individual treatment with either substance. Transmission electron microscopy revealed that the Ply2660–LL-37 combination could lead to severe cell lysis of E. faecalis. The mode of action of the Ply2660–LL-37 combination against E. faecalis was that Ply2660 degrades cell wall peptidoglycan, and subsequently, LL-37 destroys the cytoplasmic membrane. Furthermore, Ply2660 and LL-37 act synergistically to inhibit the biofilm formation of E. faecalis. The Ply2660–LL-37 combination also showed a synergistic effect for the treatment of established biofilm, as biofilm killing with this combination was superior to each substance alone. In a murine peritoneal septicemia model, the Ply2660–LL-37 combination distinctly suppressed the dissemination of E. faecalis isolates and attenuated organ injury, being more effective than each treatment alone. Altogether, our findings indicate that the combination of a phage endolysin and an antimicrobial peptide may be a potential antimicrobial strategy for combating E. faecalis.

Objectives: To characterize an NDM-1-encoding multiresistance IncHI5 plasmid from Enterobacter cloacae complex of chicken origin.Methods: Carbapenemase genes were detected by PCR and Sanger sequencing. The MICs for the E. cloacae complex isolate and its transformant were determined by the agar dilution and broth microdilution methods. Conjugation and electrotransformation were performed to assess the horizontal transferability of the carbapenemase plasmid. Plasmid DNA was isolated from the transformant and fully sequenced using Illumina HiSeq and PacBio platforms. Plasmid stability was investigated by sequential passages on antibiotic-free medium. A circular intermediate was detected by inverse PCR and Sanger sequencing.Results: Plasmid pNDM-1-EC12 carried a conserved IncHI5 backbone and exhibited an MDR phenotype. All antimicrobial resistance genes were clustered in a single MDR region. Genetic environment analysis revealed that the blaNDM-1 gene was in a novel complex integron, In469. Based on sequence analysis, the blaNDM-1-carrying region was thought to be inserted by homologous recombination. Inverse PCR indicated that an ISCR1-mediated circular intermediate can be formed. Plasmid pNDM-1-EC12 was stably maintained both in the parental strain and the transformant without selective pressure. Comprehensive analysis of IncHI5-type plasmids suggested that they may become another key vehicle for rapid transmission of carbapenemase genes.Conclusions: To the best of our knowledge, this is the first report of a fully sequenced IncHI5 plasmid recovered from an E. cloacae complex strain of food-producing animal origin. Co-occurrence of blaNDM-1 with genes encoding resistance to other antimicrobial agents on the same IncHI5 plasmid may result in the co-selection of blaNDM-1 and facilitates its persistence and rapid dissemination.

Objectives: To identify and characterize a novel tetracycline resistance gene on a multiresistance plasmid from Staphylococcus aureus SA01 of chicken origin. Methods: MICs were determined by broth microdilution according to CLSI recommendations. The whole genome sequence of S. aureus SA01 was determined via Illumina HiSeq and Oxford Nanopore platforms followed by a hybrid assembly. The new tet gene was cloned and expressed in S. aureus. The functionality of the corresponding protein as an efflux pump was tested by efflux pump inhibition assays. Results: A novel tetracycline resistance gene, tet(63), was identified on a plasmid in S. aureus SA01. The cloned tet(63) gene was functionally expressed in S. aureus and shown to confer resistance to tetracycline and doxycycline, and a slightly elevated MIC of minocycline. The tet(63) gene encodes a 459 amino acid efflux protein of the major facilitator superfamily that consists of 14 predicted transmembrane helices. The results of efflux pump inhibitor assays confirmed the function of Tet(63) as an efflux protein. The deduced amino acid sequence of the Tet(63) protein exhibited 73.0% identity to the tetracycline efflux protein Tet(K). The plasmid pSA01-tet, on which tet(63) was located, had a size of 25664 bp and also carried the resistance genes aadD, aacA-aphD and erm(C). Conclusions: A novel tetracycline resistance gene, tet(63), was identified in S. aureus. Its location on a multiresistance plasmid might support the co-selection of tet(63) under the selective pressure imposed by the use of macrolides, lincosamides and aminoglycosides.

Mammalian cathelicidins act as the potent microbicidal molecules for controlling bacterial infection, and are considered promising alternatives to traditional antibiotics. Their ability to modulate host immune responses, as well as their bactericidal activities, is essential for therapeutic interventions. In this study, we compared the bactericidal activities, antibiofilm activities and immune-modulatory properties of cathelicidins BMAP-27, BMAP-34, mCRAMP, and LL-37, and evaluated the therapeutic efficacy of the combination of BMAP-27 and LL-37 using a mouse pulmonary infection model. Our results showed that all of the four cathelicidins effectively killed bacteria via rapid induction of membrane permeabilization, and BMAP-27 exhibited the most excellent bactericidal activity against diverse bacterial pathogens. BMAP-27, mCRAMP, and LL-37 effectively inhibited biofilm formation, while BMAP-34, mCRAMP and LL-37 exerted immunomodulatory functions with varying degrees of efficacy by stimulating the chemotaxis of neutrophils, inducing the production of reactive oxygen species, and facilitating the formation of neutrophil extracellular traps. Of note, the combination of BMAP-27 and LL-37 effectively enhanced the clearance of Pseudomonas aeruginosa and reduced the organ injury in vivo. Together, these findings highlight that identifying the appropriate synergistic combination of mammalian cathelicidins with different beneficial properties may be an effective strategy against bacterial infection.