Influence of Sub-Inhibitory Dosage of Cefotaxime on Multidrug Resistant Staphylococcus haemolyticus Isolated from Sick Neonatal Care Unit

Abstract

Staphylococcus haemolyticus 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) S. haemolyticus, was investigated. A total of 19 CTX-resistant, MDR and 5 CTX-susceptible strains isolated from neonates were included. Biofilm-forming abilities of S. haemolyticus 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 (p-valueWilcoxintest—0.000008) with an increase in eDNA levels (p-valueWilcoxintest—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 S. haemolyticus isolates under the tested conditions.

Keywords: Staphylococcus haemolyticus; biofilms; cefotaxime; neonates; short-term evolution; sub-MIC.

Figure 1

Antimicrobial susceptibility and gene profiles of 19 CTX non-susceptible S. haemolyticus isolates. (A). The minimum inhibitory concentration of (MIC) of the 19 non-susceptible S. haemolyticus isolates with the inset showing the MIC of the five susceptible isolates. (B). The blaZ (858 bp), mecA (533 bp) and N-acetylglucosaminyltranferase (icaA) (166 bp) amplicons were run on 1.5% agarose gels. M; 100 bp marker, control; positive control, NTC; no template control. 16S amplicon (292 bp) served as control.

Figure 2

Cefotaxime (CTX), a commonly used ß-lactam antibiotic enhances biofilm formation among non-susceptible S. haemolyticus isolates in vitro. (A). Semi-quantitative determination of 24 h biofilm formation under known inducible conditions (tryptic soy broth with 1% glucose (TSB_glu) and 3% sodium chloride (TSB_NaCl)) and 30µg/mL CTX(TSB_CTX) using crystal violet assays. The dotted line denotes the cut-off optical density value for biofilm formation (0.25). The biofilm-forming ATCC 35984 S. epidermidis strain served as a control. (B). Biofilm supernatants run on 0.8% agarose gels to determine the presence of extracellular DNA (eDNA). M; 1000 bp marker. The lanes labelled 1, 2, 3 and 4 in each case denote TSB, TSB_glu, TSB_NaCl and TSB_CTX, respectively.

Figure 3

No change in in vitro antibiotic susceptibility among MDR, biofilm-forming S. haemolyticus isolates (JNM56C1 and JNM60C2) grown in the absence of antibiotics in short-term evolution experiments. (A). The presence of living sessile cells is confirmed by log(10)CFU counts in both isolates in the presence of 30 μg/mL CTX. The error bars represent standard deviation across three technical replicates. (B). Study design of the evolution experiment. (C). Comparison of susceptibility to ß-lactam and aminoglycoside antibiotics among ancestral and evolved populations using E-tests. (D). The blaZ (858 bp, panel 1), mecA (533 bp, panel 2) and AAC(6′)-APH(2′)(1658 bp, panel 3) and 16S (292 bp, panel 4) genes were amplified from the ancestral and evolved populations and amplicons were run on 0.8–1.5% agarose gels. M; 100/1000 bp marker, control; positive control, NTC; no template control.