6.1. Development of a capsule typing scheme for Cronobacter spp.
As discussed in Chapter 5, the bacterial capsule plays an important role in the phenotype
of Cronobacter spp., particularly as it relates to some virulence associated characteristics such as
biofilm formation. It is also possible that the variations in the bacterial capsule may contribute to
the differences in virulence that are observed for the different Cronobacter spp. Ogrodzki and
Forsythe [126] published a study on the capsular profiling of Cronobacter species, but did not
attempt to link these profiles to any particular phenotypic characteristics. Additionally, though
Ogrodzki and Forsythe [126] did examine all five of the capsular components in Cronobacter spp.,
their analysis was based solely on the presence or absence of certain genes. While this did reveal
some differences between and among the seven Cronobacter species, these capsular profiles do
little to explain the behaviour of the organisms. The analysis of the bcs gene cluster presented in
Chapter 5 went further by analysing the sequences of the genes, instead of merely their presence;
however, the analysis of the bcs gene cluster alone is not sufficient to explain all the variation
observed in colony morphology and multicellular morphotypes for C. sakazakii. As discussed
previously, this is likely due to the fact that cellulose is only one of five capsular components
observed in Cronobacter spp. [126]. In order to accurately predict phenotypes affected by capsule
formation, the sequences of all five capsular components must be taken into account.
Nonsense mutations have already been identified in the K-antigen and colanic acid gene
clusters of C. sakazakii ST8 strains [127]. As discussed in Chapter 5, these mutations may have
contributed to the observed phenotypes on infant formula (IF) agar and Congo red agar (CRA). By
analysing all five capsular gene clusters at the sequence level, it may be possible to develop a
more accurate capsular typing scheme for Cronobacter spp. using the Bacterial Isolate Genome
Sequence Database (BIGSdb) [86]. Yet, the mutations identified in the ST8 strains stress the
importance of examining not only the gene sequences, but also the corresponding protein
sequences. In contrast to the gene sequences, the protein sequences will be more closely linked
to the functionality of the gene. Analysis of the protein sequences will be able to account for both
By assigning allele numbers to the unique gene sequences and unique protein variants,
the species and strains could be sorted according to these detailed capsular profiles. From there,
phenotypic analyses could be performed to determine if there are traits that are consistent among
strains with identical or similar capsular profiles. First, IF agar and CRA could be used to assess
the differences in colony morphology as they relate to the entirety of the bacterial capsule.
Capsular profiles could also be linked to other phenotypes of interest, such as biofilm formation
and resistance to stresses that are likely to be encountered by Cronobacter spp. (e.g. desiccation,
heat, and acid). By linking these phenotypes to the capsular profiles based on both DNA and
protein sequences, it may be possible to predict if a particular strain will be a strong biofilm
producer or will be more resistant to environmental stresses before these tests are performed in
the laboratory.
Linking the capsular profiles to differences in biofilm formation could lead to a better
understanding of biofilm formation in Cronobacter spp. and other enteric bacteria. As discussed in
Chapter 5, it is unclear what role the bacterial capsule plays in biofilm formation. It is possible that
the bacterial capsule may inhibit adhesion to surfaces, leading to lower biofilm formation; however,
the capsule may be important for formation and growth of the biofilm after the cells have become
attached to the surface. Examining the different stages of biofilm formation within the context of
the capsular profile could help to elucidate the role that the capsule plays in this process.
6.2. Potential problems with this proposed typing scheme
The major difficulty with the proposal of a sequence-based capsular typing scheme is the
number of genes involved in capsule production in Cronobacter spp. For example, the O-antigen
locus contains anywhere between 7 and 13 genes [151]. Additionally, the colanic acid gene cluster
contains 20 or 21 individual genes [126]. In total, analysis of all five capsular gene clusters could
involve up to 66 gene sequences. While it may be possible to generate a laboratory assay to
sequence some of these genes, a laboratory assay to sequence all of the capsule genes would be
highly inefficient. As genome sequencing becomes cheaper and easier, it will be possible to
extract and analyse the desired sequences from the genomes of the desired strains. The BIGSdb
does have the capability to perform this type of analysis, but it must be carefully considered.
It may be suggested that it would be easier to use only a few selected genes from each
cluster to establish the capsular profile. For example, Ogrodzki and Forsythe [126] used the genes
rather than analysing all of the genes within the O-antigen locus; however, this approach is
problematic as it may miss some of the mutations that could affect phenotype. As described in
Chapter 5, C. sakazakii strain 553 lacks a small section of the bcs gene cluster. If only a few of the
genes from this cluster (e.g. bcsC, bcsB, and bcsG) were used to determine the capsular profile,
the deletion in the bcs gene cluster in strain 553 would not be reflected in its capsular profile.
Based on those three genes, strain 553 would be predicted to be cellulose positive, leading to
inconsistent results when comparing the capsular profiles to the observed phenotypes in the
laboratory. Thus, in order to accurately determine the capsular profiles of Cronobacter strains, it is
important to include all of the genes involved in production of the bacterial capsule. To do this,
genomic analysis, rather than laboratory assays, would be more efficient. Chapter 5 demonstrated
how analysis of the cellulose gene cluster could be linked to colony morphologies. Extending such
analyses to all five capsular components will lead to a better understanding of how the bacterial
capsule relates to certain phenotypic characteristics of Cronobacter spp. and other bacteria. It may
be possible to link capsular profiles to not only colony morphologies and biofilm formation, but also
other traits, particularly those behaviours that have been linked to virulence or environmental
survival. Eventually, it may be possible predict any number of phenotypic traits, based only on an
organism’s genome sequence. In order to do this, however, phenotypes of interest must first be linked to genotypes. The work presented here is a first step toward this future, but much work is
still needed to achieve accurate phenotypic prediction from genome sequences. One day, perhaps
phenotypic characterization of bacterial species and strains will become obsolete as genome