stands to benefit the most from application of WGS technologies is
the field of mycobacteriology. Because mycobacteria grow so slowly
(even the “rapidly” growing species can take up to 7 days), the use
of WGS technologies could reduce the time required for identification of mycobacterial species significantly. Additionally, since much
of the antimicrobial resistance (AMR) seen in mycobacterial species
is caused by smaller-scale mutations such as SNPs rather than larger
gene insertions that would be easier to detect (e.g., via PCR), WGS
allows the possibility of implementing drug-susceptibility testing
(DST) as a more comprehensive and cost-effective alternative to
phenotypic testing that would be much slower [8].
This chapter summarizes the process of whole-genome
sequencing and describes its applications toward the identification
and characterization of mycobacteria. As this chapter is intended to
function as a review of the concepts and general methods used in
the whole-genome sequencing of mycobacteria, detailed protocols
are not provided. For specific genome sequencing platforms, the
reader is referred to the manufacturer’s recommended protocols for
the platform to be used. Similarly, for bioinformatic techniques, the
reader is referred to the documentation for the specific software
packages and scripts to be used.
2 Identification of Mycobacterial Species Is a Challenge
Mycobacteria are characterized as aerobic to microaerophilic,
slightly curved or straight rods that are acid-fast at some stage of
their growth [9]. These bacteria are acid-fast due to the presence of
mycolic acids in their cell wall, which provides unique characteristics that make these organisms adaptable to a wide range of conditions. Such a cell wall provides a thick waxy coat that allows these
microbes to evade the immune response, resist chemical damage,
survive dehydration, and limit the effectiveness of hydrophilic antibiotics and biocides. These abilities enable some mycobacterial
species to colonize environments such as soil and water, while
others are found only in a wide variety of hosts (insects, fish,
reptiles, birds, and mammals); some are saprophytes, while others
are parasites (including obligate and opportunistic); some are fast
growers, while others grow more slowly; some grow at low temperatures, while others thrive in hot environments.
The number of mycobacterial species described has increased
greatly in the last 45 years, from approximately 30 species in 1975
[10] to approximately 190 in 2019 [11]. Many of these species
have been discovered and defined using newer laboratory techniques, which do not correlate with traditional phenotypic and biochemical methods. This poses a major challenge for
mycobacteriology laboratories in providing conclusive identification [10]. For this reason, the latest guidelines from the Clinical &
400
Marco A. Riojas et al.
the field of mycobacteriology. Because mycobacteria grow so slowly
(even the “rapidly” growing species can take up to 7 days), the use
of WGS technologies could reduce the time required for identification of mycobacterial species significantly. Additionally, since much
of the antimicrobial resistance (AMR) seen in mycobacterial species
is caused by smaller-scale mutations such as SNPs rather than larger
gene insertions that would be easier to detect (e.g., via PCR), WGS
allows the possibility of implementing drug-susceptibility testing
(DST) as a more comprehensive and cost-effective alternative to
phenotypic testing that would be much slower [8].
This chapter summarizes the process of whole-genome
sequencing and describes its applications toward the identification
and characterization of mycobacteria. As this chapter is intended to
function as a review of the concepts and general methods used in
the whole-genome sequencing of mycobacteria, detailed protocols
are not provided. For specific genome sequencing platforms, the
reader is referred to the manufacturer’s recommended protocols for
the platform to be used. Similarly, for bioinformatic techniques, the
reader is referred to the documentation for the specific software
packages and scripts to be used.
2 Identification of Mycobacterial Species Is a Challenge
Mycobacteria are characterized as aerobic to microaerophilic,
slightly curved or straight rods that are acid-fast at some stage of
their growth [9]. These bacteria are acid-fast due to the presence of
mycolic acids in their cell wall, which provides unique characteristics that make these organisms adaptable to a wide range of conditions. Such a cell wall provides a thick waxy coat that allows these
microbes to evade the immune response, resist chemical damage,
survive dehydration, and limit the effectiveness of hydrophilic antibiotics and biocides. These abilities enable some mycobacterial
species to colonize environments such as soil and water, while
others are found only in a wide variety of hosts (insects, fish,
reptiles, birds, and mammals); some are saprophytes, while others
are parasites (including obligate and opportunistic); some are fast
growers, while others grow more slowly; some grow at low temperatures, while others thrive in hot environments.
The number of mycobacterial species described has increased
greatly in the last 45 years, from approximately 30 species in 1975
[10] to approximately 190 in 2019 [11]. Many of these species
have been discovered and defined using newer laboratory techniques, which do not correlate with traditional phenotypic and biochemical methods. This poses a major challenge for
mycobacteriology laboratories in providing conclusive identification [10]. For this reason, the latest guidelines from the Clinical &
400
Marco A. Riojas et al.