Quantification and detection of Methicillin Resistant Staphylococcus aureus (MRSA) in manure, soil and air by means of a mecA specific qPCR.

Abstract

Staphylococcus aureus is a gram-positive commensal and potentially pathogenic bacterium of many different animals as well as humans. Since 1961, Staphylococcus aureus gained resistance against methicillin (MRSA) and new clones arose, including the livestock associated MRSA ST398. The current research project focuses on the emergence and distribution of MRSA in and through the environment. More specifically, it focuses on the occurrence of MRSA ST398 in agricultural soil and air, in relation to soil fertilization, as already studied in the Dutch SKBII project by means of a ST398 qPCR. This study however, only detected limited amounts of MRSA ST398 in manure and in very limited amounts in soil and air. No direct relation between manuring and the occurrence of MRSA in soil and air could be established. As the mecA gene in MRSA defines its ‘methicillin resistance’, the current threefold research project focuses on the detection of this gene in manure, soil and air, in order to screen for wider (methicillin) resistance.

First a mecA specific qPCR was developed and optimized (Part I). Primers and probes were selected and several qPCR optimization steps were completed. Eventually a SYBR Green based assay was considered best suited for further research on mecA detection. Secondly, the detection limit of this particular qPCR was defined in relation to air filter samples. (Part II). Two different kind of filters (SKC and Pall) were spiked with serial decimal dilutions of different bacteria, including MRSA ST398. After DNA extraction, the amount of recovered cells per filter was calculated and compared with the initial amount of spiked cells per filter. The detection limit of both assays is situated somewhere between 1549-15488 cells per filter. The analytical sensitivity ranges from 33%-50% at a 10-5 (~1560 cells) spiking concentration and the regression analysis revealed a highly linear assay. Also, the recovered gene copies per qPCR, correspond rather well with the theoretically predicted amount of gene copies. The precision of both SKC and Pall assays is considered fairly modest, but could have been influenced by the removal of fluid during the extraction process. The SKC and Pall filter assays showed respectively 100% and 88% specificity, hinting towards possible assay contamination. The DNA extraction protocol, corresponding to the Pall filter assay, was found to be more suited to deal with higher bacterial concentrations. Some other comments can however be made about the assays accuracy. Not only is this study a laboratory-based assay, also no influence of real particulate matter (PM) on air filters was investigated. In addition, more cells were recovered from the filters, than spiked, which however also shows that the DNA extraction process didn’t result in great DNA losses. Additionally, no inhibition testing was performed. Finally, the spiking technique itself should be redesigned for future detection limit testing. Part III of this report describes the detection of mecA in manure, air and soil samples derived from 16 Dutch pig, poultry and calf farms. However, no additional proof for the accumulation of antibiotic resistance was found in soil and air samples in comparison with the results from the MRSA ST398 qPCR assay (SKBII project). The results merely confirmed the low amount of antibiotic resistance (due to MRSA) in soil and air, except for the manure results from one farm, which indicated mecA presence not related to MRSA ST398. However, no indication for the transmission of antibiotic genes from manure to soil and air could be observed. As all samples were only tested with a qPCR, also death bacterial cells are included in the analysis, which do not contribute to the disease causing environmental bacterial load. Therefore, at this moment, no clear conclusions on specific health risks can be drawn from the findings in this report.