Application of PCR based-RFLP for species identification of ocular isolates of methicillin resistant staphylococci (MRS).

BACKGROUND & OBJECTIVE: Early detection of methicillin resistant staphylococci (MRS) from clinical specimens enables institution of appropriate antimicrobial therapy. Limited information is available on speciation of MRS. This study was undertaken to compare results of conventional and molecular methods in detection of methicillin resistance (MR) and application of PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing for speciation of ocular isolates of MRS. METHODS: A total of 110 consecutive ocular staphylococcal isolates were screened for MR. MRS was speciated by PCR-RFLP of gap gene and results were confirmed by DNA sequencing. All isolates were processed within 48 h of isolation. A single colony of bacterium, stocked as stab cultures in Hyer's and Johnson agar, was stored at 4 degrees C and sub-cultured at every 15 days interval. RESULTS: Seventy (63.6%) of 110 isolates were identified as MRS and 40 (36.4%) were MSS by conventional and molecular method (100% correlation). Of the 70 MRS, 18 (25.7%) were Staphylococcus aureus, remaining 52 (74.3%) were CNS by conventional and molecular method (100% correlation). PCR-RFLP of gap gene identified 18 (25.71%) MRS as S. aureus, 11 (15.71%) S. epidermidis, 27 (38.57%) S. haemolyticus, 6 (8.57%) S. cohnii subsp. urealyticum, 6 (8.57%) S. equorum, 1 (1.42%) S. xylosus and 1 (1.42%) S. hominis. INTERPRETATION & CONCLUSION: Overall rate of isolation MRS was 63.6 per cent and were predominantly isolated from conjunctival swab (23.6%) and donor corneal scleral rim (23.6%) of non hospitalized patients indicating their community origin. Detection of MR by mecA gene was easier and less time consuming compared to conventional methods. Speciation of MRS was possible by gap gene PCR - RFLP and the predominant MRS in our study was S. haemolyticus.

Clonal diversity of New Delhi metallobetalactamase-1 producing Enterobacteriaceae in a tertiary care centre.

PURPOSE: New Delhi metallobetalactamase-1 (NDM-1) production is a major mechanism of resistance to carbapenems among the Enterobacteriaceae and is a cause for concern in the field of microbial drug resistance. This study was performed to detect NDM-1 in Enterobacteriaceae and to determine the clonal relatedness of NDM-1 producing Escherichia coli and Klebsiella pneumoniae isolated from patients admitted in a tertiary care centre. MATERIALS AND METHODS: A total of 111 clinically significant Enterobacteriaceae isolates, resistant to cephalosporin subclass III were screened for carbapenemase production by the modified Hodge test. Minimum inhibitory concentration to imipenem and meropenem was determined and interpreted according to Clinical Laboratory Standards Institute 2011 criteria. Presence of bla NDM-1 was detected by polymerase chain reaction. To ascertain clonal relatedness, random amplification of polymorphic deoxyribonucleic acid (RAPD) was carried out for representative NDM-1 producers. RESULTS: bla NDM-1 was detected in 64 study isolates, of which 27 were susceptible to carbapenems. RAPD revealed a high degree of clonal diversity among NDM-1 producers except for a small clustering of isolates in the neonatal intensive care unit. CONCLUSION: There is extensive clonal diversity among the NDM-1 producing E. coli and K. pneumoniae. Hence, antibiotic selection pressure rather than horizontal transfer is probably an important operating factor for the emergence of NDM-1. This calls for increased vigilance, continuous surveillance and strict enforcement of antibiotic policy with restricted use of inducer drugs.

Complication of salmonella bacteremia in a case of treated fungal endophthalmitis.

This is to report a case of bacteremia caused by Salmonella typhi in a treated unilateral fungal endogenous endophthalmitis in an 18-year-old male from one of the South Asian countries. Microbiological and molecular investigations were carried out on the eviscerated material and routine blood culture was carried out. Direct examination of eviscerated material revealed the presence of fungal filaments. However, Salmonella typhi was isolated from both specimens, which was confirmed by Polymerase chain reaction targeting the 16SrRNA gene, sequencing, and random amplification of polymorphic DNA showed that they belonged to the same clone. The presence of Salmonella bacteremia in a treated unilateral fungal endophthalmitis, among young adult patients is rare and systemic symptoms should be investigated.

Fluorescent probe studies of micropolarity, premicellar and micellar aggregation of non-ionic Brij surfactants.

The dipolar nature of trans-2-[4-(dimethylamino)styryl]benzothiazole (DMASBT) in its twisted intramolecular charge transfer (TICT) excited state makes it useful as a surface probe for phenomena such as premicellar and micellar aggregation of non-ionic Brij surfactants. The process of micellization of Brij 35, Brij 58, Brij 78, and Brij 98 through the formation of smaller premicellar aggregates results in a progressive change in the nature of the DMASBT molecule and its location in the aggregates, reflecting the changes in its photophysical properties, which have been studied using steady-state fluorescence, fluorescence anisotropy, and time-correlated single-photon counting measurements. The microenvironment polarity around the DMASBT probe in the micellar phase is greater than that in corresponding premicellar phases. The orders of premicellar as well as micellar concentrations are Brij 35 > Brij 58 > Brij 98 > Brij 78. The lower fluorescence anisotropies observed in the case of Brij 78 aggregates compared to those in other Brijs studied could be due to the accessibility of a nonrigid environment as a result of a folded conformation of a part of the nonpolar long chain of surfactant molecules near the core of aggregates. Three different locations of DMASBT were noted for Brijs 35, Brij 78, and Brij 98, whereas for Brij 58 only two locations are observed. The micropolarity of the environment around DMASBT in aggregation states has been determined.

Defining the different phases of premicellar aggregation using the photophysical changes of a surface-probing compound.

Photophysical changes of a cylindrical compound undergoing twisted intramolecular charge transfer may be used as a surface probe to study the different phases of premicellar aggregate formation. The probe molecule, trans-2-[4-(dimethylamino)styryl] benzothiazole (DMASBT), attaches itself to the premicellar and the micellar aggregates of cationic, anionic, and neutral surfactants in different orientations because of its dipolar nature in the excited state. The micelle formation is preceded by a few typical rearrangements of the surfactant molecules. These events need proper inspection that can only be done by compounds that sense environmental changes by residing in the vicinity of the surface of those aggregates. Steady-state and time-resolved fluorescence spectroscopy coupled with steady-state fluorescence anisotropy measurements serve as a very useful tool to monitor premicellar aggregate formation. The dipolar interaction of DMASBT with the surface of the aggregate and its extraordinary capability to sense the polarity of the environment make it a very efficient molecule to use for the purpose.