Poster #1524 Abstract #49891 Bacteriology of Infected, Subcutaneous Tissue in Diffuse Cellulis and Foot Ulcer Infecons in Cedars Sinai Medical Center

8700 Beverly BLVD, Building 105-A Paents with Diabetes Mellitus using Cultures and Molecular Idenficaon Techniques Los Angeles, CA 90048 Dena El-sayed, MD124, Aksone Nouvong, DPM12, Paula Carlson, MS 3, Sydney Finegold, MD3, Arthur Jeng, MD1 Phone (805) 895-7761 1Olive View-UCLA Medical Center, Sylmar, California, USA, 2VA Greater Los Angeles Healthcare System, Los Angeles, California, USA, 3VA Greater Los Angeles Fax (818) 364-4573 Healthcare System and UCLA School of Medicine, Los Angeles, California, USA, 4Cedars-Sinai Medical Center, Los Angeles, California, USA Email: [email protected]

Background Results Results Table&1.&Bacteria&growing&in&superficial,&deep&and&biopsy&cultures& Table&2.&&Example&ofµbiology&in&one&patient&comparing&pyrosequencing&to&deep,& • The microbiology of diabec foot infecons, a common and complex disease, remains poorly Figure 1. Ulcer-related foot infecons • Cultures revealed fewer bacteria at deeper sample (%&of&samples)& superficial&and&biopsy&cultures! understood, and polymicrobial cultures result in unnecessarily broad anbioc coverage. Bacteria isolated in superficial ulcer cultures levels (average of 7, 7, and 3.5 in superficial swab, Study& Pyrosequencing& %&of&total&flora& Culture&identification& CFU/mL& • The 2012 guidelines on management of infected diabec foot ulcers, published by the Organism& & Percentage&(number)&of& (% of samples) deep post-debridement, and subcutaneous Participant& identification& Infecous Disease Society of America, acknowledge that infected diabec foot ulcers are patients&growing&the&common& infected skin biopsy specimens respecvely) with 26& Superficial+ Deep+ Biopsy+ Superficial+ Deep+ Biopsy+ oen polymicrobial, including gram-posive bacteria (eg. Streptococcus spp., Staphylococcus bacterial&isolates&(n=21)& 3 8 4 the predominant pathogens being MSSA, & Streptococcus*spp.& 39+ 32+ + Staphylococcus*aureus& 1.0x10 + 1.0x10 + 3.0x10 + aureus, Enterococcus spp.), aerobic gram-negave bacilli (eg. Enterobacteriaceae, & ! Superficial& Deep& Biopsy& 8 8 MSSA Streptococcus agalacae, Streptococcus anginosus & Bacteroides*spp.! 39+ 13+ + Streptococcus* 1.0x10 + 1.0x10 + + Pseudomonas aeruginosa), and anaerobes (eg. Bacteroides spp., Peptococcus spp., swab& culture& culture& anginosus& 47.6 42.9 and Streptococcus viridans in all levels (see Figure 1 Peptostreptococcus spp.). However, the queson remains as to which of these organisms are MRSA culture& & Streptococcus* 8+ 12+ + Streptococcus*oralis* 1.0x107+ 1.0x108+ + and Table 1). Enterococcus spp. and anaerobes pathogenic and which are merely colonizers. Group B Streptococcus Staphylococcus,aureus, & 57.2!(12)! 57.2!(12)! 28.5!(6)! anginosus! group& were also isolated in all levels. Of note, 7 8 • Group A Streptococcus & MSSA*& 42.9!(9)! 42.9!(9)! 19.0!(4)! & Porphyromonas* 3+ 6+ + Gemella*morbillorum& 1.0x10 + 1.0x10 + + The main eologic pathogens in diabec foot ulcer infecons are thought to be streptococci, Enterobacteriaceae were present in superficial and Staphylococcus aureus, but it is less clear what role the gram-negave bacilli such as 14.3 Streptococcus anginosus & MRSA*& 14.3!(3)! 14.3!(3)! 9.5!(2)! bennonis! 14.3 ulcer cultures but infrequently isolated in skin Streptococcus!spp.! ! 38.1!(8)! 42.9!(9)! 23.8!(5)! 3 8 Pseudomonas aeruginosa, Enterococcus spp. and anaerobes may be playing in the infecon of Enterococcus & Peptoniphilus* 2+ 12+ + Actinomyces*neuii& 1.0x10 + 1.0x10 + + biopsies (<5%). & Group!B! 19.0!(4)! 23.8!(5)! 9.5!(2)! asaccharolyticus& so ssue. Enterobacteriaceae 7 8 19 • Pyrosequencing again idenfied significantly more Streptococcus& & Gemella*morbillorum& 2+ 4+ + Actinomyces* 1.0x10 + 1.0x10 + + • Common anaerobes Furthermore it is enrely unclear which bacterial pathogen(s) are responsible for causing bacteria at all ssue levels in both paent & Group!A! 0!(0)! 0!(0)! 0!(0)! odontolyticus& diffuse, non-culturable cellulis in diabecs, as, by definion, wound cultures are not possible 8 47.6 23.8 0 populaons. Using the cut-off of >1% of total per Streptococcus& & Gemella*haemolysans& 2+ 1+ + Dermabacter*hominis& + 1.0x10 + + 7 8 in these paents. Although cellulis is generally felt to be a mono-microbial infecon, this paent isolates, pyrosequencing of subcutaneous , Streptococcus, 23.8!(5)! 19.0!(4)! 14.3!(3)! & Finegoldia*spp.& 1+ 13+ + Propionibacterium* 1.0x10 + 1.0x10 + + may or may not be true in the diabec populaon, and co-infecon may occur. ssue biopsies idenfied established pathogenic anginosus! acnes& 7 8 • The current techniques in determining the eologic pathogens in infected diabec foot ulcers bacteria in 10/21 subjects; 6 with Staphylococcus Enterococcus, & 47.6!(10)! 38.1!(8)! 38.1!(8)! & Granulicatella*spp.& 1+ 0.5+ + Staphylococcus* 1.0x10 + 1.0x10 + + epidermidis*& are problemac, where superficial cultures may be contaminated with normal skin flora. aureus, 3 with Streptococcus agalacae, 2 with Enterobacteriaceae**!, ! 14.3!(3)! 19.0!(4)! 4.8!(1)! & Enterococcus*faecalis& 1+ 1+ 4+ Bacteroides*fragilis& 2.0x107+ 2.0x109+ + Deeper cultures may have increased sensivity and specificity, however no gold standard Streptococcus anginosus. However, Common!anaerobes! & 47.6!(10)! 47.6!(10)! 42.9!(9)! & Finegoldia*magna& + 2+ 1+ Anaerococcus*vaginalis& 2.0x107+ 9.0x108+ + currently exists. Bacteria isolated in deep ulcer cultures (% of pyrosequencing did idenfy a lot of non-pathogenic (including!Bacteroides! 7 8 & Propionibacterium* + 1+ + Porphyromonas* 2.0x10 + 8.0x10 + + • The ulity of pyrosequencing in delineang the microbiology in diabec foot infecons has by-standing bacteria (See Table 2 for example). spp.,!Clostridia!spp.,! samples) spp.& bennonis& been examined, given its rapidity and its ability in idenfying pathogens that may have been Prevotella,spp.,! 6 8 & Sarcina*spp.& + 0.5+ 40+ Finegoldia*magna& 2.0x10 + 4.0x10 + + suppressed by anbioc exposure. Peptostreptococcus!spp.,! 8 and!Fusobacterium!spp.)! & Actinomyces*spp.& + 0.5+ 8+ Abiotrophia*para> + 1.0x10 + + • The goal of this study is to compare the microbiology of cellulis and infected foot ulcers in adiacens& *Staphylococcus!species!were!determined!to!be!either!MSSA!or!MRSA!by!standard! 6 diabecs via bacterial culture and molecular methods at varying sample levels to aempt to MSSA Cellulis & Flavobacterium*spp.& + + 6+ Bifidobacterium* + 2.0x10 + + 47.6 42.9 culture!techniques,!not!pyrosequencing! delineate which organisms are likely causave. MRSA • Cultures of subcutaneous aspirates of cellulis bifidum& **!Pseudomonas!spp.!not!isolated!at!any!level! 7 3 • The most direct way to invesgate this is by comparing superficial ulcer, deep ulcer, and showed no growth. & Flavobacterium* + + 6+ Gemella*haemolysans& 1.0x10 + + 1.0x10 + Group B Streptococcus ! subcutaneous ssue biopsy specimens of diabec foot ulcer infecon subjects via bacterial • RT-PCR yielded pathogenic bacteria in 44.4% of kamogawaensis& Group A Streptococcus % of samples isolang the following organisms by RT- 6 cultures and molecular analysis. For diffuse cellulis, subcutaneous aspirates were sent for 14.3 subcutaneous aspirates, idenfying Streptococcus & Ideonella*spp.& + + 5+ Anaerococcus* 7.0x10 + + + Streptococcus anginosus PCR paents with diffuse cellulis hydrogenalis& cultures and molecular analysis, which include pyrosequencing and RT-PCR. Beer 19 anginosus, MSSA, and Streptococcus agalacae in 6 50 & Flectobacillus*spp.& + + 4+ Peptoniphilus* 4.0x10 + + + idenficaon of which bacteria are present in the subcutaneous ssue (as opposed to merely Enterococcus decreasing frequency. MRSA was not idenfied 23.8 lacrimalis& (see Figure 2). 45 on the surface) may aid in furthering our understanding of which are true pathogens, which Enterobacteriaceae & Limnohabitans*spp.& + + 3+ Enterococcus*faecalis& 1.0x103+ + + • Pyrosequencing detected mulple bacteria in all 7 would be help in avoiding the unnecessary use of anbiocs, which, in turn, may help to 38.1 40 & Roseateles*spp.& + + 3+ Staphylococcus*species& + + + 19 Common anaerobes subcutaneous aspirates tested, idenfying an reduce adverse drug reacons rates, health-care costs, and emergence of resistant bacteria. 0 35 & Chryseobacterium*spp.& + + 3+ + + + + average of 15 bacteria. Using the cut-off of >1% of & Sphingomonas*spp.& + + 3+ + + + + 30 total per paent isolates, pyrosequencing idenfied & OD1*uncultured& + + 2+ + + + + common pathogenic bacteria (defined as 25 & Staphylococcus*aureus& + + 2+ + + + + Staphylococcus aureus, any B-hemolyc 20 & Staphylococcus*spp.& + + 1+ + + + + streptococci, Streptococcus anginosus group) in 3/7 % of samples isolang the & Rhodoferax*spp.& + + 1+ + + + + 15 Methods Bacteria isolated in subcutaneous ssue biopsy of subjects; 2 with Staphylococcus aureus and 1 following organisms by RT-PCR & Prochlorococcus*spp.& + + 1+ + + + + cultures (% of samples) with Streptococcus agalacae. However, 10 paents with diffuse cellulis & Herbaspirillum*spp.& + + 1+ + + + + pyrosequencing also idenfied a lot of non- 5 & Staphylococcus* + + 1+ + + + + pathogenic colonizers, such as Enterobacteriaceae, xylosus& • Single-center prospecve case series from 2010 - 2011, involving 30 diabec paents >18 0 years old: Enterococcus and anaerobes, with successful & Acidovorax*spp.& + + 1+ + + + + 19 MSSA • 9 with diffuse, non-ulcer, non-culturable lower extremity cellulis >3 cm in diameter treatment with anbiocs that lack coverage of & Ralstonia*spp.& + + 1+ + + + + these organisms (see Table 3 in handout). & Curvibacter*spp.& + + 0.5+ + + + + • 21 with an infected foot ulcer 42.9 MRSA 9.5 & Sporobacterium*spp.& + + 0.5+ + + + + • In diabec paents with diffuse, non-culturable cellulis: blood cultures and a ssue aspirate Group B Streptococcus & Phenylobacterium*spp.& + + + + + + + of the infected area were obtained, aer insllaon of a small amount of sterile normal 9.5 Group A Streptococcus & Haliea*spp.& + + + + + + + saline, which was sent for aerobic and anaerobic cultures, molecular bacterial idenficaon Streptococcus anginosus with pyrosequencing (bTEFAP) and real-me polymerase chain reacon (RT-PCR). 0 ! • In diabec paents with foot ulcer infecons, blood cultures and a superficial ulcer swab were 14.3 Enterococcus Conclusions 4.8 obtained. Debridement of the ulcer was then performed, aer which a deep ssue sample at Enterobacteriaceae • Microbiology analysis of bacteria at various ssue levels have revealed Staphylococcus aureus (primarily MSSA) and streptococci as the major pathogens in similar levels at all ssue planes (including subcutaneous skin biopsy specimens) of the infected the ulcer base was procured. Finally, a skin biopsy of the infected, erythematous so ssue Common anaerobes 38.1 diabec foot ulcer. adjacent to the ulcer was obtained. All of these samples were sent for aerobic and anaerobic • Pyrosequencing idenfied significantly more bacteria at all ssue levels in the diabec foot ulcer infecon, but the vast majority of these bacteria were non-pathogenic, by-stander organisms, likely represenng colonizaon. Thus, this technique is cultures, and molecular bacterial idenficaon with pyrosequencing (bTEFAP). limited in its ulity for idenfying pathogenic organisms in this disease state. • Descripve stascs is used to compare the bacteria isolated by culture, RT-PCR and • Culture microbiology data have shown that Enterobacteriaceae are uncommonly isolated in infected so ssue biopsy specimens (recovered in <5% of samples), thus suggesng the gram-negave bacteria are primarily colonizers and not parcipang pyrosequencing. in the so ssue infecon. Successful treatment of parcipants with anbiocs that lacked acvity against anaerobes, Enterobacteriaceae and Enterococcus, even when these were isolated, supported this noon. • PCR analysis of subcutaneous aspirates in diffuse cellulis demonstrated Streptococcus anginosus group in 45%, MSSA in 22% and Group B Streptococcus in 11%, and neither MRSA nor Group A Streptococcus were recovered in the subjects. • In summary, this study has shown Staphylococcus aureus and streptococci to be the main pathogens in infected diabec foot ulcer infecons and in diffuse cellulis. Pyrosequencing appears to be of limited ulity in idenfying the eologic pathogens, being overly sensive in recovering bystanding colonizing organisms. Enterobacteriaceae were recovered in superficial ulcers but uncommonly recovered in deep biopsy specimens, suggesng the gram-negave bacteria as colonizers.