Philadelphia, PA Materials and Methods Samples from 69 wounds from - PowerPoint PPT Presentation

James McGuire DPM, PT, LPed, FAPWHc Clinical Professor Temple University School of Podiatric Medicine Philadelphia, PA

Materials and Methods  Samples from 69 wounds from our clinic in Philadelphia, PA were analyzed by PCR and DNA sequencing analysis  The samples received real- time PCR analysis for “predominant bacteria” followed by DNA sequencing that screens for up to 25,000 known pathogens  110 different species of bacteria were identified  On average, the sampled chronic wounds demonstrated 5.3 bacteria per wound  The most commonly identified major pathogens, i.e. bacteria representing 10 percent or more of the DNA in each sample, were: Corynebacterium striatum, Staphylococcus aureus, Pseudomonas aeruginosa, Alcaligenes faecalis, Staphylococcus lugdenensis, Proteus mirabilis, and Escherichia coli, respectively

Quantitative Analysis of PCR and DNA Sequencing of Chronic Wounds From Philadelphia Courtney M. Foote, Jonathan D. Furmanek, Ebony Love DPM, James B. McGuire DPM Temple University School of Podiatric Medicine Literature Review Statement of Purpose Traditional wound cultures rely on isolation on agar and physical identification of micro-organisms that survive transport and are able to grow in the lab environment. Only 1% of The purpose of this study was to analyze the wound biome bacteria cultured can be grown easily in most hospital laboratories. Clinicians have recognized these bacteria as the infecting organisms in most wounds; however, these are just the of the Philadelphia area using DNA sequencing in order to organisms that are easiest to culture. Clinical training leads us to believe that infections are caused by one major pathogen based on standard culturing techniques; clinical tests we use better understand the infections in our region and establish a are optimized to culture planktonic bacteria rather than biofilms. It has been demonstrated that 60-90% of chronic wounds have bacteria existing in biofilms. Biofilms require specialized more targeted therapeutic approach. Traditional methods of culturing techniques, real time PCR, or DNA sequencing analysis. Panbacterial real-time PCR is an effective method of determining the total bacterial load in chronic wounds based on a analyzing chronic wound flora only sample microorganisms predetermined set of primers, and DNA sequencing from a library of thousands of known microrganisms. Bacterial communities are specific to the local environment and PCR/DNA that are easily grown on culture or in the specific database testing gives an expanded view of organisms in the local biome. It has been demonstrated in previous regional studies that epidemiological monitoring of bacterial species in the local for real time PCR. By utilizing DNA sequencing, our aim wound biome and analysis of antibiotic sensitivity profiles play an essential role in developing an antibiotic resistance policy and an effective therapeutic strategy. was to demonstrate that the colonizing organisms vary from “the usual suspects”. In this study we compared the flora Analysis and Discussion from chronic wounds using DNA sequencing results versus In our sample set, the most prevalent colonizing that of more traditional methods of testing, e.g. real time organism was Staphylococcus aureus , followed by PCR. Corynebacterium striatum, Staphylocossus lugdenensis, and Finegoldia magna, respectively Materials and Methods (Figure 2). However, the most common colonizing organism does not tell the entire story For this study, samples from 69 wounds from our clinic in since most of the wounds evaluated in this study Philadelphia, PA were analyzed by either PCR or DNA had bacteria existing in biofilms. On average, the sequencing analysis. The samples received real-time PCR sampled wounds contained 5.3 different species analysis for “predominant bacteria” followed by DNA of bacteria per wound. This biofilm must be sequencing that screens for up to 25,000 known pathogens. addressed when considering therapeutic strategy. The most ubiquitous major pathogen in our Procedure sample set, meaning 10% or more of the total We sampled 69 serial chronic wounds at Temple University DNA found in the wound, was Corynebacterium Foot and Ankle Institute. All wounds were debrided of striatum (17% ), followed by Staphylococcus aureus surface debris and slough using sterile gauze and sterile (16%), and Pseudomonas aeruginosa (14%) (Figure saline. A rough dry swab was used to collect as much surface 1). A major pathogen is defined by representing material as possible. 10% or more of the DNA found in the wound. Our study demonstrates that the most common Results Figure 1. Major Pathogens of Chronic major pathogen was not the expected “Staph or 110 different species of bacteria were identified. On average, Wounds in Philadelphia. Major pathogen is Strep” species, but Corynebacterium striatum. C. the sampled chronic wounds demonstrated 5.3 bacteria per striatum, even though frequently isolated has defined by representing 10% or more of the wound. The most common major pathogens, i.e. bacteria been overlooked in the treatment of chronic DNA found in the wound Figure 2. Most common wound colonizers representing 10 percent or more of the DNA in each sample, wounds and it’s role is not well understood. were as follows: Corynebacterium striatum, Staphylococcus aureus, Although C. striatum is often dismissed as non- Pseudomonas aeruginosa, Alcaligenes faecalis, Staphylococcus pathogenic, Patel S, et al. demonstrated four References lugdenensis, Proteus mirabilis, and Escherichia coli , respectively. cases of C. striatum osteomyelitis in the Greater 1.) Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA, Wolcott RD. BMC Microbiol. 2008 Mar 6;8:43. The most common wound colonizers were Staphylococcus Philadelphia area in patient’s with non -healing 2.) Kaeberlein T, et al. Isolating uncultivatable miroorganisms in pure culture in a simulated natural environment. Science 2002; 296(5570): 1127-29 aureus, Corynebacterium striatum, Staphylococcus lugdenensis, diabetic foot ulcers and Diphtheroids on bone 3.) James G, et al. Biofilms in chronic wounds. Wound Repair and Regeneration. 2008; 16(1),: 37-44 Finegoldia magna, Pseudomonas aeruginosa, Alcaligenes faecalis, 4.) Gentili, V., Gianesini, S., Balboni, P.G. et al. Eur J Clin Microbiol Infect Dis (2012) 31: 1523. https://doi.org/10.1007/s10096-011-1473-x culture. Until we know the predominant 5.) McGuire J. and D’Alessandro J. Combating Biofilms In The Chronic Wound. Volume 29 - Issue 8 - August 2016,:32-40 Proteus mirabilis, Peptoniphilus asaccharolyticus, Anaerococcus pathogenic bacteria present in our immediate 6.) Frikh M, et al. Profile and Antibiotic Susceptibility of Bacteria Isolates in Burn Patients Hospitalized in a Moroccan Hospital: A cross-sectional Study. vaginalis, Streptococcus agalactiae, Escherichia coli, and environment, we cannot effectively set PCR Wounds Epub 2018 Staphylococcus epidermidis , respectively. primers for more cost effective and accurate Real 7.) Patel SA, Lacovella J, Cornell RS. Corynebacterium Striatum: A Concerning Pathogen of Osteomyelitis in the Diabetic Patient. JAPMA: September 2016, Time PCR analysis. Vol. 106, No. sp1, pp. 9-9.

Prevalence  In our sample set, the most prevalent colonizing organism was Staphylococcus aureus , followed by Corynebacterium striatum, Staphylocossus lugdenensis, and Finegoldia magna, respectively

Corynebacterium: A Major Pathogen?  The most ubiquitous major pathogen in our sample set, meaning 10% or more of the total DNA found in the wound, was Corynebacterium striatum (17% ), followed by Staphylococcus aureus (16%), and Pseudomonas aeruginosa (14%) (Figure 1). Major Pathogens of Chronic Wounds in Philadelphia. Major pathogen is defined by representing 10% or more of the DNA found in the wound

Conclusion  The most common colonizing organism does not tell the entire story since most of the wounds evaluated in this study had bacteria existing in biofilms.  On average, the sampled wounds contained 5.3 different species of bacteria per wound. This biofilm must be addressed when considering therapeutic strategy  Standard culture techniques are optimized to culture planktonic bacteria rather than biofilms  It has been demonstrated that 60-90% of chronic wounds have bacteria existing in biofilms  PCR/DNA testing gives an expanded view of organisms in the local biome.