Genomic and functional characterization of novel therapeutic lytic bacteriophages targeting multidrug-resistant Enterobacter cloacae

Abstract

The alarming rates at which extensively drug-resistant (XDR) and pandrug-resistant (PDR) Enterobacter cloacae in hospitals are increasing has begun to severely limit treatment options, and thus the urgency for alternative interventions, including bacteriophage therapy. The purpose of the study was to isolate and molecularly characterize phages that can infect E. cloacae, and, furthermore, to assess the antimicrobial efficacy of the four novel lytic bacteriophages (MMRP1, MMRP2, MMRP3, and MMRP4) against antimicrobial-resistant E. cloacae isolates and to evaluate their potential as alternative therapeutic strategies. These novel phages were characterized by plaque morphology, transmission electron microscopy (TEM), host range testing, thermal and chloroform stability assays, bacterial reduction assays, and whole-genome sequencing (WGS). Among 27 clinical isolates, MDR, XDR, and PDR phenotypes were observed in 20 (74.1%), six (22.2%), and one (3.7%) isolates, respectively. All four phages produced clear lytic plaques (0.5–3.0 mm) with titers reaching up to 6 × 1010 PFU/mL, and the phage cocktail lysed 81.4% (22 of 27 isolates) of clinical isolates with high host specificity. TEM revealed that all four E. cloacae-infecting phages (MMRP1, MMRP2, MMRP3, and MMRP4) belong to the class Caudoviricetes, exhibiting icosahedral capsids, tailed morphology, and double-stranded DNA genomes, consistent with current ICTV classification criteria. Whole genome sequencing and comparative phylogenetic analysis further resolved the taxonomic placement of these phages at the family level, positioning MMRP1 within the family Demerecviridae and MMRP4 within the family Straboviridae. All phages were stable from −20 to 40 °C and were unaffected by exposure to chloroform. Phage cocktail reduced bacterial OD₆₀₀ to ≤ 0.3 within 4 h in the bacterial reduction test. WGS revealed large circular dsDNA genomes of ~132 kbp (MMRP1) and ~149 kbp (MMRP4), GC content of 38%, and modular architectures encoding structural, lytic, and replication gene modules. The most striking and highlighted suggestion that in vitro evaluation of MMRP1 and MMRP4 are highly recommended to more deeper future experimental studies to combat MDR E. cloacae nosocomial infections supported by genomic foundation and eventually, the possibility to be suitable for phage-engineering applications in clinical settings.