| Staphylococcus aureus(S.aureus)is an important food-borne zoonotic pathogen,and is used as an indicator reflecting the sanitary condition of slaughterhouses.Biofilm is a self-protection mechanism for bacteria to cope with hostile environments,and can improve bacterial tolerance and retention to internal and external environments,which will increase the risk of bacterial contamination and dissemination.It is of great significance for such fields as veterinary medicine,human medicine,and food processing.In 2019,an epidemiological investigation of S.aureus was conducted in different slaughterhouses in Hubei Province by our lab.The results showed that the positive rate in one slaughterhouse was significantly higher than in others,and isolates from different stages shared similar drug-resistant profiles and virulence factors,indicating that S.aureus was transmitted and seriously cross-contaminated along the slaughter chain.benzalkonium bromide(BB)and sodium hypochlorite(SH)are commonly used in slaughterhouses to disinfect floors,cutting tools,water,and skin.In this study,using 35 S.aureus strains from the slaughterhouse and the farm in 2019 as targets,with the sensitivity to BB and SH,as well as the ability to form biofilms as starting points,the effect of both disinfectants on S.aureus biofilm formation ability was evaluated so as to provide experimental basis for the development of scientific and effective disinfection strategy for slaughterhouses.The major results are shown as follows.1.Sensitivity to two disinfectants and ability to form biofilms of the S.aureus strainsSensitivity to both disinfectants and ability to form biofilms of S.aureus strains from different slaughter stages of samples were determined using the micro-dilution broth method and the crystal violet staining test,respectively.The results showed that the minimum bactericidal concentration(MBC)of BB ranged from 1 mg/L to 16 mg/L,and 65.71%(23/35)of the experimental strains exhibited a higher MBC value than the quality control strain ATCC 29213(2 mg/L).The experimental strain was considered insensitive when the MBC is four times or more than the quality control strain.The results showed that the proportions of insensitive strains increased with the progress of slaughtering process.The strains forming strong biofilms accounted for 45.71%of the total(16/35),in which 93.75%(15/16)were recovered from samples collected at the middle and late stages of slaughter(hair removal to cold storage).S.aureus biofilm formation ability was positively correlated with BB MBC value.For all the S.aureus strains,the MBC of SH was 0.16%of effective chlorine concentration.Analysis of the whole genome sequences of 26 S.aureus strains revealed that three sequence types(ST)were identified,with 17,6,and 1 strains of ST7,ST9,and ST2867,respectively,and the ST of other two strains was not identified.ST7 was the dominate type to form strong biofilms(15/16),and the dominant type among the isolates from the samples collected in the downstream splitting and cooling stages of slaughter chain(11/12).All the strains carried abundant biofilm-related genes,with detection rates of over 80%agrA,clfA,clfB,icaA,icaB,icaC,icaD,icaR,fnbA,fnbB,sarA,sdrC,sdrD,sdrE,sigB and nuc genes.Only two strains carried the disinfectant resistance-related gene qacL,while qacA,qacB,qacC,qacE,qacEΔ1,qacG,qacH and qacR genes were not detected.2.The effect of two disinfectants on S.aureus biofilmVarious concentrations of BB and SH were added at the early culture stages of S.aureus to study their effect on bacterial biofilm formation.The results showed that both 1/16 MBC～1 MBC of BB and 1/64 MBC～1 MBC of SH significantly inhibited the formation of bacterial biofilms(P<0.05),and the inhibitory effect on biofilm formation is related to its inhibition to bacterial growth and dependent on disinfectant concentration.The inhibitory effect of SH on S.aureus biofilm formation was obviously stronger than that of benzalkonium bromide.Further studies showed that incompletely eradicated biofilms could recover after removal of disinfectants and replenishment of nutrient conditions.Disinfectants were introduced at various phases of S.aureus biofilm formation,and the residual of biofilms was detected to determine the disinfectant’s clearance effect on biofilms.The results showed that the clearance rate of biofilms treated with 2 MBC of BB ranged from 3.81%to 14.15%in initial adhesion and proliferation phases,and only ranged from 0.75%～6.25%for 48～120 h of incubation.1/8 MBC of SH exhibited a strong ability to clear biofilm incubated for 72 h or less with a clearance rate ranging from 32.07%to 82.06%,while the clearance rates were significantly reduced for 96～120 h of incubation.The results suggesed that SH exhibited a stronger clear effect on S.aureus biofilms that BB.Ten S.aureus strains were continuously stressed with sub-lethal concentrations of BB or SH for 30 generations to assess the effect on biofilm formation ability.The results revealed that the stress of SH had no effect on the experimental strains(P>0.05).Under the stress of BB,only the#01 455 strain exhibited a dramatically impaired biofilm formation ability from strrong(OD630 of 0.662±0.039)to moderate(OD630 of 0.221 ±0.015)(P<0.01),while there was no significant effect on other strains(P>0.05).After the stress of BB,the#01 455 strain reduced the total amount of extracellular matrix and production of polysaccharides and proteins,while there was no significant change in bacterial growth ability(P>0.05),suggested that the stress of BB might decrease the ability of S.aureus to form bioflms by inhibiting the production of bacterial extracellular matrix components. |
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