Background: The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types.bla_CTX-Mgenes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given thatbla_CTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integratedbla_CTX-Mand the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements.Methods: A total of 46 clinical isolates of cefotaxime-resistantEnterobacteriaceae(1Enterobacter cloacae, 9Klebsiella pneumoniae, and 36Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes,bla_CTX-Mgenes were categorized as either chromosomal or plasmid-borne.Results: WGS-based genotyping identified 58 AMR genes, including fourbla_CTX-Malleles (i.e.,bla_CTX-M-14,bla_CTX-M-15,bla_CTX-M-27, andbla_CTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination ofbla_CTX-Mgenes. Out of 45bla_CTX-Mgene-carrying strains, 7 harbored the gene in their chromosome. In oneE. cloacaeand threeE. colistrains, chromosomalbla_CTX-M-15was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles.Conclusion: Our study revealed the co-occurrence of ISEcp1-bla_CTX-M-15and multiple AMR genes on chromosomal insertions inE. cloacaeandE. coli, suggesting that ISEcp1may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among theseEnterobacteriaceaespecies.

The demand for novel antibiotics to combat the global spread of multi drug-resistant pathogens continues to grow. Pathogenic bacteria and fungi that cause fatal human infections can also kill silkworms and the infected silkworms can be cured by the same antibiotics used to treat infections in the clinic. As an invertebrate model, silkworm model is characterized by its convenience, low cost, no ethical issues. The presence of conserved immune response and similar pharmacokinetics compared to mammals makes silkworm infection model to examine the therapeutic effectiveness of antimicrobial agents. Based on this, we utilized silkworm bacterial infection model suitable to screen the therapeutic effectiveness of various microbial culture broths and successfully identified a therapeutically effective novel antibiotic, lysocin E, which has a novel mode of action of binding to menaquinone, thus leading to membrane damage and bactericidal activity. The similar approach to screen potential antibiotics resulted in the identification of other therapeutically effective novel antibiotics, such as nosokomycin and ASP2397 (VL-2397). In this regard, we propose that the silkworm antibiotic screening model is very effective for identifying novel antibiotics. In this review, we summarize the advantages of the silkworm model and propose that the utilization of silkworm infection model will facilitate the discovery of novel therapeutically effective antimicrobial agents.

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Enterococcus faecalis is a common human gut commensal bacterium. While some E. faecalis strains are probiotic, others are known to cause opportunistic infections, and clear distinction between these strains is difficult using traditional taxonomic approaches. In this study, we completed the genome sequencing of EF-2001, a probiotic strain, using our in-house hybrid assembly approach. Comparative analysis showed that EF-2001 was devoid of cytolysins, major factors associated with pathogenesis, and was phylogenetically distant from pathogenic E. faecalis V583. Genomic analysis of strains with a publicly available complete genome sequence predicted that drug-resistance genes- dfrE, efrA, efrB, emeA, and lsaA were present in all strains, and EF-2001 lacked additional drug-resistance genes. Core- and pan-genome analyses revealed a higher degree of genomic fluidity. We found 49 genes specific to EF-2001, further characterization of which may provide insights into its diverse biological activities. Our comparative genomic analysis approach could help predict the pathogenic or probiotic potential of E. faecalis leading to an early distinction based on genome sequences.