Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

AbstractThis study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p=0.047) and 56.20% (p=0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p=0.033). Furthermore, at 72h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p=0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response

Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.

CAPRISA, 2018.Abstract available in pdf

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion

Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function

Anti-adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post-antibiotic era. Adhesins are surface-exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces, a fundamental step towards host infection. Although several Mycobacterium tuberculosis adhesins have been identified, it is predicted that numerous additional adherence-mediating components contribute to the virulence and success of this pathogen. Significant further research to discern and characterize novel M. tuberculosis adhesins is, therefore, required to gain a holistic account of M. tuberculosis adhesion to the host. This would enable the identification of potential drug and vaccine targets for attenuating M. tuberculosis adherence and infectivity. Several methods have been successfully applied to the study and identification of M. tuberculosis adhesins. In this manuscript, we review these methods, which include adherence assays that utilize wild-type and gene knockout mutant strains, epitope masking and competitive inhibition analyses, extracellular matrix protein binding assays, microsphere adhesion assays, M. tuberculosis auto-aggregation assays, and in silico analyses

Signal peptide prediction suggests Mycobacterium tuberculosis curli pilin subunit secretion via the Sec pathway may hinder MTP overexpression in Escherichia coli

Introduction Mycobacterium tuberculosis curli pili (MTP) are novel, potential TB diagnostic biomarkers as they are important virulence attributes, unique to the M. tuberculosis complex (MTBC). The production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. For example, the signal peptide of MTP governed by the classical secretion pathway may hinder the purification of the protein in E. coli systems. In this study, the secretion characteristics of MTP were determined and the cloning, expression and purification of MTP was attempted in E.coli.  Materials and methods A fragment of MTP unique to MTBC was cloned into pet101 and pGEX-6P-1 vectors. The clones were confirmed by nucleotide sequencing and expression of the protein was attempted at IPTG concentrations ranging from 0.1mM to 1mM and at temperatures between 25 °C to 37 °C. The pGEX-6P-1/mtp clone expressed protein was purified, yielding a MTP-GST fusion protein and a free GST band that were analysed by LC/MS mass spectrometry. Inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kDa and below 10 kDa, both of which were analysed by LC/MS mass spectrometry. Transcription activity of both the clones was interrogated by real time PCR on the cDNA derived from the induced clones at various time points after induction with IPTG. The signal peptide and protein secretion characteristics of the MTP protein were determined by bioinformatics analysis of the amino acid sequence using publically available software.  Results The truncated MTP fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. Expression of the pGEX-6P-1/mtp clone using 0.5 mM IPTG at 27 °C demonstrated the presence of the expected fragment at approximately 35 kDa. This was confirmed by Western Blotting using anti-GST antibodies. However, purification of MTP in adequate quantities as a pure protein fraction was unsuccessful. Mass spectrometry did not detect any fragments belonging to MTP, but only those of E.coli membrane proteins for the pet101/mtp clone and fragments of the GST tag in the case of the pGEX-6P-1/mtp clone. The bioinformatics secretion analyses of MTP predicted a strong Sec regulated secretion pathway and the absence of non-classical “mycobacterial specific” secretion.  Discussion M. tuberculosis membrane and secretory proteins often contain signal peptides. In this study, excluding the signal peptide region and using a GST tag greatly enhanced the expression of the protein in the soluble fraction. However, purification of the MTP peptide remained problematic due to a lower available peptide concentration resulting from the lower molecular weight, in the purified fraction compared to the GST tag. Alternately, the predicted Sec regulated secretion pathway may play a role in inhibition of MTP overexpression in E.coli. Thus, alternatives to E. coli expression systems or more efficient purification strategies are required for the acquisition of high quality M. tuberculosis antigens

Nosocomial transmission of tuberculosis in Africa documented by restriction fragment length polymorphism

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Analysis of clinical and microbiological data on Acinetobacter baumannii strains assist the preauthorization of antibiotics at the patient level for an effective antibiotic stewardship program

Drug resistant Acinetobacter baumannii (A. baumannii) poses serious treatment challenges and is on the rise worldwide. The Infectious Diseases Society of America/Society for Healthcare Epidemiology of America recommends preauthorization of antibiotics to ensure successful antibiotic stewardship programs (ASWPs). This study estimates and analyzes the microbiological and clinical characteristics of A. baumanii strains with differentiating criteria for sepsis versus colonization, in order to support preauthorization and assist ASWPs at the patient level.A retrospective observational study was performed from 2008 to 2014. The clinical and microbiological characteristics of A. baumannii strains were correlated to assess pathogenic status and antibiotic resistance patterns. A flow chart was produced to differentiate between sepsis and colonization amongst patient groups.A. baumannii was cultured in 2656 cases, with a prevalence of 0.9–2.4% during 7 years study periods. There was a statistically significant difference between the sepsis and colonization groups (P = 0.02). Sepsis accounted for 37–51% of A. baumanii isolates and colonisation for 49–63% (P = < 0.01). Multidrug resistant (MDR), extensive drug resistant (XDR) and pandrug resistant (PDR) A. baumannii was detected in 53–60%, 1–19% and 1% of cultures in the sepsis group, and 75%, 8–23% and 1% in the colonized group. There was a high percentage of polymicrobial infection in the sepsis group and pure growth was not always significant for sepsis.Cases of MDR and XDR A. baumannii increased over the seven-year study, while PDR strains emerged. For a successful ASWP, both clinical and microbiological information should be interpreted when establishing preauthorization/decision to treat. Keywords: MDR, XDR, PDR A. baumannii, Prevalence, Sepsis, Colonized group, Preauthorization, Antibiotic stewardship progra

Antibiotic resistance and biofilm formation of Acinetobacter baumannii isolated from high-risk effluent water in tertiary hospitals in South Africa

Objectives: Discharge of drug-resistant, biofilm-forming pathogens from hospital effluent water into mu-nicipal wastewater treatment plants poses a public health concern. This study examined the relationship between antibiotic resistance levels and biofilm formation of Acinetobacter baumannii strains isolated from hospital effluents. Methods: Antibiotic susceptibility of 71 A. baumannii isolates was evaluated by the Kirby-Bauer disk dif-fusion method. Minimum inhibitory concentrations (MICs) were determined by the agar dilution method, while the minimum biofilm eradication concentration (MBEC) was determined by the broth dilution method. Genotyping was performed for plasmid DNA. Biofilm formation was evaluated by the microtitre plate method and was quantified using crystal violet. A P-value of &lt; 0.05 was regarded as statistically significant in all tests. Results: Extensively drug-resistant (XDR) strains made up 58% of the isolates, while multidrug-resistant (MDR) and pandrug-resistant (PDR) strains made up 50% of the isolates from final effluent. The MBEC of ciprofloxacin increased by 255-fold, while that of ceftazidime was as high as 63-1310-fold compared with their respective MICs. Isolates were classified into four plasmid pattern groups with no association between biofilm formation and plasmid type ( P = 0.0921). The degree of biofilm formation was inde-pendent of the level of antibiotic resistance, although MDR, XDR and PDR isolates produced significant biofilm biomass ( P = 0.2580). Conclusion: These results suggest that hospital effluent is a potential source of MDR biofilm-forming A. baumannii strains. Appropriate treatment and disposal of effluents are essential to prevent the presence of drug-resistant pathogens in wastewater

Genome sequence of a carbapenemase-encoding Acinetobacter baumannii isolate of the sequence type 231 isolated from hospital wastewater in South Africa

Objectives The resistome, virulome, mobilome and phylogenetic relationship of the Acinetobacter baumannii isolate FG121 depicting the multilocus sequence type (ST) 231 isolated from hospital effluent water in South Africa was determined using whole-genome sequence analysis. Method A. baumannii FG121 was isolated on Leed Acinetobacter Medium (LAM) agar and the bacterial isolate was identified using the VITEK®2 platform. Antibiotic susceptibility testing was performed using Kirby–Bauer Disk diffusion method. A whole genome sequencing library was constructed from DNA extracted from the isolate using the Illumina Nextera XT library preparation kit and was sequenced using the Illumina NextSeq500 platform. Generated reads were de novo assembled using SpAdes v.3.9. The assembled contigs were annotated, and multilocus sequence type, antimicrobial resistance, and virulence genes were identified. Results The resistome was consistent with the resistance phenotype of the isolate with resistance determinants for beta-lactams, aminoglycosides, and tetracycline (blaADC-25, blaOXA-23, blaOXA-51, blaNDM-1, aph[3’]-VIa and tet[B]). Global phylogenomic analysis using BacWGSTdb revealed that the isolate belonged to the multilocus sequence type ST-231, similar to previously reported isolates from South Africa, the United States, and related to the invasive KR3831 isolate identified from Oman in 2012, suggesting the isolate might be imported from abroad. Virulome analysis predicted both virulence and biofilm-determinants of A. baumannii, which may help to establish infections in adverse conditions. Conclusion This is the first report on a carbapenemase-encoding A. baumannii ST-231 isolated from hospital effluent water. Our data will offer insight into the global phylogenetic, pathogenicity and distribution of A. baumannii in South Africa