102
(>10^9 cells/mL for E. coli). In this case, either longer incuba-
tions or smaller antibiotic concentration increases between
serial transfers may be beneficial. Growth from a 1000-fold
dilution, as described above, to full density corresponds to ten
generations of growth.- Maintaining proper sterile technique is absolutely essential,
since cross-contamination of lineages may lead to less informa-
tion about potential resistance development trajectories. Blank,
un-inoculated media control tubes should always be prepared
and incubated in parallel with experimental tubes. Regular
spraying and wiping down of equipment and workspaces with
70% ethanol is advised. Use of a laminar flow cabinet, if avail-
able, is also beneficial. - Resistance to antibiotics is often conferred fully or partially by
genetic amplification of genes on the chromosome or plas-
mids. In the absence of selection, these amplifications are rap-
idly lost from the population. In order to detect these
amplifications in sequencing data, it is important that they are
always held by selection during all the growth steps for genomic
DNA preparation. - Evolutions in liquid culture will to varying degrees generate
mixed populations. Generally sequencing single clones from a
population is preferred to sequencing the whole population.
Mixed population sequencing may be possible depending on the
number of genotypes represented in the population and the
sequencing technology used, but also may present problems both
predictable and unexpected. If a single-clone sequencing strategy
is used, it is essential that the specific clone that is sequenced be
saved. Reisolating a particular clone from a population can be
labor-intensive and success cannot be guaranteed! - The most popular whole genome sequencing technology cur-
rently for this type of analysis is that sold under the Illumina
brand. Illumina sequencing technology generates large num-
bers of short, paired reads (~75–300 bp). This type of data is
very useful in cases where a high-quality reference sequence is
available for the organism being sequenced. In this case
Illumina data can be used to find SNPs, short insertions and
deletions, and copy-number variants. In the event that the
genome being sequenced contains many repetitive sequences it
may be appropriate to use another technology, alone or in
combination with Illumina, that can generate longer contigu-
ous reads. In such instances a popular current technology is
that marketed under the PacBio brand. - For each species there are recommended quality control strains
[ 10 ] that can be purchased from the American Type Culture
Collection and these should be routinely used to ensure that the
conditions of the MIC assay are within acceptable margins.
Douglas L. Huseby and Diarmaid Hughes