Bioconversion of sodium dodecyl sulphate to rhamnolipids by transformed Escherichia coli DH5α cells-a novel strategy for rhamnolipid synthesis.

AIMS: Biological synthesis of rhamnolipids from SDS by Pseudomonas aeruginosa S15 is found to be a cost effective mode of rhamnolipid synthesis. This study aimed to attempt rhamnolipid synthesis by transformant Escherichia coli DH5α cells. METHODS AND RESULTS: Molecular analysis by curing experiments revealed that the properties of SDS based rhamnolipid synthesis were plasmid borne. Transformation of 10 kb plasmid to E. coli DH5α cells conferred rhamnolipid synthetic ability to transformant. Various genetic elements involved in SDS based rhamnolipid synthesis were analyzed using PCR based and restriction digestion based approaches. PCR amplification using primers specific for sdsA gene encoding alkylsulfatases yielded two significant amplicons viz, 1·2 kb fragment and 422 bp fragment, coding for putative dehydratase and ABC transporter respectively. Amplicon of sdsB gene lacked ability of SDS degradation and rhamnolipid synthesis. Rhamnolipid biosynthesis by transformant E. coli DH5α containing the whole of the 10 kb plasmid, was optimized to yield of 3·38 g l(-1) in 5 days of incubation. CONCLUSIONS: Plasmid encoded rhamnolipid synthesis from recombinant E. coli cells is novel and could serve as yet another promising approach among various steps adopted for safe and effective rhamnolipid synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: SDS based rhamnolipid synthesis by S15 attained a high substrate (SDS) to product (Rhamnolipid) conversion ratio. However, the use of Pseudomonas strains is always discouraged as they are opportunistic pathogens and could sometimes turn infectious. Thus, transformation of genetic elements coding SDS based rhamnolipid synthesis to nonpathogenic strains could be promising.

Detection of carbapenem resistance genes and cephalosporin, and quinolone resistance genes along with oqxAB gene in Escherichia coli in hospital wastewater: a matter of concern.

AIMS: This study was performed to detect the presence of Escherichia coli resistant to cephalosporins, carbapenems and quinolones in hospital wastewater. METHODS AND RESULTS: Wastewaters from a rural (H1) and an urban (H2) hospital were tested for E. coli resistant to cephalosporins, carbapenem and quinolones. Genes coding for chromosomal and plasmid-mediated resistance and phylogenetic grouping was detected by multiplex polymerase chain reaction (PCR) and for genetic relatedness by rep-PCR. Of 190 (H1 = 94; H2 = 96) E. coli examined, 44% were resistant to both cephalosporins and quinolones and 3% to imipenem. ESBLs were detected phenotypically in 96% of the isolates, the gene blaCTX-M coding for 87% and blaTEM for 63%. Quinolone resistance was due to mutations in gyrA and parC genes in 97% and plasmid-coded aac-(6')-Ib-cr in 89% of isolates. Only in one carbapenem-resistant E. coli, NDM-1 was detected. Nearly 67% of the isolates belonged to phylogenetic group B2. There was no genetic relatedness among the isolates. CONCLUSIONS: Hospital wastewater contains genetically diverse multidrug-resistant E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study stresses the need for efficient water treatment plants in healthcare settings as a public health measure to minimize spread of multidrug-resistant bacteria into the environment.

Drug resistance in Mycobacterium tuberculosis isolates from tuberculosis patients in Kerala, India.

OBJECTIVE: To analyse the extent of drug resistance in clinical isolates of Mycobacterium tuberculosis from patients attending various tuberculosis (TB) clinics in Kerala, India. DESIGN: Mycobacteria were isolated from sputum samples of TB patients. Isolates from 92 new and 104 retreatment cases were tested for resistance to four first-line drugs (isoniazid, rifampicin, ethambutol and streptomycin). RESULTS: Twenty-three per cent of the isolates from new cases and 14% from retreatment cases were pan-susceptible, and the rest were resistant to at least one of the drugs. Multidrug-resistant isolates accounted for 5.4% among new cases and 16.4% among retreatment cases. It should be noted that 18.5% of the isolates were mycobacteria other than tuberculosis. CONCLUSION: There is an urgent need for statewide surveys to assess the level of drug resistance using quality-assured culture and drug susceptibility services. Considering that the Revised National TB Control Programme in India has been made operational nationwide, this kind of screening should be made mandatory under the programme to effectively control the spread of TB.

Diagnostic utility of polymerase chain reaction and immunohistochemical techniques for the laboratory diagnosis of intracranial tuberculoma.

In an attempt to establish a tuberculous etiology, polymerase chain reaction (PCR) and immunohistochemical (IHC) methods were undertaken in formalin-fixed paraffin sections of ten surgical specimens of intracranial tuberculoma. The control group included an equal number of intracranial fungal granuloma. Both PCR and IHC methods did not yield false-positive results in fungal granuloma. PCR was found to be less sensitive (60%) than IHC method (80%) in this study. IHC method definitely possesses several operational advantages over PCR and is more suited to laboratories in developing countries for establishing a tuberculous etiology particularly in those patients in whom the conventional bacteriological methods did not confirm the diagnosis of tuberculoma.

Identification of genes involved in the resistance of mycobacteria to killing by macrophages.

The survival of M. leprae and M. tuberculosis in the human host is dependent upon their ability to produce gene products that counteract the bactericidal activities of macrophages. To identify such mycobacterial genes and gene products, recombinant DNA libraries of mycobacterial DNA in E. coli were passed through macrophages to enrich for clones carrying genes that endow the normally susceptible E. coli bacteria with an enhanced ability to survive within macrophages. Following three cycles of enrichment, 15 independent clones were isolated. Three recombinants were characterized in detail, and each confers significantly enhanced survival on E. coli cells carrying them. Two of the cloned genetic elements also confer enhanced survival onto M. smegmatis cells. Further characterization of these genes and gene products should provide insights into the survival of mycobacteria within macrophages and may identify new approaches of targets for combatting these important pathogens.

Expression of contact-dependent cytolytic activity by Mycobacterium tuberculosis and isolation of the genomic locus that encodes the activity.

We investigated the presence of cytolytic activity in the virulent H37Rv and attenuated H37Ra strains of Mycobacterium tuberculosis and in the vaccine strain Mycobacterium bovis BCG. The virulent strain H37Rv expressed > 3-fold more contact-dependent cytolytic activity than the attenuated strain H37Ra, and the vaccine strain M. bovis BCG did not produce cytolytic activity. We also isolated an approximately 3.2-kbp fragment of the M. tuberculosis H37Rv genome that was capable of inducing this contact-dependent hemolytic activity in a nonhemolytic strain of Escherichia coli.