Interspecific Interaction Of Common Bacterial Pathogens From Chronic Respiratory Tract Infection Under Antibiotic Background

Along with the development of disease diagnosis technology and the application of various omics methods in pathogen identification,it is suggested that most chronic infection lesions are co-colonized by multiple pathogens.Pseudomonas aeruginosa and Staphylococcus aureus are two common pathogenic bacteria which can be co-isolated from immunocompromised patients,especially from the patients suffering from hereditary pulmonary cystic fibrosis and chronic obstructive pulmonary disease.Frequent exchange of physical or chemical substances between multiple pathogens that co-colonize the same lesion can promote the development of disease and the maintenance of chronic symptoms.Antibiotics are the preferred chemical drugs for the treatment of bacterial infection.However,the effects and action mechanisms of antibiotics on the interaction between pathogenic bacteria still remain unclear.Therefore,this study mainly focused on the interspecific interaction mechanism between P.aeruginosa and S.aureus under the background of antibiotics,and conducted the following studies:(1)The minimum inhibitory concentrations of commonly used drugs for chronic respiratory tract infection against P.aeruginosa and S.aureus were determined by microbroth dilution method according to the implementation standards of antimicrobial susceptibility tests established by Clinical and Laboratory Standards Institute.The results showed that P.aeruginosa model strain PAO1 and clinical isolate COP2(PA-COP2)were resistant to tetracycline,ampicillin,streptomycin,and cefotaxime.Methicillin-resistant S.aureus COP112(MRSA-COP112)was resistant to tobramycin,gentamicin,tetracycline,ampicillin,cefotaxime and streptomycin.(2)By culturing PAO1,PA-COP2 and MRSA-COP112 on the lawn of each other or on the same plate with gradient colony distance with and without antibiotic intervention,we found that in the absence of antibiotic intervention,P.aeruginosa had a significant competitive advantage over S.aureus,while S.aureus had no significant effect on the growth of P.aeruginosa.In the presence of aminoglycoside antibiotics,MRSA-COP112 with higher resistance was able to inhibit the growth of P.aeruginosa,especially in the presence of streptomycin.(3)By co-culturing PA-COP2 and MRSA-COP112 with different ratio(1:1,1:9,9:1)on the plates supplemented with/without streptomycin,we found that the relative fitness of PA-COP2 was higher on blank plate.On streptomycin plate,MRSA-COP112 dominated the competition with PA-COP2 regardless of the initial ratio,even though it was relatively difficult for MRSA-COP112 to invade PA-COP2.In addition,a mathematical model was established based on the experimental data,and it was found that the intrinsic interaction relationship between bacterial populations with asymmetric competitive advantages could be reversed by the presence of antibiotics,and the intermittent use of antibiotics might cause periodic changes in their co-occurrence frequency.(4)To further clarify the changes in intracellular expression regulation of PA-COP2 and MRSA-COP112 in the context of streptomycin intervention,RNA-sequencing was performed on mono-cultured PA-COP2 and MRSA-COP112,and co-cultured PA-COP2 and MRSA-COP112 under different culture conditions.Compared with the untreated control group,subinhibitory streptomycin inhibited the expression of 146 genes positively regulated by the quorum-sensing(QS)system of PA-COP2,including the central regulatory genes las R and rhl R,key genes involved in the biosynthesis of pyocyanin(phz M and phz S),mex GHI-opm D operon and some other typical genes.In addition,compared with MRSA-COP112 cultured on streptomycin plate,a total of 1045 genes(412 up-regulated and 633 down-regulated)with significant expression differences were detected in the intracellular transcription of MRSA-COP112 grown on the PA-COP2 lawn under the background of streptomycin.Notably,22 up-regulated genes were related to iron-uptake.These results suggested that streptomycin inhibited the QS system of PA-COP2,and the extracellular products of PA-COP2 could enhance the iron-uptake of MRSA-COP112.(5)The effect of P.aeruginosa on the virulence of MRSA-COP112 was explored by using Caenorhabditis elegans as an infection model.The results showed that the supernatant of P.aeruginosa significantly increased the lethality of MRSA-COP112,either the supernatant of PAO1 or PA-COP2 was supplemented.Antibiotic intervention is a common treatment method for infectious diseases,and its mechanism of action has been fully studied.However,after antibiotic treatment,the changes in the regulation of intracellular expression of pathogens and the changes in pathogen interactions are still poorly understood.In this paper,the co-isolated pathogenic bacteria P.aeruginosa and S.aureus were used as models to determine the interaction relationship between bacterial pathogens with antibiotic intervention.The results showed that the inhibitory effect of P.aeruginosa on neighboring MRSA-COP112 could be reversed by the presence of subinhibitory streptomycin.The reason for this phenotype is that streptomycin can inhibit the QS system of P.aeruginosa,and the extracellular products of P.aeruginosa can increase the virulence of MRSA-COP112 and the expression of iron acquisition related genes.Therefore,this study provides an explanation for the replacement of dominant species and the coexistence of pathogens in the host with repeated antibiotic treatments,and contributes to the development of clinical diagnostic strategies and antibacterial drugs.