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Background: Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA 3 ) and five membrane components (EccB3 - EccE3 and MycP 3 ). In this study we sought to identify the sub-cellular location of EccA 3 of the ESX-3 secretion system in mycobacteria., Results: Fluorescently tagged EccA 3 localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA 3 , suggesting that EccA 3 unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA 3 associated proteins which may contribute to the localization of EccA 3 at the growing pole. EccA 3 co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified., Conclusions: Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA 3 with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA 3 is located at the site of active cell growth., (© 2022. The Author(s).)

Mycobacterium africanum consists of Lineages L5 and L6 of the Mycobacterium tuberculosis complex (MTBC) and causes human tuberculosis in specific regions of Western Africa, but is generally not transmitted in other parts of the world. Since M. africanum is evolutionarily closely placed between the globally dispersed Mycobacterium tuberculosis and animal-adapted MTBC-members, these lineages provide valuable insight into M. tuberculosis evolution. Here, we have collected 15 M. africanum L5 strains isolated in France over 4 decades. Illumina sequencing and phylogenomic analysis revealed a previously underappreciated diversity within L5, which consists of distinct sublineages. L5 strains caused relatively high levels of extrapulmonary tuberculosis and included multi- and extensively drug-resistant isolates, especially in the newly defined sublineage L5.2. The specific L5 sublineages also exhibit distinct phenotypic characteristics related to in vitro growth, protein secretion and in vivo immunogenicity. In particular, we identified a PE_PGRS and PPE-MPTR secretion defect specific for sublineage L5.2, which was independent of PPE38. Furthermore, L5 isolates were able to efficiently secrete and induce immune responses against ESX-1 substrates contrary to previous predictions. These phenotypes of Type VII protein secretion and immunogenicity provide valuable information to better link genome sequences to phenotypic traits and thereby understand the evolution of the MTBC.

The organism that causes tuberculosis in meerkats (Suricata suricatta) has been poorly characterized. Our genetic analysis showed it to be a novel member of the Mycobacterium tuberculosis complex and closely related to the dassie bacillus. We have named this epidemiologically and genetically unique strain M. suricattae.

Most current diagnostic tests for tuberculosis do not reveal the species or strain of pathogen causing pulmonary infection, which can lead to inappropriate treatment regimens and the spread of disease. Here, we report an assay for mycobacterial strain assignment based on genetically conserved mycobacterial sulfatases. We developed a sulfatase-activated probe, 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-sulfate, that detects enzyme activity in native protein gels, allowing the rapid detection of sulfatases in mycobacterial lysates. This assay revealed that mycobacterial strains have distinct sulfatase fingerprints that can be used to judge both the species and lineage. Our results demonstrate the potential of enzyme-activated probes for rapid pathogen discrimination for infectious diseases.

A modification of a highly practical human interferon-gamma release assay for the diagnosis of tuberculosis (TB), the QuantiFERON((R))-TB Gold (In-Tube Method) (QFG-IT) assay, was evaluated for diagnosing natural mycobacterial infection in rhesus macaques (Macaca mulatta). All animals in a captive colony were tested using the QFG-IT and tuberculin skin test (TST). Animals testing positive to these tests were euthanised and necropsied. Selected tissues were processed for histopathology and mycobacterial culture, and positive cultures were speciated by Mycobacterium tuberculosis complex polymerase chain reaction (PCR) and 16S rRNA gene PCR sequencing techniques. M. tuberculosis was cultured from a TST-positive/QFG-IT-positive animal which showed gross pulmonary pathology typical of TB. Additionally, Mycobacterium kansasii was cultured from a TST-negative/QFG-IT-positive animal which had no pathological or histopathological signs of mycobacterial infection. The detection of M. kansasii infection in a QFG-IT-positive animal which showed no evidence of disease indicates that this test might be a highly sensitive tool for the diagnosis of mycobacterial infection in rhesus macaques. However, these findings highlight the limitations of the QFG-IT to specifically detect infection by the pathogens M. tuberculosis and Mycobacterium bovis., (Copyright 2010 Elsevier B.V. All rights reserved.)

We report a case of pyogranulomatous pneumonia due to infection with Mycobacterium goodii in an adult female spotted hyena (Crocuta crocuta). The lungs of the animal showed consolidated, granulomatous lesions, and they were extensively and severely infiltrated. Polymerase chain reaction sequencing of isolated crude lung tissue DNA, and boiled lung culture samples, all confirmed that the causative organism was M. goodii, a recently described fast-growing organism closely related to the nonpathogenic mycobacterial species M. smegmatis. The current study illustrates that this organism can be pathogenic and cause extensive pulmonary disease.

The majority of mycobacterial plasmid vectors are derived from the pAL5000 replicon and maintained at approximately five copies per cell. We have devised a method that directly selects for high-copy-number plasmids. This involves enriching for high copy number plasmids by repeatedly isolating and retransforming plasmids from a mutant library. Using this method we have selected a copy-up version of the pAL5000 replicon. In Mycobacterium smegmatis the copy-number was shown to have increased 7-fold to between 32 and 64 copies/cell, and the plasmid remained relatively stable after 100 generations in the absence of antibiotic selection. The plasmid also has a high-copy-number phenotype in M. bovis BCG and can be used to increase expression of cloned genes, as we have demonstrated with the green fluorescent protein. The mutation was found to be the deletion of an alanine residue in the C-terminal end of the RepA replication protein. We argue that the mutation exerts its effect through altered RNA folding, thereby affecting the translationally coupled RepA-RepB expression.

Background: Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA3) and five membrane components (EccB3 – EccE3 and MycP3). In this study we sought to identify the sub-cellular location of EccA3 of the ESX-3 secretion system in mycobacteria. Results: Fluorescently tagged EccA3 localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA3, suggesting that EccA3 unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA3 associated proteins which may contribute to the localization of EccA3 at the growing pole. EccA3 co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified. Conclusions: Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA3 with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA3 is located at the site of active cell growth. [ABSTRACT FROM AUTHOR]

Background: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. Aim: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein–Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. Method: A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. Results: The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with “other genotype” strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. Conclusion: The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility. [ABSTRACT FROM AUTHOR]

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains. [ABSTRACT FROM AUTHOR]

Background Early diagnosis of simian tuberculosis (TB) is vital to prevent transmission of this disease. We evaluated the ability of the QuantiFERON-TB Gold (In-Tube Method) assay (QFG-IT) to detect TB in chacma baboons ( Papio ursinus). Methods Fifty-one baboons were tested using the Tuberculin Skin Test (TST) and the QFG-IT. Baboons testing positive, and animals exposed to infected individuals, were euthanised and subjected to necropsy. Selected tissues were processed for histopathology, mycobacterial culture and genetic speciation. Results Tuberculosis was confirmed in one TST positive/QFG-IT positive animal and one TST negative/QFG-IT positive animal. One TST positive/QFG-IT negative animal and five TST negative/QFG-IT negative animals were confirmed uninfected following necropsy. Conclusion The QFG-IT correctly detected TB in two baboons, including one TST negative individual and correctly identified six baboons as uninfected, including one TST positive individual. The QFG-IT shows promise as a sensitive, specific test for TB in chacma baboons. [ABSTRACT FROM AUTHOR]

• MILLIPLEX ® bovine multiplex assay detected African buffalo cytokines/chemokines. • Twelve of 15 cytokine targets showed significant mitogen-induced responses. • Seven cytokines were increased in TB antigen-samples from infected buffaloes. • Promising tool for screening African buffalo cell-mediated immune responses. As a recognized Mycobacterium bovis maintenance host, the African buffalo (Syncerus caffer) poses transmission risks to livestock, humans and other wildlife. Early detection of M. bovis infection is critical for limiting its spread. Currently, tests detecting cell-mediated immune responses are used for diagnosis in buffaloes. However, these may have suboptimal sensitivity or specificity, depending on the blood stimulation method. Recent evidence suggests that assays using combinations of host cytokine biomarkers may increase diagnostic performance. Therefore, this study aimed to investigate the application of a MILLIPLEX ® bovine cytokine/chemokine multiplex assay to identify candidate biomarkers of M. bovis infection in buffaloes. Whole blood from twelve culture-confirmed M. bovis- infected buffaloes, stimulated with the QuantiFERON ® TB Gold Plus in-tube system, was tested using the MILLIPLEX ® platform. Results indicated binding of bovine antibodies to fifteen buffalo cytokine/chemokine targets. Moreover, there was a significant difference in concentrations between unstimulated and TB antigen-stimulated buffalo samples for seven cytokines/chemokines included in the kit. Although these preliminary results require further investigation in larger sample sets and a comparison between M. bovis -infected and uninfected cohorts, the utility of the MILLIPLEX ® platform in a novel species was demonstrated, in addition to identifying potential African buffalo cytokines for future research. [ABSTRACT FROM AUTHOR]

The cytokine interferon gamma-inducible protein 10 (IP-10) is a sensitive biomarker of Mycobacterium bovis (M. bovis) infection in African buffaloes (Syncerus caffer). However, elevated levels of IP-10 in QuantiFERON®-TB Gold (QFT) unstimulated whole blood compromises the utility of this biomarker. In this study, IP-10 and interferon gamma (IFN-γ) concentrations in whole blood samples from M. bovis culture-confirmed buffaloes with varying degrees of pathological changes (n = 72) and uninfected controls (n = 70) were measured in the IP-10 release assay (IPRA) and IFN-γ release assay (IGRA), respectively. Findings suggest that concentrations of both cytokines in QFT Nil tubes were higher in infected buffaloes with macroscopic pathological changes consistent with bovine tuberculosis compared to uninfected controls, and IGRA values increased with more severe pathological changes in infected buffaloes (p < 0.05). Finally, in culture-confirmed buffaloes with IPRA-negative and IGRA-positive test results, most animals were also those with the most advanced pathology. We conclude that IP-10 and IFN-γ concentrations measured in QFT Nil tubes may provide insight into the presence of M. bovis pathology in infected buffaloes. Furthermore, this study highlights the value in evaluating cytokine production in both antigen-stimulated and unstimulated samples when interpreting cytokine release assay results. [ABSTRACT FROM AUTHOR]