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<i>Staphylococcus haemolyticus</i> has emerged to be a frequently encountered late-onset sepsis pathogen among newborn infants. Critical care of neonates involves substantial usage of antibiotics and these pathogens are often exposed to sub-optimal doses of antibiotics which can augment maintenance of selection determinants and a range of physiological effects, prime among them being biofilm formation. Therefore, in this study, the outcome of a sub-inhibitory dosage of a commonly prescribed third-generation antibiotic, cefotaxime (CTX), on multidrug resistant (MDR) <i>S. haemolyticus</i>, was investigated. A total of 19 CTX-resistant, MDR and 5 CTX-susceptible strains isolated from neonates were included. Biofilm-forming abilities of <i>S. haemolyticus</i> isolates in the presence of sub-optimal CTX (30 μg/mL) were determined by crystal violet assays and extracellular DNA (eDNA) quantitation. CTX was found to significantly enhance biofilm production among the non-susceptible isolates (<i>p</i>-value_Wilcoxintest—0.000008) with an increase in eDNA levels (<i>p</i>-value_Wilcoxintest—0.000004). Further, in the absence of antibiotic selection in vitro, populations of MDR isolates, JNM56C1 and JNM60C2 remained antibiotic non-susceptible after >500 generations of growth. These findings demonstrate that sub-optimal concentration of CTX induces biofilm formation and short-term non-exposure to antibiotics does not alter non-susceptibility among <i>S. haemolyticus</i> isolates under the tested conditions.