The Influence Of Iron In The Growth And Bio Film Formation Of Klebsiella Pneumoniae And ESBL Producing Klebsiella Pneumoniae

Abstract

Background: The aim of this study was to investigate the impact of different iron concentrations in the growth and biofilm formation betweenKlebsiella pneumoniae and ESBL producingKlebsiella pneumoniae
Methods: An experimental study used a sample of 12 clinical isolates of ETT aspirates from the Unit of Clinical Microbiology,Dr. Soetomo hospital, starting from October 2020 to March 2021. A growth measurement and a biofilm formation test were performed at different concentration of iron sulphate.Statistical analysis was performed using SPSS, one-way ANOVA test and post hoc LSD, with significancy level p<0.05.
Results: In the absence of iron, 0 μMol, classicK. pneumoniae showed a slower peak attainment of growth (5 McFarland), which occurred at 5 hours incubation, one hour later than the ESBL producing K. pneumoniae and ATCC 700603. With the addition of low iron concentration (1 μMol), it increased the time of peak achievement of growth rate for all strains, classic K. pneumoniae, ESBL producing K. pneumoniae and ATCC 700603. An intermediate iron sulphate concentration, 10 μMol, classic Klebsiella pneumoniaeshowed a slower rate of two peaks attainment for its log growth fase, the first peak (4 McFarland) was after five hours incubation and then the second (4.5 McFarland)occurred after 12 hours incubation, whencompare toATCC 700603. At high concentration of iron sulphate (100μMol), all strains formed gas, therefore the normal peak of log growth phase was not achieved instead an irregular fluctuating growth density curve was observed.After 24 hours incubation (day one), in the absence of iron, classic K. pneumoniae formed a moderate density (1 unit) of biofilm compare to slightly more moderate (1.3 unit) biofilm formation by ESBL producing K. pneumoniae (p=0.024) and a stronger (1.6 unit) biofilm density of ATCC 700603 (p=0.014). These biofilm densities showed the same pattern of density escalation in day two. Low (1μMol) and intermediate (10 μMol) induced all three strains into forming stronger density biofilm, whereas, high iron concentration, 100 μMol, did limit their biofilm formation.
Conclusion: ESBL producing Klebsiella pneumoniae and ATCC 700603 showed a faster growth rate and significantly stronger biofilm formation compare to classic K. pneumoniae in the depletion of iron concentration (0 μMol) (p < 0.05).Low (1 μMol) iron concentrations had proven their effect as inducer of growth and significantly forming stronger and more stable biofilm in all three strains (p> 0.05). Whereas an intermediate (10 μMol)iron concentration showed similar inducing effect in classic Klebsiella pneumoniae and ESBL producing Klebsiella pneumoniae, except ATCC 700603 tends to show more increased planktonic growth and decreasing biofilm stability in day 2 incubation (p < 0.05). A 100 μMol concentration of iron sulphate is considered high and showed an increased rate of gas formation effect which resulted in disrupted pattern of growth and a significantly slower and weaker biofilm formation in all three strains (p> 0.05, there was no significant different among three groups).