Ashok Rattan

In Vitro Low-Level Resistance to Azoles in Candida albicans Is Associated with Changes in Membrane Lipid Fluidity and Asymmetry

ABSTRACT The present study tracks the development of low-level azole resistance in in vitro fluconazole-adapted strains of Candida albicans, which were obtained by serially passaging a fluconazole-susceptible dose-dependent strain, YO1-16 (fluconazole MIC, 16 μg ml−1) in increasing concentrations of fluconazole, resulting in strains YO1-32 (fluconazole MIC, 32 μg ml−1) and YO1-64 (MIC, 64 μg ml−1). We show that acquired resistance to fluconazole in this series of isolates is not a random process but is a gradually evolved complex phenomenon that involves multiple changes, which included the overexpression of ABC transporter genes, e.g., CDR1 and CDR2, and the azole target enzyme, ERG11. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. Interestingly, fluorescent polarization measurements performed by using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed that there was a gradual increase in membrane fluidity of adapted strains. The increase in fluidity was reflected by observed change in membrane order, which was considerably decreased (decrease in fluorescence polarization values, P value) in the adapted strain (P value of 0.1 in YO1-64, compared to 0.19 in the YO1-16 strain). The phospholipid composition of the adapted strain was not significantly altered; however, ergosterol content was reduced in YO1-64 from that in the YO1-16 strain. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was also changed, with PE becoming more exposed to the outer monolayer in the YO1-64 strain. The results of the present study suggest for the first time that changes in the status of membrane lipid phase and asymmetry could contribute to azole resistance in C. albicans.

Ciprofloxacin-Resistant Neisseria meningitidis, Delhi, India

Decreased susceptibility of Neisseria meningitidis isolates to ciprofloxacin emerged from an outbreak in Delhi, India. Results of antimicrobial susceptibility testing of the meningococcal isolates to ciprofloxacin and further sequencing of DNA gyrase A quinolone-resistance–determining region confirmed the emergence of ciprofloxacin resistance in the outbreak.

Mode of Action of Ranbezolid against Staphylococci and Structural Modeling Studies of Its Interaction with Ribosomes

ABSTRACT Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively. The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity against the target pathogen.

Emergence and molecular characterization of extensively drug-resistant Mycobacterium tuberculosis clinical isolates from the Delhi Region in India.

ABSTRACT We screened 194 Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in Delhi and neighboring regions in India to identify the prevalence of extensive drug resistance (XDR) in clinical isolates. Among these, 104 isolates were found to be multidrug resistant (MDR), and 6 were identified as XDR isolates, which was later confirmed by antimicrobial susceptibility testing against the respective drug screening panel. Genotyping was carried out by amplifying and sequencing the following genes: rpoB (rifampin), katG (isoniazid), gyrA (fluoroquinolones), and rrs (amikacin, kanamycin, and capreomycin). Our analyses indicated that mutations at the hot spots of these genes were positively correlated with drug resistance in clinical isolates. The key mutation observed for rpoB was in the codon for amino acid position 531 (S531L), and other mutations were seen in the hot spot, including those encoding Q510P, L511H, D516V, and H526Y mutations. We identified S315T and R463L substitutions encoded in the katG locus. An S95T substitution encoded in the gyrA locus was the most common mutation observed in fluoroquinolone-resistant isolates. In addition, we saw D94G and D94N mutations encoded in the QRDR region. The 16S rRNA (rrs) gene encoded mainly the A1401G mutation and an additional mutation, G1484T, resulting in ribosomal modifications. Taken together, the data in this report clearly establish the presence of phenotypically distinct XDR strains in India by molecular profiling and further identify specific mutational hot spots within key genes of XDR-TB strains.

Synthesis and biological activity of novel oxazolidinones

A number of 5-substituted derivatives of Ranbezolid, a novel oxazolidinone were synthesized. Antibacterial activity of the compounds against a number of sensitive and resistant bacteria showed promising results.

Multidrug resistantSalmonella typhi in Delhi

In 1990, we isolated 158 strains ofSalmonella typhi from blood cultures of patients suffering from typhoid fever. Seventy nine (50%) of these isolates were found to be simultaneously resistant to chloramphenicol, ampicillin and cotrimoxazole. These strains were also resistant to streptomycin and tetracycline, but sensitive to gentamicin, amikacin and cephalexin. The minimum inhibitory concentrations of chloramphenicol and trimethoprim for a representative number of these strains were found to be >1024 ug/ ml and >128 ug/ml respectively. Majority of the multidrug resistant (MDR) strains tested against cefotaxime (23/23), ciprofloxacin (38/38) and amoxycillin plus clavulanic acid (23/24) were sensitive to these drugs.

Group B meningococcal meningitis in India.

The first case of infection with Group B meningococcus in India is reported. The patient was a 4-month-old boy who presented with meningitis and died within 6 h of admission. Gram stain of CSF showed meningococci and latex particle agglutination test on CSF was strongly positive for Neisseria meningitidis serogroup B. The CSF was also positive for meningococcus by polymerase chain reaction using primers NM1 and NM6, which amplify a 650 bp region of the dihydropteroate synthase (dhps) gene of N. meningitidis.

Evaluation of a safe sputum processing method for detecting tuberculosis.

AIMS--To evaluate a safe sputum processing method for detection of tuberculosis in developing countries. METHODS--A sample processing method was developed in which acid fast bacilli were killed with 1% sodium hypochlorite and concentrated by flotation on a layer of xylene before staining by the Ziehl Neelsen or auramine O methods. RESULTS--Best results were obtained by auramine O staining after flotation. Staining by the Ziehl Neelsen method after flotation gave better results than direct Ziehl Neelsen staining without flotation. CONCLUSIONS--The flotation method with Ziehl Neelsen staining offers advantages for smear preparation in the tuberculosis control programmes of developing countries.

Antibacterial multifilament nylon sutures.

Multifilament nylon fibers were made antibacterial by dopping with iodine. Nylon fibers were immersed in acetone solution of iodine for 48 hours at room temperature for dopping of iodine. It was observed that iodine uptake by the nylon fibers increased with the increase in concentration of iodine in the solution. Antibacterial activity of these iodine dopped samples was evaluated by measuring the zone of inhibition. The bacterial species used for this study were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. Iodine dopped fibers exhibited good antibacterial activity against these bacterial species. Release of iodine in distilled water is sustained for about 30 days. Antibacterial activity of the fibers decreases with the release of iodine in water. Ultra-violet and visible spectroscopic studies showed that tri-iodide ions were released from the dopped samples in the aqueous medium. These I3- ions might be responsible for the observed antibacterial activity. Fiber shrinks on iodine dopping leading to increase in the denier of the fiber. However effect of iodine dopping on the breaking load of fibers is not significant.

Diagnosis of tuberculosis by polymerase chain reaction

For the diagnosis of extrapulmonary tuberculosis in adults and all forms of tubercular infections in children, microscopic and cultural techniques have (seen shown to be inadequate. Many serological techniques have been employed for non culture diagnosis of tuberculosis. Early promising results have repeatedly given way to subsequent findings of non-specificity. Major mycobacterial antigens have been shown to be heat shock proteins which are highly conserved in nature.DNA probes for tuberculosis are specific but have a sensitivity equivalent to AFB smear examination. Polymerase Chain Reaction (PCR) with its ability to selectively amplify DNA fragments of interest offers a potentially powerful technique for the rapid, specific and sensitive diagnosis of tuberculosis. Samples from partially treated patients could be culture negative but can be detected by PCR.