Rapid identification of microbes in positive. blood cultures by matrix-assisted laser. desorption/ionisation time-of-flight (Maldi-

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1 JCM Accepts, published online ahead of print on 8 August 03 J. Clin. Microbiol. doi:0.8/jcm Copyright 03, American Society for Microbiology. All Rights Reserved Rapid identification of microbes in positive blood cultures by matrix-assisted laser desorption/ionisation time-of-flight (Maldi- Tof) mass spectrometry- ( Vitek MS biomérieux.) Arnold G W Foster. Department of Microbiology, Pathology North, NSW Health Pathology, John Hunter Hospital, Newcastle, 305 NSW AUSTRALIA Address correspondence to Arnold Foster, Arnold.Foster@hnehealth.nsw.gov.au Downloaded from on September 9, 08 by guest

2 ABSTRACT Sepsis is a major cause of mortality worldwide, in community and hospitalized patients. There is a wide range of pathogens and the correct identification and correct antimicrobial therapy is critical to ensure an optimal clinical outcome. With the recent introduction of matrix-assisted laser desorption-ionization time-of-flight (Maldi-Tof) mass spectrometry, rapid identification of bacteria and fungi is now possible. The purpose of this study was to develop a rapid technique for identifying organisms in positive blood cultures using the Vitek MS (biomérieux). This technique is a lysis centrifugation method which involved a four-step wash and centrifugation procedure. A total of 53 monomicrobial positive blood cultures ( Bactec plus aerobic, anaerobic and paediatric bottles) were tested using the Vitek MS- (Knowledge base Version.0) with an overall 9.% and 88.% of organisms identified to genus and species level respectively. For 6 Gram positive bacteria, 96.3% and 9.9% were identified to genus and species level respectively; for 9 gram negative bacteria, 84.7% and 83.7% were identified to genus only and species level respectively. The results obtained using this method demonstrate that the Vitek MS can be used for rapid and effective identification of bacteria from positive blood cultures with in minutes after the positive signal has been provided by the Bactec FX ( Becton-Dickenson) blood culture instrument. This will lead to faster administering of appropriate antimicrobial therapy and increase the chance for an optimal clinical outcome for the patient. INTRODUCTION Sepsis is one of the most common causes of death in seriously ill patients. About 8 million cases occur worldwide each year with a mortality rate of around 30%. () The first 4 hours of patient care is critical for appropriate antimicrobial therapy and Downloaded from on September 9, 08 by guest

3 mortality increases by approximately 7% for every hour a septic patient remains untreated or is receiving inappropriate antimicrobial therapy. () The present turnaround time for identification of the causative organism, from the time of positivity by the blood culture instrument, is about 8-4 hours. Faster identification technology - such as Maldi-Tof mass spectrometry provides faster and more accurate identification of bacteria and fungi. The objective of this work was to use the Vitek MS (bioméreiux) to attempt to identify bacteria directly from positive blood culture bottles within minutes of the time of the positive signal by the Bactec FX ( Becton- Dickenson) blood culture machine. Various methods and techniques both commercial and in-house have been used for direct identification of microorganisms from positive blood culture broths, for example Sepsityper (Bruker), saponin method for bacterial extraction, (3,4 ) lysis centrifugation methods (9) and serum separator method. (5) All these techniques show better results for the identification of the Gram negative bacilli compared to Gram positive organisms including the differentiation of Staphylococci species. This new technique not only maintains the high level of identification for Gram negative bacilli as seen in previous methods, but also achieves and high level of identification in Gram positive organisms including Staphylococci species, with 7/8 (99.%) of all Staphylococci identified to species level. Materials and Methods A mixture of 53 positive aerobic Bactec Plus + aerobic/f, anaerobic Bactec Lytic/0 anaerobic/f and paediatric bactec Peads Plus/F blood culture bottles were incubated in an automated Bactec FX blood culture instrument (Becton Dickenson Cockeysville Downloaded from on September 9, 08 by guest 3

4 93 94 Maryland, USA). As a new positive bottle was detected, broths from the bottles were assessed using the Vitek MS (biomérieux Australia) For each positive blood culture bottle, 7mls of broth was removed and added to 3 mls of wash buffer No.- (.5ml of sterile distilled water and 0.5ml of Spree multi action lemon washing detergent. (Colgate Palmolive Pty Ltd. Sydney, NSW, AUSTRALIA. Composition/information on ingredients, Sodium laureth sulfate 0-<5%, Lauramidopropyldi methylamine oxide 0-<5%, Myristamidopropyl amine oxide 0-<%, Tetrasodium EDTA0-<%, Formaldehyde <0.0%, Isothiazolinones <0.00%. ) (6) then centrifuged for minutes at 3000g. The supernatant was removed and the pellet resuspended in 0mls of wash buffer No.-(9.5ml sterile distilled water and 0.5ml of Spree washing detergent concentrate) then centrifuged for minutes at 3000g. The same procedure was then followed with wash buffer No.3 (0mls sterile distilled water containing 0.g N-acetyl-L-cysteine) and washed with 0mls sterile distilled water. The supernatant was removed, then ul of pellet placed on a Maldi-Tof slide, and 0.5ul formic acid (VITEK MS-FA) added to each well and air dried. ul of matrix (VITEK MS CHCA) was added and air dried. The slide was run in the Maldi-Tof instrument (biomérieux VITEK MS ) to obtain the identification. The procedure took approximately 30 minutes. Each isolate was then identified from culture plate the following day using Maldi-Tof (biomérieux VITEK MS), Vitek and other laboratory tests to confirm identification of the isolate. All organisms were placed onto four wells of a Vitek MS slide. If the identification of two or more wells were given with a high confidence level, this was considered as a acceptable identification. Misidentification result was given when two or more Downloaded from on September 9, 08 by guest 4

5 8 9 identifications out of the four wells had a high confidence level but didn t match the identification given from the Vitek MS form the subculture the following day No identification result was given when two or more identifications received a poor confidence level or no identification was given by the Vitek MS in all 4 wells. Results Of the 53 monomicrobial positive blood cultures tested, 9.% were successfully identified to genus level and 88.% to species level. Of the 6 Gram positive organisms 55/6 (96.3%) were identified to genus level and 48/6(9.9%)were identified to species level. 8 Staphylococcus spp. 7/8 (99.%) were identified to species level. All 45 (00%) of the Staphylococcus aureus were identified correctly and differentiated from coagulase negative Staphylococcus. Streptococcus spp. 8/ (80.%) were identified to genus level and 3/ (6.9%) identified to species level. Of the 9 Gram negative organisms identified, 78/9 (84.7%) were correctly identified to genus level and 77/9(83.7%) were identified to species level. (see table ) 6 positive blood cultures were not included in the study due to mixed growth on subculture. A further eight positive cultures were not identified due to absence of the organisms from the Vitek MS knowledge base version.0. Only / 53 (0.8%) microbes were misidentified which was Streptococcus pneumoniae and one Klebsiella pneumoniae misidentified as Enterobacter aerogenes, and 0/53 (7.9%) broths no identification was obtained by the Vitek MS (Biomereiux knowledge base: version.0). See table for list of unidentified and misidentified organisms. (see table.) Downloaded from on September 9, 08 by guest 5

6 4 43 Only monomicrobial cultures were used in this study so 6 broths were excluded due to mixed subcultures and were considered to be of no clinical importance Discussion Problems in previous studies were the identification of Gram positive organisms such as Staphylococcus spp. and Streptococcus spp. Various techniques and methods using commercial available kits currently available, such as Sepsityper kit from (Bruker Daltonics Billerica, Massachusetts, USA.) [3,4,7] showed poor results in identification of Gram positive organisms including Staphylococci, this has been overcome with the present technique. However, misidentification of S. pneumoniae caused by a lack of data in the database on the Vitek MS (knowledge base version.0) software was also evident in this and other studies. (8,9) Difficulties with the identification of encapsulated organisms (eg. Klebsiella spp.) experienced in previous studies(0) have also been overcome using the present technique. With all 45 (00%) Staphylococcus aureus and 8/8 (98.8%) of coagulase negative Staphylococcus identified to species level. This is of major clinical importance as it enables the clinician to distinguish between Staphylococcus aureus sepsis and possible skin contamination due to other Staphylococci. The cost of this technique was ($.38 AUD) per positive broth tested, which is cheaper than the Sepsityper (Bruker)($5.5 US) () and it also has the same turn around time as the Sepsityper -30 to 45 minutes. Downloaded from on September 9, 08 by guest 6

7 This new technique is now being used routinely in our laboratory and we are currently using it as screening test in a trial study using the Xpert MRSA/SA BC kit (Cepheid- Sunnyvale, USA) for identification of Staphylococcus aureus and MRSA direct from positive blood culture broths. The retail price of the Xpert MRSA/ SA BC kit is $85.00 (AUD) (). In a period of months our laboratory had 50 positive blood cultures with Staphylococci isolated. Out of that 50, Staphylococcus aureus was isolated in 50 positive blood culture broths and the remaining 00 broths contained coagulase negative Staphylococci. If all 50 positive blood cultures were tested using the Xpert MRSA/ SA BC kit the cost would be $,750 (AUD). With the use of this new technique as a screening test only 50 positive broths would be tested reducing the cost to $4,50(AUD) giving a saving of $8,6(AUD) after the cost of the 00 coagulase negative Staphylococcus screening tests has been included. It has been demonstrated that rapid identification, such as this technique, can significantly decrease the period of hospital stay for the patient and decrease hospital costs.(3,4) CONCLUSION Our results demonstrate that it was possible to obtain rapid and accurate identification of bacteria direct from positive blood cultures using the Vitek MS (Knowledge base version.0). Using this lysis centrifugation technique, it was possible to identify the causative organism approximately minutes after a new blood culture was detected as positive by the Bactec FX (Becton-Dickenson) machine. Organism identification would therefore be available for the medical staff at virtually the same Downloaded from on September 9, 08 by guest 7

8 90 9 time as the Gram stain result. This would enable important decisions to be made on the clinical significance of the isolate and the use of appropriate antimicrobial therapy This method will allow prompt differentiation between Staphylococcus aureus and other Staphylococci (the majority of which are contaminants) and provide early clinically important information and reduce inappropriate use of antibiotics. It has also been demonstrated that this method can provide large savings in the cost of rapid identification of Staphylococci species in positive blood cultures. The only limitations of this procedure were mixed cultures, broths from patients with very high white cell counts as seen in CML patients not being able to obtain clean spun pellet resulting in no identification been made by the Vitek MS and the current database of the Vitek MS (knowledge base Version.0.) Overall, our data suggested that this method will provide considerable patient benefit and contribute to reducing mortality due to septicaemia. ACKNOWLEDGMENTS The author would like to thank Dr Stephen Graves, Dr Chris Ashhurst-Smith, Dr Patrick Harris and Dr Mahesh Menon who contributed to this study is various ways. REFERENCES. B Schaaf, J Kruse, J Rupp, Reinert RR, Droemann D, Zabel P, Ewig S, Dalhoff K. Sepsis severity predicts outcome in community-acquired pneumococcal pneumonia. 007;30: Cited in article Diagnosis of bloodstream infection: present and future biomérieux May 0. Downloaded from on September 9, 08 by guest 8

9 Kumar, Anand MD; Roberts, Daniel MD; Wood, Kenneth.E DO; Bruce Light, MD; Joseph E, Parrillo, MD; Satendra Sharma, MD; Robert Suppes BSc; Daniel Feinstein, MD; Sergio Zanotti, MD; Leo Taiberg, MD; David Gurka, MD;Aseem Kumar, PhD; Mary Cheang, MSc. Duration of hypotension before intiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. 006; Vol 34(6): Jonathan H.K. Chen, Pak-Leung Ho,3, Grace S.W. Kwan, Kevin K.K. She, Gilman K. H. Slu, Vincent C.C. Cheong Yam,3 Direct Bacterial Identification in Positive Blood Cultures using two commercial MALDI- TOF mass spectrometry systems. J. Clin Microbiol. June 03 vol. 5no Meex C, Neuville F, Descy J, Huynen P, Hayette MP, De Mol P, Melin P. Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction. J Med Microbiol. 0 Nov;6(Pt ):5-6. Doi: /jmm Epub 0 Jul Moussaoui W, Jaulhac B, Hoffman AM, Ludes B, Kostrezewa M, Riegel P, Prevost G. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clin Microbiol Infect. 00 Nov;6():63-8. doi:0./j x Klein S, Zimmermann S, Koehler C, Mischnik A, Alle W, Bode K. Integration of MALDI-TOF mass spectrometry in blood-culture diagnostic. A fast effective approach. 0 [ Epub] PMID: Cited in article Diagnosis of Downloaded from on September 9, 08 by guest 9

10 bloodstream infection: present and future biomérieux May Jen Kok, Lee C. Thomas, Thomas Olma, Sharon C.A. Chen, and Jonathan R. Iredell. Identification of Bacteria Culture Broths Using Matrix-Assisted Laser Desorption- Ionization Sepsityper and Time of Flight Mass Spectrometry.0;6(8):e Lindsay G. Stevenson, Steven K. Drake, and Patrick R. Murray. Rapid Identification of Bacteria in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of flight Mass Spectrometry. 00; 48(): Guy Prod hom, Alain Bizzini, Christain Durussel, Jacques Billie and Gilbert Greub. MALDI-TOF mass spectrometry for direct bacterial identification from positive blood culture pellets. 00 doi:0.8/jcm Philippe R. S. Lagacé-Wiens a,b,heather J. Adam a,b, James A. Karlowsky a,b, Kimberly A. Nichol b, Amanda A. Webb b, Crystal Miller b and Michelle J. Alfa a,b Identification of Blood Culture Isolates Directly from Positive Blood Cultures by use of Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry and Commercial Extraction Systems: Analysis of Performance, Costs, and Turnaround Time. J. Clin Microbiol. October 0 vol. 50 no Jane Davies a,b,*, Claire L. Gordon a,b, *, Steven Y. C. Tong a,c, Robert W. Baird b and Joshua S. Davis a,c Impact of Results of a Rapid Staphylococcus aureus Diagnostic Test on Prescribing of Antibiotics for Patients with Clustered Gram-Positive Cocci in Blood Cultures J. Clin. Microbiol. June 0 vol. 50 no Downloaded from on September 9, 08 by guest 0

11 Katherine K. Perez, PharmD; Randall J. Olsen, MD, PhD; William L. Musick, PharmD; Patricia L. Cernoch, BS; Lames R. Davis, PhD; Geoffrey A. Land, PhD; Leif E. Peterson, PhD; James M. Musser, MD, PhD. Intergating Rapid Pathogen Identification And Antimicrobial Stewardship Significantly Decreases Hospital Costs.0doi: /arpa OA 4. Agnés Ferroni,*, Stéphanie Suarez, Jean-Luc Beretti, Brunhilde Dauphin, Emmanuelle Billie,3, Julie Meyer 3, Marie-Elisabeth Bougnoux,3, Alexandre Alanio, Patrick Berche,3 and Xavier Nassif,3. J. Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Clin. Microbiol. May 00 vol. 48 no Downloaded from on September 9, 08 by guest

12 Table. Results obtained by the Vitek MS of 53 positive blood cultures. Gram Positive Number of isolates tested Number identified species / genus Staphylococci Staphylococcus aureus Staphylococcus capitis Staphylococcus warneri Staphylococcus epidermidis Staphylococcus hominis Staphylococcus haemolyticus Staphylococcus simulans Staphylococcus lugdunensis Staphylococcus auricularis Staphylococcus caprae Streptococci Streptococcus pyogens Streptococcus mitis/oralis Streptococcus dysgalactiae Streptococcus pneumoniae /45 3/3 4 /4 37/ /5 / / / / Total = 6 / 7-(99.%)- to species level / 0/3 / 0/0 Downloaded from on September 9, 08 by guest Streptococcus agalactiae / Streptococcus parasangunis / Streptococcus salivarius 0/0

13 Table continued Number of isolates tested Number identified species/genus Streptococcus anginosis 0/ Enterococcus faecalis 5 5/5 Enterococcus faecium Others Micrococcus luteus Propionibacterium acnes Bacillus licheniformis Corynebacterium pseudodiptheriticum Bacillus cereus gp. Bacillus simplex Rothia ssp. Finegoldia magna Enterobacteriaceae Escherichia coli Proteus mirabilis Proteus penneri/vulgaris Pseudomonas aeruginosa /3 / / / / 0/3 / 0/ / 43/43 / 0/ 3/3 Downloaded from on September 9, 08 by guest Klebsiella pneumoniae 0/0 Klebsiella oxytoca 3 3/3 Enterobacter aerogenes / Citrobacter freundii 0/0 3

14 Acinetobacter lwoffii / Acinetobacter radioresistans / Stenotrophomonas 0/0 Providencia rettgeri Enterobacter cancerogenus Enterobacter cloacae/asburiae Serratia marcescens Acinetobacter baumannii Others Niesseria meningiditis Leptotrichia ssp. Bacteroides vulgatus / / / / / / 0/0 / Downloaded from Bacteroides thetaiotaomicron Bacteroides fragilis Helicobacter species Environmental Gram negative bacilli 6 Total= 53 / / 0/0 0/0 Total = 3/53- (88.%) to species level and 33/53- (9.%) to genus level. on September 9, 08 by guest 4

15 Table. Unidentified Misidentified Staphylococcus haemolyiticus Streptococcus pneumoniae Bacillus cereus gp. Streptococcus dysgalactiae Propionibacterium acnes Streptococcus salivarius Escherichia coli Klebsiella pnuemoniae Citrobacter freundii - (ID as Streptococcusmitis/oralis) - (ID as Enterobacter aerogenes) Downloaded from Stenotrophomonas maltophilia Leptotrichia species Helicobacter Species Unidentified environmental contaminants not on Vitek MS database. - not on Vitek MS data base 6 not on Vitek MS data base Total = 0 Total = on September 9, 08 by guest 5

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