Object: Staphylococcus aureus (S. aureus) is one of the main causative agents of mastitis in dairy cows. Due to the massive use of antibiotics, more and more Staphylococcus aureus are showing multi-drug resistance, which poses a great challenge to the clinical treatment of dairy cow mastitis. In this context, the use of phages to combat bacterial infections has been increasingly researched and applied. However, in phage therapy, in addition to the fact that pathogenic bacteria will develop resistance to phages and affect the effectiveness of treatment, phages will have complex interactions with pathogenic bacteria, and their mechanisms are still unclear. In this study, on the basis of drug resistance analysis of isolates of Staphylococcus aureus of mastitis origin from dairy cows in a large-scale dairy farm in Xinjiang, phages were isolated with the Staphylococcus aureus isolates as the host bacteria, and their biological properties and genomic characteristics were analysed. One strain of multi-drug-resistant Staphylococcus aureus of mastitis origin from dairy cows and one lysogenous phage were taken as the objects of the study, and the morphology change of the strains before and after phages infection was investigated. By analyzing the morphological changes of the strain before and after phage infection, the growth ability, biofilm formation ability and transcriptomic changes of the strain under the pressure of lysogenic phage and/or antimicrobial drugs, the interactions between Staphylococcus aureus and lysogenic phage were preliminarily explored, which will provide theoretical basis for the development and application of phage preparations in the future.
Methods:
1. Staphylococcus aureus was isolated and identified from milk samples of cows suffering from mastitis collected from a large-scale dairy farm in Xinjiang region. The susceptibility of the isolates to commonly used antimicrobial drugs was determined by the K-B method and the MIC method, and the carriage of drug-resistant genes of the isolates was detected by PCR.
2. Using the S. aureus isolates as host bacteria, the lytic phage was isolated and purified from the sewage samples of dairy farms by using the spot-drop method and the double-layer agar plate method, the lysogenic phage was induced in these isolates by using mitomycin C; the morphology of phage was observed by transmission electron microscopy. The biological characteristics of phage, such as the lysate spectrum of phage, the optimal multiplicity of infection, the one-step growth curve, the thermal stability, the acid and alkali tolerance, and ultraviolet tolerance, etc. were investigated. The whole genomes of the lytic and lysogenic phages were sequenced and comprehensively analyzed by using several online software and tools, and the whole genomes were also be comparative genomics analyzed and genetic evolutionary analyzed.
3. The morphological changes of the strains after the lysogenic phage infection wrere determined by electron microscopy, in vitro bacterial growth curves and drug sensitivity tests. The inhibitory effect on the ability of Staphylococcus aureus growth and biofilm formation were analyzed by determining the growth capacity and biofilm formation of Staphylococcus aureus in the conditions of lysogenic phage and / or different antimicrobial drugs (methicillin and tetracycline), to analyse the effect of lysogenic phage on the vitro growth ability and biofilm formation ability of S. aureus, and to assess the interaction between lysogenic phage and antibacterial drugs. S. aureus during different growth periods under the condition of lysogenic phage infection were transcriptome sequenced to analyze the gene expression profiles of S. aureus , in order to explore the interaction mechanism between S. aureus and its lysogenic phage.
Results:
1. 74 strains of Staphylococcus aureus were isolated from 138 milk samples of dairy cows with mastitis, with an isolation rate of 53.62%. 90.5% of the isolates were multi-resistant strains, which showed varying degrees of resistance to 15 antimicrobial drugs. the resistance rates of these strains to vancomycin, methicillin, sulfisoxazole, lincomycin, penicillin, and enrofloxacin were 97.30%, 82.43%, 79.73%, 71.62%, 67.57%, 67.57%, respectively. Strains resistant to three or more antimicrobial drugs accounted for 90.5%, strains resistant to nine or more antimicrobial drugs accounted for 56.8%, and one isolate was resistant to all examined antimicrobial drugs. A total of seven drug-resistant genes, including blaZ, linA, ermB, aacA-aphD, aac6-aph2, Aph(3')-a and tetM, were detected in S. aureus isolates of dairy cow mastitis origin, among which the detection rate of aminoglycoside-resistant gene aacA-aphD was the highest, 8.10%, and the detection rates of the remaining resistance genes ranged from 2.70% to 4.05%.
2. 3 strains of Staphylococcus aureus phages were isolated in this experiment, among which, 2 strains were lytic phages, named P50-1 and P74, and 1 strain was a lysogenic phage named P51. 3 strains of phages belonged to the order of tailed phages and the family of long-tailed phages. the lytic rates of phages P51, P50-1, and P74 against 78 strains of Staphylococcus aureus were 34.62%, 20.51%, and 25.64%, respectively. the optimal MOI of P51, P50-1, and P74 were 0.1, 0.01, and 0.01, respectively; the average lytic amounts were about 83 PFU/cell, 72 PFU/cell and 86 PFU/cell, respectively. 3 phage strains still had high activity in the conditions of the temperature ≤50℃ and the pH 6.0 ~ 10.0, and 3 phage strains could still retain part of their activity under prolonged UV irradiation. The whole genome sizes of lytic phage P50-1 and lysogenic phage P51 were 43895bp and 42263bp, respectively; the GC contents were 33.65% and 33.85%, respectively. 2 phages strains did not carry drug resistance genes and virulence genes, which were safe at the gene level. 2 phages strains had many genes with unknown functions in their genomes. Comparative genomics analysis revealed that P50-1 had the highest homology with Staphylococcus aureus phage StauST398-2, and P51 had the highest homology with Staphylococcus aureus phage vB_SauS_320. Genetic evolutionary analysis based on the large subunits of the terminal enzymes and the major coat proteins revealed that P50-1 was closest to S. aureus phage phiSa2wa_st72; P51 was closest to S. aureus phage R4 and vB SauS phi2, respectively.
3. Transmission electron microscopy observation revealed that after the lysogenic phage infection, the size of the test strain J57a was significantly smaller, the biofilm around the bacterium was significantly reduced, and there was no significant change in the growth ability in vitro, but the sensitivity to four kinds of antimicrobial drugs such as clindamycin, furantoin, cefepime, and cefotaxime was enhanced. Both lysogenic phage P51 and different concentrations of antimicrobial drugs (methicillin and tetracycline) significantly inhibited the growth of J57. The inhibitory ability of Phage P51 on J57 was stronger than antimicrobial drugs. The inhibitory ability of antimicrobial drugs on J57 was positively correlated with its concentration. A certain concentration of methicillin would assist in enhancing the inhibitory ability of phage P51 on J57; however, a certain concentration of tetracycline would antagonise the inhibitory ability of phage P51 on J57. Lysogenic phage P51 significantly inhibited the biofilm formation of J57. Transcriptomic analysis revealed that lysogenic phage P51 could induce the up-regulation of 8 resistance genes, such as aminoglycoside ant6ie and tetracycline mepa, and the down-regulation of 12 resistance genes, such as glycopeptide vanra and vanrg, in S. aureus J57. P51 and methicillin synergistically induced the up-regulation of penicillin-resistant genes in J57 at the plateau stage; tetracycline can induce the up-regulation of 8 resistance genes, such as tetracycline tet38, aminoglycoside aadd, aad9ib, in J57 for a long period of time, and the expression level of resistance genes in J57 at the group of P51 and tetracycline was not significantly different from that of the tetracycline-treated group. Under the action of lysogenic bacteriophage P51, Staphylococcus aureus J57 synthesizes a large amount of biosynthesis related enzymes during the incubation period, preparing for the bacteriophage to synthesize its own related proteins in the host bacteria; During the lysis burst stage, the LysM peptidoglycan binding domain (J57_GM0000759), a protein related to bacterial cell wall degradation, is upregulated during the lysis process, assisting in the large-scale lysis of bacteria by bacteriophages. Conclusion:
1. Isolates of Staphylococcus aureus of mastitis origin from cows in a large-scale dairy farm in Xinjiang region carried multiple drug-resistant genes, had a high rate of resistance to common antimicrobial drugs, and showed multi-drug resistance. The resistance phenotype of S. aureus isolates from dairy cow mastitis sources had a low compliance rate with resistance genes.
2. The three phage strains isolated in this experiment had wide lysis spectrum, strong lytic ability, strong thermal stability and alkali resistance, and a certain degree of tolerance to ultraviolet rays. All three phage strains did not have drug-resistance genes and virulence genes in the whole genome, and the safety at the gene level was good.
3. After the lysogenic phage infection, the formed biofilm of the strains was significantly reduced, and the sensitivity to some antimicrobial drugs was enhanced. There are different interactions between phages and different antibiotics in inhibiting the growth of S. aureus. Lysogenic phages could induce the up-regulation of the expression of several antibiotic resistance genes in S. aureus; the ability of lysogenic phages to regulate the expression of S. aureus resistance genes varied under different antibiotic pressures.
谷歌
火狐
