Håvard Valvatne

Pyrazinamide Resistance among South African Multidrug-Resistant Mycobacterium tuberculosis Isolates

ABSTRACT Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Waynes assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.

Isoniazid and rifampicin resistance-associated mutations in Mycobacterium tuberculosis isolates from Yangon, Myanmar: implications for rapid molecular testing

OBJECTIVES To evaluate the frequency and nature of mutations in genes associated with resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates collected from Yangon, Myanmar. METHODS Ninety-six isoniazid-resistant M. tuberculosis isolates, including 29 multidrug-resistant isolates, were analysed for mutations in the rpoB, katG, inhA, oxyR and ahpC genes. RESULTS Mutations in the rpoB gene were detected in 25 (86.2%) of the 29 rifampicin-resistant isolates. Of the 96 isoniazid-resistant isolates, 61 (63.5%) had mutations in codon 315 of the catalase-peroxidase-encoding gene (katG). Mutations in codon 315 were observed at a higher frequency in the multidrug-resistant isolates than in the isoniazid-resistant isolates (86.2% versus 53.7%, respectively, P = 0.003). Mutations in the oxyR-ahpC promoter region and in the inhA gene were observed in 14.6% and 2.1% of the isolates, respectively. Genotyping performed on the 96 M. tuberculosis isolates revealed a total of 94 different genotyping patterns. A distinct genotypic pattern was found in 92 isolates, whereas 4 isolates belonged to two clusters with identical genotypes, suggesting that the majority of the isolates were not from an outbreak of a single drug-resistant clone. CONCLUSIONS This study provides the first molecular characterization of isoniazid- and rifampicin-resistant M. tuberculosis isolates from Myanmar and gives information on the molecular basis for rifampicin and isoniazid drug resistance in M. tuberculosis. The study generates useful information for the development of potential rapid molecular drug susceptibility tests.

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.

Clonal clustering and colonization factors among thermolabile and porcine thermostable enterotoxin‐producing Escherichia coli

A considerable proportion of enterotoxigenic Escherichia coli (ETEC) do not possess identifiable colonization factors (CFs). Genetic fingerprint analyses based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) showed that 9 of 10 such CF‐negative isolates which produced the thermolabile and the porcine thermostabile enterotoxin could be divided into three clusters. Following transformation with a plasmid harbouring the gene encoding CfaR, a positive regulator for several ETEC adhesins, three of the six strains in the first cluster expressed coli surface antigen 20 (CS20). No CFs were identified on the two transformed strains in the second cluster while the transformants of the two strains in the last cluster expressed CS12, the N‐terminal amino acid sequence of which was deciphered. The study illustrates the potential of using genetic fingerprinting to group ETEC into clusters of strains with genes encoding different CFs and confirms the ability of CfaR to induce the expression of several different CFs.

Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex

The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome.

Mycobacterium tuberculosis Mce1 protein complex initiates rapid induction of transcription of genes involved in substrate trafficking.

The mammalian cell entry (Mce)1 protein complex has an important role during the initial phase of a Mycobacterium tuberculosis (M. tuberculosis) infection. Murine macrophages were infected with M. tuberculosis H37Rv or Δ-mce1 H37Rv, and total RNA was isolated from the host cells at 15, 30 and 60 min, and 4 and 10 h post-infection. With the aim of studying the role for the Mce1 protein complex on host gene expression, the RNA was hybridized onto 44 K whole-genome microarrays. Selected genes were verified by reverse-transcriptase quantitative PCR (RT-QPCR). ‘Transport’ was the most overrepresented biological process during the first hour post H37Rv infection. Five genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3, (5.5-fold)) involved in substrate trafficking were verified by RT-QPCR to be upregulated by >2-fold 1 h post H37Rv infection. By 1 h post Δ-mce1 H37Rv infection, only Abca1 and Slc6a12 were upregulated by >2-fold. A number of other genes, which may be directly involved in substrate trafficking or share the same transcription, were found to have expression profiles similar to the genes involved in substrate trafficking. The Mce1 protein complex has a significant role in the transcriptional activation of genes involved in substrate trafficking during the initial phase of an M. tuberculosis infection.

Expression of colonization factor antigen I fimbriae by enterotoxigenic Escherichia coli; influence of growth conditions and a recombinant positive regulatory gene

Enterotoxigenic Escherichia coli (ETEC) may spontaneously lose the positive regulatory cfaR gene and thereby the capacity to express colonization factor antigen I (CFA/I). A recombinant plasmid harbouring the cfaR gene was transformed into cfaR‐negative mutant ETEC strains. CFA/I expression of wild‐type and cfaR‐transformed ETEC cultivated in different liquid media was quantified. At 37°C, a high level of CFA/I expression from wild‐type and cfaR‐transformed strains was observed after growth in CFA broth. Transformation enhanced CFA/I expression only marginally. The transformant cultures showed a considerable variation in CFA/I expression which was paralleled by the proportion of individual bacteria producing CFA/I. This heterogeneity could be explained by a variable tendency to structural CFA/I gene loss among individual cfaR‐transformed bacteria.