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Usefulness of matrix-assisted laser desorption

ionization time-of-flight mass spectrometry
to identify pathogens, including polymicrobial
samples, directly from blood culture broths
This article was published in the following Dove Press journal:
Infection and Drug Resistance
19 April 2017
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Maya Hariu 1,2 Abstract: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (TOF-
Yuji Watanabe 1,2 MS) is now widely used to detect pathogens in clinical settings in Japan. Here, we report the
Nozomi Oikawa 1,2 ability of TOF-MS to detect bacteria from blood culture (BC) broths, and compare the efficacy
Masafumi Seki 1 of TOF-MS to that of conventional culture methods. Bacteria were correctly detected from 63
monomicrobial samples within 80 minutes; results matched those obtained by conventional BC
1
Division of Infectious Diseases and
Infection Control, 2Laboratory for methods, although the conventional methods took 2–3 days. In addition to the 63 monomicrobial
Clinical Microbiology, Tohoku Medical samples, another three polymicrobial samples were tested; notably, the infecting bacteria were
and Pharmaceutical University not correctly identified in two of these three samples. To better assess the TOF-MS detection of
Hospital, Sendai, Japan
polymicrobial samples, we tested various ratios of mixed broth samples, including combinations
of the bacteria that we were unable to detect in clinical samples. Combinations of Enterobacter
cloacae and Pseudomonas aeruginosa were correctly detected at a culture ratio of 2:1, but not in
the 3:1 mixture. These results suggested that TOF-MS is a strong tool for the rapid and correct
detection of pathogens from monomicrobial BC samples, though results need to be carefully
checked when handling known or suspected polymicrobial samples.
Keywords: bacteremia, polymicrobial sample, Enterobacter cloaca, Pseudomonasa aeruginosa

Introduction
Sepsis, a host-wide infection by bacteria, often presents as a multiple-organ dysfunction
and results in a 10–40% mortality rate. Bacteremia is typically diagnosed by microbio-
logical tests, usually consisting of blood cultures (BCs).1 The presence of pathogens in
the bloodstream is detected in only 4–12% of all BCs in hospitals; the identification
of pathogens via BC can take 2–3 days and sometimes yields false-negative results.1,2
Therefore, rapid bacterial identification from BCs is of great interest, especially for
sepsis cases. Examples of other identification techniques include polymerase chain
reaction, fluorescence in situ hybridization, and high-throughput DNA sequencing.2–5
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
(TOF-MS) allows identification of most of the pathogenic bacteria grown on agar
Correspondence: Masafumi Seki
Division of Infectious Diseases and plates from isolated colonies within a few minutes, and has proven efficiency and
Infection Control, Tohoku Medical and reproducibility.6–8 Plasma represents up to 55% of the total blood volume, and offers
Pharmaceutical University Hospital,
1-12-1 Fukumuro, Miyagino-ku, Sendai,
a good opportunity to recover grown bacteria, as microbes are mostly present in the
Miyagi 983-8612, Japan extracellular compartment.
Tel +81 22 983 1221
Fax +81 22 983 1232
In this study, we performed identification of bacteria in BCs by TOF-MS, and
Email seki@hosp.tohoku-mpu.ac.jp compared our results with those obtained by conventional BC methods. Among the
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clinical samples tested, few contained two distinct bacte- for TOF-MS identification according to MBT Sepsityper
rial species, but the polymicrobial samples represented the kit instruction manual.
biggest challenge for TOF-MS identification. Therefore, we
further examined several combinations of bacteria at various Mixture of bacteria in various ratios for
ratios in an effort to better assess the utility of TOF-MS for analysis by TOF-MS
such samples. To examine the ability of TOF-MS to distinguish the poly-
microbial samples, we prepared several types of bacterial
Patients and methods mixtures in various ratios, ranging from 1:9 to 4:1, and then
Samples performed the BC broth analysis as described. The selected
Blood specimens from adult patients were systematically bacteria were the following: a: Escherichia coli, b: Pseudo-
collected and cultured using the Signal Blood Culture monas aeruginosa, c: Enterococcus faecalis, d: Staphylococ-
System (Oxoid; Thermo Fisher Scientific, Waltham, MA, cus aureus, e: Streptococcus pneumoniae, f: Staphylococcus
USA), including both anaerobic and aerobic resins. Bottles epidermidis, and g: Enterobacter cloacae. The combinations
containing 8–10 mL of whole blood were incubated in MIR- were as follows: 1: a+b, 2: a+c, 3: a+d, 4: a+e, 5: a+f, 6: b+c,
553 instruments (SANYO, Moriguchi, Japan) for 5–7 days 7: b+d, 8: b+e, 9: b+f, 10: b+g, 11: c+d, 12: c+e, 13: c+f, 14:
for the standard incubation protocol, or for fewer days if an d+e, 15: d+f, 16: e+f, and 17: f+g.
earlier positive signal was detected. The direct identifica-
tion of bacteria by TOF-MS from both positive aerobic and Results
anaerobic bottles (Bruker Daltonics, Billerica, MA, USA) Detection of pathogens from the patient
was performed serially in parallel with the routine protocol samples by TOF-MS
at the clinical laboratory of Tohoku Medical and Pharmaceu- We tested 463 blood samples originating from anaerobic/
tical University Hospital; these tests were performed from aerobic blood vials. For this comparison of TOF-MS results
July 2014 to June 2015. After subculturing on agar plates, with those obtained by routine techniques, 66 samples were
any positive BC was simultaneously assessed for bacterial positive for bacteria, and 20 distinct species were detected
identification using conventional procedures, including in the overall study (Tables 1 and 2). MALDI-TOF BioTyper
Walkaway panels (Beckman Coulter, Brea, CA, USA), and was able to identify most or all of these specimens at the
if necessary, with complementary individual biochemical or species level.
enzymatic tests. A total of 63 samples were monomicrobial cases, with E.
This study was approved by the Committee for Clinical coli (n=18), S. aureus (n=10), and S. epidermidis (n=8) detected
Scientific Research of Tohoku Medical and Pharmaceutical most frequently (Table 1). Average score values were 2.465 for
University Hospital in July 08 and December 10, 2015 (No. E. coli, and >2.0 for both S. aureus and S. epidermidis. For all
ID2015-2-008 and ID2015-2-037, respectively), and all the bacteria-positive sample, average scores were 2.408 for
patients whose blood specimens were used provided written Gram-negative rods and 2.281 for Gram-positive cocci.
informed consent.
Polymicrobial BCs
Separation of blood cells and bacteria Routine classical identification detected three polymicrobial
and protein extraction protocol and samples representing six isolates; however, TOF-MS identi-
identification of bacteria fied only four bacteria in these three samples using the identi-
We used the MALDI Sepsityper kit (Bruker Daltonics). In fication methods provided by BioTyper (Table 2). Specifically,
brief, approximately 1 mL of cultured sample was recovered BioTyper called two of these three cases as monomicrobial
from a given positive BC bottle, transferred to a microfuge samples (Case 2 and Case 3; Table 2).
tube, and combined with 200 µL lysis buffer. After mix- In fact, polymicrobial samples were suspected when
ing by vortexing for 10 seconds, tubes were centrifuged at MALDI BioTyper proposed two alternative species that were
10,000 g for 2 minutes at room temperature, and superna- identified with score values >1.7. For such cases, BioTyper
tants were removed. We then resuspended each pellet by allows the superimposition and comparison of the sample
pipetting with 1 mL washing buffer before recentrifuging spectrum with library spectra in various mixture ratios
the samples for 1 minute at 10,000 g. After removal of the (Figure 1).
supernatant, the bacterial pellet was treated according to We detected both E. cloacae and P. aeruginosa (Case
the standard ethanol/formic acid protein ­extraction protocol 2, Table 2) from clinical samples when we mixed at 2:1

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Dovepress TOF-MS in blood culture-positive samples

Table 1 Bacteria detected from blood culture broth by time-of- ­therapy.2,9 Any delay in identification remains unsatisfac-
flight mass spectrometry matched with the conventional method tory for clinicians, who may be forced to initiate antimi-
Detected pathogens (n=63) Number Score value crobial therapy prior to precise diagnosis.1 Shortening the
Escherichia coli 18 2.465 delay in providing appropriate therapy may strengthen
Staphylococcus aureus 10 2.387
therapeutic specificity, and is expected to have a beneficial
Staphylococcus epidermidis 8 2.141
Enterococcus faecalis 3 2.426 effect on the recovery of patients. The high sensitivity of
Klebsiella pneumoniae 3 2.226 TOF-MS in detecting peptides and the possibility of iden-
Pseudomonas aeruginosa 3 2.399 tifying bacteria with software such as BioTyper has been
Staphylococcus caprae 2 2.175
shown to permit high-quality microbial identification by
Staphylococcus hominis 2 2.201
Streptococcus agalactiae Group B 2 2.461 TOF-MS.10
Streptococcus constellatus 2 2.199 Here, we demonstrated the identification of bacteria
Aerococcus urinae 1 2.149 from BCs using MALDI-TOF BioTyper, which allowed
Corynebacterium striatum 1 2.304
Enterobacter agglomerans 1 2.374
95.5% correct, single-step identifications among a total of
Enterobacter cloacae 1 2.283 20 microorganisms from 66 clinical blood samples, including
Klebsiella ornithinolytica 1 2.380 three polymicrobial samples, starting from small volumes of
Proteus mirabilis 1 2.395
BC. Monomicrobial samples were correctly identified at the
Staphylococcus capitis 1 2.179
Streptococcus anginosus 1 2.451 species level in 100% of cases. All bacteria were identified
Streptococcus bovis 1 2.355 within the first 2–3 hours following BC positivity.
Streptococcus pyogenes (Group A) 1 2.289 Hansen et al reported that TOF-MS analysis did not
produce scores high enough for species identification in two
Table 2 Detected or non-detected polymicrobial cases by time- bacteremia cases that presented with diverticular diseases;
of-flight mass spectrometry (TOF-MS) instead, the infecting bacteria were identified by the sequenc-
Case Pathogens detected by Pathogens detected Score ing method,11 as we have previously reported.3,12,13 However,
conventional culture by TOF-MS value
the infecting bacteria investigated by Hansen et al were
1 Streptococcus constellatus + S. constellatus + 2.104
relatively rare Gram-positive cocci, such as Ruminococcus
Klebsiella pneumoniae K. pneumoniae
2 Enterobacter cloacae + E. cloacae 2.325 species.11 Furthermore, treatment following rapid organism
Pseudomonas aeruginosa identification by TOF-MS has been shown to lead to reduced
3 Staphylococcus epidermidis S. epidermidis 2.167 mortality in severe sepsis cases after implementation of
Enterococcus faecalis
TOF-MS in combination with BC.14 Furthermore, TOF-MS
recently has been shown to reduce the use of wide-spectrum
ratio ­(Figure 1A), but E. cloacae alone in the 3:1 mixture antibiotics in infection control when employed as part of an
­(Figure 1B). Therefore, we next performed TOF-MS analysis antimicrobial stewardship program.15 These results suggest
on samples that combined several pairs of bacteria at various that rapid and accurate identification of microbes by TOF-MS
mixture ratios, including the same combinations as detected can contribute to better treatment and outcomes for patients
in Cases 2 and 3 (Table 3). We found that TOF-MS was able with infectious diseases in clinical settings.
to detect the combined bacteria at ratios ranging from 1:3 However, identification by TOF-MS is limited by the
to 1:1 (Table 3). species and some specific situations; notably, polymicrobial
cases may not be correctly diagnosed by TOF-MS. For poly-
Discussion microbial samples, the observed profile may represent the
New technologies, such as TOF-MS, have revolutionized mixed profiles of two distinct bacteria, with both showing
the identification of bacteria. TOF-MS is fast, reliable, and significant scores (Table 2). Such a situation will require
inexpensive, and the majority of clinically relevant bacteria closer examination in the TOF-MS context. In these cases,
can be identified at the species level.6–8 the corresponding BCs will need to be carefully checked
In contrast, even though BC is considered to be the stan- at the next isolation plate (typically grown for testing
dard for the detection of microorganisms in blood samples, antimicrobial susceptibility), to distinguish the presence of
identification by BC remains dependent on several different additional bacterial isolates for subsequent identification, if
factors, including bacterial density, bacterial adaptation necessary. This follow-up evaluation may help to validate the
to new growth environments, individual rates of growth, initial status of the blood samples, if not precluded earlier
competition between bacteria, and initial a­ ntimicrobial by Gram staining.

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Enterobacter cloacae 20105

A + Pseudomonas aeruginosa 19955

1.0

0.5
Relative intensity

0.0

–0.5

–1.0

2 4 6 8 10 12
3
m/z (10 )

Pseudomonas aeruginosa 8147

B 1.5

1.0
Relative intensity

0.5

0.0

–0.5

–1.0

–1.5
2 4 6 8 10 12
3
m/z (10 )
Figure 1 Identification of polymicrobial results displayed within the BioTyper 2.0 graphic view in the case of Enterobacter cloacae and Pseudomonas aeruginosa at ratios of 2:1
(A) and 3:1 (B), respectively. The mass spectra show the difference in peaks (presence or absence) and their intensities between the sample spectrum and those of bacteria
identified at first pass by BioTyper. The upper part of the figure within the inner windows shows the unknown spectrum containing perfectly matching peaks (0–200 ppm)
in green, imperfectly matching peaks (200–500 ppm) in yellow, and non-matching peaks in red. The lower part (blue) shows the dedicated main spectrum included in the
database. In our case, both E. cloacae and P. aeruginosa were identified correctly at a score value of 2.580 in the 2:1 mixture ratio (A), but only E. cloacae was identified (at
a score value of 2.407) in the 3:1 mixture ratio (B).

Christner et al reported that BioTyper scores >1.5 were in ­polymicrobial samples is a challenge. Therefore, if the
essential for the identification of 8% of the isolates, but presence of more than one pathogen is suspected, it may be
that work did not consider the possibility of polymicrobial better to try to test the various mixture ratios, as demonstrated
samples.16 Moussaoui et al tested a new protocol for ­bacterial in the present study.
identification from BC broths, but only 10 of a total of 50 In conclusion, we examined the analytical ability of TOF-
isolates from 21 polymicrobial samples were identified in MS and found that the technique yielded valid ­identification
that work.6 La Scola and Raoult reported the identification for >95% of bacteria derived from monomicrobial and
of only one of each mixture of species for 18 samples among polymicrobial BC samples. Even in polymicrobial cases,
22 bacteria-positive BC broths that contained two or more analysis of corrected mixture ratios of combinations of the
different species.7 No species was identified in two of those candidate infecting bacteria may facilitate detection of indi-
polymicrobial samples, and false species identifications vidual species, as we have shown in the present study. This
were obtained in two cases.7 Using an in-house saponin rapid identification by TOF-MS is expected to lower costs
lysis method (in place of the MALDI Sepsityper kit), Meex for clinical laboratories and to provide better information to
et al were able to identify only one of each pair of isolates physicians, facilitating targeted antimicrobial therapy and
in six separate polymicrobial BCs.8 These results suggest potentially contributing to a decrease in antimicrobial pres-
that the identification by TOF-MS of two or more bacteria sure in hospitals.

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Dovepress TOF-MS in blood culture-positive samples

Table 3 Detected bacteria in various combination ratios by time-of-flight mass spectrometry

Combinations Ratio
1:9 1:8 1:7 1:6 1:5 1:4 1:3 1:2 1:1 2:1 3:1 4:1
1 b b b b b Both Both Both Both Both Both a
2 Both Both Both Both Both Both Both Both a a a a
3 d d d d d Both Both Both a a a a
4 e Both Both Both Both Both Both Both a a a a
5 f f f f f f Both Both Both a a a
6 Both Both Both Both Both Both Both Both Both Both Both b
7 d Both Both Both Both Both Both Both Both Both Both b
8 e e e e Both Both Both Both Both b b b
9 f f f Both Both Both Both Both Both Both Both b
10 g g g g g g g Both Both Both Both b
11 d d d d d d Both Both Both Both Both c
12 e e e e e Both Both Both c c c c
12 f f f f f f Both Both Both Both Both c
14 e e e e e Both Both Both d d d d
15 f Both Both Both Both Both Both Both Both Both d d
16 f f f f f f Both Both Both Both e e
17 g g g g g g g g g Both Both f
Notes: a: Escherichia coli, b: Pseudomonas aeruginosa, c: Enterococcus faecalis, d: Staphylococcus aureus, e: Streptococcus pneumoniae, f: Staphylococcus epidermidis, and g: Enterobacter
cloacae; 1=a:b, 2=a:c, 3=a:d, 4=a:e, 5=a:f, 6=b:c, 7=b:d, 8=b:e, 9=b:f, 10=b:g, 11=c:d, 12=c:e, 13=c:f, 14=d:e, 15=d:f, 16=e:f, and 17=f:g.

Disclosure 8. Meex C, Neuville F, Descy J, et al. Direct identification of bacteria from

BacT/ALERT anaerobic positive blood cultures by MALDI-TOF-MS:
This work was supported by the Japanese Society for the MALDI Sepsityper kit versus an in-house saponin method for bacterial
Promotion of Science Grant-in-Aid for Scientific Research extraction. J Med Microbiol. 2012;61(Pt 11):1511–1516.
9. Schelonka RL, Chai MK, Yoder BA, Hensley D, Brockett RM, Ascher
26461158 (to MS). The authors report no conflicts of inter- DP. Volume of blood required to detect common neonatal pathogens.
est in this work. J Pediatr. 1996;129(2):275–278.
10. Mellmann A, Bimet F, Bizet C, et al. High interlaboratory reproduc-
ibility of matrix-assisted laser desorption ionization-time of flight mass
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