| Staphylococcus aureus is an important opportunistic human,animal and foodborne pathogen which poses a serious health threat by its ability to cause significant morbidity and mortality due to bacteremia,pneumonia and soft tissue infections.S.aureus can cause infection in a wide range of hosts including human,livestock,poultry,pets and wild animals.The zoonotic pathogen can be transmitted though animal derived food products and cause a great threat to public health.The emergence and dissemination of methicillin-resistant strains of S.aureus(MRSA)of animal origin non-sensitive to most β-lactams is of great concern.MRSA is continuously evolving with increased resistance to several antibiotics which results in failure of antibiotic therapies.The resistant bacteria contain a number of mobile genetic elements which ensure evolution of the bacteria in changing environment through horizontal gene transfer.Some regulatory genes control expression of various virulence and resistant factors and it is very important to understand their regulatory mechanisms.Several studies have been conducted to check the prevalence of methicillin-resistant strains of Staphylococcus aureus(MRSA)in animals and animal-derived food products but limited data are available regarding their virulence and associated gene expression profile.Therefore,this study was designed to find out highly resistant and virulent strain,and then have deep insights into its resistance and virulence mechanisms through transcriptome analysis.In the first study,antibiotic resistance and virulence of MRSA and methicillinsensitive S.aureus animal isolates were determined in vitro by agar dilution,biofilm formation,adhesion,invasion and intracellular survivability assays.In addition,the pathogenicity of these isolates was examined in a murine model of S.aureus sepsis.MRSA1679 a,a strain isolated from chicken,was observed to be highly virulent,in cell culture and in mouse model,and exhibited extensive resistant profile.Secondly,in order to identify relevant genes potentially involved in the development of virulence and multiple antibiotic resistance,a differential gene expression profile of a highly virulent zoonotic MRSA isolate and a human reference strain(ATCC29313)using Illumina-based transcriptome and quantitative RT-PCR analyses was compiled.Virulence factors such as adhesion and capsular proteins were highly up-regulated while those for toxins and exoenzymes were downregulated.Biofilm-associated genes were also highly expressed in the MRSA strain.Up-regulation of crucial regulators of virulence like sarA and KdpDE was observed and seemed to play an important role in repression of exotoxin and induction of adhesion and cell wall proteins leading to strong biofilm production.In addition to resistance genes like blaZ,ermC and femA,up-regulation of vraS and multidrug ABC transporter genes contributed to multidrug resistance in the tested MRSA strain.Fluoroquinolone resistance was attributed to mutational changes in gyrA and par C genes.In addition to several stress-related genes,expression of many carbohydrate metabolism genes was also high which reflected the bacterial response to environmental changes.Many of mobile genetic elements were also differentially expressed,and contributed to the resistance and virulence regulation in the MRSA strain.Our findings from comparison between transcriptome analysis of MRSA1679 a and ATCC 29213 suggested that many virulence and resistance determinants in S.aureus are controlled by a complex network of various regulators.Expression of these factors is changed according to the environmental conditions by the global regulators,and sarA is the most important of those as it adds to pathogenicity of the bacteria and ensures its survival in diverse environment.Moreover,mobile genetic elements also affect the expression of many genes and ultimately,the phenotypic characteristics of S.aureus.Concerning our findings from first two studies and keeping in view the importance of the drug for treatment of infections,a second transcriptome study was aimed at understanding the response of highly virulent and resistant MRSA1679 a strain to subinhibitory concentrations(1/2 MIC)of cefquinome by global gene expression analysis.Cefquinome is a fourth generation cephalosporin and has been developed especially for veterinary use.It has been registered for use in many counties around the world and used for for treatment of many infections such as respiratory diseases,foot rot and acute mastitis in cattle,calf septicemia,metritis-mastitis-aglactia syndrome in sow,respiratory disease of horse and foal septicemia.The mechanism of MRSA resistance to cefquinome is still not established and need to be confirmed.So,we designed this study to check the effect of cefquinome on the transcriptome of MRSA1679 a.The poultry strain was treated with sub-inhibitory concentration of cefquinome and then,compared the transcriptome of treated strain with that of original wild strain.Results of transcriptome analysis indicated that efflux pumps(QacA,NorB,Bcr and ABCb)were up-regulated in MRSA1679 a as resistance character to extrude cefquinome.Penicillin binding preotein was over-expressed to confer resistance against this β-lactam antibiotic.Adhesion and biofilm-forming capacity of the MRSA was enhanced in addition to overexpression of many stress-related genes.Carbohydrate metabolism,and secretion and transport activity was significantly increased in MRSA.As a whole,genome of the bacteria got involved to overcome the stress induced by cefquinome and showed great genetic potential to survive in the challenging environment.So,this study gives us a profound understanding of MRSA1679 a potential to be an important pathogen and resistance pattern of MRSA against cefquinome.Further systematic studies taking into account molecular approaches are needed to demonstrate the multiple antibiotic resistance mechanisms of pathogens exposed to various antibiotics through combination therapy. |
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