The Guinea-Bissau family of Mycobacterium tuberculosis complex revisited

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048).     

Study of Mycobacterium tuberculosis Complex Genotypic Diversity in Malaysia Reveals a Predominance of Ancestral East-African-Indian Lineage with a Malaysia-Specific Signature

Background

Tuberculosis (TB) still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC) isolates causing the disease is important to determine the effectiveness of the control and surveillance programs.

Objectives

This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database.

Methods

Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe.

Results

Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT) designation in the database (the latter included 6 newly created SITs). The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster) as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (p<0.01). However, no statistically significant differences were noted when comparing drug resistance vs. major lineages, nor between gender and clades.

Conclusions

The ancestral East-African-Indian (EAI) lineage was most predominant followed by the Beijing lineage. A comparison of strains with those prevailing in neighboring countries in South Asia, East Asia and South East Asia underlined the phylogeographical specificity of SIT745 for Malaysia, and its probable ongoing evolution with locally evolved strains sharing a specific signature characterized by absence of spacers 37, 38, and 40. Pending complementary genotyping confirmation, we propose that SIT745/EAI-SOM is tentatively reclassified as SIT745/EAI-MYS.     

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.     

Mycobacterium tuberculosis Population in Northwestern Russia: An Update from Russian-EU/Latvian Border Region

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA −15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia.     

Molecular Epidemiology of Tuberculosis in Kaohsiung City Located at Southern Taiwan, 2000-2008

Background

We present the first comprehensive analysis of Mycobacterium tuberculosis (MTB) isolates circulating in southern Taiwan. In this 9-year population-based study, the TB situation in the Kaohsiung region was characterized by genotypic analysis of 421 MTB isolates.

Methods

All 421 isolates of MTB were analyzed by spoligotyping and MIRU-VNTR typing. Drug-resistance patterns were also analyzed.

Results

The percentage of EAI (East African-Indian) strains increased across sampling years (2000–2008) in southern Taiwan, whereas the proportion of Beijing lineages remained unchanged. Clustering was more frequent with EAI genotype infections (odds ratio = 3.6, p<0.0001) when compared to Beijing genotypes. Notably, MTB resistance to streptomycin (STR) had significantly increased over time, but resistance to other antibiotics, including multidrug resistance, had not. Three major genes (gidB, rpsL and rrs) implicated in STR resistance were sequenced and specific mutations identified.

Conclusions

This study revealed that EAI strains were highly transmissible and that STR resistance has increased between 2000 and 2008 in Kaohsiung, Taiwan.     

Distinct Modes of Transmission of Tuberculosis in Aboriginal and Non-Aboriginal Populations in Taiwan

Tuberculosis incidence among aborigines is significantly higher than for Han Chinese in Taiwan, but the extent to which Mycobacterium tuberculosis (MTB) strain characteristics contribute to this difference is not well understood. MTB isolates from aborigines and Han Chinese living in eastern and southern Taiwan, the major regions of aborigines, were analyzed by spoligotyping and 24-loci MIRU-VNTR. In eastern Taiwan, 60% of aboriginal patients were ≤20 years old, significantly younger than the non-aboriginal patients there; aborigines were more likely to have clustered MTB isolates than Han Chinese (odds ratio (OR) = 5.98, p<0.0001). MTB lineages with high clustering were EAI (54.9%) among southern people, and Beijing (62.5%) and Haarlem (52.9%) among eastern aborigines. Resistance to first-line drugs and multidrug resistance (MDR) were significantly higher among eastern aborigines (≥15%) than in any other geographic and ethnic group (p<0.05); MDR was detected in 5 of 28 eastern aboriginal patients ≤20 years old. Among patients from the eastern region, clustered strains (p = 0.01) and aboriginal ethnicity (p = 0.04) were independent risk factors for MDR. The lifestyles of aborigines in eastern Taiwan may explain why the percentage of infected aborigines is much higher than for their Han Chinese counterparts. The significantly higher percentage of the MDR-MTB strains in the aboriginal population warrants close attention to control policy and vaccination strategy.     

Multi-Locus Variable Number of Tandem Repeat Analysis for Rapid and Accurate Typing of Virulent Multidrug Resistant Escherichia coli Clones

One hundred E. coli isolates from Norway (n = 37), Sweden (n = 24), UK (n = 20) and Spain (n = 19), producing CTX-M-type - (n = 84), or SHV-12 (n = 4) extended spectrum β-lactamases, or the plasmid mediated AmpC, CMY-2 (n = 12), were typed using multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes (MTCs). A strong agreement between the MLST profile and MLVA typing results was observed, in which all ST131-isolates (n = 39) and most of the STC-648 (n = 10), STC-38 (n = 9), STC-10 (n = 9), STC-405 (n = 8) and STC-23 (n = 6) isolates were clustered distinctly into MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate tool for genotyping isolates of globally disseminated virulent multidrug resistant E. coli lineages, including ST131.     

Genetic diversity of Mycobacterium tuberculosis isolates from Tibetans in Tibet, China

Background

Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited.

Methodology/Principal Findings

In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, n = 522). Shared-type (ST) 1 was the most dominant genotype (88.89%, n = 512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set.

Conclusions/Significance

The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis.     

The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

Background

Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.

Methodology/Principal Findings

Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.

Conclusion

The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.     

Genotypic diversity and drug susceptibility patterns among M. tuberculosis complex isolates from South-Western Ghana

Objective

The aim of this study was to use spoligotyping and large sequence polymorphism (LSP) to study the population structure of M. tuberculosis complex (MTBC) isolates.

Methods

MTBC isolates were identified using standard biochemical procedures, IS6110 PCR, and large sequence polymorphisms. Isolates were further typed using spoligotyping, and the phenotypic drug susceptibility patterns were determined by the proportion method.

Result

One hundred and sixty-two isolates were characterised by LSP typing. Of these, 130 (80.25%) were identified as Mycobacterium tuberculosis sensu stricto (MTBss), with the Cameroon sub-lineage being dominant (N = 59/130, 45.38%). Thirty-two (19.75%) isolates were classified as Mycobacterium africanum type 1, and of these 26 (81.25%) were identified as West-Africa I, and 6 (18.75%) as West-Africa II. Spoligotyping sub-lineages identified among the MTBss included Haarlem (N = 15, 11.53%), Ghana (N = 22, 16.92%), Beijing (4, 3.08%), EAI (4, 3.08%), Uganda I (4, 3.08%), LAM (2, 1.54%), X (N = 1, 0.77%) and S (2, 1.54%). Nine isolates had SIT numbers with no identified sub-lineages while 17 had no SIT numbers. MTBss isolates were more likely to be resistant to streptomycin (p<0.008) and to any drug resistance (p<0.03) when compared to M. africanum.

Conclusion

This study demonstrated that overall 36.4% of TB in South-Western Ghana is caused by the Cameroon sub-lineage of MTBC and 20% by M. africanum type 1, including both the West-Africa 1 and West-Africa 2 lineages. The diversity of MTBC in Ghana should be considered when evaluating new TB vaccines.     

The Pattern of Cytokine Production In Vitro Induced by Ancient and Modern Beijing Mycobacterium tuberculosis Strains

It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1β. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.     

Genetic Diversity of Mycobacterium tuberculosis from Guadalajara,Mexico and Identification of a Rare Multidrug Resistant Beijing Genotype

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.     

First Molecular Epidemiology Study of Mycobacterium tuberculosis in Kiribati

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.     

Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTI_n−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.     

Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Background

The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/Principle Findings

Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion

This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.     

A Country-Wide Study of Spoligotype and Drug Resistance Characteristics of Mycobacterium tuberculosis Isolates from Children in China

Background

Tuberculosis (TB) is still a big threat to human health, especially in children. However, an isolation of Mycobacterium tuberculosis culture from pediatric cases remains a challenge. In order to provide some scientific basis for children TB control, we investigated the genotyping and drug resistance characteristics of M. tuberculosis isolates from pediatric cases in China.

Methodology/Principal Findings

In this study, a total of 440 strains including 90 from children (<15 years), 159 from adolescents (15–18 years) and 191 from adults (>18 years) isolated in 25 provinces across China were subjected to spoligotyping and drug susceptibility testing. As a result, Beijing family strains were shown to remain predominant in China (85.6%, 81.1% and 75.4% in three above groups, respectively), especially among new children cases (91.0% vs. 69.6% in previously treated cases, P = 0.03). The prevalence of the Beijing genotype isolates was higher in northern and central China in the total collection (85.1% in northern and 83.9% in central vs. 61.6% in southern China, P<0.001) and a similar trend was seen in all three age groups (P = 0.708, <0.001 and 0.025, respectively). In adolescents, the frequencies of isoniazid (INH)-resistant and ethambutol (EMB)-resistant isolates were significantly higher among Beijing strains compared to non-Beijing genotype strains (P = 0.028 for INH and P = 0.027 for EMB). Furthermore, strong association was observed between resistance to rifampicine (RIF), streptomycin (STR) and multidrug resistance (MDR) among Beijing compared to non-Beijing strains in previously treated cases of children (P = 0.01, 0.01 and 0.025, respectively).

Conclusion/Significance

Beijing family was more prevalent in northern and central China compared to southern China and these strains were predominant in all age groups. The genetic diversity of M. tuberculosis isolates from children was similar to that found in adolescents and adults. Beijing genotype was associated with RIF, STR and MDR resistance in previously treated children.     

Population Distribution of Beta-Lactamase Conferring Resistance to Third-Generation Cephalosporins in Human Clinical Enterobacteriaceae in The Netherlands

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne β-lactamases that confer resistance to third-generation cephalosporins. The epidemiology of these bacteria is not well understood, and was, therefore, investigated in a selection of 636 clinical Enterobacteriaceae with a minimal inhibitory concentration >1 mg/L for ceftazidime/ceftriaxone from a national survey (75% E. coli, 11% E. cloacae, 11% K. pneumoniae, 2% K. oxytoca, 2% P. mirabilis). Isolates were investigated for extended-spectrum β-lactamases (ESBLs) and ampC genes using microarray, PCR, gene sequencing and molecular straintyping (Diversilab and multi-locus sequence typing (MLST)). ESBL genes were demonstrated in 512 isolates (81%); of which 446 (87%) belonged to the CTX-M family. Among 314 randomly selected and sequenced isolates, bla _CTX-M-15 was most prevalent (n = 124, 39%), followed by bla _CTX-M-1 (n = 47, 15%), bla _CTX-M-14 (n = 15, 5%), bla _SHV-12 (n = 24, 8%) and bla _TEM-52 (n = 13, 4%). Among 181 isolates with MIC ≥16 mg/L for cefoxitin plasmid encoded AmpCs were detected in 32 and 27 were of the CMY-2 group. Among 102 E. coli isolates with MIC ≥16 mg/L for cefoxitin ampC promoter mutations were identified in 29 (28%). Based on Diversilab genotyping of 608 isolates (similarity cut-off >98%) discriminatory indices of bacteria with ESBL and/or ampC genes were 0.994, 0.985 and 0.994 for E. coli, K. pneumoniae and E. cloacae, respectively. Based on similarity cut-off >95% two large clusters of E. coli were apparent (of 43 and 30 isolates) and 21 of 21 that were typed by belonged to ST131 of which 13 contained bla _CTX-M-15. Our findings demonstrate that bla _CTX-M-15 is the most prevalent ESBL and we report a larger than previously reported prevalence of ampC genes among Enterobacteriaceae responsible for resistance to third-generation cephalosporins.     

Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates

Background

Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are opportunistic pathogens and serve as a large reservoir of staphylococcal cassette chromosome mec (SCCmec). Characterization of SCCmec in MR-CoNS can generate useful information on the mobilization and evolution of this element.

Methodology/Principal Findings

Non-repetitive MR-CoNS clinical isolates (n = 84; 39 S. epidermidis, 19 S. haemolyticus, 9 S. hominis, 6 S. capitis, 4 S. warneri, 2 S. cohnii, 2 S. saprophyticus, 1 S. kloosii, 1 S. simulans and 1 S. massiliensis) were collected. All isolates could grow on plates with 4 mg/L cefoxitin and all had mecA as detected by PCR. Strain typing using RAPD and ERIC-PCR revealed that almost all isolates were of different strains. SCCmec typing was performed using multiplex PCR published previously. For isolates in which SCCmec could not be typed, the mec complex classes were determined by additional PCR and the ccr genes were amplified with published or newly-designed primers and then sequenced. SCCmec types were assigned for 63 isolates by multiplex PCR and were assigned for 14 other isolates by PCR targeting mec and ccr. Among 77 isolates with determined SCCmec types, 54 had a single type, including type III (n = 19), IV (n = 14), V (n = 10), II (n = 2), I (n = 1), VIII (n = 1) and five unnamed types (n = 7), while 23 isolates had two types, III+V (n = 12), II+V (n = 8), II+IV (n = 2) or IV+V (n = 1). The five unnamed types were assigned UT1 (class A mec, ccrA1/ccrB4), UT2 (class C1 mec, ccrA4/ccrB4), UT3 (class A mec, ccrA5/ccrB3), UT4 (class C2 mec, ccrA2/ccrB2 plus ccrC1) and UT5 (class A mec, ccrA1/ccrB1 plus ccrC1).

Conclusions/Significance

SCCmec types III, IV and V were prevalent in MR-CoNS and many isolates could harbor more than one type. Several new types of SCCmec were identified, highlighting the great genetic diversity and the need of developing classification schemes for SCCmec in MR-CoNS.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1?) Type

Background

India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited.

Methodology/Principal Findings

Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients'' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes.

Conclusion

CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates.