Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections View Full Text


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Article Info

DATE

2016-11-08

AUTHORS

Vibeke Børsholt Rudkjøbing, Trine Rolighed Thomsen, Yijuan Xu, Rachael Melton-Kreft, Azad Ahmed, Steffen Eickhardt, Thomas Bjarnsholt, Steen Seier Poulsen, Per Halkjær Nielsen, Joshua P. Earl, Garth D. Ehrlich, Claus Moser

ABSTRACT

BackgroundNecrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture.MethodsIn this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples.ResultsFor 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods.Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum.ConclusionThe study emphasizes that many pathogens can be involved in NSTIs, and that no specific “NSTI causing” combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI. More... »

PAGES

652

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12879-016-1976-2

DOI

http://dx.doi.org/10.1186/s12879-016-1976-2

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1004343137

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/27821087


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29 schema:description BackgroundNecrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture.MethodsIn this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples.ResultsFor 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods.Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum.ConclusionThe study emphasizes that many pathogens can be involved in NSTIs, and that no specific “NSTI causing” combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI.
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35 schema:keywords Acinetobacter baumannii
36 Fusobacterium necrophorum
37 MethodsIn
38 MicroSeq
39 NSTI
40 NSTIs
41 PCR
42 S. pyogenes
43 Sanger sequencing
44 Streptococcus pneumoniae
45 Streptococcus pyogenes
46 abundance
47 additional microorganisms
48 analysis
49 atypical findings
50 baumannii
51 biosensor
52 clinicians
53 clone libraries
54 combination
55 concordant results
56 construction
57 culture
58 debridement
59 destruction
60 diversity
61 exists
62 failure
63 faster turnaround time
64 findings
65 fluorescence
66 fungi
67 gene clone libraries
68 gold standard
69 group
70 group of infections
71 growth
72 half
73 high specificity
74 hybridization
75 identification
76 identification of microorganisms
77 identification of pathogens
78 infection
79 library
80 life
81 life-threatening infections
82 method
83 microbial diversity
84 microbial pathogens
85 microorganisms
86 molecular methods
87 most samples
88 multiple molecular methods
89 necrophorum
90 necrosis
91 organ failure
92 pathogens
93 patients
94 pneumoniae
95 pyogenes
96 pyrosequencing
97 quantitative PCR
98 rRNA gene clone libraries
99 rapid destruction
100 rapid results
101 relative abundance
102 results
103 samples
104 sequencing
105 shock
106 situ hybridization
107 soft tissue
108 soft tissue infections
109 species exists
110 specificity
111 standard culture
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114 subsequent Sanger sequencing
115 such methods
116 supplemental use
117 surgical samples
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