Diabetic Foot Infections IDWeek 2015 poster

 Poster #1524 Abstract #49891 Cedars Sinai Medical Center 8700 Beverly BLVD, Building 105-­‐A Los Angeles, CA 90048 Phone (805) 895-­‐7761 Fax (818) 364-­‐4573 Email: [email protected] Bacteriology of Infected, Subcutaneous Tissue in Diffuse Celluli2s and Foot Ulcer Infec2ons in Pa2ents with Diabetes Mellitus using Cultures and Molecular Iden2fica2on Techniques 124
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Dena El-­‐sayed, MD , Aksone Nouvong, DPM , Paula Carlson, MS3, Sydney Finegold, MD3, Arthur Jeng, MD1 1Olive View-­‐UCLA Medical Center, Sylmar, California, USA, 2VA Greater Los Angeles Healthcare System, Los Angeles, California, USA, 3VA Greater Los Angeles Healthcare System and UCLA School of Medicine, Los Angeles, California, USA, 4Cedars-­‐Sinai Medical Center, Los Angeles, California, USA Background •  The microbiology of diabe3c foot infec3ons, a common and complex disease, remains poorly understood, and polymicrobial cultures result in unnecessarily broad an3bio3c coverage. •  The 2012 guidelines on management of infected diabe3c foot ulcers, published by the Infec3ous Disease Society of America, acknowledge that infected diabe3c foot ulcers are oIen polymicrobial, including gram-­‐posi3ve bacteria (eg. Streptococcus spp., Staphylococcus aureus, Enterococcus spp.), aerobic gram-­‐nega3ve bacilli (eg. Enterobacteriaceae, Pseudomonas aeruginosa), and anaerobes (eg. Bacteroides spp., Peptococcus spp., Peptostreptococcus spp.). However, the ques3on remains as to which of these organisms are pathogenic and which are merely colonizers. •  The main e3ologic pathogens in diabe3c foot ulcer infec3ons are thought to be streptococci, and Staphylococcus aureus, but it is less clear what role the gram-­‐nega3ve bacilli such as Pseudomonas aeruginosa, Enterococcus spp. and anaerobes may be playing in the infec3on of soI 3ssue. •  Furthermore it is en3rely unclear which bacterial pathogen(s) are responsible for causing diffuse, non-­‐culturable celluli3s in diabe3cs, as, by defini3on, wound cultures are not possible in these pa3ents. Although celluli3s is generally felt to be a mono-­‐microbial infec3on, this may or may not be true in the diabe3c popula3on, and co-­‐infec3on may occur. •  The current techniques in determining the e3ologic pathogens in infected diabe3c foot ulcers are problema3c, where superficial cultures may be contaminated with normal skin flora. Deeper cultures may have increased sensi3vity and specificity, however no gold standard currently exists. •  The u3lity of pyrosequencing in delinea3ng the microbiology in diabe3c foot infec3ons has been examined, given its rapidity and its ability in iden3fying pathogens that may have been suppressed by an3bio3c exposure. •  The goal of this study is to compare the microbiology of celluli3s and infected foot ulcers in diabe3cs via bacterial culture and molecular methods at varying sample levels to akempt to delineate which organisms are likely causa3ve. •  The most direct way to inves3gate this is by comparing superficial ulcer, deep ulcer, and subcutaneous 3ssue biopsy specimens of diabe3c foot ulcer infec3on subjects via bacterial cultures and molecular analysis. For diffuse celluli3s, subcutaneous aspirates were sent for cultures and molecular analysis, which include pyrosequencing and RT-­‐PCR. Beker iden3fica3on of which bacteria are present in the subcutaneous 3ssue (as opposed to merely on the surface) may aid in furthering our understanding of which are true pathogens, which would be help in avoiding the unnecessary use of an3bio3cs, which, in turn, may help to reduce adverse drug reac3ons rates, health-­‐care costs, and emergence of resistant bacteria. Methods •  Single-­‐center prospec3ve case series from 2010 -­‐ 2011, involving 30 diabe3c pa3ents >18 years old: •  9 with diffuse, non-­‐ulcer, non-­‐culturable lower extremity celluli3s >3 cm in diameter •  21 with an infected foot ulcer •  In diabe3c pa3ents with diffuse, non-­‐culturable celluli3s: blood cultures and a 3ssue aspirate of the infected area were obtained, aIer ins3lla3on of a small amount of sterile normal saline, which was sent for aerobic and anaerobic cultures, molecular bacterial iden3fica3on with pyrosequencing (bTEFAP) and real-­‐3me polymerase chain reac3on (RT-­‐PCR). •  In diabe3c pa3ents with foot ulcer infec3ons, blood cultures and a superficial ulcer swab were obtained. Debridement of the ulcer was then performed, aIer which a deep 3ssue sample at the ulcer base was procured. Finally, a skin biopsy of the infected, erythematous soI 3ssue adjacent to the ulcer was obtained. All of these samples were sent for aerobic and anaerobic cultures, and molecular bacterial iden3fica3on with pyrosequencing (bTEFAP). •  Descrip3ve sta3s3cs is used to compare the bacteria isolated by culture, RT-­‐PCR and pyrosequencing. Results Results Figure 1.
Bacteria isolated in superficial ulcer cultures (% of samples) MSSA MRSA Group B Streptococcus Group A Streptococcus Streptococcus anginosus Enterococcus Enterobacteriaceae Common anaerobes 42.9
47.6
14.3
14.3
19
47.6
23.8
0
Bacteria isolated in deep ulcer cultures (% of samples) MSSA MRSA Group B Streptococcus Group A Streptococcus Streptococcus anginosus Enterococcus Enterobacteriaceae Common anaerobes 42.9
47.6
14.3
19
23.8
38.1
19
0
Bacteria isolated in subcutaneous 2ssue biopsy cultures (% of samples) 19
42.9
9.5
9.5
0
14.3
4.8
38.1
MSSA MRSA Group B Streptococcus Group A Streptococcus Streptococcus anginosus Enterococcus Enterobacteriaceae Common anaerobes Ulcer-­‐related foot infec2ons •  Cultures revealed fewer bacteria at deeper sample levels (average of 7, 7, and 3.5 in superficial swab, deep post-­‐debridement, and subcutaneous infected skin biopsy specimens respec3vely) with the predominant pathogens being MSSA, Streptococcus agalac3ae, Streptococcus anginosus and Streptococcus viridans in all levels (see Figure 1 and Table 1). Enterococcus spp. and anaerobes were also isolated in all levels. Of note, Enterobacteriaceae were present in superficial ulcer cultures but infrequently isolated in skin biopsies (<5%). •  Pyrosequencing again iden3fied significantly more bacteria at all 3ssue levels in both pa3ent popula3ons. Using the cut-­‐off of >1% of total per pa3ent isolates, pyrosequencing of subcutaneous 3ssue biopsies iden3fied established pathogenic bacteria in 10/21 subjects; 6 with Staphylococcus aureus, 3 with Streptococcus agalac3ae, 2 with Streptococcus anginosus. However, pyrosequencing did iden3fy a lot of non-­‐pathogenic by-­‐standing bacteria (See Table 2 for example). Celluli2s •  Cultures of subcutaneous aspirates of celluli3s showed no growth. •  RT-­‐PCR yielded pathogenic bacteria in 44.4% of subcutaneous aspirates, iden3fying Streptococcus anginosus, MSSA, and Streptococcus agalac3ae in decreasing frequency. MRSA was not iden3fied (see Figure 2). •  Pyrosequencing detected mul3ple bacteria in all 7 subcutaneous aspirates tested, iden3fying an average of 15 bacteria. Using the cut-­‐off of >1% of total per pa3ent isolates, pyrosequencing iden3fied common pathogenic bacteria (defined as Staphylococcus aureus, any B-­‐hemoly3c streptococci, Streptococcus anginosus group) in 3/7 of subjects; 2 with Staphylococcus aureus and 1 with Streptococcus agalac3ae. However, pyrosequencing also iden3fied a lot of non-­‐
pathogenic colonizers, such as Enterobacteriaceae, Enterococcus and anaerobes, with successful treatment with an3bio3cs that lack coverage of these organisms (see Table 3 in handout). Table&1.&Bacteria&growing&in&superficial,&deep&and&biopsy&cultures& Table&2.&&Example&of&microbiology&in&one&patient&comparing&pyrosequencing&to&deep,&
(%&of&samples)&
superficial&and&biopsy&cultures!
Organism&
&
!
Staphylococcus,aureus,
&
&
Streptococcus!spp.!
&
&
,
&
&
MSSA*&
MRSA*&
!
Group!B!
Streptococcus&
Group!A!
Streptococcus&
Streptococcus,
anginosus!
&
!
&
Percentage&(number)&of&
patients&growing&the&common&
bacterial&isolates&(n=21)&
Superficial& Deep&
Biopsy&
swab&
culture&
culture&
culture&
57.2!(12)! 57.2!(12)! 28.5!(6)!
42.9!(9)!
42.9!(9)! 19.0!(4)!
14.3!(3)!
14.3!(3)! 9.5!(2)!
38.1!(8)!
42.9!(9)! 23.8!(5)!
19.0!(4)!
23.8!(5)! 9.5!(2)!
0!(0)!
0!(0)!
0!(0)!
23.8!(5)!
19.0!(4)!
14.3!(3)!
Enterococcus,
47.6!(10)! 38.1!(8)! 38.1!(8)!
Enterobacteriaceae**!,
14.3!(3)!
19.0!(4)! 4.8!(1)!
Common!anaerobes!
47.6!(10)! 47.6!(10)! 42.9!(9)!
(including!Bacteroides!
spp.,!Clostridia!spp.,!
Prevotella,spp.,!
Peptostreptococcus!spp.,!
and!Fusobacterium!spp.)!
*Staphylococcus!species!were!determined!to!be!either!MSSA!or!MRSA!by!standard!
culture!techniques,!not!pyrosequencing!
**!Pseudomonas!spp.!not!isolated!at!any!level!
!
% of samples isola2ng the following organisms by RT-­‐
PCR pa2ents with diffuse celluli2s 50
45
40
35
30
25
20
15
10
5
Study&
Participant&
26&
&
&
Culture&identification&
CFU/mL&
Deep+
32+
13+
Biopsy+
+
+
8+
12+
+
3+
6+
+
2+
12+
+
Actinomyces*neuii&
1.0x10 +
&
Streptococcus*
anginosus!
Porphyromonas*
bennonis!
Peptoniphilus*
asaccharolyticus&
Gemella*morbillorum&
Staphylococcus*aureus&
Streptococcus*
anginosus&
Streptococcus*oralis*
group&
Gemella*morbillorum&
2+
4+
+
&
&
Gemella*haemolysans&
Finegoldia*spp.&
2+
1+
1+
13+
+
+
&
Granulicatella*spp.&
1+
0.5+
+
&
&
&
1+
+
+
1+
2+
1+
4+
1+
+
&
&
Enterococcus*faecalis&
Finegoldia*magna&
Propionibacterium*
spp.&
Sarcina*spp.&
Actinomyces*spp.&
+
+
0.5+
0.5+
40+
8+
&
Flavobacterium*spp.&
+
+
6+
&
+
+
6+
&
Flavobacterium*
kamogawaensis&
Ideonella*spp.&
Actinomyces*
odontolyticus&
Dermabacter*hominis&
Propionibacterium*
acnes&
Staphylococcus*
epidermidis*&
Bacteroides*fragilis&
Anaerococcus*vaginalis&
Porphyromonas*
bennonis&
Finegoldia*magna&
Abiotrophia*para>
adiacens&
Bifidobacterium*
bifidum&
Gemella*haemolysans&
+
+
5+
&
Flectobacillus*spp.&
+
+
4+
Limnohabitans*spp.&
Roseateles*spp.&
Chryseobacterium*spp.&
Sphingomonas*spp.&
OD1*uncultured&
Staphylococcus*aureus&
Staphylococcus*spp.&
Rhodoferax*spp.&
Prochlorococcus*spp.&
Herbaspirillum*spp.&
Staphylococcus*
xylosus&
Acidovorax*spp.&
Ralstonia*spp.&
Curvibacter*spp.&
Sporobacterium*spp.&
Phenylobacterium*spp.&
Haliea*spp.&
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
3+
3+
3+
3+
2+
2+
1+
1+
1+
1+
1+
Anaerococcus*
hydrogenalis&
Peptoniphilus*
lacrimalis&
Enterococcus*faecalis&
Staphylococcus*species&
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1+
1+
0.5+
0.5+
+
+
+
+
+
+
+
+
&
&
&
&
&
&
&
&
&
Conclusions %&of&total&flora&
Superficial+
39+
39+
&
&
&
&
&
&
&
% of samples isola3ng the &
following organisms by RT-­‐PCR &
pa3ents with diffuse celluli3s &
&
0
Pyrosequencing&
identification&
Streptococcus*spp.&
Bacteroides*spp.!
Superficial+
3
1.0x10 +
8
1.0x10 +
Deep+
8
1.0x10 +
8
1.0x10 +
Biopsy+
4
3.0x10 +
+
1.0x107+
1.0x108+
+
1.0x107+
1.0x108+
+
3
1.0x10 +
8
+
1.0x10 +
7
1.0x10 +
8
+
+
1.0x107+
1.0x108+
1.0x108+
+
+
1.0x107+
1.0x108+
+
7
9
2.0x10 +
7
2.0x10 +
7
2.0x10 +
2.0x10 +
8
9.0x10 +
8
8.0x10 +
+
+
+
2.0x106+
+
4.0x108+
1.0x108+
+
+
+
2.0x10 +
+
1.0x107+
+
1.0x103+
7.0x10 +
+
+
4.0x106+
+
+
1.0x103+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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+
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+
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+
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+
+
+
+
+
+
+
+
+
+
6
6
!
•  Microbiology analysis of bacteria at various 3ssue levels have revealed Staphylococcus aureus (primarily MSSA) and streptococci as the major pathogens in similar levels at all 3ssue planes (including subcutaneous skin biopsy specimens) of the infected diabe3c foot ulcer. •  Pyrosequencing iden3fied significantly more bacteria at all 3ssue levels in the diabe3c foot ulcer infec3on, but the vast majority of these bacteria were non-­‐pathogenic, by-­‐stander organisms, likely represen3ng coloniza3on. Thus, this technique is limited in its u3lity for iden3fying pathogenic organisms in this disease state. •  Culture microbiology data have shown that Enterobacteriaceae are uncommonly isolated in infected soI 3ssue biopsy specimens (recovered in <5% of samples), thus sugges3ng the gram-­‐nega3ve bacteria are primarily colonizers and not par3cipa3ng in the soI 3ssue infec3on. Successful treatment of par3cipants with an3bio3cs that lacked ac3vity against anaerobes, Enterobacteriaceae and Enterococcus, even when these were isolated, supported this no3on. •  PCR analysis of subcutaneous aspirates in diffuse celluli3s 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 diabe3c foot ulcer infec3ons and in diffuse celluli3s. Pyrosequencing appears to be of limited u3lity in iden3fying the e3ologic pathogens, being overly sensi3ve in recovering bystanding colonizing organisms. Enterobacteriaceae were recovered in superficial ulcers but uncommonly recovered in deep biopsy specimens, sugges3ng the gram-­‐nega3ve bacteria as colonizers.