| Objective: In this study,16 Sr RNA high-throughput sequencing technique was used to identify the microflora of MRSA infectious wounds and to compare the difference of microflora composition between MRSA infectious wounds and adjacent skin,to clarify the microflora and beneficial resident bacteria in MRSA infectious wounds and to provide a theoretical basis for bacterial intervention therapy of MRSA infectious wounds.Methods:1.Patients with MRSA infectious wounds in Hunan Provincial People’s Hospital from August 2019 to August 2020 were selected.Sterile swabs were used to obtain samples from the wound site and the adjacent site(5cm from the wound site)of the subjects,in which the wound site sample was the experimental group(group D)and the adjacent site sample was the control group(group N).The clinical data of the patients were collected.2.DNA extracted from the samples was sequenced by 16 S r RNA high-throughput sequencing technology.The OTUs were formed according to the similarity of the sequences of 97%,and compared with the species classification database to complete the bioinformatic analysis.3.The reliability of sampling and sequencing results was evaluated according to species accumulation curve and dilution curve.4.Alpha diversity analysis was used to compare the abundance and species diversity of microflora between the wound group and the para-wound group.5.Beta diversity analysis was used to compare the differences in the microflora composition between the wound group and the para-wound group.6.Based on the taxonomic information of species,the microflora of the wound group and the para-wound group were compared at the phylum and genus level,and the possible beneficial bacteria were found.Results:1.A total of 33 samples in the two groups were sequenced,and a total of 1563254 effective sequences were obtained.After quality control and filtering,1398131 optimized sequences were obtained.According to the 97% similarity,1069 OTUs,can be classified into 20 phyla,36 classes,65 orders,122 families,298 genera and 340 species.2.The accumulation curve and dilution curve show that the sampling quantity was sufficient,the sequencing data was reasonable,and the follow-up data could be analyzed.3.The results of Alpha diversity analysis showed that the Chao index,ACE index and Shannon index in group D were lower than those in group N(P<0.05),while the Simpson index in group D was higher than that in group N(P<0.05).The results showed that the species richness and species diversity in group D were significantly lower than those in group N(P<0.05).4.The results of Pco A analysis in Beta diversity analysis showed that the samples of the two groups were far apart,and the composition structure of microflora was different.By Anosim analysis,the statistical value R > 0,indicating that the difference between groups was more significant than that within groups(P<0.05),and there were significant differences in microflora composition between the two groups.5.At the gate level,the composition of the dominant microflora of the two groups was the same,but the abundance was different.At the genus level,there were differences in the composition and abundance of dominant microflora between the two groups.The Staphylococcus in group D was significantly higher than that in group N.The results of Spearman analysis showed that the change of species diversity in the wound group was significantly correlated with most of the different microflora(P<0.01),and negatively correlated with the abundance of Staphylococcus.Lactobacillus could be detected in both groups,and there was a significant difference between the two groups(P<0.001).The abundance of Lactobacillus in group N was higher than that in group D.Conclusion:1.There were significant differences in species diversity and microflora composition between MRSA infected wound site and adjacent wound site.The species richness and species diversity of wound site were lower than those of adjacent wound site.2.The species diversity of MRSA infectious wounds decreased significantly,which was related to the significant increase of Staphylococcus.Staphylococcus may lead to the decline of other resident bacteria by competing for limited survival resources.3.Lactobacillus were abundant in the site adjacent to the wound and were beneficial resident bacteria in the skin.They may have the potential to antagonize MRSA and are beneficial to the recovery of the wound.Objective: To explore the antibacterial effect and anti-biofilm effect of Nisin alone and its combination with berberine hydrochloride on methicillin-resistant Staphylococcus aureus through in vitro and animal experiments,to provide a theoretical basis for the treatment of MRSA infective wounds with Nisin combined with berberine hydrochloride.Methods:1.The minimal inhibitory concentrations(MIC)of Nisin and berberine hydrochloride to MRSA were determined by micro broth dilution technique,respectively.2.The MIC of Nisin combined with berberine hydrochloride against MRSA was determined by micrdilution checkerboard techniques.The combined effect of the two drugs was evaluated by fractional inhibitory concentration index(FICI).3.The dynamic picture of antibacterial activities of Nisin and berberine hydrochloride alone or in combination was determined by the Time-Kill curves method.4.The destruction and inhibition of Nisin,berberine hydrochloride and Nisin+ berberine hydrochloride on MRSA biofilm were verified by crystal violet staining.5.The model of MRSA infected wound in mice was established and divided into 5 groups: Nisin group,berberine hydrochloride group,Nisin+berberine hydrochloride group,normal saline group and gentamicin group.After successful modeling,the drug was given on the same day and continued until the wound healed.The curative effect of each group was evaluated by observing the general condition of mice,wound healing,wound bacterial load and pathological sections.Results:1.The MIC of Nisin and berberine hydrochloride to MRSA was 2μg/m L and 64μg/m L,respectively.2.The MIC of Nisin combined with berberine hydrochloride on MRSA was 0.5μg/m L(1 / 4×MIC)and 16μg/m L(1 / 4×MIC),FICI = 0.5),respectively,indicating a synergistic effect.3.The Time-Kill curve showed that both Nisin and berberine hydrochloride had concentration-dependent antimicrobial effects on MRSA.Compared to the two drugs used alone,the combination can show a bactericidal effect in a shorter time.4.Anti-biofilm effect test: both Nisin and berberine hydrochloride showed concentration dependence on the anti-biofilm effect of MRSA,and the combination of Nisin and berberine hydrochloride showed more substantial anti-biofilm effect.5.Results of animal experiments:(1)General condition: The spirit,diet,excretion and activity of mice in the Nisin group,berberine hydrochloride group,Nisin+ berberine hydrochloride group and gentamicin group were regular,while spirit in the normal saline group were poor and activity decreased.The body weight of mice in each group increased with time.(2)Results of wound healing: The wound size of the Nisin+ berberine hydrochloride group was significantly smaller than that of the Nisin group,berberine hydrochloride group and normal saline group on the 5th,10 th and 15 th day after administration.There was a significant difference in wound healing rate between the Nisin+ berberine group and those of the three groups(P<0.05),but there was no significant difference between the Nisin+ berberine group and the gentamicin group(P>0.05).(3)Results of wound bacterial load: The bacterial load of the Nisin+ berberine hydrochloride group was lower than that of the Nisin group,berberine hydrochloride group and the normal saline group on the5 th,10th and 15 th day after administration(P<0.01).On the 10 th and15th day,the bacterial load of the Nisin+ berberine group was lower than that of the gentamicin group(P<0.05).(4)Pathological results: On the10 th day,there were few inflammatory cells,fibroblasts and neovascularization in the berberine hydrochloride group and the Nisin group.A large number of fibroblasts and neovascularization could be seen in the Nisin+berberine hydrochloride group and the gentamicin group.A large number of inflammatory cells infiltration and necrotic pus cells were observed on the wound surface of mice in the normal saline group,and the new granulation tissue was less.Conclusion:1.Both Nisin and berberine hydrochloride have an antibacterial effect on MRSA.2.The combination of Nisin and berberine hydrochloride has a synergistic effects,and the combination of Nisin and berberine hydrochloride can reduce the concentration of both and shorten the action time.3.Both Nisin and berberine hydrochloride can break the mature biofilm and inhibit the formation of biofilm,and the combined application has a more substantial effect.4.Both Nisin and berberine hydrochloride can inhibit the growth of bacteria in MRSA infected wounds and promote wounds healing,and the effect of the combined group is better. |
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