In vitro efficacy of bacteriophage and colistin combinations in eradicating biofilm formed by colistin-resistant Enterobacter cloacae complex

In the case of dry antibiotic pipelines and the emergence of resistance against last-resort antibiotics, such as colistin, alternative medicinal techniques have been necessitated. In this context, the potential of phages as a viable alternative to antibiotics is a beacon of hope. Phage and antibiotic combination therapy is a compelling solution to the problem of phage or bacterial mutant generation. In most studies, phage and antibiotic combinations were used simultaneously. Only a few phages have been recently utilised before antibiotics, significantly reducing the bacterial load in vitro and in vivo. Therefore, we investigated the additive action of phage antibiotic combinations in preventing biofilm formation against three different colistin-resistant clinical isolates of the Enterobacter cloacae complex, specifically Enterobacter cloacae and Enterobacter hormaechei, at various time points with varying phage concentrations. We used different phage concentrations and sub-MICs of colistin combinations to determine the optimal phage concentration that shows synergy against three different clinical isolates of the Enterobacter cloacae complex. We also investigated the optimal time for applying antibiotics after phage treatment to eradicate Enterobacter cloacae complex in both planktonic and biofilm states. The cross-colistin susceptibility of colistin-resistant strain after co-treatment with SIM (Phage antibiotic simultaneous) and AF (Antibiotic added 8 h before phage) was also evaluated using the broth dilution method. In brief, applying phage before 8 h or 6 h of colistin, i.e. PF (Phage first followed by antibiotics) treatment at a dosage of 10⁶ PFU/mL, was an effective sequential therapy for eliminating the biofilm and the planktonic form of Enterobacter cloacae complex. The Institutional Ethics Committee of Banaras Hindu University (Institute of Medical Sciences), Varanasi, with reference number Dean/2024/IAEC/6876, has approved this in vitro study.