molecular biology research

Molecular Biology Research in Bangladesh in collaboration with

National and International Institutes and Organizations

Presented By –Muhammad Asaduzzaman, B.Pharm (DU), M.Pharm (DU),

Lecturer, Dept. of Pharmacy, East West UniversityFor –

Dr. Abul Kalam Azad Chowdhury Professor, Dept. of Clinical Pharmacy & Pharmacology, University of Dhaka

andFormer Vice Chancellor, University of Dhaka

1. Biotechnology to solve some local health problems: arsenicosis, molecular characterization of pathogens, emergence of multi drug resistance, development of drugs for multidrug resistant pathogens

2. Antibiotic Susceptibility and Molecular Characterization of Salmonella Paratyphi A Isolated in Bangladesh

3. Emergence of Optochin-resistant Streptococcus pneumoniae: Implications for Diagnosis and Management of Pneumococcal Diseases

4. Molecular Characterization of Tubercle bacilli of ancestral and modern type

5. Antioxidants (a recipe of vitamins, β-carotene, selenium ; and Applephenone® in detoxification of Arsenic induced oxidative injury in rabbits

6. Pachypodol, a flavonol from the leaves of Calycopteris floribunda, inhibits the growth of CaCo colon cancer cell line in vitro

7. Antibacterial activity of two limonoids from Swietenia mahagoni against MDR bacterial strains

List of work done and work in progress

Biotechnology to solve some local health problems: arsenicosis, molecular characterization of pathogens, emergence of multi drug

resistance, development of drugs for multidrug resistant pathogens

A.K. Azad Chowdhury, Sheikh Zahir Raihan, M. Asaduzzaman, Nishat Nasrin and Nahid Akhter, A. K. M. Shahidur Rahman, Mohammad Shawkat Ali, Dept. of Clinical Pharmacy & Pharmacology, University of Dhaka, Dhaka-1000

&Golam Hasan Rabbani, Mahbubur Rahman, and Kaiser Ali Talukder, Internation Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka

presented in the Conference on - “Promotion of Biotechnology in Bangladesh: National and

International Perspectives”, April 6-8, 2007, Dhaka, Bangladesh

Antibiotic Susceptibility and Molecular Characterization of Salmonella Paratyphi A

Isolated in BangladeshK. A. Talukder1, M. Asaduzzaman2, Z. Islam1, M. Aslam1, A. S. Mondol1, I. J. Azmi1, M. A. Islam1 and A.K.A. Chowdhury2

1. ICDDR,B, 2. Dept. of Clinical Pharmacy and Pharmacology, University of Dhaka

Enteric fever (both typhoid and paratyphoid fevers) continues to be a major public health problem even after the development and advent of newer antimicrobial drugs.

Salmonella enterica serovar Paratyphi A is the second most common cause of enteric fever after S. Typhi in developing countries including Bangladesh.

The total number of typhoid fever cases was estimated to be 21.7 million with 216,510 deaths per year. The global annual incidence of paratyphoid fever was found to be 5.4 million (Crump et al., 2004).

Recently, in in Asia gives there is an increase in paratyphoid fever and, in the first five weeks of 2006, the number of infections of Salmonella Paratyphi A and Salmonella Typhi increased in the UK (Nair et al., 2006).

Current, effective vaccines against typhoid fever do not work against paratyphoid fever and moreover some strains of Salmonella Paratyphi A have become resistant to the current antibiotics of choice, called fluoroquinolones.

Conference on - “Promotion of Biotechnology in Bangladesh: National and International Perspectives”, April 6-8, 2007, Dhaka, Bangladesh


Isolated in Bangladesh

Objective:

The major objective of this study was to characterize Salmonella Paratyphi A strains using phenotypic and genotypic traits. A total of 524 strains of Salmonella Paratyphi A were isolated at Clinical Microbiology Laboratory from patients attending the Dhaka treatment center of ICDDR,B as well as from patients referred from other clinics and hospitals in Dhaka between January 1999 and June 2004.

Methodology:

Of these, 31 strains were characterized extensively using serotyping, antibiotic resistance analysis, plasmid profile analysis and pulsed-field gel electrophoresis (PFGE) and DNA sequencing was performed to investigate the resistance mechanism against fluroquinolone antibiotics.


K. A. Talukder1, M. Asaduzzaman2, Z. Islam1, M. Aslam1, A. S. Mondol1, I. J. Azmi1, M. A. Islam1 and A.K.A. Chowdhury2




Results:

Among the 31 strains, 32% (n=10) were resistant to nalidixic acid. Strains that showed reduced susceptibility to ciprofloxacin by disk diffusion method, had elevated MIC value (0.5µg/ml).

Analysis for mutation in the gyrA and parC genes in the QRDR by sequencing revealed a point mutation in the gyrA gene at codon 83 (TCC to TTC, which substitutes phenylalanine for serine) whereas no mutations were found in parC gene.

Of 31 strains, 16 (52%) S. Paratyphi A strains harbored 35 MDa middle-ranged plasmid which were found to be non-conjugative.

Pulse-field gel electrophoresis (PFGE) showed that most of the strains (83%) were clonal regardless of antimicrobial sensitivity patterns.






Results:

KD-1487

ATCC 9150

Ser

Phe

Fig. BLAST sequence result showing single amino acid change in peptide sequences between test (KD-1487) and reference (ATCC 9150) strains






Results:

Fig. PFGE showing clonal relationship among the Salmonella Paratyphi A strains

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Fig. Agarose gel electrophoresis showing presence of 35 MDa plasmid among the Salmonella Paratyphi A strains




Emergence of Optochin-resistant Streptococcus pneumoniae: Implications for Diagnosis and

Management of Pneumococcal DiseasesM. Rahman1, H. Rashid1, N. Nasrin2, K. Zaman1, N. Nahar1, and A.K.A. Chowdhury2


Acute respiratory tract (ARI) infections are now the principal causes of death in children under five years of age throughout the world including developing countries.

Pneumonia accounts for up to 30% of all deaths in children less than 5 years of age (Leowski, 1986) and for 70% of the deaths (about 8,000) in children who die each day (Anonymous, 1990).

ARI is the leading cause of death for infants in Bangladesh and in most developing country settings, accounting for 25-33% of deaths. New, safe effective vaccines have been developed, but the burden of pneumococcus in Bangladesh is unclear.

Pneumonia is a frequent occurrence with incidence rates of at least 1 per 10 children per year in rural settings and >1 episode per child per year in urban settings.



Management of Pneumococcal Diseases

Even in ideal situations pneumococci are difficult to isolate, and so the vast majority of pneumococcal infections are unrecognized. And consequently treatment failure occurs due to Misdiagnosis or wrong diagnosis.

The optochin susceptibility test remains the primary and, in some cases, the only method in clinical microbiology laboratories to differentiate S. pneumoniae from α-hemolytic viridans streptococci.

However, emergence of optochin resistance in S. pneumoniae results in misidentification of pneumococci, lowering their isolation rate that jeopardizes the diagnosis, treatment and prevention of pneumococcal diseases.


M. Rahman1, H. Rashid1, N. Nasrin2, K. Zaman1, N. Nahar1, and A.K.A. Chowdhury2




Objective:

The main objective was to observe the emergence of optochin-resistant S. pneumoniae and also to indicate the necessity genotypic method for diagnosis.

Methodology:

Fifteen Hundred nasopharyngeal swabs of mothers and infants enrolled in the study were subjected to the tests used to identify S. pneumoniae such as colony morphology, gram staining, optochin susceptibility and bile solubility tests and finally confirmed by lytA (autolysin) PCR.

Antimicrobial susceptibility test and MIC value detection were also performed to see the emergence of drug-resistance pattern among the strains.






Results:

In total, 111 optochin-resistant α-hemolytic streptococci strains were detected. When subjected to bile solubility test, 37 (33.3%) optochin-resistant, bile soluble S. pneumoniae were obtained, as other α-hemolytic-streptococci were not bile soluble.

An S. pneumoniae-specific lytA gene PCR was positive in all 37 isolates confirming their identification. Thus, the optochin susceptibility test failed to differentiate S. pneumoniae from other streptococcal species, showing its decreasing sensitivity and specificity.

Optochin-resistant S. pneumoniae had significant co-resistance to other antimicrobial agents (64.86% were resistant to penicillin, 78.37% to co-trimoxazole, 78.37% to ciprofloxacin, 45.95% to tetracycline and 27.03% to azithromycin/erythromycin) and multi-drug-resistance (resistant to 3 drugs, 64.86%).






Results:

Percentage of optochin resistant S. pneumoniae

91%

7% 2% Total No. of samples

Optochin resistantisolatesOptochin resistant S.pneumoniae

Fig. Percentage of isolation of optochin resistant S. pneumoniae.

Fig. Percentage of isolation of optochin resistant S. pneumoniae.




Molecular Characterization of Tubercle bacilli of ancestral and modern type

Saira Banu1, Nahid Akhter2 and A.K.A. Chowdhury2


Molecular characterization of the two types of Tubercle bacilli (modern and ancestral) by molecular typing of the genomes collected from different places such as, prison, district hospitals and TB clinics of Dhaka city.

Comparative study of three finger-printing methods as tool for epidemiological studies of M. tuberculosis:

1. Deletion analysis

2. Spoligotyping method

3. MIRU-VNTR

(The work is still in progress)

Antioxidants (a recipe of vitamins, β-carotene, selenium ; and Applephenone® in detoxification of

Arsenic induced oxidative injury in rabbits

• Inorganic arsenic contamination of drinking water from various sources has been the cause of chronic arsenic poisoning in many countries including Bangladesh

• Arsenic concentrations in drinking water in Bangladesh have been found to be higher than WHO recommended standards of .01mg/L.

• The mechanisms of arsenic toxicity in man are poorly understood, although the involvement of oxidative stress, DNA damage, and genetic abnormalities has been suggested.

• During oxidative metabolism, arsenic produces free radicals such as superoxides anion (O2

‑) and hydrogen peroxide (H2O2) which induce lipid peroxidation and cell death.

Golam Hasan Rabbani1, Sheikh Zahir Raihan2, and A K Azad Chowdhury2


Journal of Environmental Science and Health Part A- Toxic/Hazardous Substances & Environmental Engineering , A38 (1), 273-287, 2003


Arsenic induced oxidative injury in rabbitsGolam Hasan Rabbani1, Sheikh Zahir Raihan2, and A K Azad Chowdhury2


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Figure. Arsenic-induced changes in whole blood GSH, serum TBARS, and serum NOx concentrations.

Pre-arsenic

Post-arsenic



Arsenic induced oxidative injury in rabbitsGolam Hasan Rabbani1, Sheikh Zahir Raihan2, and A K Azad Chowdhury2


Figure. Effects of polyphenols and the recipe of vitamins on whole blood GSH and serum NOx concentration in arsenic-intoxicated rabbits. Changes due to treatment are expressed as percentage increase or decrease from the values obtained in the arsenic-intoxicated rabbits.

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Pachypodol, a flavonol from the leaves of Calycopteris floribunda, inhibits the growth of

CaCo colon cancer cell line in vitro

• Calycopteris floribunda Lam., commonly known as ‘goichia lata or goache lata’, is a

large climbing woody shrub from Bangladesh, and well distributed in a number of other south-east Asian countries. Traditionally, C. floribunda has been used in colic, as an antihelminthic, astringent and carminative, and for the treatment of diarrhoea, dysentery, jaundice and malaria in many countries including Bangladesh.

• Pachypodol (5,4′-dihydroxy-3,7,3′-trimethoxyflavone) has been isolated from the leaves of C. floribunda by repeated column chromatography on silica gel, and the structure confirmed by spectroscopic means.

• The general toxicity of pachypodol was determined by the brine shrimp lethality assay• The cytotoxic potential of this flavonoid has been evaluated by the Promega’s CellTiter

96 Non-Radioactive Cell Proliferation Assay using the CaCo-2 colon cancer cell line (IC50 = 185.6 μM).

Phytother. Res. 2008 Published online in Wiley InterScience (www.interscience.wiley) DOI:10.1002/ptr.2539

Hosne Ara Ali, A K Azad Chowdhury, Abul K M Rahman, Tomasz Borkowski, Lutfun Naharand Satyajit Sarker. Phytother. Res. 22:1684-87. 2008

http://www.interscience.wiley/

Pachypodol, a flavonol from the leaves of Calycopteris floribunda, inhibits the growth of

CaCo colon cancer cell line in vitroHosne Ara Ali, A K Azad Chowdhury, Abul K M Rahman, Tomasz Borkowski, Lutfun Nahar

and Satyajit Sarker. Phytother. Res. 22:1684-87. 2008

Fig. Structure of pachypodol (1) isolated from Calycopterisfloribunda

Table. General toxicity and cytotoxicity of pachypodol, respectively, towards brine shrimps and the CaCo-2 colon cancer cells

Phytother. Res. 2008 Published online in Wiley InterScience (www.interscience.wiley) DOI:10.1002/ptr.2539

http://www.interscience.wiley/

Antibacterial activity of two limonoids from Swietenia mahagoni against MDR bacterial strains

• Solvent partitioning followed by column chromatography of the MeOH extract of the seeds of Swietenia mahagoni afforded two limonoids, swietenolide (1) and 2-hydroxy-3-O-tigloylswietenolide (2).

• The compounds were identified by spectroscopic means. The antibacterial activity of these compounds was assessed against eight MDR bacterial strains (clinical isolates) by the conventional disc diffusion method.

A. K. M. Shahidur Rahman, A. K. Azad Chowdhury, Husne-Ara Ali, Sheikh Z. Raihan, Mohammad S. Ali, Lutfun Nahar and Satyajit D. Sarker. J Nat Med. 63:41-45. 2009

J Nat Med (2009) 63:41–45 DOI 10.1007/s11418-008-0287-3

Current Interests

• Study of polymorphism of PPARG genes and its association with the pathogenesis and progression of type 2 diabetes mellitus

• Study of polymorphism in CYP genes of Bengali population, cloning of the genes in E. coli for studying drug metabolism and isolation of metabolites for future drug developments

Study of polymorphism of PPARG genes and its association with the pathogenesis and

progression of type 2 diabetes mellitusColin N A Palmer ([email protected] ), Thomas Friedberg ([email protected] ), Sabina Yeasmin, Munirul Alam, S. Monira, Muhammad Asaduzzaman, and A.K.A. Chowdhury

Study of the molecular basis of metabolic syndrome has revealed that malfunctioning of peroxisome-proliferator-activated receptors (PPARs) is responsible for the syndrome (includes type-2 diabetes mellitus) which is characterised by abdominal obesity, atherogenic dyslipidemia, elevated blood pressure, insulin resistance with or without glucose intolerance.

An insulin resistant state following nuclear peroxisome proliferator activated receptors (PPARs) deactivation is the key phase of metabolic syndrome development.

The study of polymorphisms of the genes of PPARs, especially PPARG gene, is very important to understand the pathogenesis of type 2 diabetes which in turn will help design effective drugs to control it.

Type 2 Diabetes mellitus is on the rise in Bangladesh10, it is important to study the polymorphism in PPRAG genes in the Bengali population as the genetic polymorphisms are closely related to the ethnic groups and races.

mailto:[email protected]


Study of polymorphism in CYP genes of Bengali population, cloning of the genes in E. coli for

studying drug metabolism and isolation of metabolites for future drug developments

Interindividual variations in the efficacy and toxicity of drugs are dependent on the

polymorphisms of the genes producing enzymes (CYPs) responsible for the metabolism of the drugs.

Polymorphism in the genes of CYPs causes varied drug response. The polymorphism of the CYP genes has been worked out in many races and ethnic groups throughout the world but very little in Bengali population.

The study has been designed to find out the polymorphism in PPARG and selected CYP genes in Bengali population and their effect on the pathogenesis of the type 2 diabetes mellitus and that on the efficacy and toxicity of antidiabetic drugs (thiazolidine diones).

Colin N A Palmer ([email protected] ), Thomas Friedberg ([email protected] ), Sabina Yeasmin, Munirul Alam, S. Monira, Muhammad Asaduzzaman, and A.K.A. Chowdhury



molecular biology research

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