The Impact of a Customized MALDI-TOF Library for Environmental

Technical Sheet
The Impact of a Customized
MALDI-TOF Library for Environmental
Microbial Identifications
Executive Summary
While a number of microbial identification systems are available to the pharmaceutical market for compendial testing, these platforms tend
to underperform, because they do not support the broad range of microorganisms encountered in environmental monitoring programs.
The reference libraries are often biased toward clinical, rather than environmental, organisms. A prime example is Bruker Daltonic’s
MALDI-TOF Biotyper® microbial identification platform. Here, we illustrate a systematic approach for the inclusion of new library entries,
with the priority on adding the most frequently encountered environmental species first. The additional entries created using this approach
increased the percentage of environmental isolates identified using the Biotyper platform from 73.1% to 83.7% (n=13,786 and 15,095).
While low cost and rapid turnaround time remain strong advantages for MALDI-TOF microbial identification platforms, our data demonstrate
that building a reference database with environmental organisms in mind is essential for its suitability to nonclinical applications.
Introduction
Methods
The Accugenix® brand supports the pharmaceutical, medical
device, nutraceutical, personal care and cosmetics industries,
helping to maintain environmental monitoring programs and
product failure investigations or other compendial testing. Having
identified more than a million samples over the past decade,
our services are well suited to examine the efficacy of microbial
identification platforms for environmental monitoring applications,
often using in-house 16S gene sequencing to compare and
evaluate systems. Since we first offered our AccuPRO-ID®
microbial identifications using Bruker Daltonic’s MALDI-TOF
Biotyper platform in 2010, we have processed over 70,000
samples on the system and continue to optimize the platform to
better accommodate environmental isolates. This study assesses
the suitability of the Biotyper platform for identifying environmental
isolates, demonstrates our systematic approach to broadening
the reference library and shows the impact of adding new library
entries on reportability for these industrial sectors.
All data presented in this study were generated from unknown
samples received for microbial identification. Samples submitted
for AccuPRO-ID® MALDI-TOF identification were processed
using a direct formic acid lysis method and/or the extract method
(per Accugenix SOPs and the manufacturer’s instructions) and
analyzed using Bruker’s MALDI-TOF and Biotyper software.
Samples with a “match factor” greater than 1.75 and separated
by >0.1 were ascribed probable species-level identifications. All
samples that failed to produce an identification using Biotyper
were submitted for AccuGENX-ID® 16S rDNA sequencing,
where sequence data were manually assembled and compared
against the validated and proprietary Accugenix 16S reference
library. Qualified data analysts determined identifications and
confidence levels. 16S-based sequence identification is used as
the reference method for this study.
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© 2013, Charles River Laboratories International, Inc.
Technical Sheet
Results
Figure 1: Diversity of Species Routinely Identified
Log (Frequency of Occurrence)
90%
95%
99%
Figure 3: Systematic Approach for the Inclusion of New
MALDI-TOF Library Entries
100%
100,000
Samples
10,000
1,000
Spotting
100
10
1
1
500
1000
1500
2000
MALDI-TOF MS
Species Count
The number of unique species identified by 16S sequencing
(x-axis) versus the frequency at which these species were
encountered (y-axis) (n=233,366). Vertical lines (dark blue)
designate the number of species that make up the top 90%, 95%,
99% and 100% of species identified. These results indicate that a
wide range of species are isolated from environmental sources.
Figure 2: Species Diversity of MALDI-TOF
Fall-Through Samples
Log (Frequency of Occurrence)
90%
95%
Spectra Acquisition /
Analysis
Library Comparison
99% 100%
1,000
100
Species-Level
Identification
10
1
1
500
1000
16S DNA
Sequencing
1500
Species Count
The number of unique species (x-axis) versus the number of times
these species failed to generate an identification (y-axis) using the
MALDI-TOF platform (n=12,199). Vertical lines (dark blue) indicate
the number of species that makeup the top 90%, 95%, 99% and
100% of fall-through species. These results indicate that when
the Biotyper system is used for environmental monitoring, the fallthrough samples represent an extensive number of species.
No Reportable
Identification
Report
Report & Identification
of MALDI-TOF
“Fall-Through” Samples
All samples that fail to generate an identification using the MALDITOF platform are submitted for 16S sequence identification. By
identifying all MALDI-TOF “fall-throughs,” we have the unique
opportunity to improve the MALDI-TOF Biotyper reference library.
Endotoxin and Microbial Detection
The Impact of a Customized MALDI-TOF
Library for Environmental Microbial Identifications
Technical Sheet
100
90
80
70
60
50
Missing
Present
40
30
20
10
0
Average
Figure 6: New Entries Target the Most Frequently Occurring
Species
Top 95% Encountered
Species (Percent)
Species (Percent)
Figure 4: Proportion of Fall-Through Species Present
and Missing from the Reference Library
100
90
80
70
60
50
40
30
20
10
0
Average - Weighted for
Frequency of Fall Through
The percent of MALDI-TOF fall-through species (y-axis)
represented by at least one MALDI-TOF library entry
(present=dark pink) or are not yet included in the library
(absent=light pink) (n=1,242). This comparison was completed
by disregarding the fall-through occurrence of each species (left),
as well as weighting for fall-through occurrence (right). These
data indicate that the inclusion of new species, and to a lesser
extent redundant species-level entries, is needed to improve the
reference library for environmental monitoring applications.
66.0%
96.6%
Biotyper
v3.3.1.2
Accugenix
v13.03
Missing
Present
Of the 559 species constituting the top 95% of species
encountered, the percent of species that are present (darker pink)
or absent (lighter pink) is compared against the manufacturer’s
library(left) and the Accugenix library (right). These data indicate
that library entries targeting the most frequently encountered
species seen are developed first, effectively tailoring the library
toward the environmental microorganisms important to the
industries we serve.
Figure 7: Source of Library Entries Leading to a
MALDI-TOF Identification
Figure 5: Number of Library Entries and Frequency
of Additions
Number of Library Entries
6000
5000
Biotyper
(63.1%)
4000
3000
1000
0
13
20
12
20
11
20
10
20
Year
Matching
Biotyper
(47.6%)
Discrepant
Biotyper
(18.1%)
Total Entries
Biotyper Entries
Accugenix Entries
2000
Accugenix
(36.9%)
No Other
Match
(34.3%)
The number of MALDI-TOF library entries (y-axis) created by the
manufacturer (orange), Accugenix (light blue) and combined
(dark blue), plotted versus time (x-axis). While the Accugenix
MALDI-TOF library currently only comprises ~15% of the entire
database, it is updated approximately 2-3 times more frequently
than the manufacturer’s updates (see entry markers) to include
microbes from environmental origins.
Of all samples that resulted in an identification (n=13,786), the
source of the top-matching library entry (Biotyper in dark pink,
Accugenix in light pink) was evaluated (left-hand pie chart).
Accugenix library entries were the top-match on 36.9% of all
reported identifications. Given that Accugenix entries make up
only ~15% of the entire library (Fig. 5) but are the top-match
on nearly 37% of all identifications, Accugenix entries are the
top match more than twice as often as Biotyper entries. Of all
samples reported with an Accugenix entry as the top match (righthand pie chart), Biotyper’s library would have reported the same
identification in 47.6% of the cases, but a discrepant identification
or no reportable identification in 18.1% and 34.3% of the cases,
respectively. Together, these data indicate that the Accugenix
library increases identification reportability and accuracy for
samples from environmental origins.
Endotoxin and Microbial Detection
The Impact of a Customized MALDI-TOF
Library for Environmental Microbial Identifications
Technical Sheet
Samples Tested (Percent)
Figure 8: Impact of a Customized Database on
Reportable Identifications
100
90
80
70
60
50
40
30
20
10
0
26.9%
16.3%
No Reportable
Identification
Reportable
Identification
Biotyper
v3.3.1.2
Accugenix
v13.03
Of all recent samples submitted for identification using the MALDITOF platform (n=15,095), the percent of samples resulting in no
reportable identification (light pink) and a reportable identification
(dark pink) was determined when using the manufacturer’s
database (left) and Accugenix’s customized database (right).
When using Accugenix’s library entries for environmental microbial
identifications, there is a 10.6% increase in reportable samples,
which translates into a 39.4% decrease in the non-reportable rate.
Discussion
While the number of microbial species frequently encountered in
the environmental arena is rather daunting (Fig. 1), inclusion of
these species in any microbial identification platform’s reference
library is critical to increasing reporting and/or improving a
platform’s accuracy for non-clinical applications. Using the
systematic approach presented here (Fig. 3), the species that
most frequently fail to generate an identification using Biotyper
are pinpointed by 16S-sequencing all fall-through samples
(Fig. 4), enabling the addition of these fall-through species to
the Accugenix reference library (Fig. 6). To date, these library
entries generate identifications more than twice as frequently
as the entries provided by the manufacturer, confirming that the
most frequent fall-throughs are added to the Accugenix reference
library first. On a similar note, these additional library entries
generate nearly 37% of all identifications, increase the number of
identifications by more than 10% and effectively reduce the nonreportable rate by 39.4% (Fig. 8). With future library releases, the
percentage of samples generating an identification will continue
to rise. That being said, the current percentage of fall-through
samples is 16.3%, which indicates that backing the MALDITOF platform with Accugenix 16S sequencing is still required
to reliably generate identifications for samples originating from
environmental sources.
Glossary of Terms
AccuGENX-ID® – genotypic identification method utilizing
comparative DNA sequencing of the 16S gene of bacteria for
identification with the highest accuracy and reportable rate
AccuPRO-ID® – polyphasic approach utilizing MALDI-TOF
and AccuGENX-ID 16S rDNA sequencing, assuring the highest
accuracy and reportable rate
Environmental monitoring – program designed to demonstrate
compliance and control of a manufacturing environment,
providing a baseline microbial profile of a facility and allowing for
tracking and trending to source contaminations or excursions
Genotypic – identification method using DNA sequence data or
fragment analysis
Library/Database – validated data set containing known
microorganism entries used to compare against unknown
microbial data for identifications
MALDI-TOF – matrix-assisted laser desorption/ionization timeof-flight mass spectrometry method which produces a ribosomal
protein spectrum that can be used for identification
Phenotypic – identification method using biochemical or
physiological data
Polyphasic – method consisting of two or more phases
Proteotypic – identification method that uses a ribosomal protein
spectral fingerprint
rDNA – DNA that encodes for the ribosomal RNA subunits in
an organism; after the rRNA sequence is transcribed from the
rDNA, it assembles into the ribosomal complex, e.g., the
bacterial 16S rRNA
Ribosome – complex molecule responsible for protein synthesis
consisting of multiple RNAs and proteins assembled into the
structure
North America: 1.877.274.8371 • Europe: 00 800 15 78 97 43
email: askcharlesriver@crl.com
www.criver.com
© 2013, Charles River Laboratories International, Inc.