High-Level Resistance to Quaternary Ammonium Compounds in Clinical MRSA Isolates

Background: Quaternary ammonium compounds (QACs) are microbicides commonly used in hard surface disinfectants and in alcohol-free hand sanitizers. There is an extensive body of research regarding resistance of microorganisms to QACs. Resistance to QACs typically occurs through efflux pumps, which are found in both gram-positive and gram-negative bacteria.  At least 5 different QAC efflux (qac) genes have been described in Staphylococcus aureus; which may be located together with antibiotic resistance genes on transferable elements such as plasmids and transposons.  The majority of studies to date have focused on minimum inhibitory concentration (MIC) data and the current dogma is that the resistance conferred by qac genes is small and well below in-use concentrations. 

Objective: The objective of this study was to determine whether clinical MRSA strains exhibit resistance to killing by the QAC benzethonium chloride (BZE) in short contact times relevant to hand sanitizers.  A second objective was to determine whether resistance to killing by BZE correlates with the presence of qac genes.

Methods: Eleven strains of Staphylococcus aureus (8 MRSA and 3 MSSA) were used. An isogenic pair (RN4420 and RN4220/pGO1) was included to examine the influence of the qacC gene on QAC resistance. Standard PCR was used to detect the presence of qac genes (qacA/B, qacC, qacF, qacG, qacH). MICs were performed by the microdilution method. In vitro Time-Kill experiments were carried out according to ASTM E 2315 using a 15-second contact time.

Results: BZE MICs for S. aureus strains ranged from 2 to 8 µg/ml. Higher MICs were associated with the presence of a qac gene. The concentrations of BZE required to achieve complete kill in 15-second time kill experiments were much higher and ranged from 150 to >4800 µg/ml (0.15%  to >0.48%).  Notably, USA400 was not killed at the highest concentration of BZE tested (0.48%), which is above the typical concentrations used in QAC-based hand sanitizers (0.1%-0.2%).  We did not find a strong correlation between the presence of a qac gene and the concentration of BZE required to kill S. aureus.

Conclusions: These results demonstrate that MICs are not appropriate for determining resistance to QACs.  Time-Kill experiments revealed that the concentrations of BZE required to kill MRSA in short contact times can approach or exceed those used in alcohol-free hand sanitizers.  Because no correlation was found between BZE resistance and the presence of qac genes, a novel resistance mechanism may be involved.  Further research is needed to determine whether MRSA strains are resistant to BZE-based products in actual use and whether these strains resist killing by QAC based hard surface disinfectants.