Biomedical subjects

Izhar U H Khan

Publications and source records attributed to Izhar U H Khan.

7 recordsLinked to original sources

PubMed2005

Occurrence and characterization of multiple novel genotypes of Mycobacterium immunogenum and Mycobacterium chelonae in metalworking fluids.

Rapidly growing mycobacteria colonize metalworking fluids, leading to contamination of occupational environments and exposure-related respiratory illnesses in machine workers. Lately, it has been emphasized that these fluids are colonizable by a single genotype of a rapidly growing mycobacterium species, Mycobacterium immunogenum. Here, we report on the genotypic diversity of mycobacteria in these fluids, including isolation and characterization of multiple novel genotypes of two distinct species, Mycobacterium chelonae and M. immunogenum. Using agar culturing and Mycobacterium-specific PCR, 13 mycobacterial isolates were recovered from 100 geographically diverse in-use metalworking fluid samples. Based on restriction fragment length polymorphism of PCR products, DNA sequencing (hsp65 gene segment), and phylogenetic analysis of 16S-23S rDNA internal transcribed spacer (ITS) sequences, six isolates were identified as M. immunogenum and seven as M. chelonae; an additional isolate from metalworking fluid diluent water was identified as M. diernhoferi. Genomic DNA macro-restriction fragment pattern analysis, using pulsed-field gel electrophoresis with XbaI and SpeI restriction digestions, showed intraspecies variation among the isolates of M. immunogenum and M. chelonae. Visual and computer-assisted dendrogram analysis of the XbaI macro-restriction patterns revealed three novel genotypes of M. immunogenum and two of M. chelonae, whereas SpeI macro-restriction patterns revealed only two genotypes for each isolate. None of the identified genotypes matched the reportedly dominant one of M. immunogenum from metalworking fluids. Both mycobacterial species are prevalent in metalworking fluids and there is a considerable strain-level genetic diversity within them.

Bacterial Proteins↗

PubMed2005

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline.

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.

Alveolitis, Extrinsic Allergic↗

PubMed2005

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis.

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

Bacterial Typing Techniques↗

PubMed2004

A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA).

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.

Bacterial Proteins↗

PubMed2004

Real-time PCR assays for genus-specific detection and quantification of culturable and non-culturable mycobacteria and pseudomonads in metalworking fluids.

Genus-specific real-time PCR assays were developed and optimized for the direct culture-independent detection and quantification of Mycobacteria and Pseudomonads in contaminated metalworking fluids (MWF) and the results were compared with conventional culturing using selective media. It included optimization of the direct DNA isolation from the fluid matrix and the amplification conditions using genus-specific primers. Mycobacterium-specific primers based on 65-kDa heat shock protein (hsp) gene, and Pseudomonas-specific primers based on 16S rRNA gene were used. A standard curve was developed each for the two model bacterial species Mycobacterium immunogenum and Pseudomonas fluorescens, representing two important genera frequently isolated from MWF. A minimum quantification limit of 10 cells/ml was achieved although as low as 1 cell/ml yielded a detectable amplicon signal. Of the twenty MWF field samples contaminated with mixed microflora, only two samples yielded putative colonies of Mycobacteria and Pseudomonads by culturing method, while seven samples responded to the genus-specific real-time PCR detection and quantification for each genus. In contrast to the low culturable counts, the real-time PCR based cell counts ranged from 1.3 x 10(2) to 5.5 x 10(5)cells/ml and 5.2 x 10(2) to 7.0 x 10(5)cells/ml for Mycobacteria and Pseudomonads, respectively, indicating a significant non-culturable fraction in the fluids, for the two genera. This is the first application of real-time PCR protocol to MWF samples for detection and quantification of total (culturable and non-culturable) Mycobacteria and Pseudomonads without culturing.

Bacterial Proteins↗

PubMed2004

Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis.

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i). a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii). an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii). a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.

Bacteriolysis↗

PubMed2003

DNA-based methodologies for rapid detection, quantification, and species- or strain-level identification of respiratory pathogens (Mycobacteria and Pseudomonads) in metalworking fluids.

Mycobacteria and pseudomonads occurring in modern metalworking fluids (MWF) have been implicated in occupational health hazards as causal agents for hypersensitivity pneumonitis (HP) and other respiratory illnesses in machine workers exposed to these fluids and their aerosols. Unlike the conventional cultural and biochemical methods, which are often slow and ambiguous and detect only culturable cells, DNA-based methods offer a time-saving alternative for reliable detection and identification of both culturable and nonculturable bacteria in MWF and for selective quantification of individual genera of pathogens of interest in these fluids. This is the first report on DNA-based direct detection of mycobacteria and pseudomonads in MWF without culturing. Genus-specific PCR approach was successfully applied for screening of field MWF samples originating from different industrial users for detection of mycobacteria or pseudomonads including both culturable and nonculturable cells. PCR in combination with amplicon DNA sequencing led to the identification of Mycobacterium chelonae, Pseudomonas nitroreducens, and an undefined Pseudomonas species from these fluids. Genome fingerprinting by pulsed-field gel electrophoresis (PFGE) on Mycobacterium isolates further showed that the isolates represented three strains of M. chelonae although the possibility of one of the strains being clonal with M. immunogenum cannot be excluded. In parallel efforts, a quantitative competitive PCR method developed based on the Pseudomonas-specific PCR was applied to quantify total P. fluorescens cells in contaminated metalworking fluid and MWF aerosol without culturing. The DNA-based protocols developed in this study will allow rapid screening of field MWF samples for the presence of both culturable and nonculturable cells and thus facilitate effective fluid management and timely exposure assessment.

DNA Fingerprinting↗