Biomedical Engineering Reference
In-Depth Information
may be influenced by lateral gene transfer (LGT) and may lead to incorrect
identifications. To compensate for possible LGT events, it has been suggested that
multilocus sequence analysis (MLSA) of at least five housekeeping genes from
diverse chromosomal loci and with wide distribution among taxa is required to reli-
ably distinguish a species from related taxa [
230
]. After a more thorough evalua-
tion, however, Konstantinidis and co-workers [
231
] concluded that three genes are
sufficient to anticipate the possible effects of LGT in MLSA-based identification
schemes. For LAB, MLSA based on the combined sequence analysis of the genes
atpA, pheS,
and
rpoA
has been successfully explored for species identification of
enterococci [
229
] , lactobacilli [
67
] , leuconostocs [
232
] , and pediococci [
233
] . For
sequence-based differentiation of LAB at strain level, multilocus schemes typically
include six or seven housekeeping genes. The resulting multilocus sequence typing
(MLST) approach has so far mainly been applied to study community structure,
evolution and phylogeography of bacterial pathogens [
234
] . A few MLST schemes
have been specifically developed for
Lactobacillus
species, including
Lb. casei
[
235,
236
] ,
Lb. plantarum
[
237
] and
Lb. salivarius
[
238
] .
5.4.2
Culture-Independent Approaches
The first approaches used to identify sourdough microorganisms independent of
culturing relied on the use of oligonucleotide probes targeting ribosomal gene
sequences specific for individual species or groups of species. The majority of these
probe-based methods made use of partial 16S rRNA gene sequences that were
identified as molecular signatures unique to specific LAB species [
203,
239
] .
Gradually, the relatively laborious probe hybridizations were replaced by faster
community PCR assays using species-specific oligonucleotide primers. The success
of both approaches strongly depended on rigorous
in silico
probe or primer design
and required in vitro and in vivo validation using taxonomically well-characterized
type and reference strains and spiked sourdough samples, respectively. Species-
specific PCR primers complementary to signature sequences in the 16S or 23S
rRNA gene or in the 16S-23S rRNA intergenic spacer region have been applied for
the culture-independent identification of sourdough LAB species [
26,
156,
161,
240-
242
]. By combining multiple sets of primers, several typical sourdough LAB
species can be simultaneously detected. In this way, Settanni and co-workers [
156
]
developed a two-step multiplex community PCR assay that enabled rapid
identification of up to 16
Lactobacillus
species in sourdough samples. The introduc-
tion of real-time PCR technology has allowed one to further increase the sensitivity
of PCR-based identification assays and enables the simultaneous detection and
quantification of food microorganisms [
243
]. For this purpose, SYBR Green-based
real-time PCR assays based on the detection of the
pheS
gene have been used for
source tracking of
Lb. plantarum
and
Lb. sanfranciscensis
in traditional sourdoughs
and their production environments [
155
] .
