Bjarnsholt, Thomas

Eickhardt-Sørensen, Steffen Robert

Høiby, Niels

Lorenzen, Jan

Moser, Claus

Nielsen, Per Halkjær

Thomsen, Trine Rolighed

Wolff, Tine Yding

English

    Aalborg UniversitetDirect sequencing and RipSeq interpretation as a tool for identification ofpolymicrobial bacteremiasWolff, Tine Yding; Eickhardt-Sørensen, Steffen Robert; Moser, Claus ; Bjarnsholt, Thomas;Nielsen, Per Halkjær; Høiby, Niels; Lorenzen, Jan; Thomsen, Trine RolighedPublication date:2012Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Wolff, T. Y., Eickhardt-Sørensen, S. R., Moser, C., Bjarnsholt, T., Nielsen, P. H., Høiby, N., Lorenzen, J., &Thomsen, T. R. (2012). Direct sequencing and RipSeq interpretation as a tool for identification of polymicrobialbacteremias. Poster presented at European Congress of Clinical Microbiology and Infectious Diseases, London,United Kingdom.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: December 26, 2020  RipSeq analysis of the sequencing chromatograms revealed bacterial DNA in all 15 samples and for 12 samples (80%) DNA from at least two different species was identified. The results of the RipSeq analysis corresponded well with the cultivation data and the clone libraries, and real-time PCR confirmed several of the findings. However for some samples, bacteria were identified by one method that could not be confirmed by any of the other methods.    Direct sequencing and RipSeq interpretation as a tool for identification of polymicrobial bacteremia    Tine Y Wolff1, Steffen Eickhardt2, Claus Moser2, Thomas Bjarnsholt2, Per H Nielsen3, Niels Høiby2, Jan Lorenzen1, Trine R Thomsen1,3   1: The Danish Technological Institute, Life Science Division, Kongsvang Allé 29, 8000 Aarhus C, Denmark. 2: Department of Clinical Microbiology, Rigshospitalet, University Hospital of Copenhagen,  Juliane Maries Vej 22, 2100 Copenhagen, Denmark. 3: Department of Biotechnology, Chemistry, and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, 9000 Aalborg, Denmark.  BACKGROUND AND AIM  Direct sequencing has become an important supplementary tool for identification of microorganisms in culture-negative infections. However, the combination of broad-range PCR and direct sequencing is not compatible with polymicrobial samples, as it gives mixed sequencing chromatograms. The commercially available tool RipSeq Mixed separates chromatograms resulting from up to three different species. In a previous study, 293 blood samples were examined by cultivation based methods and direct sequencing for comparison. For 15 samples direct sequencing was invalid despite that one or more species were identified by cultivation.  In this study the chromatograms of these 15 samples were analyzed using RipSeq Mixed to see if this would affect the outcome of direct sequencing.       MATERIALS & METHODS  Clone library Establishment of 16S rRNA gene clone library and subsequent sequencing. Cultivation Aerobic and anaerobic blood cultivation was performed using the BACTEC 9240 system.   CONCLUSION RESULTS     • Analysis of sequencing chromatograms with RipSeq Mixed revealed DNA from 1-3 different bacterial species in all 15 samples where direct sequencing was initially invalid.   • RipSeq Mixed thereby improved the performance of direct sequencing considerably.  • Generally there is a risk of detecting clinically irrelevant DNA residing in the sample when applying DNA based methods. To make sure that only active microorganisms are detected, the less stabile RNA could be targeted instead of DNA. However, this study is based on culture-positive samples and therefore the findings are assumed to result from active bacteria.             This work was supported by the Danish PWT Foundation- Investment in Public Welfare Technology (ABT-fonden) and the Ministry of Science Technology and Innovation.  We thank Masumeh Chavoshi, Maria Kristin Bjørnsdottir and Pia Poss for valuable technical assistance.  Quantitative PCR (qPCR) Species specific determination of gene copy numbers. ACKNOWLEDGEMENTS Direct 16S sequencing The 3130xl Genetic Analyzer and MicroSeq system (Applied Biosystems) was applied on culture-positive samples. Table 1: Fifteen blood samples found to be polymicrobial by cultivation but negative by direct sequencing were analyzed by Ripseq Mixed. The RipSeq algorithm assigns a similarity score to each result, and results below 99.3% identity were left out. The RipSeq results are listed according to the score. For selected samples clone libraries were constructed and real-time PCR performed. “(an)” and “(ae)” indicate anaerobic and aerobic blood cultivation, respectively. “CoNS”: Coagulase-negative staphylococci. “n.n.”: nearest neighbor to the BLAST hits referred to as uncultured bacteria.  Sample Cultivation Direct sequencing and RipSeq Mixed Clone library Real-time PCR 1 (an) Citrobacter freundii Enterococcus faecalis 1: C.freundii/gillenii/youngae/ Enterobacter asburiae 2: E. faecalis 3: Veillonella parvula 31 clones: 5 C. freundii, 3 E. faecalis, 3 V. dispar, 20 uncultured bacteria (n.n.: V. dispar, V. parvula and C. freundii) Positive for E. faecalis 1 (ae) C. freundii E. faecalis 1: C. freundii/gillenii 2: E. faecalis Clone library not constructed Positive for E. faecalis 2 (ae) E. faecium CoNS 1: E. faecium 32 clones: 10 E. faecium, 9 Enterococcus sp., 13 uncultured bacteria (n.n.: E. faecium) Positive for Staphylococcus sp. 3 (ae) Klebsiella oxytoca Staphylococcus aureus Micrococcus sp. 1: Enterobacter cloacae/ludwigii/ K. oxytoca 2: S. aureus 20 clones: 2 Klebsiella sp., 7 Staphylococcus sp., 11 uncultured bacteria (n.n.: S. aureus) Positive for S. aureus 3 (an) K. oxytoca S. aureus 1: C. youngae/E. cloacae/ludwigii/ K. oxytoca 2: S. aureus 22 clones: 1 Klebsiella sp., 1 Staphylococcus sp., 11 uncultured bacteria (n.n.: S. aureus)  Positive for S. aureus 4 (ae) E. cloacae Acinetobacter baumanii S. epidermidis Bacillus cereus 1: Aeromonas hydrophila 2: E. cloacae/hormachei 23 clones: 1 A. veronii, 1 Enterobacter sp., 6 Bacillus sp., 6 B. cereus, 3 B. anthracis, 6 uncultured bacteria (n.n.: E. hormaechei and E. cloacae) Positive for Staphylococcus sp. 4 (an) E. cloacae A. baumanii 1: V. dispar/parvula 2: E. coli/Shigella boydii 22 clones: 2 Enterobacter sp., 2 A. hydrophila, 5 Aeromonas sp., 13 uncultured bacteria (n.n.: A. hydrophila, E. hormaechei and E. cloacae) Positive for Staphylococcus sp. 5 (an) E. coli K. pneumonia E. faecalis 1: B. anthracis/cereus/thuringiensis 2: E. cloacae/hormachei/E. coli/albertii/ S. boydii/dysenteriae/sonnei 3: Kluyvera ascorbata 39 clones: 1 Escherichia sp., 6 Clostridium clostridioforme, 9 Veillonella sp., 5 V. dispar, 1 Enterococcus sp., 1 E. faecalis, 1 Clostridium sp., 15 uncultured bacteria (n.n.: V. parvula and C. clostridioforme) Positive for K. pneumoniae Negative for E. faecalis 6 (ae) Streptococcus mitis Gemella haemolysans 1: S. mitis/genomosp. C1/oralis/parasanguinis/sp. 2: S. oligofermentans 3: Streptococcus sp. (oral taxon 056) 40 clones: 1 S. parasanguis, 4 G. haemolysans, 3 Staphylococcaceae sp., 32 uncultured bacteria (n.n.: S. mitis, G. haemolysans, Granulicatella adiacens, S. parasanguis, Actinomyces sp.) Not analyzed 6 (an) S. mitis G. haemolysans S. salivarius 1: S. mitis 2: Abiotrophia para-adiacens 3: G. haemolysans Clone library not constructed Not analyzed 7 (an) Propionibacterium acnes 1: P. acnes 2: S. aureus Clone library not constructed Negative for S. aureus and Staphylococcus sp. 8 (an) S. epidermidis Candida albicans 1: S. epidermidis Clone library not constructed Positive for Staphylococcus sp. Negative for C. albicans. 9 (ae) S. aureus Hemolytic streptococci grp. B 1: S. aureus 2: S. agalactiae Clone library not constructed Positive for S. aureus 10 (an) K. pneumoniae E. faecium 1: K. pneumoniae Clone library not constructed Positive for K. pneumoniae 11 (ae) S. oralis CoNS 1: S. mitis/oralis 2: S. epidermidis Clone library not constructed Not analyzed 

Direct sequencing and RipSeq interpretation as a tool for identification of polymicrobial bacteremias

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Direct sequencing and RipSeq interpretation as a tool for identification of polymicrobial bacteremias

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