Mechanisms On Inducible Oxacillin Resistance Of Pork-derived Staphylococcus Sciuri

Staphylococcus sciuri is one of the opportunistic pathogen which can cause a variety of infections and diseases.In recent years,the detection rate of Staphylococcus sciuri in the whole process of livestock production and clinic is increasing gradually.The unreasonable use of antibiotics in animal husbandry,resulting in the pressure of antibiotic screening which stimulate the emergence and spread of Staphylococcus sciuri antibiotic resistance.As it is difficult to cure the bacterial infection of livestock,the quality of animal husbandry products suffers.Therefore,it is of positive significance for the development of animal husbandry to elucidate the development of antibiotic resistance of Staphylococcus sciuri from animal husbandry and to reveal its genetic mechanism through molecular biology and genomics methods.The vast majority of S.sciuri contains mecA1,the evolutionary origin of mecA,but is still sensitive to ?-lactams(OS-MRSS,oxacillin-susceptible mecA1-positive S.sciuri).It is known that methicillin-sensitive Staphylococcus aureus with mecA can be induced by antibiotics to develop methicillin-resistance,but antibiotic induced resistance to OS-MRSS is still poorly studied.So it is important to clarify the induced?-lactams resistance of OS-MRSS for the study of the development of resistance and an effective antibiotic treatment.(1)52 pathogenic bacteria were isolated and identified,of which 75%(39 isolates)were staphylococci and 25%(13 isolates)were Enterococcus faecalis.Among 39staphylococci isolates,there were 27 S.aureus(including 4 MRSA),5 S.sciuri,5 S.saprophyticus,and 2 S.pasteurii 29 samples of pork at retail.(2)Three OS-MRSS isolates were isolated with an oxacillin MIC=1?g/mL and induced with oxacillin for 10 consecutive days.Further mechanisms were performed by several molecular and omics methods.A highly?-lactam resistance of OS-MRSS could be induced(MIC>256?g/mL)by oxacillin and inherited stably within a certain time.After PCR and qRT-PCR validation,it was confirmed that the induced drug-resistant bacteria did not have a two-component regulatory system to regulate the expression of mecA1,and the copy number and expression level of mecA1 did not change significantly after induction.The heterologous expression test of mecA1 proved that both Newman-mecA1 and RN4220-mecA1 were sensitive to oxacillin.This study believed that the induced resistance of Staphylococcus sciuri?-lactam was not caused by mecA1.(3)In order to further explore the cause of induced?-lactam antibiotics resistance of mecA1 positive Staphylococcus sciuri,multiple molecular and omics methods were used to further investigate the mechanism of action.Firstly,the whole genome of Staphylococcus sciuri NWAF26 and induced?-lactam resistance bacterium NWAF26~R was sequenced and their whole genome sequences were analyzed.Based on comparative genomic analysis,it was suggested that the induced resistance to?-lactam in Staphylococcus sciuri was not caused by mecA1 or any other gene mutation.We carried out a transcriptome analysis between NWAF26 and NWAF26~R eventually.We found the expression of four genes in NWAF26~R involved in the pentose phosphate pathway were up-regulated by an average of4.5-fold.In addition,the expression of Mal X(EIIC)and Glv A,were up-regulated by 11.2and 13.3 fold,respectively,which could increase the carbon source supply for pentose phosphate pathway.The up-regulation of these metabolic pathways can effectively increase the production of ribulose-5p.TarJ encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol-5p from ribulose-5p,and TarI encodes a cytidylyl transferase for the synthesis of CDP-ribitol from ribitol-5p and CTP.Under the action of TarJ(up-regulated for 1.9-fold)and TarI(up-regulated for 1.8-fold),ribulose-5p is transformed into CDP-ribitol,which is used to synthesize new wall teichoic acid(WTA)by TarL.The nascent WTA will be translocated to the outer surface of the cell membrane by TagGH(up-regulated by 4.9-fold and 3.4-fold,respectively),finally leading to an increase of WTA in the cell wall.Another analysis showed that ltaS,a key factor mediating lipoteichoic acid synthesis,was also up-regulated by 2.6-fold.While the expression of peptidoglycan hydrolase LrgA was down-regulated by 101.4-fold.Changes in the above pathways will promote the increase of extracellular teichoic acid,and then the increase of the thickness of the bacterial cell wall.(5)After induction,the strain showed a lag in growth rate,changes in intracellular zinc and iron ion concentrations,decreased adhesion ability,and decreased biofilm thickness.Based on transcriptome analysis,we believe that these phenotypes are achieved by altering the expression of genes related to bacterial growth and energy supply pathways,metal ion transport pumps,and bacterial extracellular adhesion factors.In this study,oxacillin was found for the first time to induce the production of?-lactam antibiotics in Staphylococcus sciuri,and this resistance was stably inherited over a time frame.This induced resistance was not caused by mecA1 or mutations in arbitrary genes.This induced resistance may be due to thickening of the bacterial cell wall.The development of induced resistance to?-lactam antibiotics was accompanied by phenotypes such as delayed growth,changes in intracellular metal ion concentration,reduced adhesion capacity and reduced biofilm formation in the induced resistant strains.Induced resistant bacteria develop resistance to antibiotics that target other parts of the bacteria.These phenomena are often overlooked in drug sensitivity testing in clinical microbiology laboratories and require special attention.Therefore,the results of this study are a good reference for the prevention and control of Staphylococcus scruri.