Susceptibility Of Benzalkonium Bromide And Characterization Of Quaternary Ammonium Compound Resistance Gene In Aeromonas Isolated From Integrated Pond

Aeromonas spp. is mainly conditional pathogens bacteria in aquaculture. It can cause disease outbreaks, causing serious economic losses to the aquaculture industry.In order to prevent and control diseases caused by Aeromonas, quaternary ammonium disinfectant has been widely used. In the announcement of Ministry of Agriculture No. 1435 the benzalkonium bromide is approved for use in aquaculture production. In this paper we study the susceptibility of Aeromonas spp. to benzalkonium bromide and detect the qac genes to know the sensitivity of the isolatesto benzalkonium bromide and the rate of qac genes that the isolates carried. This study is to provide the reference for application of scientific and effective of benzalkonium bromide in aquaculture.Two standard strains-ATCC 25922 and ATCC 7966 were selected, using the American Journal of Clinical and Laboratory Standards Institute(CLSI) micro dilution method to determine the minimal inhibitory concentration( MIC) and minimal bactericidal concentration( MBC) with different inoculum size and different concentration of Muller-Hinton broth. The results show that the MICs were not significantly different with the inoculum size chang from 0.5×103 CFU/m L to 0.5×108 CFU/m L. When the MH broth concentration increased from 10% to 100%, the dfference of the MICs were not significant neither. Therefore the following study we use the 100 percent M-H broth and the inoculum size of the CLSI recommended.We collected the sample from an Integrate pond in Foshan Guangdong Province in June 2013. We isolated 121 Aeromonas, respectively from manure(64), pond water(20), pond sediment(14) and fish(23). We detected the minimal inhibitory concentration(MIC) and minimal bactericidal concentration(MBC) of these strains. The results show that MIC of ATCC 25922 was 8mg/L; the MIC of ATCC 7966 was 16mg/L. MICs rang of the strains from pig were 8~128mg/L; the MIC rang of the strains from pond were 8~64mg/L. The MIC50 and MIC90 of the strain from pig were 8.9mg/L and 30.0mg/L; The MIC50 and MIC90 of the strain from fish were 22.3mg/L and 39.2mg/L. 99.1 percent of the isolats had a ratio(MBC/MIC) below 4. It show that benzalkonium bromide still had a good sterilization effect. However the value of the MICs higher than the concentration of significantly higher than the concentration recommend by Ministry of Agriculture.According to the reference, we chosed 6 kinds qac(Quaternary ammonium compound, qac) genes. We detected and analysised the qac gene of 121 Aeromonas by PCR method. The results showed: 119(98.4%) carried qac genes, 52(43.0%) carries two kinds qac gene; 23(19.0%) carries three kinds qac gene; 25(20.7%) carry four kinds qac gene; 1(0.8%) carries five kinds qac gene; MIC50 and MIC90 of qac A/B positive strains were 30.6mg/L and 52.9mg/L, MIC50 and MIC90 of qac H positive strains were 32.0mg/L and 64.0mg/L, higher than other qac gene positive strains’ MIC50 and MIC90. The qac A/B and qac H gene may be associated with the resistance of the Aeromonas to benzalkonium bromide. The high detection rate and multiple carry phenomena of qac genes, suggesting that Aeromonas have a risk of resistance to benzalkonium bromide.