Genetic signatures of Mycobacterium tuberculosis Nonthaburi genotype revealed by whole genome analysis of isolates from tuberculous meningitis patients in Thailand.

Genome sequencing plays a key role in understanding the genetic diversity of Mycobacterium tuberculosis (M.tb). The genotype-specific character of M. tb contributes to tuberculosis severity and emergence of drug resistance. Strains of M. tb complex can be classified into seven lineages. The Nonthaburi (NB) genotype, belonging to the Indo-Oceanic lineage (lineage 1), has a unique spoligotype and IS6110-RFLP pattern but has not previously undergone a detailed whole genome analysis. In addition, there is not much information available on the whole genome analysis of M. tb isolates from tuberculous meningitis (TBM) patients in public databases. Isolates CSF3053, 46-5069 and 43-13838 of NB genotype were obtained from the cerebrospinal fluids of TBM Thai patients in Siriraj Hospital, Bangkok. The whole genomes were subjected to high throughput sequencing. The sequence data of each isolate were assembled into draft genome. The sequences were also aligned to reference genome, to determine genomic variations. Single nucleotide polymorphisms (SNPs) were obtained and grouped according to the functions of the genes containing them. They were compared with SNPs from 1,601 genomes, representing the seven lineages of M. tb complex, to determine the uniqueness of NB genotype. Susceptibility to first-line, second-line and other antituberculosis drugs were determined and related to the SNPs previously reported in drug-resistant related genes. The assembled genomes have an average size of 4,364,461 bp, 4,154 genes, 48 RNAs and 64 pseudogenes. A 500 base pairs deletion, which includes ppe50, was found in all isolates. RD239, specific for members of Indo Oceanic lineage, and RD147c were identified. A total of 2,202 SNPs were common to the isolates and used to classify the NB strains as members of sublineage 1.2.1. Compared with 1,601 genomes from the seven lineages of M. tb complex, mutation G2342203C was found novel to the isolates in this study. Three mutations (T28910C, C1180580T and C152178T) were found only in Thai NB isolates, including isolates from previous study. Although drug susceptibility tests indicated pan-susceptibility, non-synonymous SNPs previously reported to be associated with resistance to anti-tuberculous drugs; isoniazid, ethambutol, and ethionamide were identified in all the isolates. Non-synonymous SNPs were found in virulence genes such as the genes playing roles in apoptosis inhibition and phagosome arrest. We also report polymorphisms in essential genes, efflux pumps associated genes and genes with known epitopes. The analysis of the TBM isolates and the availability of the variations obtained will provide additional resources for global comparison of isolates from pulmonary tuberculosis and TBM. It will also contribute to the richness of genomic databases towards the prediction of antibiotic resistance, level of virulence and of origin of infection

Intact pks15/1 in Non–W-Beijing Mycobacterium tuberculosis Isolates

To determine whether intact pks15/1 is unique to the W-Beijing family, we investigated 147 Mycobacterium tuberculosis strains with different IS6110 genotypes. Intact pks15/1 was found in 87.8% of cerebrospinal fluid and 84.9% of sputum isolates. It was found not only in W-Beijing strains (≈97%) but also in other genotypes (38.5%–100%)

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics

Technical Report on critical concentrations for drug susceptibility testing of isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine)

The critical concentrations for culture-based phenotypic drug susceptibility testing (DST) to first-line anti-TB drugs have been revised by the the World Health Organization (WHO). Critical concentrations for rifampicin have been lowered while those for isoniazid have been maintained at the present level. This update helps address the discordance observed between phenotypic and molecular methods to detect rifampicin resistance and improves the accuracy of DST. As a result patients with TB will have a more accurate diagnosis. This document is the outcome of a Technical Expert Group meeting convened by WHO in 2020 to assess the results of a systematic review of published literature on critical concentrations for DST of the most important first-line anti-TB drugs, isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine). These critical concentrations had not been revised since 2008. New evidence over the past decade showed that critical concentrations used for phenotypic methods to detect rifampicin resistance may incorrectly classify strains with certain mutations. The following media were considered: Löwenstein-Jensen (LJ), Middlebrook 7H10 (7H10), Middlebrook 7H11 (7H11) and BACTEC™ Mycobacterial Growth Indicator Tube™ 960 (MGIT). Guidance has been provided to resolve discordance between genotypic and phenotypic results for these drugs and areas for further research have been highlighted. DST methods continue to have a very important role to identify resistance not detected by molecular assays and to support the interpretation of molecular assays results. However, they require sophisticated laboratory infrastructure, qualified staff and strict quality assurance procedures.Peer reviewe

A first assessment of the genetic diversity of Mycobacterium tuberculosis complex in Cambodia

<p>Abstract</p> <p>Background</p> <p>Cambodia is among the 22 high-burden TB countries, and has one of the highest rates of TB in South-East Asia. This study aimed to describe the genetic diversity among clinical <it>Mycobacterium tuberculosis </it>complex (MTC) isolates collected in Cambodia and to relate these findings to genetic diversity data from neighboring countries.</p> <p>Methods</p> <p>We characterized by 24 VNTR loci genotyping and spoligotyping 105 <it>Mycobacterium tuberculosis </it>clinical isolates collected between 2007 and 2008 in the region of Phnom-Penh, Cambodia, enriched in multidrug-resistant (MDR) isolates (n = 33).</p> <p>Results</p> <p>Classical spoligotyping confirmed that the East-African Indian (EAI) lineage is highly prevalent in this area (60%-68% respectively in whole sample and among non-MDR isolates). Beijing lineage is also largely represented (30% in whole sample, 21% among non-MDR isolates, OR = 4.51, CI_95%[1.77, 11.51]) whereas CAS lineage was absent. The 24 loci MIRU-VNTR typing scheme distinguished 90 patterns with only 13 multi-isolates clusters covering 28 isolates. The clustering of EAI strains could be achieved with only 8 VNTR combined with spoligotyping, which could serve as a performing, easy and cheap genotyping standard for this family. Extended spoligotyping suggested relatedness of some unclassified "T1 ancestors" or "Manu" isolates with modern strains and provided finer resolution.</p> <p>Conclusions</p> <p>The genetic diversity of MTC in Cambodia is driven by the EAI and the Beijing families. We validate the usefulness of the extended spoligotyping format in combination with 8 VNTR for EAI isolates in this region.</p

Genomic evidence supporting the clonal expansion of extensively drug-resistant tuberculosis bacteria belonging to a rare proto-Beijing genotype.

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major public health issue in Thailand. The high prevalence of modern Beijing (Lineage 2.2.1) strains has been associated with multi- and extensively drug-resistant infections (MDR-, XDR-TB), complicating disease control. The impact of rarer proto-Beijing (L2.1) strains is less clear. In our study of thirty-seven L2.1 clinical isolates spanning thirteen years, we found a high prevalence of XDR-TB cases (32.4%). With ≤ 12 pairwise SNP distances, 43.2% of L2.1 patients belong to MDR-TB or XDR-TB transmission clusters suggesting a high level of clonal expansion across four Thai provinces. All XDR-TB (100%) were likely due to transmission rather than inadequate treatment. We found a 47 mutation signature and a partial deletion of the fadD14 gene in the circulating XDR-TB cluster, which can be used for surveillance of this rare and resilient M. tuberculosis strain-type that is causing increasing health burden. We also detected three novel deletion positions, a deletion of 1285 bp within desA3 (Rv3230c), large deletions in the plcB, plcA, and ppe38 gene which may play a role in the virulence, pathogenesis or evolution of the L2.1 strain-type