Sehrish Firyal

Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrug-resistant Mycobacterium tuberculosis isolates from Pakistan.

The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin-resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan.

DNA typing of Pakistani cattle breeds Tharparkar and Red Sindhi by microsatellite markers

Microsatellite markers are used for any individual identity and breed characterization in animals that is an efficient and successful way of investigation. They are used for multiple purposes as genetic detectors including, rapid mutation rate, high level of polymorphism, and range of variety of microsatellite markers available. A panel of 19 microsatellite markers was developed for breed characterization in Tharparkar and Red Sindhi breeds of cattle in Pakistan. Forty four blood samples of cattle (each breed) were collected from Department of Livestock Management, Sindh Agriculture University, Tandojam, Tando Qaiser, Tharparkar Cattle Farm Nabi sar Road, Umer Kot, Sindh, and Govt. Red Sindhi Cattle Breeding Farm, Tando Muhammad Khan Pakistan. Breed characterization was 100% successful. Average PIC, He and Power of Exclusion values were found to be 0.91, 0.62 and 13.28, respectively. Pattern of allelic frequencies of most of the microsatellite markers were clearly distinct between two breeds. As a result of present study a reliable, efficient and very informative panel of microsatellite markers was successfully developed which was capable to interpret individual identity, forensic cases and breed characterization in cattle. This facility is ready to be provided to local cattle breeder at commercial level for DNA testing of cattle. This study will also be highly helpful for breed conservation of cattle. In addition this study can also become a basis to open up new disciplines of animal forensics in Pakistan.

Molecular classification of Pakistani collared dove through DNA barcoding

Pakistan is bestowed by a diversified array of wild bird species including collared doves of which the taxonomy has been least studied and reported. DNA barcoding is a geno-taxonomic tool that has been used for characterization of bird species using mitochondrial cytochrome c oxidase I gene (COI). This study aimed to identify taxonomic order of Pakistani collared dove using DNA barcoding. Purposely herein, we present a phylogenetic analysis of Pakistani collared dove based on 650 base pairs of COI gene sequences. Analysis of phylogenetic tree revealed that Pakistani collared dove shared a common clade with Eurasian collared dove (Streptopelia decaocto) and African collared dove (Streptopelia roseogrisea) which indicated a super-species group in Streptopelia genus. This is the first report of molecular classification of Pakistani collared dove using DNA barcoding.

In vitro activity of Nigella sativa against antibiotic resistant Salmonella enterica

Salmonellosis is a major food-borne disease worldwide and antimicrobial resistance in Salmonella is a public health problem. Phytochemicals are alternative therapeutics to treat antibiotic resistant Salmonella. Biochemically identified Salmonella enterica of human and poultry origin (n = 10) were confirmed by polymerase chain reaction. Susceptibility to antibiotics was determined by Kirby Bauer disc diffusion method. In-vitro anti-salmonella activity of N. sativa essential oil and extracts (aqueous and methanol) was determined against antibiotic resistant isolates by well diffusion and micro broth dilution method. Cytotoxic potential of N. sativa was observed by MTT assay. In S. eneterica the highest resistance (100%) was detected against nalidixic acid and ampicillin followed by oflaxacin (80%), tetracycline, co-trimoxazole and amoxicillin (60%), ciprofloxacin (40%) and gentamicin (20%). Methanol extract of N. sativa produced zone of inhibition from 35 ± 1.00 to 17 ± 1.00 with mean MIC value ≥562.5 ± 384.1 μg/mL. Essential oil showed antibacterial activity with zone of inhibition from 20 ± 1.00 to 14 ± 1.00 mm and mean MIC value ≥1000.0 ± 322.7 μg/mL. Aqueous extract had no anti-salmonella activity. MTT results showed more than 50 percent cell survival at concentrations >625 and >1250 μg/mL for methanol extract and essential oil of N. sativa respectively; concentrations less than cytotoxic values required for anti-salmonella activity. It was concluded that N. sativa had in-vitro activity against S. enetrica and can be used as therapeutic.

In-Silico Analysis of Gene Als2 Genetic Variants Identified in the Affected Horses and Humans with Motor Neuron Disease

ABSTRACT Motor neuron disease (MND) is a spontaneous neurologic disorder of humans and adult horses, which results from the degeneration of motor neurons in the spinal cord and brain stem. The etiology of MND disease in the South Asian region is not well understood. To achieve the objective of the study, blood samples were collected from both affected humans and horses. DNA was purified by inorganic method; subsequently, ALS2 gene was sequenced both in humans and horses of Pakistani origin diagnosed with MND. In case of affected individuals, by targeted sequencing, we identified two novel ALS2 mutations, the missense substitution c.194T>C (p.Phe65Ser) located in the first RCC1 domain of ALS2 and the nonsense mutation c.2998delA (p.Ile1000*) located in the PH domain of ALS2. In case of affected horses, we identified two missense substitution mutations c.247G>A (p.Ile83Val) and c.914T>G (p.Leu305Arg) in the ALS2 gene. In‐silico analysis was performed by PolyPhen‐2 and SIFT of identified genetic variants to predict whether a substitution of specific amino acid affects protein function and to understand the pathogenicity of identified genetic variants. Our results suggest that genetic variants identified in human families were found to be pathogenic, whereas missense substitutions identified in horse ALS2 gene were not causing MND in affected horses. HighlightsDegeneration of motor neurons in the spinal cord and brain stem causes motor neuron disease (MND) in adult horses.In this study, ALS2 gene was sequenced both in humans and horses of Pakistani origin diagnosed with MND.In humans, two novel ALS2 mutations, the missense substitution located in the first RCC1 domain of ALS2 and the nonsense mutation located in the PH domain of ALS2 were recorded; whereas in horses, two missense substitution mutations in the ALS2 gene were found.Genetic variants were found to be pathogenic, whereas missense substitutions identified in horse ALS2 gene were not causing MND in affected horses.

Detection of methicillin-resistant Staphylococcus aureus of poultry and human sources

Staphylococcus aureus(S. aureus ) is a common gram positive anaerobic facultative pathogen of human and birds; and mostly resides on skin, wounds, mucous membranes, alimentary & urogenital tracts, soft tissue infections. Resistance in S. aureus against antibiotics has been increasingly reported though depending on the clonal lineage. Among the resistant strains methicillin resistant S. aureus (MRSA) is a major threat to the community as its genome is more proneto evolution. The emergence of MRSA is distressing in community that embodies a model for emergence of this uncontrollable super bugs. The molecular based detection and characterization of MRSA has been unstudied in the local population of human and poultry. In the present study50 isolates (n= 42 were recovered from poultry external naries, respiratory tract and feathers) and (n= 08 were recovered from naries and hands of poultry workers) were used after biochemical and bacteriological identifications. MecA gene (527 bp)was amplified for the detection of MRSA followed by and sequencing using specially designed primers. The obtained sequences were compared with reported data and phylogenetic analysis was done. Comparative analysis indicated 2 polymorphic sites in the local isolates. Phylogenetic analysis showed that local poultry isolates were mono-phyletically claded with S. aureus MRSA and shared the same clade with human MRSA isolates. This indicated that this organism has a zoonotic potential. This is the first report of detection and sequencing of MRSA isolates recovered from poultry and human samples.