Abstract Book of EAVLD2012 - eavld congress 2012

S4 - O - 02
BIOLOG GENERATION III, MATRIX ASSISTED LASER DESORPTION/IONISATION TIME-OF-FLIGHT
(MALDI-TOF) MASS SPECTROMETRY AND 16S rRNA GENE SEQUENCING FOR THE
IDENTIFICATION OF BACTERIA OF VETERINARY INTEREST
Peter Wragg 1 , Luke Randall 2 , Adrian Whatmore 3
1 Animal Health and Veterinary Laboratories Agency, Laboratory Services Department, Penrith, United Kingdom
2,3 Animal Health and Veterinary Laboratories Agency, Department of Bacteriology, Weybridge, United Kingdom
Biolog, Maldi-ToF, 16S rRNA
Introduction
The veterinary bacteriologist is currently presented with a wide
variety of technologies offering potential solutions to problems in
the identification of bacteria.
16S rRNA sequencing is perhaps the most familiar of the genomic
approaches. Nonetheless, genomic methods are not without
limitations and may suffer similar database validation issues to
phenotypic systems (1,2). Automated or semi-automated
metabolic methods have developed rapidly in recent years, with
multi-analyte systems offering extended databases, although
frequently less well populated with veterinary taxa. Biolog’s
Generation III identification system employs a novel method of
detecting substrate utilisation and an extensive database,
including veterinary isolates (3). Matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF) has
become widely recognised as one of the most adaptable of the
chemotaxonomic methods (4). Veterinary strains are wellrepresented
on manufacturer’s databases. Database
enhancement initiatives are ongoing for all three technologies in a
number of research facilities.
This study was designed to assess the performance of the above
methods for the identification of a limited but typical range of
referral isolates of veterinary significance (n=66) by comparison to
partial 16S rRNA DNA sequencing, with a view to indicating
potential suitability as a ’front-line’ identification tool.
Materials & methods
The isolates used in this study consisted of field strains referred
for determinative bacteriology by Regional Laboratories of the
Animal Health and Veterinary Laboratories Agency (AHVLA),
although a number of strains from the National Culture Type
Collection (NCTC, Colindale, London) and the American Type
Culture Collection (ATCC, Manassas, Virginia) were also included.
Each field isolate had previously been identified by conventional
biochemical approaches. Cultures were prepared on 5% sheep
blood-Columbia agar (Oxoid, Basingstoke, UK), incubated either
aerobically or in 7.5% carbon dioxide at 37 °C for 18-24h,
according to cultural characteristics, and tested blind by each of
the three methods as follows.
Biolog Generation 3:
Inocula were prepared according to manufacturer’s instructions
(Biolog, Hayward, CA). Growth patterns were measured using a
Microstation reader and analysed using Microlog software.
Matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF):
Samples were prepared using standard Bruker protocols (Bruker
Daltonik GmbH, Bremen, Germany) (4) and analysed using a
Bruker Autoflex II machine. Spectra were analysed with MALDI
Biotyper software version 2.0.10.0 in comparison with reference
spectra (MALDI Biotyper reference library version 3).
16S rRNA gene sequencing:
Ribosomal RNA sequence identification was based on a minimum
600bp sequence corresponding to the V2-V4 regions of the gene.
Sequences were compared with both NCBI and RDP databases
with criteria of a >99% similarity to the type strain of a
taxonomically valid species being used as identification criteria for
species. If matches were below 99% or there were matches >99%
to multiple taxonomically valid species identification was given to
the genus level only.
In the majority of cases, referral for determinative bacteriology
within AHVLA requires identification to species level. A scoring
system was devised, using either 16S sequencing or the
NCTC/ATCC identity as the ‘gold standard’. Where sequencing
lacked species information for comparison, identification was
scored at genus level only in all methods.
Results
Scores of 3, 1 and 0 were assigned for identifications to species,
genus and ’ no identification’, respectively.
Table 1 summarises the performance of each system.
Table 1: Performance of identification systems
Numbers of strains identified Biolog MALDI 16S sequencing
To genus 28 30 29
To species 27 27 36
No ID 11 9 1
Incorrect genus 0 0 0
Incorrect species 0 0 0
Overall score 109 111 137
Scores for MALDI and Biolog may have improved if alternative
phenotypic data had been considered in determining true identity.
Discussion & conclusions
Scores for MALDI and Biolog were broadly comparable, although
a number of taxa were identified more specifically by particular
techniques. A number of isolates proved refractory either by
MALDI-TOF or Biolog, although local enhancements to databases
would minimise these occurrences.
Whilst each methodology offers unique advantages, other factors
which require consideration are the accessibility of the technology,
including start-up and running costs, the quality and ease of
supplementation of the manufacturer’s database and the
requirement for centralisation of resources and expertise which
may be implicit in the choice of technique.
Acknowledgements
We thank the AHVLA Regional Laboratories at Bury St Edmunds,
Starcross and Sutton Bonington for provision of field strains, Mark
Koylass and the Central Sequencing Unit at AHVLA Weybridge.
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