Lipase‐catalyzed kinetic resolution of novel antitubercular benzoxazole derivatives

Novel benzoxazole derivatives were synthesized, and their antitubercular activity against sensitive and drug‐resistant Mycobacterium tuberculosis strains (M. tuberculosis H₃₇Rv, M. tuberculosis sp. 210, M. tuberculosis sp. 192, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii) was evaluated. The chemical step included preparation of ketones, alcohols, and esters bearing benzoxazole moiety. All racemic mixtures of alcohols and esters were separated in Novozyme SP 435‐catalyzed transesterification and hydrolysis, respectively. The transesterification reactions were carried out in various organic solvents (tert‐butyl methyl ether, toluene, diethyl ether, and diisopropyl ether), and depending on the solvent, the enantioselectivity of the reactions ranged from 4 to >100. The enzymatic hydrolysis of esters was performed in 2 phase tert‐butyl methyl ether/phosphate buffer (pH = 7.2) system and provided also enantiomerically enriched products (ee 88‐99%). The antitubercular activity assay has shown that synthesized compounds exhibit an interesting antitubercular activity. Racemic mixtures of alcohols, (±)‐4‐(1,3‐benzoxazol‐2‐ylsulfanyl)butan‐2‐ol ((±)‐ 3a ), (±)‐4‐[(5‐bromo‐1,3‐benzoxazol‐2‐yl)sulfanyl]butan‐2‐ol ((±)‐ 3b ), and (±)‐4‐[(5,7‐dibromo‐1,3‐benzoxazol‐2‐yl)sulfanyl]butan‐2‐ol ((±)‐ 3c ), displayed as high activity against M. scrofulaceum, M. intracellulare, M. fortuitum, and M. kansasii as commercially available antituberculosis drug‐Isoniazid. Moreover, these compounds exhibited twice higher activity toward M. avium (MIC 12.5) compared with Isoniazid (MIC 50).     

Molecular diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates from patients with tuberculosis in Honduras

Background  

Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe.     

Biofilm formation by Mycobacterium avium isolates originating from humans, swine and birds

Background  

Mycobacterium avium includes the subspecies avium, silvaticum, paratuberculosis and hominissuis, and M. avium subspecies has been isolated from various environments all over the world including from biofilms in water distribution systems. The aim of this study was to examine isolates of M. avium subsp. avium and M. avium subsp. hominissuis of different origin for biofilm formation and to look for correlations between biofilm formation and RFLP-types, and to standardise the method to test for biofilm formation. In order to determine the best screening method, a panel of 14 isolates of M. avium subsp. avium and M. avium subsp. hominissuis, were tested for their ability to form biofilm in microtiter plates under different conditions. Subsequently, 83 additional isolates from humans, swine and birds were tested for biofilm formation. The isolates were tested for the presence of selected genes involved in the synthesis of glycopeptidolipids (GPLs) in the cell wall of M. avium, which is believed to be important for biofilm formation. Colony morphology and hsp65 sequvar were also determined.     

Few amino acid positions in <Emphasis Type="Italic">rpoB</Emphasis> are associated with most of the rifampin resistance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mutations in rpoB, the gene encoding the β subunit of DNA-dependent RNA polymerase, are associated with rifampin resistance in Mycobacterium tuberculosis. Several studies have been conducted where minimum inhibitory concentration (MIC, which is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited) of rifampin has been measured and partial DNA sequences have been determined for rpoB in different isolates of M. tuberculosis. However, no model has been constructed to predict rifampin resistance based on sequence information alone. Such a model might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates.     

Characterisation of <Emphasis Type="Italic">mycobacteria</Emphasis> isolated from slaughter cattle in pastoral regions of Uganda

Background  

Bovine tuberculosis is a zoonotic problem in pastoral cattle and communities in Uganda. Tuberculin tests in pastoral cattle had shown a high herd but low animal prevalence, with a high proportion of avian reactors. No work had been done to identify the mycobacterial species involved. The objective of the study was to isolate and characterise Mycobacterial species causing tuberculous lesions in slaughtered animals. Lesioned organs compatible with bovine tuberculosis in slaughtered cattle from pastoral areas in Uganda were collected and cultured to isolate mycobacteria. AccuProbe culture identification kits for the Mycobacterium tuberculosis complex, M. avium complex and M. avium were used to identify the isolates. Spoligotyping and Insertion Sequence (IS) 1311 and IS1245 Restriction Fragment Length Polymorphism analysis (RFLP) were used to further characterise the isolates.     

Evolution of two distinct phylogenetic lineages of the emerging human pathogen <Emphasis Type="Italic">Mycobacterium ulcerans</Emphasis>

Background  

Comparative genomics has greatly improved our understanding of the evolution of pathogenic mycobacteria such as Mycobacterium tuberculosis. Here we have used data from a genome microarray analysis to explore insertion-deletion (InDel) polymorphism among a diverse strain collection of Mycobacterium ulcerans, the causative agent of the devastating skin disease, Buruli ulcer. Detailed analysis of large sequence polymorphisms in twelve regions of difference (RDs), comprising irreversible genetic markers, enabled us to refine the phylogenetic succession within M. ulcerans, to define features of a hypothetical M. ulcerans most recent common ancestor and to confirm its origin from Mycobacterium marinum.     

Non‐Tuberculosis mycobacterium speciation using HPLC under Revised National TB Control Programme (RNTCP) in India

Aims

Non‐Tuberculous Mycobacteria (NTM) are ubiquitous in nature. The data on prevalence of NTM under the RNTCP is scarce. Many NTM species have clinical significance, and hence their identification and speciation are important.

Methods and Results

It is a cross‐sectional study conducted at the five RNTCP accredited culture and drug susceptibility testing (CDST) laboratory. The culture isolates from AFB positive but Immunochromatographic test negative samples were taken for identification and speciation using HPLC. Of the total 266 isolates only 164 isolates had a second sample received at the laboratory. The speciation was done using HPLC for those isolates. The type of species identified are: 26·8% (44) were Mycobacterium chelonae, 12·8% (21) were Mycobacterium fortuitum, 9% (15) were Mycobacterium gordonae, 9% (15) were Mycobacterium tuberculosis complex, 6·1% (10) were Mycobacterium kansasii, 4·9% (8) were Mycobacterium simiae, 2·4% (4) were Mycobacterium thermophile, 1·2% (2) were Mycobacterium gastri, 0·6% (1) were Mycobacterium scrofulaceum, 0·6% (1) were Mycobacterium avium and 4·9% (8) isolates had chromatogram which was un‐interpretable.

Conclusion

Identification and its speciation of NTM are not routinely done under TB control programme. Since HPLC could identify 95% of isolates belonging to 10 species, the speciation of NTM using HPLC should gain importance in the diagnosis of disease caused by NTM.

Significance and Impact of Study

NTM are emerging as important causative agents of pulmonary and extra pulmonary disease, the ability to recognize disease caused by NTM and subsequently treat such disease has become increasingly important. The identification of NTM up to its species level should gain importance in all TB reference Laboratories.     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

Pulsed-field gel electrophoresis profile homogeneity of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> isolates from cattle and heterogeneity of those from sheep and goats

Background  

Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied.     

Genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor

Background  

Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia.     

Comparative analysis of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> isolates from cattle,sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing

Background  

Mycobacterium avium subsp. paratuberculosis (Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and SnaBI-SpeI pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar).     

The <Emphasis Type="Italic">Mycobacterium marinum mel2</Emphasis> locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species

Background  

Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood.     

Assessing the effectiveness of low‐pressure ultraviolet light for inactivating Mycobacterium avium complex (MAC) micro‐organisms

Aims: To assess low‐pressure ultraviolet light (LP‐UV) inactivation kinetics of Mycobacterium avium complex (MAC) strains in a water matrix using collimated beam apparatus. Methods and Results: Strains of M. avium (n = 3) and Mycobacterium intracellulare (n = 2) were exposed to LP‐UV, and log₁₀ inactivation and inactivation kinetics were evaluated. All strains exhibited greater than 4 log₁₀ inactivation at fluences of less than 20 mJ cm^?2. Repair potential was evaluated using one M. avium strain. Light repair was evaluated by simultaneous exposure using visible and LP‐UV irradiation. Dark repair was evaluated by incubating UV‐exposed organisms in the dark for 4 h. The isolate did not exhibit light or dark repair activity. Conclusions: Results indicate that MAC organisms are readily inactivated at UV fluences typically used in drinking water treatment. Differences in activation kinetics were small but statistically significant between some tested isolates. Significance and Impact of the Study: Results provide LP‐UV inactivation kinetics for isolates from the relatively resistant MAC. Although UV inactivation of Mycobaterium species have been reported previously, data collected in this effort are comparable with recent UV inactivation research efforts performed in a similar manner. Data were assessed using a rigorous statistical approach and were useful towards modelling efforts.     

Virulence-related <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp <Emphasis Type="Italic">hominissuis</Emphasis> MAV_2928 gene is associated with vacuole remodeling in macrophages

Background  

Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.     

Comparison of the Virulence for Mice of Mycobacterium avium and Mycobacterium intracellulare Identified by DNA Probe Test

The virulence of various serovars of Mycobacterium avium and M. intracellulare identified by DNA probe test was compared with each other. We found species- and serovar-dependencies of M. avium complex (MAC) virulence to mice in terms of mortality, incidence of lung lesions and bacterial load in the visceral organs, as follows. First, human- or environment-derived M. intracellulare was more virulent for mice, as compared to M. avium isolated from patients or environmental sources. Second, the virulence of MAC isolates belonging to serovars 1, 8, 9 (M. avium), 14 and 16 (M. intracellulare) is in the order of serovars 16 > 14 > 8 > 1 > 9. These aspects were different from those for MAC virulence to human and bird, swine and cattle.     

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis

Aims

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Methods and Results

The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti‐TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4–5 days, the response differences between the sensors made of drug‐sensitive isolates are distinguishable from the sensors made of drug‐resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti‐TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para‐aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions.

Conclusions

To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode‐based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Significance and Impact of the Study

The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O₂ electrode array microbial sensor reactor to enable a high‐throughput drug resistance analysis.     

Characterization of <Emphasis Type="Italic">Campylobacter</Emphasis> phages including analysis of host range by selected <Emphasis Type="Italic">Campylobacter</Emphasis> Penner serotypes

Background  

The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans.     

Rapid detection of laboratory cross-contamination with Mycobacterium tuberculosis using multispacer sequence typing

Background  

The ability to culture Mycobacterium tuberculosis from clinical specimens serves as the gold standard for the diagnosis of tuberculosis. However, a number of false-positive diagnoses may be due to cross-contamination of such specimens. We herein investigate such episode of cross-contamination by using a technique known as multispacer sequence typing (MST). This technique was applied to six M. tuberculosis isolates prepared within the same laboratory over a two-week period of time.