Analysis Of The Chronic Wound Microbiota By High-Throughput Full-Length Sequencing Technology

Background: Chronic wound refers to the surface wound in which the body cannot restore its anatomical and functional integrity through the normal repair process,and enters a state of pathological inflammation under the action of various factors.It includes diabetic foot ulcers,pressure ulcers and venous ulcers,which have high disability and mortality rates.Infection is one of the important reasons for the difficult healing of diabetic chronic wounds.The microbial composition of diabetic wounds has attracted the attention of scholars at home and abroad for a long time.However,whether there is a difference in the distribution of bacterial flora between diabetic wounds and chronic wounds caused by other reasons,and whether diabetes will affect the structure of bacterial flora of wounds are still controversial.At present,the diagnosis of chronic wounds in clinical practice mainly relies on swab culture method,which has the advantages of mature technology,long history,and low cost,but it also has limitations such as long culture time and low sensitivity.In recent years,molecular diagnosis represented by high-throughput sequencing has attracted more and more attention.Different from culture method,which depends on the culture environment,high-throughput sequencing can not only improve the resolution of species identification,but also improve the accuracy of the identification of microbial species composition in the sample,so as to more truly restore the microbial community structure in the sample,which is an important research direction in the field of molecular diagnosis in the future.In this study,we used swab culture method and high-throughput sequencing method to detect 33 patients with chronic wounds,and compared the similarities and differences of the two detection methods,so as to provide reference for clinical practice.Chapter 1 Analysis of the Chronic Wound Microbiota by 16 S rRNA Gene-Based Pyrosequencing and the Impact of DiabetesObjective: To analyze the similarities and differences in the distribution of bacterial flora in chronic wounds between diabetic and non-diabetic patients,and to evaluate the effect of diabetes on the structure of bacterial flora.To analyze the relationship between the distribution of bacterial flora in chronic wounds and its possible related clinical indicators,so as to provide reference for clinical empirical application of antibiotics.Methods: 33 patients with chronic wounds who were hospitalized in the Department of Wound Surgery,the First Affiliated Hospital of Dalian Medical University from March 2022 to December 2022 were enrolled.The patients were divided into two groups according to whether they were complicated with diabetes.Group A included13 patients with chronic wounds without diabetes,and group B included 20 patients with chronic wounds with diabetes.The relevant clinical indicators were collected,and the swab specimens of the wounds were collected for gene-based pyrosequencing.Results:1.A total of 437,646 sequences were obtained,including 168,671 sequences in group A and 268,975 sequences in group B.A total of 1844 OTUs were obtained by clustering,including 662 otus in group A,with an average of 51 otus per case.Group B: 1186,with an average of 59.2.Alpha diversity analysis Shannon index and Simpson index were both>0.05,suggesting that there was no significant difference in diversity and evenness between the two groups.Beta diversity analysis MRPP index>5;The PCo A map based on Weighted-Unifrac showed that the sample distance between the two groups was close,indicating that there was no significant difference in community distribution between the groups.3.At the phylum level,Proteobacteria was the most abundant phylum in both groups,followed by Firmicutes and Bacteroidetes.At the genus level,Staphylococcus was the most abundant in both groups,followed by Pseudomonas and Prevotella.At the species level,Staphylococcus aureus and Staphylococcus epidermidis were the most abundant,followed by Citrobacter freundii and Enterococcus faecalis.The abundance of Enterococcus faecalis in group A was the highest,followed by Serratia marcescens and Staphylococcus aureus.In group B,Citrobacter freundii was the most abundant,followed by Staphylococcus epidermidis and Staphylococcus aureus.There was no significant difference in species distribution between the two groups(P>0.05).4.By spearman analysis,the distribution of wound bacteria was not related to age,gender and glycosylated hemoglobin;Prevotella＿timonensis,Corynebacterium＿aurimucosu and greater Fingolidia may be positively correlated with the course of disease.Hemoglobin was positively correlated with Staphylococcus epidermidis,and negatively correlated with Streptococcus＿sp＿HMSC034A12,Streptococcus medius,Solobacterium＿moorei.Staphylococcus aureus and Corynebacterium jasminoides were positively correlated with white blood cell level.Enterococcus faecalis and Bacteroides fragilis were positively correlated with procalcitonin level,while Oligotrophomonas maltophilia was negatively correlated with procalcitonin level.Alaligenes faecalis was positively correlated with albumin level,while Prevotella＿timonensis was negatively correlated with albumin level.In other words,the occurrence of Prevotella＿timonensis is higher when the disease duration is longer and the albumin level is lower.Conclusions:1.There was no significant difference in the diversity and structure of bacterial flora in chronic wounds between diabetic and non-diabetic patients.2.The most abundant strain in chronic wounds was Staphylococcus aureus,followed by Staphylococcus epidermidis.3.The distribution of bacteria in the wound was not related to age,gender and glycosylated hemoglobin.When the course of disease was long,Prevotella＿timonensis,Corynebacterium＿aurimucosu and great Fingolder bacteria were more likely to appear;When the white blood cell increased,Staphylococcus aureus and Corynebacterium jasminoides infection were more likely.Enterococcus and Bacteroides fragilis are more likely to occur when procalcitonin is elevated.When the course of disease is long and the albumin level is low,the infection of Prevotella＿timonensis should be considered.Chapter 2 A Comparison of 16 S rRNA Gene-Based Pyrosequencing and Swab Culture of Chronic WoundsObjective: To compare the similarities and differences of 16 S rRNA gene-based pyrosequencing and swab culture in detecting the microbiota of chronic wounds.Methods: 33 patients with chronic wounds who were hospitalized in the Department of Wound Surgery,the First Affiliated Hospital of Dalian Medical University from March 2022 to December 2022 were enrolled.Two swab specimens were collected from the wound,one for aerobic culture and the other for high-throughput sequencing.Results: Staphylococcus aureus and Staphylococcus epidermidis were the most common species,which were detected in 21.2%(7/33)and 18.2%(6/33)of the samples,respectively.The 16 SrRNA assay yielded 1844 OTUs,with an average of 56 outs per sample.The most abundant species were Staphylococcus aureus and Staphylococcus epidermidis,followed by Citrobacter freundii and Enterococcus faecalis.At the species level,78.9%(15/19)of the cultured bacteria were successfully detected by molecular assays,whereas only 6.0%(15/248)of the sequence-derived bacteria were successfully cultured.Staphylococcus aureus,Staphylococcus epidermidis,Citrobacter freundii,which were cultured more frequently,all had higher relative abundance in sequencing,and bacteria with higher relative abundance were more likely to be detected by culture.Anaerobic bacteria that play an important role in the wound can only be detected by sequencing.Conclusions: The sequencing method has a higher sensitivity than the culture method and has unique advantages for the detection of anaerobes.Bacteria that are highly abundant in the microbiota are more likely to be detected by culture.