Inhibiting biofilm formation by Klebsiella pneumoniae B5055 using an iron antagonizing molecule and a bacteriophage

Abstract

Background: Success of biofilm dwelling bacteria in causing persistent and chronic infections is attributed to their resistance towards antibiotics and immune defences. Free iron is critical for the growth of biofilm associated bacteria. Therefore in the present study, the effect of limiting iron levels by addition of divalent Co[II] ions in combination with a bacteriophage was used for preventing/disrupting Klebsiella pneumoniae biofilms.

Results: A significantly higher reduction (p < 0.005) in bacterial numbers in the younger as well as older biofilms treated with Co[II] and depolymerase producing phage in combination was observed in comparison to when either of the agents was used alone. The role of phage borne depolymerase was confirmed, as an insignificant eradication of biofilm by non-depolymerase producing bacteriophage in combination with cobalt ions was observed. The results of viable count were further confirmed by visual examination of biofilms.

Conclusion: From the study it can be concluded, that iron antagonizing molecules and bacteriophages can be used as adjunct therapy for preventing biofilm development.

Figure 1

Kinetics of biofilm formation by K. pneumoniae B5055 grown in minimal media (M9) with or without supplementation of FeCl₃. *p < 0.05 (10 μM FeCl₃ vs control group).

Figure 2

Kinetics of biofilm formation by K. pneumoniae B5055 grown in minimal media (M9) containing cobalt salt (CoSO₄) or FeCl₃ alone and in combination. *p < 0.05 (10 μM FeCl₃ + 500 μM CoSO₄ vs 10 μM FeCl₃), ^¶p < 0.05 (10 μM FeCl₃ + 500 μM CoSO₄ vs 500 μM CoSO₄).

Figure 3

Kinetics of biofilm formation by K. pneumoniae B5055 grown in minimal media (M9) supplemented with 10 μM FeCl₃ and treated with 500 μM cobalt salt (CoSO₄) and bacteriophage (KPO1K2)/ (NDP) alone as well as in combination. *p < 0.05 [(10 μM FeCl₃ +500 μM CoSO₄ + Ø(KPO1K2) vs 10 μM FeCl₃/10 μM FeCl₃+ 500 μM CoSO₄/10 μM FeCl₃+ Ø(KPO1K2)], **p < 0.005 [(10 μM FeCl₃ +500 μM CoSO₄ + Ø(KPO1K2) vs 10 μM FeCl₃/10 μM FeCl₃+ 500 μM CoSO₄/10 μM FeCl₃+ Ø(KPO1K2)], ^#p < 0.05 [(10 μM FeCl₃ + Ø(KPO1K2) vs 10 μM FeCl₃], ^$p < 0.05[(10 μM FeCl₃ +500 μM CoSO₄) vs 10 μM FeCl₃], ^!p > 0.05[(10 μM FeCl₃ +500 μM CoSO₄ + Ø(NDP) vs 10 μM FeCl₃+ 500 μM CoSO₄].

Figure 4

Kinetics of biofilm formation (on cover slips) by K. pneumoniae B5055 grown in minimal media (M9) supplemented with 10 μM FeCl₃ and treated with 500 μM cobalt salt (CoSO₄) and bacteriophage (KPO1K2) alone as well as in combination. **p < 0.005 [(10 μM FeCl₃ +500 μM CoSO₄ + Ø(KPO1K2) vs 10 μM FeCl₃/10 μM FeCl₃+ 500 μM CoSO₄/10 μM FeCl₃+ Ø(KPO1K2)], *p < 0.05 [(10 μM FeCl₃ +500 μM CoSO₄ + Ø(KPO1K2) vs 10 μM FeCl₃+ 500 μM CoSO₄], ^#p < 0.005 [(10 μM FeCl₃ +500 μM CoSO₄ + Ø(KPO1K2) vs 10 μM FeCl₃/10 μM FeCl₃+ Ø(KPO1K2)].

Figure 5

K. pneumoniae B5055 biofilm developed on coverslip (a/a´) 3/7 day biofilm grown in 10 μM FeCl₃ supplemented media (b/b´) 3/7 day biofilm grown in 10 μM FeCl₃ + 500 μM cobalt salt supplemented media (c/c´) 3/7 day biofilm grown in 10 μM FeCl₃ supplemented media followed by treatment with phage KPO1K2 (d/d´) 3/7 day biofilm grown in 10 μM FeCl₃ + 500 μM cobalt salt supplemented media followed by treatment with phage KPO1K2.