The M. tuberculosis complex has 8 independent lineages ( L1-8 ) [ 5 ], which vary in their geographic distribution and spread, with L1 ( Indo-Oceanic ) and L2 ( East Asian ) the prevailing ones circulating in Thailand [ 6–10 ]. The modern Beijing sublineage ( L2.2.1 ; AAF3 ) has been continuously reported to be associated with clonal expansion of MDR-TB [ 6, 7, 11 ]. In contrast, the “ Manu-ancestor ” or “ proto-Beijing ” genotype ( spoligotype 523, ancestry 2.1 ; [ 9 ] ) has a lower prevalence [ 6, 10 ], with few reports, including signal detection in the Chiba prefecture in Japan [ 12 ] and Guangxi state of Southern China [ 13 ]. recently, we noticed a electric potential infection event involving proto-Beijing strains causing XDR-TB [ 7 ]. To understand if there is farther transmittance of the L2.1 strain and any relate XDR-TB clonal expansion, we have analysed the WGS from thirty-seven isolates collected over 13-year period from 14 provinces of Thailand. tuberculosis ( TB ), caused by Mycobacterium tuberculosis, is one of the top 10 causes of deathrate globally. The ball-shaped problem of drug-resistant TB, particularly multi ( MDR-TB ) and extensive ( XDR-TB ) forms, is complicating disease control [ 1 ]. terabyte in Thailand remains high with an estimate 153 per 100,000 populations while around 50,167 cases were bacteriologically confirmed in 2018. An calculate 4000 MDR/RR-TB, including 1312 confirm, were among advise pneumonic terabit cases [ 1 ]. treatment for patients with drug-resistant TB is prolonged, expensive and outcomes are poor. Drug resistance in M. tuberculosis is about entirely due to mutations including unmarried nucleotide polymorphism ( SNPs ) in genes coding for drug-targets or -converting enzymes [ 2, 3 ]. Whole-genome sequence ( WGS ) platforms are being used to understand the mutations underlying drug resistance and to characterize transmission, where M. tuberculosis isolates ( sourced from unlike hosts ) that have near-identical genomic variation are most likely to have arisen from a recent transmission consequence [ 4 ]. Using WGS, molecular epidemiologic studies can investigate the cause of emerging drug resistance and focus on the contribution of likely genetic and environmental risk factors across a population to plan for TB see. The L2.1 isolates ( nitrogen = 56 ) consisted of 37 strains from our solicitation and 19 globally distributed. The quality of their succession read was checked using FastQC ( v0.11.7 ) [ 20, 21 ]. Sequencing reads were mapped to the M. tuberculosis H37Rv reference genome ( NC_000962.3 ) using BWA-MEM software [ 22 ]. The sets of map paired-end reads were joined using the SAMtools suite. Variants were called using GATK software ( v3.8 ) with HaplotypeCaller in -ERC GVCF manner for generating gVCF files. Multiple gVCF files were aggregated and combined into one GVCF file using GATK GenotypeGVCFs. only the one nucleotide polymorphism ( SNPs ) from VCF end product were extracted using SelectVariants tool and filtered using VariantFiltration [ 23 ]. The snpEff software ( v4.2 ) was used for variant note [ 24 ]. SNPs determined in the repetitive regions, paralogous gene families, and drug-resistant genes were discarded using VCFtools [ 25 ]. SNPs with allelic frequencies less than 75 % or read-depth less than 10 reads were removed. The resulting set of high-confidence SNPs were used to construct a phylogenetic tree based on the utmost likelihood method acting and 1000 bootstrap replicates using RAxML ( v8.0.0 ) software [ 26 ]. iTOL software was used for visualizing the phylogenetic trees [ 27 ]. Drug-resistance profiles ( and lineages ) were predicted in-silico using TBProfiler ( v2.0 ) [ 15 ]. Spoligotypes were predicted using SpoTyping [ 28 ]. The in-silico psychoanalysis for 31 know regions of differences ( RDs ) was performed by RD-Analyzer [ 18 ]. DELLY software [ 29 ] was used to predict big geomorphologic variants ( SVs ) using minimum paired-end map timbre 20 and standard deviation units as 3 times. High-quality SVs had to be supported by paired-end and schism reads. All variants across the samples were merged into a single file using BCFtools software. All putative genetic markers were manually reviewed for accuracy using Integrated Genomic Viewer software [ 30 ], and, if considered as true form they were included for depiction. clinical isolates sourced from 725 terabyte patients across 42 of 77 provinces from Thailand were classified as MDR-TB, pre-XDR, XDR-TB and pan-susceptible by applying phenotypical drug susceptibility testing ( DST ) assays using drug-containing MiddleBrook 7H10 agar ( M7H10 ). The 723 culturable isolates were from the retrievable stock cultures of 1415 MDR-, pre-XDR and XDR-TB from the Drug-Resistant Tuberculosis Research Fund Laboratory. These isolates were obtained from pneumonic terabit patients diagnosed between 2005 and 2012 and covering 42 of 76 provinces in Thailand. All isolates were sub-cultured on Loewenstein-Jensen media and incubated at 37°C for four weeks. Multiple loopfulls of M. tuberculosis colonies were taken and DNA extraction was carried out using the cetyltrimethylammonium bromide-sodium chloride method acting [ 14 ]. Genomic DNAs were sequenced using Illumina platforms, across a number of sites, including Novogene AIT ( Singapore ), Genomic Institute Singapore ( GIS ), London School of Hygiene & Tropical Medicine, and Mahidol University. We applied the TBProfiler creature [ 15 ] to the WGS data to in-silico strain-type, and identified 35 ( 4.8 % ) isolates as L2.1 strains. To determine the genetic similarity between presently and recently circulating L2.1 causing XDR-TB, two holocene L2.1 XDR-TB isolates ( 2015 and 2017 ) from Kanchanaburi state were blended into this study. here L2.1 collection ( north = 37 ) represents the largest number of L2.1 strains from Thailand. The raw sequence data for the 37 samples are deposited in NCBI ’ s Sequence Read Archive ( SRA ) database ( BioProject ID PRJNA63078 ). They were analysed together with other 19 L2.1 M. tuberculosis strains obtained from public data ; 16 isolates from high prevalence area ( China ), one sequester from the neighbor state ( Laos ), and two well-characterized L2.1 isolates from the Sanger Institute [ 16–19 ]. The accession numbers of all 56 genomes are listed in Table S1. The 37 L2.1 M. tuberculosis were isolated from Thai-patients, across 19 districts among 14 provinces, between 2005 and 2017 by the Drug-Resistant Tuberculosis Research Fund, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok ( affected role no.1–19 and 22–37 ) and the National Reference Tuberculosis Laboratory ( affected role no. 20 and 21 ). epidemiologic linkage between patients was not documented.

social organization variations ( SVs ) were called relative to the M. tuberculosis H37Rv address genome. thirty-nine high-quality SVs were detected. Three common deletion regions across all combined dataset of L2.1 isolates were detected with sizes between 827 and 4824 bp ( table 1 ), all exclusive to the sub-lineage. The largest genome SV involved a omission of plcA ( Rv2351c ) and PPE38 ( Rv2352c ), and a partial deletion of plcB ( Rv2350c ). There were deletions unique to the clusters ( MDR-TB 2 ; pre-XDR 2 ). Twelve isolates belonging to the XDR-TB cluster contained one singular deletion in the Rv1058 region, which was not found in any early isolates. During the study period, the first XDR-TB case ( patient no. 9 ) was infected with an L2.1 XDR-TB clone from Kanchanaburi in 2005. subsequent events have revealed that the XDR-TB clone has emerged endlessly, of which 12 had at least one isolate over a span of 13 years, and been restricted to within 180 km across four provinces from the first font. potential transmission clusters were revealed by determining the pairwise SNP distance between the 37 Thai-isolates. The pairwise genic distance varied between 0 and 381 SNPs ( Table S3 ). Using a 12 SNP cut-off [ 31 ], 43.2 % of patients belonged to 3 unlike possible clusters, including : ( i ) an XDR-TB bunch with a maximum size of 12 isolates ( human body 3 ( B ) ), ( two ) 2 pre-XDR isolates, and ( three ) 2 MDR-TB isolates. Mutation patterns associated with underground were besides unique for each bunch ( Table S4 ), providing electric potential evidence of infection. The MDR-TB cluster ( n = 2 ; no.14 ( 2008 ) and 16 ( 2009 ) ) was composed of patients from the same zone and had identical SNP patterns, including mutations known to be creditworthy for drug resistor ( rpoB Ser450Leu ; katG Ser315Thr ). Within the pre-XDR bunch ( newton = 2 ; no.4 ( 2008 ) and 6 ( 2012 ) ), there was merely 1 SNP difference ( a synonymous mutant difference between isolates 4 and 6 ), and the established drug-resistance mutations were identical ( katG Ser315Thr ; rpoB Ser450Leu ; rpsL Lys88Arg ; embB Gly406Asp ; pncA Tyr103His ; gyrA : Asp94Asn ; thyA deletion 3071595-3077080 ). For the XDR-TB bunch ( n = 12 ), the pairwise genic distance varied between 1 and 11 SNPs. Nine ( 75.0 % ) XDR-TB were isolated from Kanchanaburi state, including Thamaka ( normality = 5 ; no.9 ( 2005 ), 12 ( 2007 ), 15 ( 2008 ), 20 ( 2015 ), and 21 ( 2017 ) ), Muang ( newton = 2 ; no.11 ( 2007 ) and 13 ( 2008 ) ), Bophloi ( n = 1 ; no.10 ( 2006 ) ) and Thamuang ( nitrogen = 1 ; no.19 ( 2012 ) ) districts. The other three isolates collected from affected role in Chachoengsao ( no.7 in 2008 ), Rachaburi ( no.31 in 2012 ), and Suphaburi ( no.35 in 2011 ). Ten ( 83.3 % ) XDR isolates had identical resistance mutation profiles ( katG Ser315Thr ; rpoB Ser450Leu ; embB Met306Ile ; pncA Ile90Ser ; rrs : 1401a > deoxyguanosine monophosphate ; gyrA Asp94Gly ; folC Glu40Gly ). XDR-TB samples from Chachoengsao ( no.7 ), and Suphanburi ( no.35 ) were located 174 and 64.7 kilometres, respectively, away from earlier isolates. They had identical drug-resistant mutations to the majority of the XDR-TB cluster, except for the addition of the Leu527Val mutant in the ( rifampicin compensatory ) rpoC gene. The late isolate ( no. 20 ; class 2015 ) had an extra mutation ( thyX −16C > T ) related to para-aminosalicylic acid resistor. These results suggest that there was a long-run infection of local clusters of XDR-TB, which carried common drug-resistance mutations, thereby explaining its success as a spreader [ 32–34 ]. To investigate the within and between country outspread of L2.1, an analysis of the genome variation of all 56 isolates ( Thailand, China, Laos, and others ) was performed. A total of 3685 high-confidence SNPs were identified across 56 M. tuberculosis isolates, including 3206 ( 87.0 % ) in coding regions ( 1141 silent, 2028 missense, and 37 nonsense ), 12 in non-coding and 467 in intergenic regions ( Table S2 ). Across the 56 isolates, 2484 ( 67.4 % ) SNPs were identified in single isolates. A phylogenetic tree based on the 3685 SNPs identified revealed that Thai-isolates were largely interleaved with early countries including China ( CHA ), Laos ( LAO ), and others ( NA ) ( Figure 2 ). All 12 ( 100 % ) XDR-TB isolates were clustered together and were distinguished from the others by at least 47 SNPs. Twenty-seven of 47 SNPs had missense mutations. This large and durable monophyletic clade with high bootstrap support ( 100 % ) included XDR-TB cases from Kanchanaburi ( newton = 9 ). The clade was besides interleaved with XDR-TB isolates from Ratchaburi ( nitrogen = 1 ), Chachoengsao ( n = 1 ), and Supanburi ( n = 1 ) ( Figure 3 ( A ) ). The time range between the first and last isolate was 13 years ( 2005–2017 ). furthermore, the results revealed that 2 ( 28.6 % ) pre-XDR and 2 ( 12.5 % ) MDR-TB patients identified were limited to one geographic district and separated by at least 90 SNPs and 60 SNPs, respectively, from any other survey isolates. This resultant role implies that this detail strive was circulating locally. A genotypical clustering rate of 43.2 % was found among patients who were infected with L2.1 breed, revealing the high flat of transmission in this population. The genotypical drug-resistance profile of each isolate is shown in Figure 2. In silico spoligotyping classified the 37 L2.1 strains into 12 genotypes, and the most common spoligotype ( 23/37 ; 62.2 % ) was ride 523 ( 777777777777771 ). Of these SIT 523 isolates, 12 ( 52.2 % ) were XDR-TB, and were sourced from 4 provinces ; Chachoengsao ( newton = 1 ), Kanchanaburi ( nitrogen = 9 ), Ratchaburi ( n = 1 ), Suphanburi ( n = 1 ) ( Figures 1 and 2 ). All isolates harboured the extend RD105 deletion but had RD207, RD181 and pks15/1 regions intact.

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The overall rate of MDR-TB individuals with pre-XDR and XDR-TB are high. Twelve isolates ( 32.4 % ) are XDR-TB whereas 7 isolates ( 18.9 % ) are pre-XDR. sixteen isolates are MDR-TB ( 43.2 % ) and the remaining 2 isolates are pan-susceptible. Demographic datum of each patient is shown in ( table 1 and Table S1 ). The L2.1 incident rate ( north = 37 or 5.1 % ) was high compared with studies in the Chiangrai province, Northern Thailand ( 1.0 % ) [ 10 ] and Guangxi state of southern China ( 0.2 % ) [ 13 ]. unfortunately, our collection had no isolates from Chiangrai. Within province the highest number of L2.1 strive was observed in Kanchanaburi ( n = 13 ), followed by Nakhon Ratchasima ( normality = 5 ), Buriram and Bangkok ( nitrogen = 3 ) province .

Discussion

The holocene emergence of drug-resistant TB infections in Thailand has been attributed to strains in M. tuberculosis linage 2, particularly the “ modern Beijing ” ( Lineage 2.2.1 ) sub-lineage. M. tuberculosis from this sub-lineage are highly virulent causing disease outbreaks, with escape from the effects of BCG inoculation, they disseminate efficiently, and easily acquire antibiotic immunity [ 6, 7, 9, 35 ]. The more rare “ proto-Beijing ” ( L2.1 ) appears to be transmitting, and has a high aptness to be XDR-TB. In our survey, the number of MDR-TB individuals with XDR-TB is ∼6 times greater than the WHO ball-shaped estimate ( 6.2 % ) [ 1 ], and all are clustered. Poor treatment adhesiveness is not the only divisor that contributes to drug resistance, but besides the failure in controlling transmission of drug-resistant strains. This study was a hospital-based retrospective analysis included the retrospective and passive nature of case-finding, based lone on retrievable isolates from stock cultures during 2005–2012 ; therefore, they were a located of public toilet isolates and may not be representative of all isolates from the community, and we can not exclude some unintended bias. additionally, TB database information of Thailand during 2005–2012 was not well developed, and the capacity of the testing ground to perform phenotypical drug susceptibility test was besides express. however, the number of L2.1 isolates retrieved is the largest to date from a single state, and the underlying sampling from 725 culturable M. tuberculosis covers 42 of 77 provinces [ 2, 6, 7, 17 ]. here we reported the presence of L2.1 isolates, including in Bangkok, Buriram, Chachoengsao, Chonburi, Kanchanaburi, Nakhonratchasima, Nongkhai, Phetchabun, Phrae, Ratchaburi, Rayong, Saraburi, Suphanburi, and Suratthani, but not Chiangrai provinces [ 7, 10 ]. WGS data were integrated with routinely clinical information to identify the putative TB bunch. unfortunately, epidemiologic information was not available in this study. The consolidation of social-network analysis with high-resolution bacterial genome sequencing would provide traceable information in evaluating TB transmittance [ 36 ]. The 12-SNP distance was proposed by using studies in depleted incidence TB settings [ 31 ], and seems reasonable for our study, set in the context of low L2.1 population prevalence and the residential district transmission investigation. Using clustering threshold of 12 SNPs, we defined 3 distinct alone clonal clusters which carried particular drug-resistant mutation radiation pattern for each ringer ( Table S4 ), implying diverse evolution histories for the individual population. A high bunch rate of 43.2 % among L2.1 infected patients highlighted the high transmissibility of proto-Beijing strains. Two of the three clusters identified had one pair of strains and emerged in a particular region. Among the largest possible bunch groupings consisting of twelve XDR-TB transmission clusters, at least seven putative transmission events were postulated to underlie the largest bunch on the basis of a shared social sic and WGS genotyping, in which each individual isolate was supposed to be accumulated genetic diversity within-host [ 31 ]. Nine isolates were retrieved from patients in Kanchanaburi province, of which four district hospitals were involved in four putative transmittance events ; 5 ( 41.7 % ) isolates from Thamaka, 2 isolates from Muang, 1 isolate from Tha muang, and 1 isolate from Bo phloi district. The early 3 events were possibly infected through social contact in Chachoengsao ( affected role no.7 ), Rachaburi ( affected role no.31 ), and Supanburi ( affected role no.35 ). however, the electric potential of clonal expansion reported here would switch care towards a rare genotype causing XDR-TB in Kanchanaburi province, not only in a detail hospital but through the high preponderance of MDR-TB, specially within L2.2.1 ; AAF3 [ 7, 11, 36, 37 ]. The miss of transfer of these hyper-resistant strains to other geographic locations may be associated with adaptation to local-host population, as seen in L1 strains [ 9 ] or other factors which need to be explored in the future.

forty-seven SNPs and a 78 bp deletion in fadD14 ( encoded fatty-acid–CoA ligase ) were deliver in all XDR-TB clonal spreaders, but not other isolates studied. notably, 27 ( of the 47 ) SNPs resulted in a missense mutation and were potentially subject to lifelike excerpt. The familial markers shared by the successful XDR-TB bunch could be related to virulence or used to screen for the universe of antibiotic-resistant bacteria. specific resistance-conferring mutations and strain genic background are often associated with a seaworthiness price [ 32, 33 ]. The local clusters of XDR-TB carry park drug-resistance mutations, potentially explaining its successful dissemination phenotype more promptly than other genotypes. inescapably, host familial setting besides influences the response to mycobacterial activity [ 6, 17 ]. The consecutive appearance of XDR-TB bunch conferred overgrowth their ancestral drug-susceptible clone that become indiscernible in this study. Mutations found within the XDR-TB bunch indicate a noteworthy adaptation capability during infection. Further, the factors influencing transmission remain ailing silent, but as WGS becomes routine in a clinical set, it will be possible to reconstruct the transmission chains and assess the host and M. tuberculosis genic and non-genetic factors affecting transmissibility. We detected three fresh deletion positions which may play a function in the virulence, pathogenesis or development of the L2.1 strain-type. Poorer bacterial growth has been observed in presence of azole in M. tuberculosis strains with CYP144 hard [ 38 ]. Azole drugs may have activity against the L2.1 sift. Further, a deletion of 1285 bp within desA3 and oxidoreductase Rv3230c may cause the L2.1 strain to be immune to the second-line anti-TB drug isoxyl [ 39 ]. boastfully deletions in the plcB, plcA, and ppe38 gene were besides detected. The deletion of ppe38 and loss of ESX-5 substrates secretion as found in advanced Beijing sub-lineages may have increased their virulence and contributed to their ball-shaped spread [ 40 ]. Whereas partial derivative deletion of phospholipase C encoded by plc genes ( plcA, B, C and D ) may have a character in assisting the bacteria to escape from phagosomal containment, as found in Listeria monocytogenes or Clostridium perfringens [ 41 ]. This may cause lower virulence than a mod Beijing sub-lineage. overall, our survey has revealed that M. tuberculosis sub-lineage L2.1 is geographically restricted but has a great leaning to be XDR-TB and transmit. We anticipate that revealing the XDR-TB effect of sub-lineage L2.1 will lead to more far-flung surveillance and much-needed epidemiologic studies. The solid recommendation from the Thailand national TB control program to implement N95 or equivalent dissemble wearing for active TB patients particularly MDR-, pre-XDR and XDR-TB patients will be an extra measure to prevent TB transmittance during intensive treatment .