Mechanisms of Linezolid Resistance in Mycobacteria.

Mycobacteria form some of the most notorious and difficult-to-treat bacterial pathogens. As a group, they are intrinsically resistant to many commonly used antibiotics, such as tetracyclines and beta-lactams. In addition to intrinsic resistances, acquired multidrug resistance has also been observed and documented in Mycobacterium tuberculosis (MTB), Mycobacterium leprae and non-tuberculous mycobacteria (NTM). To combat multidrug resistant infections by these pathogens, innovative antimicrobials and treatment regimens are required. In this regard, linezolid, an oxazolidinone introduced for clinical use just two decades ago, was added to the therapeutic armamentarium for drug-resistant mycobacteria. It exhibits antibacterial activity by binding to the 50S ribosomal subunit and inhibiting protein synthesis. Unfortunately, linezolid resistance has now been documented in MTB and NTM, in many parts of the world. Most linezolid-resistant mycobacterial strains show mutations in the ribosome or related genes, such as in the rplC, rrl and tsnR genes. Non-ribosomal mechanisms appear to be rare. One such mechanism was associated with a mutation in fadD32, which encodes a protein that plays an important role in mycolic acid synthesis. Mycobacterial efflux proteins have also been implicated in linezolid resistance. This review summarises current knowledge of genetic determinants of linezolid resistance in mycobacteria, with the aim of contributing information that could facilitate the discovery of new therapeutic approaches to overcome, delay or avoid further developments of drug resistance among these important pathogens.

Mutations in Genes Encoding 23S rRNA and FadD32 May be Associated with Linezolid Resistance in Mycobacteroides abscessus.

Linezolid is one of the antibiotics used to treat the Mycobacteroides abscessus infection. However, linezolid-resistance mechanisms of this organism are not well understood. The objective of this study was to identify possible linezolid-resistance determinants in M. abscessus through characterization of step-wise mutants selected from a linezolid-susceptible strain, M61 (minimum inhibitory concentration [MIC]: 0.25 mg/L). Whole-genome sequencing and subsequent PCR verification of the resistant second-step mutant, A2a(1) (MIC: >256 mg/L), revealed three mutations in its genome, two of which were found in the 23S rDNA (g2244t and g2788t) and another one was found in a gene encoding the fatty-acid-CoA ligase FadD32 (c880t→H294Y). The 23S rRNA is the molecular target of linezolid and mutations in this gene are likely to contribute to resistance. Furthermore, PCR analysis revealed that the c880t mutation in the fadD32 gene first appeared in the first-step mutant, A2 (MIC: 1 mg/L). Complementation of the wild-type M61 with the pMV261 plasmid carrying the mutant fadD32 gene caused the previously sensitive M61 to develop a reduced susceptibility to linezolid (MIC: 1 mg/L). The findings of this study uncovered hitherto undescribed mechanisms of linezolid resistance in M. abscessus that may be useful for the development of novel anti-infective agents against this multidrug-resistant pathogen.

RshA mutations contributing to tigecycline resistance in Mycobacteroides abscessus.

Tigecycline is an important rescue antibiotic for many bacterial infections. In Mycobacteroides abscessus, tigecycline resistance has been associated with dysregulated stress response caused by aberrations in the interaction of the SigH and RshA factors. In this study, two tigecycline-resistant mutants of M. abscessus (CL5A and CL6A) with mutations in the rshA gene were studied using gene complementation, RT-qPCR and the bacterial adenylate cyclase two-hybrid (BACTH) system. The results supported the premise that mutations in the rshA interrupt the RshA-SigH interaction to cause the overexpression of the sigH gene that leads to tigecycline resistance or reduced susceptibility.

Genetic Determinants of Tigecycline Resistance in Mycobacteroides abscessus.

Mycobacteroides abscessus (formerly Mycobacterium abscessus) is a clinically important, rapid-growing non-tuberculous mycobacterium notoriously known for its multidrug-resistance phenotype. The intrinsic resistance of M. abscessus towards first- and second-generation tetracyclines is mainly due to the over-expression of a tetracycline-degrading enzyme known as MabTetX (MAB_1496c). Tigecycline, a third-generation tetracycline, is a poor substrate for the MabTetX and does not induce the expression of this enzyme. Although tigecycline-resistant strains of M. abscessus have been documented in different parts of the world, their resistance determinants remain largely elusive. Recent work on tigecycline resistance or reduced susceptibility in M. abscessus revealed the involvement of the gene MAB_3508c which encodes the transcriptional activator WhiB7, as well as mutations in the sigH-rshA genes which control heat shock and oxidative-stress responses. The deletion of whiB7 has been observed to cause a 4-fold decrease in the minimum inhibitory concentration of tigecycline. In the absence of environmental stress, the SigH sigma factor (MAB_3543c) interacts with and is inhibited by the anti-sigma factor RshA (MAB_3542c). The disruption of the SigH-RshA interaction resulting from mutations and the subsequent up-regulation of SigH have been hypothesized to lead to tigecycline resistance in M. abscessus. In this review, the evidence for different genetic determinants reported to be linked to tigecycline resistance in M. abscessus was examined and discussed.

Concentrated specimen smear microscopy utilising a polymer membrane sandwich filtration vessel for the detection of acid-fast bacilli in health facilities in Sabah, East Malaysia.

A simple, ready-to-use concentrated specimen smear microscopy method employing a nanometer silicon polyvinylidene fluoride (PVDF) polymer membrane sandwich filtration vessel to concentrate acid-fast bacilli (AFB) in samples (SFV-CSSM, Hunan-Tech New Medical System Co. Ltd. China) was compared with direct sputum smear microscopy (DSSM) to determine its performance using culture on modified Ogawa agar as reference. The results for 4114 clinical samples collected from health facilities in Sabah were interpreted with reference to culture results, sample collection-transportation conditions and clinical data including responses to anti-TB drug treatment. The SFV-CSSM showed higher sensitivity than DSSM (79.4% versus 60.5%) and less background interference. Its ability to detect low levels of AFB at an affordable cost makes it an excellent tool for the screening of pauci-bacillary samples as well as for active case finding in TB control programs.

A simple spreadsheet-based method for relative quantification using quantitative real-time PCR.

Relative quantification is a popular analysis in gene expression studies using quantitative real-time PCR (qPCR). However, the calculation steps using the major algorithms for this analysis are rather complicated. In this study, we developed an easy-to-use spreadsheet-based method for relative quantification. The inputs from end-users are the efficiencies of both target and reference genes and the Cq values of those genes from cases and controls. This method performed normalization (with one or more reference genes), calculation of fold change of gene expression, and statistical analysis to analyze the difference between the groups in a step-by-step manner, which would allow the end-users to understand how the analysis arrived at the conclusion. Four previously published data sets with different experimental designs were used as examples. The calculated results were concordant with the results computed by the Relative Expression Software Tool (REST) 2009, a popular tool for relative quantification. Altogether, our method, which offers easy-to-understand calculation steps and does not require specialized instruments, software, or expertise to operate, would be a useful tool for students, educators, and scientists in the field of molecular biology.

Mutations in atpG2 may confer resistance to gentamicin in Listeria monocytogenes.

Introduction Listeriosis, a foodborne infection caused by Listeria monocytogenes, could lead to febrile listerial gastroenteritis and a more invasive form which is often associated with a high mortality and hospitalisation rate. Gentamicin, used as an adjunct therapy with ampicillin, remains the treatment of choice for this life-threatening and invasive infection.Gap statement Nevertheless, there is little data on gentamicin resistance determinants in L. monocytogenes.Aim In this study, we selected and characterised B2b, a gentamicin-resistant mutant derived from L. monocytogenes ATCC 19115 to determine the target(s) of resistance in L. monocytogenes after exposure to gentamicin.Methodology Whole-genome sequencing was carried out to identify the mutation site(s) and possible mechanism(s) of resistance. The mutant was characterised using antimicrobial susceptibility testing and PCR. For biological verifications, complementation and allelic exchange mutagenesis were carried out.Results We found that the gentamicin resistance in B2b was caused by a 10 bp deletion in atpG2 which encodes a gamma subunit of the ATP synthase in L. monocytogenes. Using atpG2 PCR, various other mutations were identified in other gentamicin resistant mutants derived from ATCC 19115. In addition, the mutation from B2b, when introduced into L. ivanovii, also caused gentamicin resistance in this Listeria species.Conclusion Hence, atpG2 mutations appear to be important determinants of gentamicin resistance not only in L. monocytogenes but possibly also in other Listeria species.

A stop-gain mutation in sigma factor SigH (MAB_3543c) may be associated with tigecycline resistance in Mycobacteroides abscessus.

Introduction. Tigecycline is currently acknowledged to be one of the most effective antibiotics against infections caused by Mycobacteroides abscessus.Gap statement. The genetic determinants of tigecycline resistance in M. abscessus are not well understood.Aim. In this study, we characterized a tigecycline-resistant M. abscessus mutant, designated CL7, to identify the potential resistance mechanism.Methodology. CL7 was characterized using antimicrobial susceptibility testing, whole-genome sequencing, PCR and RT-qPCR. For biological verification, gene overexpression assays were carried out.Results. Whole-genome sequencing and the subsequent gene overexpression assays showed that CL7 harboured a stop-gain mutation in MAB_3543 c, which may be responsible for the tigecycline resistance phenotype. This gene encodes an orthologue of SigH, which is involved in the positive regulation of physiological stress response and is negatively regulated by the RshA anti-sigma factor in Mycobacterium tuberculosis. We hypothesized that the MAB_3543 c mutation may disrupt the interaction between SigH and RshA (MAB_3542 c). RT-qPCR analyses revealed the upregulation of MAB_3543 c and other key stress response genes, which has previously been shown to be a hallmark of SigH-RshA bond disruption and tigecycline resistance.Conclusion. The MAB_3543c mutation may represent a novel determinant of tigecycline resistance in M. abscessus. The findings of this study will hopefully contribute to our knowledge of potential tigecycline resistance mechanisms in M. abscessus, which may lead to better diagnostics and treatment modalities in the future.

A single-gene approach for the subspecies classification of Mycobacteroides abscessus.

The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.

Tigecycline resistance may be associated with dysregulated response to stress in Mycobacterium abscessus.

Previously, we characterized 7C, a laboratory-derived tigecycline-resistant mutant of Mycobacterium abscessus ATCC 19977, and found that the resistance was conferred by a mutation in MAB_3542c, which encodes an RshA-like protein. In M. tuberculosis, RshA is an anti-sigma factor that negatively regulates the SigH-dependent heat/oxidative stress response. We hypothesized that this mutation in 7C might dysregulate the stress response which has been generally linked to antibiotic resistance. In this study, we tested this hypothesis by subjecting 7C to transcriptomic dissection using RNA sequencing. We found an over-expression of genes encoding the SigH ortholog, chaperones and oxidoreductases. In line with these findings, 7C demonstrated better survival against heat shock when compared to the wild-type ATCC 19977. Another interesting observation from the RNA-Seq analysis was the down-regulation of ribosomal protein-encoding genes. This highlights the possibility of ribosomal conformation changes which could negatively affect the binding of tigecycline to its target, leading to phenotypic resistance. We also demonstrated that transient resistance to tigecycline could be induced in the ATCC 19977 by elevated temperature. Taken together, these findings suggest that dysregulated stress response may be associated with tigecycline resistance in M. abscessus.

A mutation in anti-sigma factor MAB_3542c may be responsible for tigecycline resistance in Mycobacterium abscessus.

In this study, we characterized 7C, a spontaneous mutant selected from tigecycline-susceptible Mycobacterium abscessus ATCC 19977. Whole-genome sequencing (WGS) was used to identify possible resistance determinants in this mutant. Compared to the wild-type, 7C demonstrated resistance to tigecycline as well as cross-resistance to imipenem, and had a slightly retarded growth rate. WGS and subsequent biological verifications showed that these phenotypes were caused by a point mutation in MAB_3542c, which encodes an RshA-like protein. In Mycobacterium tuberculosis, RshA is an anti-sigma factor that negatively regulates the heat/oxidative stress response mechanisms. The MAB_3542c mutation may represent a novel determinant of tigecycline resistance. We hypothesize that this mutation may dysregulate the stress-response pathways which have been shown to be linked to antibiotic resistance in previous studies.

Multiphasic strain differentiation of atypical mycobacteria from elephant trunk wash.

Background. Two non-tuberculous mycobacterial strains, UM_3 and UM_11, were isolated from the trunk wash of captive elephants in Malaysia. As they appeared to be identical phenotypes, they were investigated further by conventional and whole genome sequence-based methods of strain differentiation. Methods. Multiphasic investigations on the isolates included species identification with hsp65 PCR-sequencing, conventional biochemical tests, rapid biochemical profiling using API strips and the Biolog Phenotype Microarray analysis, protein profiling with liquid chromatography-mass spectrometry, repetitive sequence-based PCR typing and whole genome sequencing followed by phylogenomic analyses. Results. The isolates were shown to be possibly novel slow-growing schotochromogens with highly similar biological and genotypic characteristics. Both strains have a genome size of 5.2 Mbp, G+C content of 68.8%, one rRNA operon and 52 tRNAs each. They qualified for classification into the same species with their average nucleotide identity of 99.98% and tetranucleotide correlation coefficient of 0.99999. At the subspecies level, both strains showed 98.8% band similarity in the Diversilab automated repetitive sequence-based PCR typing system, 96.2% similarity in protein profiles obtained by liquid chromatography mass spectrometry, and a genomic distance that is close to zero in the phylogenomic tree constructed with conserved orthologs. Detailed epidemiological tracking revealed that the elephants shared a common habitat eight years apart, thus, strengthening the possibility of a clonal relationship between the two strains.

The mRNA expression of soluble urokinase plasminogen activator surface receptor in human adipose tissue is positively correlated with body mass index.

suPLAUR is the transcript variant that encodes the soluble form of the urokinase plasminogen activator surface receptor (suPLAUR). This soluble protein has been shown to enhance leukocyte migration and adhesion, and its circulatory level is increased in inflammatory states. In this pilot study, we used RNA-Seq to examine the splicing pattern of PLAUR in omental adipose tissues from obese and lean individuals. Of the three transcript variants of the PLAUR gene, only the proportion of suPLAUR (transcript variant 2) increases in obesity. After removing the effects of gender and age, the expression of suPLAUR is positively correlated with body mass index. This observation was validated using RT-qPCR with an independent cohort of samples. Additionally, in our RNA-Seq differential expression analysis, we also observed, in obese adipose tissues, an up-regulation of genes encoding other proteins involved in the process of chemotaxis and leukocyte adhesion; of particular interest is the integrin beta 2 (ITGB2) that is known to interact with suPLAUR in leukocyte adhesion. These findings suggest an important role for suPLAUR in the recruitment of immune cells to obese adipose tissue, in the pathogenesis of obesity.

Identification of new genomospecies in the Mycobacterium terrae complex.

Members of the Mycobacterium terrae complex are slow-growing, non-chromogenic acid-fast bacilli found in the natural environment and occasionally in clinical material. These genetically closely-related members are difficult to differentiate by conventional phenotypic and molecular tests. In this paper we describe the use of whole genome data for the identification of four strains genetically similar to Mycobacterium sp. JDM601, a newly identified member of the M. terrae complex. Phylogenetic information from the alignment of genome-wide orthologous genes and single nucleotide polymorphisms show consistent clustering of the four strains together with M. sp. JDM601 into a distinct clade separate from other rapid and slow growing mycobacterial species. More detailed inter-strain comparisons using average nucleotide identity, tetra-nucleotide frequencies and analysis of synteny indicate that our strains are closely related to but not of the same species as M. sp. JDM601. Besides the 16S rRNA signature described previously for the M. terrae complex, five more hypothetical proteins were found that are potentially useful for the rapid identification of mycobacterial species belonging to the M. terrae complex. This paper illustrates the versatile utilization of whole genome data for the delineation of new bacterial species and introduces four new genomospecies to add to current members in the M. terrae complex.

Comparative genomic analysis of Mycobacterium iranicum UM_TJL against representative mycobacterial species suggests its environmental origin.

Mycobacterium iranicum is a newly reported mycobacterial species. We present the first comparative study of M. iranicum UM_TJL and other mycobacteria. We found M. iranicum to have a close genetic association with environmental mycobacteria infrequently associated with human infections. Nonetheless, UM_TJL is also equipped with many virulence genes (some of which appear to be the consequence of transduction-related gene transfer) that have been identified in established human pathogens. Taken all together, our data suggest that M. iranicum is an environmental bacterium adapted for pathogenicity in the human host. This comparative study provides important clues and forms the basis for future functional studies on this mycobacterium.

First Whole-Genome Sequence of Mycobacterium iranicum, a Newly Reported Mycobacterial Species.

Mycobacterium iranicum is a new species of nontuberculous mycobacterium reported in 2013. Here, we describe the first whole-genome sequence of this species, that of M. iranicum strain UM_TJL, isolated from a patient in Malaysia.