| BackgroundIn 2015,415 million people had diabetes,and this number is expected to rise to 642 million by 2040.With a rapid increasing number of diabetes patients,the chronic complications resulted from diabetes need to be prevented and promptly treated.Diabetic foot is one of the most common chronic complications in diabetes.Patients with diabetic foot ulcers have a 15%-27%risk of underlying a lower extremity amputation.Infection is a frequent cause lead to pool healing for diabetic foot ulcers.The risk of amputation was 154.5 times greater in patients with diabetes who had an infection than in those who did not,and the mortality reach up to 50%after amputations.Infections need to be reliably diagnosed and promptly controlled to reduce the risk of amputations.Consequently,it is of great value if we can improve the effectiveness of empirical antibiotic treatment,the reliability of sampling techniques for microbial cultures and also the accuracy of identification of pathogenic bacteria for patients with diabetic foot infections(DFIs).Conventional culture and analysis of antibiotic susceptibility are time consuming(3 to 5 days on average).They cannot guide the clinicians to select a targeted antibiotic timely.An effective empirical antibiotic treatment is of great importance for patients with DFIs.A research reported that the initial antibiotic regimen for 56%of patients was changed,mainly because of a mismatch with the culture susceptibility results.Previous studies have indicated that there was a significant difference in distribution of pathogens among wounds of different Wagner grades.Another study proved that the varying climates and regions also could result in significantly different in pathogenic bacteria for patients with respiratory tract infections.In addition,with an increasing proportion of antibiotic abuse,the resistance rate to antibiotics was changed more and more fast.The difference in distribution of pathogens has not been assessed with respect to varying types of ulcers for patients with DFIs.Furthermore,previous studies ignored the fact that the climates could influence the growth of microorganisms.The studies about the antibiotic resistance change of pathogens were not much.The effectiveness of empirical antibiotic treatment will be improved greatly if we know well about the distribution and antibiotic susceptibility of pathogens from infected diabetic foot wounds in different climates.IDSA and IWGDF suggested that patients with DFIs should be obtained an appropriate specimen for culture from all infected wounds so as to guide antibiotic therapy.IWGDF proposed that clinicians should obtain cultures preferably of a tissue specimen as a gold standard rather than a swab for the identification of pathogens.However,deep tissue biopsy has a probable risk of injuring surrounding tissues.Superficial wound swabbing is still applied more widely in clinical practice because it is easy to perform.The reliability of these two sampling techniques for the microbiological diagnosis of DFIs is still a debated issue.Most researches considered swabs are often contaminated with normal skin flora or colonizers,and they may result in failure to identify deep-tissue pathogens.Another study has indicated that there is no need for biopsy,as there are no significant differences regarding the bacterial species isolated between swab and tissue samples.The concordance between culture results from swab and tissue cultures has not been assessed with respect to PEDIS infection grade.It will be of great value for selecting a reliable sampling technique for diabetic foot wounds if we reappraise the concordance between swab and tissue culture results according to varying PEDIS grades.So far,the diagnosis of pathogenic bacteria from infected diabetic foot wounds was based on the conventional culture.However,there are some limitations in this technology as follow:(1)It is time consuming;(2)Only about 2%of all known bacteria are able to be cultured in the laboratory,due to their fastidious growth requirements;(3)Bacterial culture did not necessarily reveal the most abundant or clinically important organisms,because microbes in clinical infected specimens exist in complex communities lacked oxygen and nutriment rather than as pure cultures.16S rRNA gene sequencing can provide more definitive taxonomic classification in comparison to culture-based approached for many organisms,while proving less time consuming and labor intensive.So far,the Ion Torrent Personal Genome Machine(PGM)sequencing is the fastest platform in running speed(2.3-7.3 hours),and was demonstrated the method is sufficiently rapid and accurate for identification of bacteria from cystic fibrosis sputum samples.Further explore the feasibility and accuracy of the PGM technology for rapid diagnosis of bacterial pathogens from infected diabetic foot wounds is of great value.Furthermore,previous study indicated that skin supports the growth of commensal bacteria,which protect the host from pathogenic bacteria both directly and indirectly.The comparison of microbial community between wounds and intact skin was helpful to speculate the morbigenous microorganisms in diabetic foot ulcers and probable probiotics in skin.These studies will bring a novel target and view to prevent and treatment for patients with DFIs.Chapter 1 Distribution and antibiotic resistance change of pathogens from infected diabetic foot wounds in recent 15 yearsObjectivesTo explore the distribution of pathogens causing infections in the diabetic foot patients and analyze the drug susceptibility so as to provide guidance for reasonable clinical use of antibiotics,and to improve the effectiveness of empirical antibiotic treatment.Patients and Methods1.PatientsA retrospective study was carried out on the microbial culture and antibiotic susceptibility from 580 patients with DFIs hospitalized in Nanfang Hospital from January 2000 to December 2014.All the patients enrolled were accorded with diagnostic criteria of DM proposed by WHO 1999,and of DFI proposed by IDSA.2.Methods(1)Grouping:580 patients were divided into three following groups according to PEDIS grading system:grade2(n=96),grade3(n=314),grade4(n=170).All ulcers were classified as neuropathic ulcers(n=339),ischemic ulcers(n=48)and neuroischemic ulcers(n=193)according to have lower limb neuropathy and arteriopathy or not.Seasons was classified by pentad mean temperature method:Spring(February to March),Summer(April to October)and Autumn(November to January).Then the distribution of pathogens in different groups was analyzed.And the data was divided into two phases according to the variation trend of drug resistance as follow:2000 to 2010,2011 to 2014,then analysis was carried out on the change of drug resistance in recent 15 years.(2)Statistical analysis:Statistical analysis was performed with Statistical Package for Social Sciences version 19.The distribution of data was evaluated for normality by the Kolmogorov-Smirnov test.Normally distributed variables were expressed as the mean ± standard deviation.Variables without normal distribution were mainly expressed as the median and interquartile range.Qualitative variables were expressed as constituent ratio and compared using the X2 test.Logistic regression analysis was used to analyze the influenced factors to distribution of pathogens in wounds with DFIs.Statistically significant differences were indicated by a P value of<0.05.Results1.Logistic regression analysis indicated that the distribution of pathogens in infected diabetic foot was influenced by both PEDIS grades(P<0.001)and seasons(P<0.01).The influence of ulcer types to pathogenic strains was not significant statistically(P>0.05).2.In grade 2 wounds,the dominant bacterial flora was Gram-positive bacteria,while Gram-negative bacteria were the most frequently isolated in grade 4 wounds.The distribution of pathogens was influenced by seasons in PEDIS grade 3 wounds.Gram-negative bacterium were the most frequently isolated in summer(55.5%)while gram-positive bacterium were the most common in spring or autumn(60.1%)(P<0.05).With an increasing PEDIS grade,the composite infections increased in each season(P<0.001).3.Among Gram-positive bacterium,Staphylococcus was dominating floras(44.7%),they were totally resistant to penicillin and ampicillin while vancomycin and linezolid were the most effective agents(100%).The resistance rate to amoxicillin/clavulanate was increased from 15.0%to 48.1%in 15 years(P<0.05).Among gram-negative bacterium,Enterobacteriaceae was dominating floras(61.3%),they were highly resistant to ampicillin(91.3%)while meropenem were the most effective agents(99.5%).The resistance rate to cefotaxime is obviously increased from 29.2%to 43.7%in 15 years(P<0.05).Conclusions1.We can treat the diabetic foot patients with antibiotics empirically according to antibiotics susceptibility of different pathogens.And the type of pathogens could be speculated based on PEDIS grades and also seasons.2.The resistance of pathogens to a part of antibiotics in recent 4 years was much higher than those in previous 11 years.Among them,p-lactam and cephalosporin antibiotics were the most prominent in increased trend of resistance rate.It is essential to pay attention to pathogen survey and use antibiotics more rationally.Chapter 2 A comparison of tissue versus swab culturing for infected diabetic foot woundsObjectivesTo assess the clinical usefulness of swabbing in comparison with tissue biopsy so as to select a more valuable sampling techniques for the microbiological diagnosis of diabetic foot infections.Patients and methods1.PatientsWe enrolled 56 consecutive diabetic patients with clinically infected foot ulcers.The patients were hospitalized at the Department of Endocrinology and Metabolism of Nanfang Hospital affiliated with Southern Medical University from October 2014 to July 2015.All the patients enrolled were accorded with diagnostic criteria of DM proposed by WHO 1999,and of DFI proposed by IDSA.The study was approved by the ethics committee of Nanfang Hospital and informed consent was obtained from all patients enrolled.2.Methods(1)Grouping:Fifty-six patients with diabetic foot infection were divided into the 3 following groups according to PIDES infection classification:grade 2(n=10),grade 3(n=29),and grade 4(n=17).(2)Swab-specimens collection:Each wound was swabbed using the Levine technique,(3)Tissue-specimens collection:A deep tissue specimen of 4 mm in diameter was obtained from the base of the ulcer via punch biopsy(4)Microbial culture:Cultures were processed following the same standard procedures between swab and tissue samples(5)Statistical analysis:Comparisons among quantitative variables were performed by one-way ANOVA(for normally distributed variables)and the independent-samples Kruskal-Wallis H test(for variables without normal distribution).Qualitative variables were compared using the X2test.Variation trends of variables among three groups were evaluated by Linear Regression(for quantitative variables with normally distribution),Spearman correlation test(for quantitave variables without normally distribution),and Linear-by-Linear association(for qualitative variables).Others were as same as Chapter 1.3 Results1.A total of 81 microorganisms(an average of 1.4 per wound)were isolated from both the swab and tissue specimens from 56 wounds.There was little variation in the diagnosis of monomicrobial versus polymicrobial infection(P>0.05).2.The organisms isolated from specimens by ulcer swabbing and by tissue biopsy were identical in 80.0%of wounds of Grade 2,whereas this proportion decreased to 31.0%and to 29.4%in grade 3 and grade 4 wounds,respectively(P<0.05).3.Compared with culture results from tissue samples,the sensitivity and specificity for the identification of all types of Gram-positive bacteria by swab cultures were more than 60%and 80%,respectively.Among the Gram-negative organisms,the sensitivity for the identification of E.coli,Morganella,and Citrobacter by swab cultures was very low(33.3%).Some pathogens isolated from the deep tissue specimens(such as Serratia,Acinetobacter,Ralstonia pickettii and Klvyvera ascorbata)were not isolated from the swabs.Conclusions2.Swab cultures may be reliable for identifying pathogens in diabetic foot wounds classified as grade 2.2.It is advisable to culture deep-tissue specimens in wounds with grade>3 because swab cultures have a high risk of missing pathogens,especially Gram-negative bacteria.Chapter 3 Rapid diagnosis of bacterial pathogens in diabetic ulcers and analysis of microbial diversity in contralateral intact skin based on 16S rRNA gene high-throughput sequencingObjectivesTo explore the feasibility of PGM 16S rRNA gene sequencing based on PGM technology for rapid diagnosis of bacterial pathogens in DFIs.Concordance between sequencing results from swab and tissue specimens was performed to evaluate the reliability of ulcer swabbing for microbiological diagnosis of DFIs.The comparison of microbial community between wounds and skin was performed to speculate the major morbigenous microorganism in wounds and also probable probiotics in skin.Patients and methods1.PatientsA total of 10 patients with diabetic foot infections hospitalized at the Department of Endocrinology and Metabolism of Nanfang Hospital from January 2015 to July 2015 were recruited for this study.All the patients enrolled were accorded with diagnostic criteria of DM proposed by WHO 1999,and of DFI proposed by IDS A.The study was approved by the ethics committee of Nanfang Hospital and informed consent was obtained from all patients enrolled.2.Methods(1)Specimens collection:Each wound was swabbed using the Levine technique(S Group,labeled as S1,S2...S9,S10).After swabbing,a deep tissue specimen was obtained from the base of the ulcer via punch biopsy(T Group,labeled as T1,T2...T9,T10).The microorganisms in skin were sampled by swabbing the area of intact contralateral skin.(2)Microbial culture:As same as Chapter 2.(3)DNA extraction:Genomic DNA was extracted from all samples by using the QIAamp DNA Mini Kit according to the instructions with minor modifications.(4)PCR amplification of 16S rRNA genes:The V1-V2 hypervariable region of the 16S rRNA gene was amplified using the bacterial universal primer 27F and 338R.The PCR products were run on an agarose gel and purified using the AxyPrepDNA Gel Extraction Kit.Equal quantities of each sample were pooled for sequencing.Sequencing was carried out on the Ion Torrent PGM system(Life Technologies)using Ion318 chip.(5)Statistical Analysis:Following sequencing,data were trimmed and optimized using unique.seqs command.These sequences were aligned with the SILVA rRNA database.Subsequently,we clustered the sequences into 97%similarity operational taxonomic units(OTUs)as a basis for further analysis of microbial diversity.The comparison of alpha-diversity measurements among three groups were performed by one-wayANOVA followed by Tukey's HSD test,using SPSS version 19.0.A heatmap analysis was performed to visualize the 50 most abundant genus,using R package "vegan".A Venn diagram containing shared and unique species among three groups was performed using R programming language.In addition,the principal component analysis(PCA)was evaluated using R programming language and visualized using PC-ORD.Furthermore,The LDA Effect Size Analysis was carried out to compare the bacterial communities between T Group and C Group,using LEfSe program.Results1.Compared with the conventional culture results(an average of 1.5 per specimen),the mean number of bacterial genus identified by 16S rRNA gene(an average of 66.1 per specimen)was significantly more.In addition,the sequencing results contained all pathogenic bacteria diagnosed by conventional culture.However,80%of the most dominant pathogens diagnosed by 16S rRNA sequencing were in disagreement with conventional culture results.2.The alpha bacterial diversity of diabetic foot wounds(including S Group and T Group)is significantly higher than that of contralateral skin,whatever at the OTUs level,genus level or Shannon Index(P<0.001).However,there is no significant difference in number of OTUs,genus and also Shannon Index between S Group and T Group(P>0.05).3.The Venn diagram demonstrated that the proportion of species shared between C and T Group,C and S Group,S and T Group were only 11.4%,11.0%and 31.0%,respectively.In addition,the PCA plot illustrated clear and significant separation of samples from wounds and contralateral intact skin.Furthermore,some wound samples taken by swabbing were close to the corresponding tissue samples(such as S1 and T1),while others were highly divergent(such as S3 and T3),4.As shown in 16S rRNA gene sequencing results,Staphylococcus had the greatest relative abundance in contralateral intact skin(44.4%),following Corynebacterium(18.2%).And the most dominant bacteria in 70%of swab specimens and 50%of deep-tissue specimens from wounds were anaerobion(including Fusobacterium,Prevotella,Bacteroides and Porphyromonas),which were not identified by conventional culture.5.The LEfSe analysis dmonstrated that the abundance of Staphylococcus,Actinobacteria and Propionibacteriaceae etc.in contralateral intact skin was significantly higher than that in diabetic foot wounds.On the other hand,the anaerobic bacteria Bacteroidales and Lactobacillales played a crucial role in bacterial communities of diabetic foot wounds.Conclusions1.The results of our study have highlighted PGM sequencing as a promising tool for bacteriological diagnosis of DFIs for the first time.2.It is necessary to sample the deep-tissue specimens from wounds of UT grade 3 for microbiological diagnosis of DFIs.3.The abundance of Staphylococcus,Actinobacteria and Propionibacteriaceae etc.in contralateral intact skin was significantly higher than that in diabetic foot wounds,indicating some importance within micro-ecology of intact skin.On the other hand,the anaerobic bacteria Bacteroidales and Lactobacillales played a crucial role in bacterial communities of diabetic foot wounds. |
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