MS Research at Environmental Microbiology & Molecular Ecology Laboratory

Department of Microbiology, University of Dhaka.

 

 

 

       

 

 

 

ISOLATION & MOLECULAR CHARACTERIZATION OF SHIGELLA  FROM FOOD & WATER IN SLuM AREAS OF BANGLADESH

 

 

 

 

 

Introduction

Shigella spp is one of the most life-threatening pathogen that is responsible for approximately 10% of total diarrhoeal cases (Stoll et al., 1982). In developing countries, shigellosis is most common in children less than 5 years old and is usually spread by the excreta of infected individuals either directly by faecal-oral route or by water, contaminated food or flies. Shigella spp was isolated most frequently in the winter (October – January) and in the hot summer (April – May) with high morbidity and mortality rates in developing countries like Bangladesh. According to World Health Organization (WHO) each year about 163 million peoples are affected by shigellosis epidemics in developing countries and of them 1.1 million episodes (0.7%) result in death. it was found that Shigella  flexneri was dominating over other species in case of shigellosis. The prevalence of shigellosis elucidated that in between 1990-2001 (data collected in International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR,B) 49% of the total cases were due to S. flexneri and S. dysenteriae comprised only 28%. Shigellosis is a waterborne disease and considered as priority waterborne disease by World Health Organization (WHO). In Asia and the Pacific, fecal pollution is one of the most serious problems, affecting both surface water and ground water bodies and leading to a tenacious persistence of water born diseases. The estimated uploading of biological pollution of the aquatic environment may increase up to 18 times than the present status. Therefore it could be easily deemed that aquatic environment might play the fundamental rule as sink for Shigella spp. But yet to date no successful protocol has been set up for detection of Shigella spp form the environmental samples. This is because when they are cultivated in conventional culture media that are mostly designed for clinical organisms, a large number of environmental microbial cells have been reported to remain in a “viable but non-culturable” (VBNC) state (Colwell et al.; 2000). This means that although the cells are viable, they cannot divide or grow in conventional culture media, but will be able to grow when introduced into human intestine (Colwell et al., 1996). The proponents of the VBNC concept emphasize that owing to unfavourable conditions e.g. stress, injury, starved etc a large population of cells do not recover in culture media. The idea is that given optimal pH, temperature, nutrients, growth stimulators and imposing most favourable growth conditions or repairing the injured cell by designing such media with verities of organic infusions so that the recovery of the microbial population will be increased and this ultimately lead to devise culture media that support the growth of VBNC population such as Shigella spp

Shigellosis occurs as a disease endemic in Bangladesh, and at least three large epidemics caused by Shigella dysenteriae type 1 have occurred between 1972 and 1994, causing high morbidity and mortality, particularly in children (Chen et al., 1980; Katz et al., 1986). In Bangladesh, the predominant species of the genus Shigella are S. flexneri and S. dysenteriae type 1; there are other two species of Shigella named Shigella sonnei and Shigella boydii which are also potential enteric pathogens. In many developing countries with inadequate sanitation, fecal contamination of environmental waters by enteric pathogens is very common. In Bangladesh alone, Shigella dysentery causes 75,000 deaths among the children of younger than five years annually during peak epidemic years and 35,000 deaths in non-epidemic years (Bannish and Woltyniak, 1991). Therefore it is very pertinent to understand that whether Shigella spp can survive and persist in the environment in absence of primate host.

 

General Objective

Designing of a complete protocol for detection and isolation of Shigella spp from the aquatic environment and foods. Its molecular characterization and determining the ultimate impact of environmental parameters on the survival and growth of Shigella spp.

 

Specific Objectives

1.         To design pre-enrichment and enrichment culture technique in order to increase the recovery of Shigella species from the aquatic environment.

2.         To isolate the predominant clinical Shigella isolates.

3.         To design appropriate microcosm models for survival of Shigella spp.

4.         Plasmid profile analysis and comparison of the environmental and clinical isolates of Shigella spp.

5.         Determination of the protein profile of the Shigella spp in various growth stages when exposed to deferent environmental stresses.

6.         Isolation and characterization of  protein profile of Shigella spp and its relation to VBNC.

7.         Development of antibody against the late protein of Shigella spp.

8.         Assessment of gene expression of Shigella spp under environmental conditions.

9.         Microbial community structure analysis of Shigella spp in the environment through PCR-DGGE method.

10.       Determination of the organization of virulent genes among the environmental isolates of Shigella spp.

 

Materials and Methods  

Enrichment and Isolation Through Culture Technique

The recovery of Shigella spp from the environmental sample is still a mystery.  Most enteropathogenic bacteria are subjected to various strains and stresses when discharged into the environment. As a result recovery declines

For this purpose the initial aim will be the designing of enrichment technique for increasing the yield of Shigella spp and its close relatives in the samples. A unique enrichment technique will be designed in such a way that the first enrichment will enhance the recovery of total bacterial popultion and the second enrichment will targeting the enrichment of Shigella spp in the samples. To get a particular type of bacteria from any sample we usually use selective plate, these selective plates have some sort of toxicity against the other bacterial population. There is a great probability that this selective toxicity may affect the target bacterial population since they are very fragile in the environment. To solve this problem, our plan is to design a selective media that also permit the growth of such fragile cells present in environmental samples.

Colony blot Hybridization

Colony blot hybridization is the latest way to isolate specific type of bacteria from a mixed culture by using probe specific to the bacteria. In case of colony blot hybridization, the enriched broth samples will be plated on non-selective media then colony blots will be prepared and subjected to hybridization with specific probe or a double hybridization can also be followed. It is fact that not all non-selective media support the growth of total bacterial population present in the sample. So an attempt will also be taken to design new non-selective media to support the maximum recovery of the bacterial population using aerobic, anaerobic and micro aerobic conditions.

Usually ipaH is used for the identification of Shigella spp ( Faruque et al.; 2002). Labeled ipaH probe will be used for this purpose. The colonies that will show positive signal in colony blot hybridization will then be subjected to various biochemical tests for reconfirmation of the isolates to be Shigella spp.

Polymerase Chain Reaction (PCR)

After colony blot hybridization the isolated strains will then be subjected to PCR for identification of the isolates. ipaH probe will be used for colony blot hybridization but it is also reported to be present in certain type of Escherichia coli.  To decipher this question polymerase chain reaction (PCR) will be carried out to identify the presence of ial, virG, ipaBCD genes among the isolates as these genes are the virulence specific genes of Shigella spp.

Plasmid profile analysis

The preliminary isolates of Shigella spp will then be subjected to plasmid profile analysis according to the procedure given by Kado and Liu (1981). The presence of 140 MDa invasive plasmid will be used as a marker specific for Shigella spp. Plasmid profile analysis will also help to characterize the divergence of isolated strains.     

Microcosms study of Shigella spp in laboratory and in the environment  

Non-culturablity of the environmental Shigella spp is a common phenomenon. But very few information are available about why and how this pathogen undergoes VBNC state. For this purpose a set of microcosms together with the known Shigella spp will be designed in the laboratory as well as a special devise will be set in the aquatic environment with some known clinical and reference strains of Shigella in such a way that the devise will permit only the natural condition but not the other autochthonous flora of the biosystem. These researches will be focused on the metabolic state of the Shigella spp from culturable to VBNC in the environment through in situ analysis. For this purpose, the designed devise will be light penetrable 1L enclosures with dialysis membrane to allow continuous equilibration with nutrients and other chemicals outside the enclosures will be filled with filter-sterilized pond or lake water and specific shigellae strain will be added to the system at a concentration of 106 CFU/ml. This devise will be kept in a typical eutrophic aquatic ecosystem at a depth of 0.25 m.  Sample will be taken from there time to time.

Molecular analysis of strain viability

A portion of the collected sample will be used for determination of the viability of the inoculated strain through culture technique and direct viable count through microscope, the rest of the sample will be preserved in RNA later which will be analysed later by DNA-microarray technique or RT-PCR. The results from DNA microarray and RT-PCR analysis will elucidate the expression of genes in the viable cells. This will provide enough information about how, why and whether Shigella persists in the environment as VBNC state? The viability of the microorganism will also be determined through substrate uptake experiments, in which uptake of 3H-labeled thymidine and 14C-labeled glucose and acetate will be measured. Fluorescence microscopy with AODC, ITN or DAPI will also be applied for instant detection and enumeration of total viable cells in the samples

 Protein profile analysis

The protein content of the Shigella outer membrane structure at VBNC state and the culturable state will be determined to know the difference between the protein content in the cell wall structure. The protocol for this purpose will be followed as described by Merrell et al. (2001) and gradually monoclonal antibody against the protein of VBNC Shigella spp cell wall that differs from that of the culturable Shigella spp will be developed in order to detect VBNC Shigella spp afterwards. 

 

 

 

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