Comparative characterisation of genotypically different clones of MRSA in the production of biofilms

Abstract

The ability to adhere and produce biofilms is characteristic of enhanced virulence among isolates of methicillin-resistant Staphylococcus aureus (MRSA). The aim of the study is to find out whether these characteristics are consistently similar among isolates variations of MRSA. The study used 30 various isolates of MRSA belong to 13 spa types and 5 MLST types and determined the aggregation, the adherence, and the production of biofilms and slime for each isolate. The methods used to evaluate these characteristics were a modified Congo red agar assay (MCRA), a microtiter plate assay (MPA), high-magnification light microscopy, scanning electron microscopy (SEM), and PCR. The study found that isolates belonging to similar Spa, SCCmec, and ST types have similar abilities to produce biofilms; however, their ability to produce slime on CRA was found to be different. Moreover, isolates that have different Spa types showed high variation in their ability to produce biofilms. The results of light microscope revealed the isolates that produced strong and weak biofilms and formed similar aggregation on the glass surfaces. SEM results showed that all 30 MRSA isolates that were tested were 100% positive for biofilm formation, although to varying degrees. Further testing using PCR confirmed that 100% of the 30 isolates tested were positive for the presence of the icaADBC, fnbA, eno, ebps, clfA, and clfB genes. The prevalence of fib, cna, fnbB, and bbp in MRSA clones was 90, 93.33, 53.33, and 10%, respectively. This study indicate that differences in biofilm production capacities are caused by the differences in surface protein A (Spa) type and are not due to differences in MLST and SCCmec types.

Figure 1

Total biofilm formation of different clinical MRSA isolates. The bacteria were grown in 6-well tissue culture plates containing TSB supplemented with 1% glucose. The cells that adhered to the plate surface after washing were visualised by staining with safranin. The clones were considered as highly, strongly, moderately, weakly, and nonadherent based upon their absorbance as measured by a spectrophotometer.

Figure 2

A chart displaying each spa type measures the quantity of biofilm using a microtiter plate assay for all isolates of this spa type represented as dots. Data represent the mean of triplicate determinations of the increase in bacterial adhesion. An OD value of 0.5 was selected as the cut-off point; any OD value above the cutoff OD was considered positive for biofilm formation. The isolates of one clone have similar quantitative biofilm-forming properties. The isolates of different clones have different quantitative biofilm forming properties. (a) Indicates to the single frequently isolates belonging to different spa typing. (b) Indicates to the three similar isolates of one clone. (c) Indicates to the two similar isolates of one clone. +: Indicates to the positive biofilm reference strain. −: Indicates to the negative biofilm reference strain.

Figure 3

Direct visualisation of bacterial autoaggregation in liquid culture media of strongly, moderately, and weakly adherent isolates on a glass coverslip surface after a 48 h incubation using a colony counter lens (a) and the Nikon Eclipse light microscope (b).

Figure 4

Direct visualisation of bacterial auto-aggregation in liquid culture media of the non-adherent Staphylococcus epidermidis ATCC 12228 reference strain on a glass coverslip surface after a 48 h incubation using a colony counter lens (a) and the Nikon Eclipse light microscope (b).

Figure 5

A scanning electron micrograph illustrating the appearance of thick extracellular products in strongly, moderately, and weakly adherent isolates (a) and the absence of those products in the non-adherent Staphylococcus epidermidis ATCC 12228 reference strain (b).

Figure 6

A scanning electron micrograph revealing variable degrees of heavy biofilm layer production. Highly and strongly adherent isolates produced a large thick layer (a), and moderately and weakly adherent isolates formed a lower degree of a close network among themselves (b), while negative biofilm producers showed an absence of network layers (c).