Ellipsometric-based novel DNA biosensor for label-free, real-time detection of Bordetella parapertussis.

Pertussis (or whooping cough) is a contagious disease mainly affecting infants and children and predominantly caused by Bordetella pertussis followed by Bordetella parapertussis. B. parapertussis causes a milder cough but usually symptomatically appears like B. pertussis infection. Thus the epidemiology of illness caused by B. parapertussis is not well understood. In this study, a sensitive and specific method for the rapid diagnosis of B. parapertussis is presented. The covalent immobilization of thiol-terminated DNA oligonucleotides (ss DNA SAM) on a silicon surface by disulfide bond formation is investigated with atomic force microscopy (AFM) and ellipsometry. The measurements indicated an average layer thickness of 5 ± 0.84 nm for 2 µg/µl concentration and 24 h incubation time. This thickness changed to 8.4 ± 0.92 nm for the same concentration (2 µg/µl) by altering the incubation time to 48 h. Ellipsometric data recorded before and after hybridization of B. parapertussis revealed an increase in mean grain area from 91 nm2 to 227 nm2 and a change in the refractive index from 1.489 to 1.648 for 2 µg/µl B. parapertussis, respectively. This change in the refractive index was used to evaluate the amount of adsorbed molecules and their density. The results showed that the density of adsorbed molecules increased from 0.2 to 0.97 g/cm3 after B. parapertussis attachment, respectively. To confirm the hybridization of B. parapertussis to ss DNA SAM, the ds DNA SAM was denatured and the ss DNA SAM surface was reproduced with an average height variation of 6.42 ± 0.75 nm. This showed the stability of the DNA film that can be tuned by varying the concentration and incubation time, thus providing a robust method for the label-free detection of B. parapertussis other than routinely used PCR detection.

Molecular detection identified a type six secretion system in Campylobacter jejuni from various sources but not from human cases.

AIM: To determine the presence of the T6SS in Campylobacter jejuni from diverse sources. METHODS AND RESULTS: The recently identified type VI secretion system (T6SS) is a bacterial injection machinery that plays a role in virulence, symbiosis, bacterial interactions and environmental stress responses. This system has been recently discovered in the major enteric pathogen Camp. jejuni. In this study, we used multiplex PCR (mPCR), based on conserved genetic markers of the T6SS, to screen 366 Pakistani Camp. jejuni isolates from humans, poultry, cattle, wildlife or waste-water sources. We identified the T6SS in isolates from all of these sources except humans. The overall prevalence of the T6SS among the isolates was 17/366 (4·6%) and the T6SS positive isolates clustered into four different groups. Transcription of the T6SS genes, determined using RT-PCR, was observed in bacteria cultured at 37 or 42°C but not in 37°C cultures adjusted to pH3. CONCLUSIONS: Campylobacter jejuni isolates harbouring T6SS markers genes were identified in livestock and non-livestock sources but in this study we did not identify human diarrhoeal isolates which possessed the T6SS. We demonstrated down-regulation of T6SS in an acidic environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study questions the role of the T6SS in human diarrhoeal disease. Moreover this study did not identify a clear association of Camp. jejuni isolates harbouring T6SS with any of the niches tested. Our study highlights the need to establish the role of the T6SS in environmental survival or virulence.

Bordetella parapertussis outbreak in Bisham, Pakistan in 2009-2010: fallout of the 9/11 syndrome.

Pertussis or whooping cough is a highly contagious community disease mainly caused by Bordetella pertussis and B. parapertussis. We report a minor outbreak of whooping cough (2009-2010) in symptomatic subjects from Bisham, near Swat, Khyber Pukhtoonkhawa province, Pakistan. Interestingly, our results show that all the culture-positive isolates (n = 21) collected from children (average age 3·46 years), were identified as B. parapertussis after routine identification tests and PCR IS481, IS1001 and IS1002. Furthermore, in the affected patients, none had received immunization with diphtheria-pertussis-tetanus (DTPw) vaccine. Therefore, the possibility of the re-emergence of the disease due to limitation of basic health services as a result of the political unrest due to the 9/11 situation is also examined. Moreover, we discuss the importance of vaccinating both adults and children with DTPwPaw vaccine containing both organisms for better protection.

Novel fluorescent protein from Hydnophora rigida possesses green emission.

Fluorescent proteins are a family of proteins capable of producing fluorescence at various specific wavelengths of ultra violet light. We have previously reported the identification and characterization of a novel cyan fluorescent protein (HriCFP) from a reef coral species, Hydnophora rigida. In search of new members of the diverse family of fluorescent proteins, here we report a new green fluorescent protein (HriGFP) from H. rigida. HriGFP was identified, cloned, expressed in Escherichia coli and purified to homogeneity by metal affinity and size exclusion chromatography. The dynamic light scattering and gel filtration experiments suggested the presence of monomers in solution. The peptide mass fingerprint on the purified protein established the identity of HriGFP. HriGFP had excitation peak at 507 nm and emission peak at 527 nm. HriGFP was similar to HriCFP except the last 16 amino acid sequence at the C-terminal; however, they have shown least similarity with other known fluorescent proteins. Moreover the computational model suggests that HriGFP is a globular protein which consists of 6 α-helices and 3 ß-sheets. Taken together our results suggested that HriGFP is a novel naturally occurring fluorescent protein that exists as a monomer in solution.

Virulence factors profile of drug-resistant Escherichia coli isolates from urinary tract infections in Punjab, Pakistan.

Escherichia coli is considered to be the main causative agent of urinary tract infections (UTIs). The primary objective of this study was to investigate the spectrum of five virulence factors among drug-resistant clinical E. coli isolates associated with pyelonephritis and cystitis. A total of 101 samples were positive for E. coli (42 from pyelonephritis cases and 59 from cystitis cases) out of 457 urine samples of patients. Among toxins, haemolysin and secreted autotransporter toxin are found more frequently in isolates causing pyelonephritis (p < 0.020) than cystitis (p < 0.083). The frequent occurrence of P-pili, S-fimbria and protein involved in intestinal colonisation was noted among E. coli isolates associated with pyelonephritis. Overall, the study suggests that clinical isolates associated with pyelonephritis are more virulent than those associated with cystitis and diversified association with various antimicrobial resistance phenotypes was noted.

Novel fluorescent protein from Hydnophora rigida possess cyano emission.

Currently, a broad diversity of fluorescent proteins among marine organisms range from cyano-red emissions. Fluorescent proteins differ in their DNA sequences from green fluorescent protein (GFP). We identified cDNA encoding the gene of a new protein from the reef coral Hydnophora rigida of the Merulinidae family. Both the spectral properties and putative primary sequence of the protein has been determined. The cloned cDNA encode peptide we call HriCFP is comprised of 134 amino acids. It has characteristics of a cyano fluorescent protein (HriCFP) and its sequence is markedly different from known GFP from the hydroid jellyfish Aequorea victoria. HriCFP was cloned, expressed, purified and exist as monomer. The peptide mass finger print on the purified protein confirmed identity of HriCFP.

High proportion of multi-drug resistant Mycobacterium tuberculosis in Saudi Arabia.

289 patients with TB, presented to a single tertiary care unit in Saudi Arabia between 1989 and 1994. The isolate from each patient was tested for in vitro susceptibility to rifampicin, isoniazid, ethambutol, pyrazinamide and streptomycin. 25 patients (8.7%) had isolates resistant to at least 1 anti-tuberculous drug. Single drug resistance (SDR)-mainly isoniazid-occurred in 14, and resistance to at least 2 drugs (multi-drug resistance-MDR) in 11, of which 8 were due to both isoniazid and rifampicin. Previous drug treatment occurred significantly more often in patients with MDR (8/11), than SDR (1/14) (p = 0.0021). A literature review of another 5571 patients from Saudi Arabia with TB revealed an incidence of resistance of M. tuberculosis isolates to at least 1 anti-tuberculous drug tested, of between 5.9% and 44%. The overall percentage of patients with resistant tuberculosis (including our own patients) was 14.9%. Resistance to streptomycin (8.9%), isoniazid (6.6%), and rifampicin (6.1%) were the commonest reported. There were as many patients with MDR as there were SDR. A history of previous anti-tuberculous treatment was found in over 40%. The high rate of anti-tuberculous resistance in Saudi Arabia may be due to poor supervision of anti-TB treatment, the embryonic healthcare system, over-the-counter antibiotic availability, treatment of endemic diseases such as brucella with rifampicin etc., a large migrant work force, and possibly increased toxicity of anti-tuberculous drugs secondary to the high incidence of chronic liver disease in the country.