Restricting ciprofloxacin-induced resistant variant formation in biofilm of Klebsiella pneumoniae B5055 by complementary bacteriophage treatment

The in vitro efficacy of lytic bacteriophage as a therapeutic agent against Klebsiella pneumoniae biofilm, alone or in combination with ciprofloxacin, was studied. The pathogenic potential of the resistant variants formed during the treatment was evaluated. A lytic bacteriophage in combination with ciprofloxacin was used for the treatment of K. pneumoniae B5055 biofilm. The efficacy and the frequency of resistant variant formation were estimated after respective treatments. The resistant variants were characterized for their virulence potential. Bacteriophage alone was able to eradicate the biofilm effectively and no significant difference was observed in its ability to eradicate biofilm in combination with ciprofloxacin. However, combination treatment using ciprofloxacin and bacteriophage significantly arrested the emergence of resistant variants. The small number of variants that developed had a lower propensity to form biofilms, produced small amounts of cell-associated capsular polysaccharide and demonstrated increased susceptibility to mouse peritoneal macrophages. Altered morphology and changed pattern of the outer membrane proteins of bacterial isolates were also observed. The combination treatment not only killed the bacteria, but also restricted the formation of resistant variants significantly as compared with individual treatments. Hence, a combination of bacteriophage and ciprofloxacin offers an effective strategy to combat the emergence of treatment-associated resistance.