Characterization of Small Colony Variants of Klebsiella pneumoniae: Correlation with Antibiotic Resistance and Biofilm Formation

Background: Small colony variants (SCVs) of bacterial pathogens are smaller, slow-growing variants which often pose a challenge to the clinical microbiologist in their identification and characterization. SCVs are receiving much attention in recent years due to their association with several types of chronic infections. In this study, we aimed to develop a suitable culture media for high frequency generation and stable maintenance of SCV of Klebsiella pneumoniae. We also intended to compare different phenotypic characteristics such as growth, antibiotic resistance pattern, and biofilm-forming potential of SCVs with the original parental strain. Methods: We used Mueller-Hinton agar containing the extract of clove (Syzygium aromaticum) for the generation of SCV. Antibiotic sensitivity was determined using disk diffusion method and minimum inhibitory concentration determinations using microdilution method. Biofilm formation was assessed using crystal violet dye binding assay. Results: Mueller-Hinton agar (MHA) containing clove (Syzygium aromaticum) extract (10% volume/volume; MHA-C10) supported generation of SCV from K. pneumoniae at high frequency. SCVs were smaller in colony size and grew slowly in comparison to the wild-type original strain. In addition, SCVs exhibited increased resistance to aminoglycoside group of antibiotics (gentamicin and kanamycin). Crystal violet dye binding spectrophotometric method showed increased biofilm formation potential by SCVs in comparison to their parental counterparts. Conclusions: The findings of this study show that MHA-C10 can be used as a bacterial culture media for the formation of SCV by K. pneumoniae. SCVs, thus, generated on MHS-C10 exhibited typical characteristics of SCVs.