allow for rapid implementation of infection control measures
[8]. This section reviews just a few of the WGS-enabled techniques
with respect to AMR for mycobacteria.
5.3.1 Drug Resistance
Mechanisms and Genotypic
Detection
Resistance to antimicrobial drugs can arise in bacteria through four
primary routes: lateral transfer of genes that confer resistance
mechanisms, mutations in the genes that encode proteins targeted
by the drugs, upregulation of efflux pumps to expel drugs from the
cell, and intrinsic methods such as low permeability of cell walls
[151]. Although recent lateral gene transfer is rare within the
mycobacteria [14, 152], the other mechanisms have been documented in different mycobacterial species. The unique cell wall of
the species in this genus confers low permeability to solutes including many standard antibiotics [153], and efflux pumps have been
shown in many mycobacterial species such as M. abscessus and
M. smegmatis to be involved in drug resistance [154–156].
Genotypic detection of resistance involves characterizing mutations or gene presence/absence with resistance to a certain drug.
For mycobacteria, this tends to be characterization of mutations in
specific genes or in promoters. Efflux pump-mediated resistance
can sometimes be detected this way, if mutations in the promoters
increase pump activity and thus increase resistance. However, if no
genetic change is related to efflux pump-mediated resistance, genotypic prediction will not be suitable.
The best characterized resistance mechanism in M. tuberculosis
is against rifampicin (RIF) [157, 158]. Mutations in rpoB, specifically in the rifampicin resistance determining region (RRDR), can
predict RIF resistance with 97.5% sensitivity and 98.8% specificity
[159]. This limited genetic range enables rapid detection of RIF
resistance through either sequencing for the rpoB gene or assessing
wild-type probe binding patterns in this region, which is the basis of
genotyping methods such as Cepheid GeneXpert and Xpert Ultra,
GenoType MTBDRplus v2.0, and Genoscholar NTM + MDRTB
II [160–162]. However, cases of RIF resistance with no associated
rpoB mutation have been documented, especially in M. smegmatis,
potentially due to efflux pump activity [163, 164]. This means that
the accuracy of genotypic methods for detecting this resistance is
species-specific and requires a greater understanding of the underlying mechanisms outside of M. tuberculosis. Of note, until recently,
rpoB numbering followed the E. coli system, meaning the gene
position numbers were off by 81 bp. Current approaches all use
the correct numbers based on the sequence of the rpoB gene from
M. tuberculosis H37Rv [165]. This can make comparisons and
collating of resistance mutations difficult, especially from older
research papers.
Similar to RIF resistance, many other M. tuberculosis drug
resistances [especially isoniazid (INH) and fluoroquinolones
(FQs)] can be determined by sequencing of specific genes
Mycobacterial ID and Characterization Using WGS
441
[8]. This section reviews just a few of the WGS-enabled techniques
with respect to AMR for mycobacteria.
5.3.1 Drug Resistance
Mechanisms and Genotypic
Detection
Resistance to antimicrobial drugs can arise in bacteria through four
primary routes: lateral transfer of genes that confer resistance
mechanisms, mutations in the genes that encode proteins targeted
by the drugs, upregulation of efflux pumps to expel drugs from the
cell, and intrinsic methods such as low permeability of cell walls
[151]. Although recent lateral gene transfer is rare within the
mycobacteria [14, 152], the other mechanisms have been documented in different mycobacterial species. The unique cell wall of
the species in this genus confers low permeability to solutes including many standard antibiotics [153], and efflux pumps have been
shown in many mycobacterial species such as M. abscessus and
M. smegmatis to be involved in drug resistance [154–156].
Genotypic detection of resistance involves characterizing mutations or gene presence/absence with resistance to a certain drug.
For mycobacteria, this tends to be characterization of mutations in
specific genes or in promoters. Efflux pump-mediated resistance
can sometimes be detected this way, if mutations in the promoters
increase pump activity and thus increase resistance. However, if no
genetic change is related to efflux pump-mediated resistance, genotypic prediction will not be suitable.
The best characterized resistance mechanism in M. tuberculosis
is against rifampicin (RIF) [157, 158]. Mutations in rpoB, specifically in the rifampicin resistance determining region (RRDR), can
predict RIF resistance with 97.5% sensitivity and 98.8% specificity
[159]. This limited genetic range enables rapid detection of RIF
resistance through either sequencing for the rpoB gene or assessing
wild-type probe binding patterns in this region, which is the basis of
genotyping methods such as Cepheid GeneXpert and Xpert Ultra,
GenoType MTBDRplus v2.0, and Genoscholar NTM + MDRTB
II [160–162]. However, cases of RIF resistance with no associated
rpoB mutation have been documented, especially in M. smegmatis,
potentially due to efflux pump activity [163, 164]. This means that
the accuracy of genotypic methods for detecting this resistance is
species-specific and requires a greater understanding of the underlying mechanisms outside of M. tuberculosis. Of note, until recently,
rpoB numbering followed the E. coli system, meaning the gene
position numbers were off by 81 bp. Current approaches all use
the correct numbers based on the sequence of the rpoB gene from
M. tuberculosis H37Rv [165]. This can make comparisons and
collating of resistance mutations difficult, especially from older
research papers.
Similar to RIF resistance, many other M. tuberculosis drug
resistances [especially isoniazid (INH) and fluoroquinolones
(FQs)] can be determined by sequencing of specific genes
Mycobacterial ID and Characterization Using WGS
441