Characterisation and secretion mechanism of Bordetella pertussis autotransporter proteins

The identification and characterisation of new virulence determinants of 5. pertussis is providing important information for understanding the colonisation and survival strategies of the microorganism. B. pertussis deploys a range of surface-associated components to enable its successful colonisation of the host. Bap-5 has been identified as a new member of the B. pertussis autotransporter family of proteins that includes PRN, BrkA, TCF and Vag-8, largely due to its homology at the C-terminus and some other similar regions such as the RGD (integrin-binding) and SGXG (glycosaminoglycan-binding) motifs. The bap-5 gene also exists in B. bronchiseptica and B. parapertussis. Characteristic upstream regulatory sequences such as a ribosome-binding site were not seen in bap-5, but a potential heptameric BvgA-binding motif was identified. The expression of Bap-5 was confirmed by RT-PCR and Western blotting and was shown to be bvg dependent. Although Bap-5 does not possess a typical signal sequence like pertactin (PRN), its surface localisation was confirmed by agglutination and immunofluorescence assays. A potential role for Bap-5 in infection was studied by generating Bap-5 deficient mutants in two strains of B. pertussis. An allelic exchange procedure with the suicide vector pSS1129 carrying the bap-5 gene disrupted with a kanamycin-resistance cassette was used. PCR and Southern blotting confirmed the replacement of the wild-type bap-5 gene with the mutated version. Moreover, SDS-PAGE and Western blotting of outer-membrane preparations of B. pertussis Taberman wild-type and its Bap-5-deficient mutant showed a clear difference in their outer-membrane profile at ~79.9kDa presumably representing the unprocessed form and bands at ~65 kDa and ~16 kDa may represent the processed forms of the protein. The Bap-5 characterisation studies showed that the Taberman Bap-5-deficient strain was less able than the parent strain to colonise the lower respiratory tract of mice and adhesion studies (in vitro) showed that the Taberman parent was better in adhering to certain cell types than the Bap-5-deficient mutant

Comparative analysis of organophosphate degrading enzymes from diverse species

Different types of organophosphorous compounds constitute most potent pesticides. These chemicals attack the nervous system of living organisms causing death. Different organisms produce enzymes to degrade these chemicals. These enzymes are present in simple microorganisms from archaea, bacteria to complex eukaryotes like humans. A comparison of representative eight shortlisted enzymes involved in the degradation and inactivation of organophosphates from a wide range of organisms was performed to infer the basis of their common functionality. There is little sequence homology in these enzymes which results in divergent tertiary structures. The only feature that these enzymes seem to share is their amino acid composition. However, structural analysis has shown no significant similarities among this functionally similar group of organophosphate degrading enzymes

Antibiotic susceptibility profiling and virulence potential of Campylobacter jejuni isolates from different sources in Pakistan

AbstractObjectiveTo determine antibiotic resistance patterns and virulence potential of Campylobacter jejuni (C. jejuni) isolates from clinical human diarrheal infections, cattle and healthy broilers.MethodsAntibiotic sensitivity patterns of C. jejuni isolates were determined by Kirby Bauer Disc Diffusion assay. These isolates were then subjected to virulence profiling for the detection of mapA (membrane–associated protein), cadF (fibronectin binding protein), wlaN (beta–l,3–galactosyltransferase) and neuAB (sialic acid biosynthesis gene). Further C. jejuni isolates were grouped by random amplification of polymorphic DNA (RAPD) profiling.ResultsA total of 436 samples from poultry (n=88), cattle (n=216) and humans (n=132) from different locations were collected. Results revealed percentage of C. jejuni isolates were 35.2% (31/88), 25.0% (54/216) and 11.3% (15/132) among poultry, cattle and clinical human samples respectively. Antibiotic susceptibility results showed that similar resistance patterns to cephalothin was ie. 87.0%, 87.1% and 89%among humans, poultry and cattle respectively, followed by sulfamethoxazole+trimethoprim 40.0%, 38.7% and 31.0% in humans, poultry and cattle and Ampicillin 40%, 32% and 20% in humans, poultry and cattle respectively. Beta–lactamase activity was detected in 40.00% humans, 20.37% cattle and 32.25% in poultry C. jejuni isolates. CadF and mapA were present in all poultry, cattle and human C. jejuni isolates, wlaN was not detected in any isolate and neuAB was found in 9/31 (36%) poultry isolates. RAPD profiling results suggested high diversity of C. jejuni isolates.ConclusionsDetection of multidrug resistant C. jejuni strains from poultry and cattle is alarming as they can be potential hazard to humans. Moreover, predominant association of virulence factors, cadF and mapA (100% each) in C. jejuni isolates from all sources and neuAB (36%) with poultry isolates suggest the potential source of transmission of diverse types of C. jejuni to humans

Post-Operative Complications of Surgery for Chronic Subdural Hematoma (SDH) and Prevention

Objective:  The study aimed to determine the rate and type of complications during surgery for treatment of chronic subdural hematoma and assess ways for their prevention. Material and Methods:  A total of 50 patients of chronic SDH were selected from the Neurosurgery Department of Bahawal Victoria Hospital. Patients were treated surgically with a single burr hole evacuation under local anesthesia, introduced a subdural drain, nursed in a head-down position for 24 hours, and given plenty of fluids orally and intravenous route. The surgical technique involved a formation of a single burr hole at the point of maximum density. Results:  Out of 50, 43 patients recovered smoothly postoperatively and discharged on the 7th postoperative day. Two patients were re-operated due to inadequate evacuation or reaccumulation. One patient developed subdural empyema post-operatively and expired in spite of good antibiotic cover. In one patient subdural drain penetrated the brain parenchyma resulting in dysphasia. Another patient formed an intracerebral hematoma due to irrigation of the cavity with pressure. One patient with GCS 4/15 developed seizures postoperatively and expired after one hour. One patient developed gross subdural tension pneumocephalus after removing the subdural drain was re-operated and recovered. Conclusion:  Single burr hole evacuation of chronic SDH under local anesthesia is the most accepted surgical treatment. Using proper aseptic surgical techniques, the introduction of the minimum necessary length of the subdural catheter to avoid penetration into the brain parenchyma, followed by careful irrigation of the subdural cavity can help prevent complications

Normal Pressure Hydrocephalus: Selection of Patients for Shunt Placement

Objective: To determine the criteria in our setup, for patients with normal pressure hydrocephalus (NPH), based on clinical and radiological findings.Material and Methods: This is multiple center study, including departments of Neurosurgery at LGH, Lahore / KEMU, Lahore. Over 25 years, 240 patients were included purely on clinical criteria of poor memory, sphincter loss, gait apraxia and Evan’s ratio ≥ 0.30 on CT or MRI. All patients had shunting procedures.Results: Hundred and ninety patients were improved and all of them had significant reduction in Evan’s ratio i.e. ≥ 0.06. Only 10 patients had infection out of whom 8 lost to follow up while 2 had shunt revision.Conclusion: Shunting procedures especially VP shunt shows good results if selection is stringent and no co-morbidity is associated

Draft Genome Sequence of the Enteropathogenic Bacterium Campylobacter jejuni Strain cj255.

The enteropathogen Campylobacter jejuni is a global health disaster, being one of the leading causes of bacterial gastroenteritis. Here, we present the draft genome sequence of C. jejuni strain cj255, isolated from a chicken source in Islamabad, Pakistan. The draft genome sequence will aid in epidemiological studies and quarantine of this broad-host-range pathogen

Gaseous and soil OCPs and PCBs along the Indus River, Pakistan: spatial patterns and air–soil gradients

This study presents first-hand information on the occurrence of persistent organic pollutants (POPs) in the ambient air and surface soil along the Indus flood-plain, Pakistan. The sampling campaign was conducted at 15 site locations during 2014–15, along the Indus River (approximately 1300 km). Composite surface soil samples (N = 15) and passive air samples (N = 15) were collected for the estimation of gaseous POPs as well as air–soil exchange to evaluate the POP emission and distribution or dispersion patterns, source tracking, and contribution of the local and regional sources towards POP accumulation in the Indus River system. Among the studied POPs, levels of DDTs and PCBs were noticeably higher in ambient air (50–560 and 10–1100 pg m−3) and in soil (0.20–350 and 1.40–20 ng g−1), respectively. Regarding the spatial patterns, higher DDT concentrations (ng g−1) were detected in the air and soil samples collected from the wet mountain zone (WMZ) (p \u3c 0.05), followed by the alluvial riverine zone (ARZ), low-lying mountain zone (LLZ), and frozen mountain zone (FMZ). The PCB data did not exhibit significant differences (p \u3e 0.05) for the air samples, while PCB concentrations were significantly higher (p \u3c 0.05) in soil from the LLZ, which may be associated with rapid urbanization and industrial activities in this area. The air–soil exchange of DDTs and PCBs showed net volatilization at most of the studied sites except for a few samples from the FMZ and WMZ. Results of this study about air–soil exchange gradients indicate the long range regional atmospheric transport (LRAT) of POPs to the colder areas (FMZ) of Pakistan, where these act as a secondary source of POPs in these areas

Freely dissolved Organochlorine Pesticides (OCPs) and Polychlorinated Biphenyls (PCBs) along the Indus River Pakistan: Spatial pattern and Risk assessment

Freely dissolved OCPs and PCBs were measured by using polyethylene passive samplers at 15 sites during 2014 throughout the stretch of the Indus River to investigate the spatial pattern and risk assess. Levels (pg/L) of dissolved ∑OCPs and ∑PCBs ranged from 34 to 1600 and from 3 to 230. Among the detected OCPs, dissolved DDTs (p,p′-DDE, followed by p,p′-DDT) predominated with levels of 0.48 to 220 pg/L. The order of occurrence for other studied OCPs was as follows: HCB, endosulfans, chlordanes, and HCHs. Spatially, dissolved (pg/L) ∑OCPs varied (p \u3c 0.05) as the following: surface water of the alluvial riverine zone (ARZ) showed the highest levels (114) followed by the frozen mountain zone (FMZ) (52.9), low-lying zone (LLZ) (28.73), and wet mountain zone (WMZ) (14.43), respectively. However, our zone-wise PCB data did not exhibit significant differences (p \u3e 0.05). Principal component analysis/multilinear regression results showed pesticide usage in the crop/orchard fields and health sector, electric and electronic materials, and widespread industrial activities as the main source of OCPs and PCBs along the Indus River. Our results showed that OCPs and PCBs contaminated water intake, playing an important role towards the considerable cancer/non-cancer risk (HI and CR values) along the Indus River Flood-Plain