Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniae and E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

Recombinant bacteriophage T4 displaying key epitopes of the foot-and-mouth disease virus as a novel nanoparticle vaccine.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that has caused significant economic losses in the livestock industry. Peptide vaccines engineered with the protective epitopes of FMDV have provided a safer alternative for disease prevention than the traditional inactivated vaccines. However, the immunogenicity of the peptide is usually poor and therefore an adjuvant is required. Here, we showed that recombinant T4 phages displaying the B-cell epitope of the FMDV VP1 protein (VP1130-158), without additional adjuvants, induced similar levels of antigen-specific IgG1 but higher levels of IgG2a compared to the peptide vaccine. Incorporation of a CD4+ T cell epitope, either 3A21-35 of FMDV 3A protein or P2830-844 of tetanus toxoid, further enhanced the immunogenicity of VP1-T4 phage nanoparticles. Interestingly, the extrinsic adjuvant cannot enhance the immunogenicity of the nanoparticles, indicating the intrinsic adjuvant activities of T4 phage. Furthermore, the recombinant T4 phage can be produced on a large scale within a short period of time at a relatively low-cost using Escherichia coli, heralding its potential in the development of a safe and effective FMDV vaccine.