ABSTRACT
Genus Klebsiella from faeces of sloth bears was screened by using culture morphology, Gram’s staining, biochemical tests and polymerase chain reaction. Our results showed that out of 60 samples collected, 22 samples (36.67%) were cultured on Klebsiella Selective Agar Base with Klebsiella Selective Supplement and Gram’s stain revealed rod-shaped Gram-negative organism with purple-magenta colony - like colonies. The biochemical tests of cultured samples revealed negative to indole production and methyl red test, positive to Voges-Proskauer test, positive to Simmon citrate utilization test, negative to H2S production and that produced acid over acid reaction in TSI agar and positive to urea production in cultured samples. All Klebsiella species isolates were sensitive to azithromycin followed by enrofloxacin and resistant to clindamycin and methicillin. The gyrA gene was amplified by PCR for the genus Klebsiella and found to be positive of 36.67%. This study may provide information for developing strategies in the future in the control of Klebsiella species infections in sloth bears
ABSTRACT
A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.