Burkholderia pseudomallei is generally classified as a gram negative, facultative anaerobic, motile and rod shaped bacteria (Wiersinga et al., 2006, Holden et al., 2004, Dance, 2002). It is formerly known as Bacillus pseudomallei or Pseudomonas pseudomallei, and is later re-named as Burkholderia pseudomallei in 1992 based on 16S rDNA sequence (Currie et al., 2004). Burkholderia pseudomallei has been documented by many researchers as a known causative agent of melioidosis, a disease that can cause acute illness in both humans and animals (Gilad et al., 2007).
B. pseudomallei is motile, due to the existence of lophotrichous flagella (DeShazer et al., 1997). This bacterium has the ability to grow on various types of organic matters such as in carbohydrates, amino acids and fatty acids. At the same time, the morphology the B. pseudomallei colony can be positively identified although it may vary from rough to smooth wrinkled and may either shown to have white or brown pigmentations (Stone, 2007). It can grow in any growth medium with temperatures ranging from 180C to 420C, with the optimum temperature being 37oC (Tong et al., 1996). Nevertheless, the B. pseudomallei has been shown to have a rapid growth curve at 420C in the growth medium and this in turn can cause a depletion or shortage of nutrients within 48 hours and will eventually form a layer of sediment on the plate.
The optimal pH for the growth of bacteria was reported to be in the range of pH 5 to 8. It was also reported that rapid bacterial inactivation occurs when the pH is below 4.5 (Tong et al., 1996). In addition, studies have shown that B. pseudomallei is able to survive when exposed to solutions with less than 2.5% of sodium chloride. However, the bacteria tend to be inactive when exposed to solutions that are more than 2.5% sodium chloride (Inglis and Sagripanti, 2006). This is opposed to the study done by Pumirat et al. (2009), which mentioned that B. pseudomallei can survive in a salty concentration of more than 2.5%. So far, no other studies were conducted on the survival rate of the particular bacteria in seawater but there were reports of melioidosis in survivors of the tsunami disaster that occurred on the 26th December 2004. Those infected were believed to have directly contacted B. pseudomallei through their lungs by ingesting contaminated flood water supply (Chierakul et al., 2005). There were reports regarding the use of chlorine to treat drinking water to eliminate the B. pseudomallei. Studies conducted by Howard and Inglis (2003), showed that a concentration of 1000 ppm of chlorine can eradicate B. pseudomallei in drinking water, though this may not be commercially viable as 1000 ppm of chlorine may cause more harm to human health instead. Furthermore, this bacterium can only survive for 7.75 minutes after exposure to UV light (Tong et al., 1996).
The genome of B. pseudomallei strain K96243, a clinical isolate from Thailand had been sequenced and analysed by Wellcome Trust Sanger Institute, United Kingdom. B. pseudomallei has main two chromosomes, namely, chromosome 1 and chromosome 2 which are 4.1Mb and 3.2Mb in size respectively (Holden et al., 2004). A large percentage of coding sequence in chromosome 1 has been associated with core functions like cell growth and metabolism while chromosome 2 entails more extensive coding sequences that are involved in accessory functions such as survival and adaptation to the environment (Holden et al., 2004). In addition, the National Center for Biotechnology Information currently has listed another 20 genome sequences and among the 20, three were fully annotated. The three strains are strains 1106a, 1710a (Thai clinical isolates) and 668 (an Australian clinical isolate) (Natalie et al., 2009).
The genomic DNA of B. pseudomallei strain K96243 has 16 genomic islands that take up to 6% of the whole genome (Holden et al., 2004). Tumapa et al. (2008) demonstrated that there is variation in the presence of genomic islands among different B. pseudomallei isolates. However, the presence of a specific island does not correlate with the virulence of the isolate. The paper by Tumapa et al. (2008) presented only a few representative islands and strains. There is a possibility that other studies may identify striking differences in terms of genomic characteristics between clinical and environmental isolates. By performing phylogenetic studies, it was demonstrated that B. pseudomallei is not so closely related to B. thailandensis than to B. mallei. This suggested that the evolution of B. pseudomallei is more recent compared to other members of the Burkholderia genus (Ou et al., 2005).
By comparing the genomic, transcriptional and proteomic levels of strain B. pseudomallei K96243 and B. pseudomallei 15682, there were significant intrinsic differences between both strains (Ou et al., 2005). From the data obtained, it was suggested that horizontal gene transfer or gene loss events had occurred as about 43% of the gene expression differences were associated with genes that are not present in one or the other strain. Furthermore, about 38 % of the global proteomic differences were attributed to strain-specific isoforms of proteins expressed in these two strains (Ou et al., 2005). These findings correlate with the results obtained in another two studies that observed the genome-wide variability between B. pseudomallei K96243 and 1026b strains (DeShazer, 2004) and between B. pseudomallei K96243 and B. mallei (Fushan et al., 2005). From all these findings gathered, it can be deduced that variation in phenotypes that are related to growth rate, environmental resistance and virulence may be due to the molecular variation that in different B. pseudomallei strains (Ou et al., 2005).
B. pseudomallei is reported to thrive through saprophytic means. It inhibits stagnant waters or soils, for example, in paddy fields or in flooded areas (Chaowagul et al., 1989). This explains why it is was prevalent among rice farmers and helicopter pilots during the Vietnam War, as most of them were exposed to contaminated soil through ingestion and open wounds (Sanford, 1995).
Incidence of melioidosis that occurred in certain parts of the world however, remains woefully unknown. This is especially in third world countries. This could be due to the fact that some reported cases were poorly documented, under reported or even due to limited resources and unsuitable facilities in some laboratories (Dance, 2002).
One of the reasons why the study of B. pseudomallei generated a world wide interest is due to the fact that it is a potential agent of melioidosis, which can indirectly cause a broader spectrum of chronic illnesses in both humans and animals alike (Gilad et al., 2007). So far, there have been reports showing that animals such as birds, dogs, goats, kangaroos, pigs, camels, horses, cats, rats and dolphins are being infected by melioidosis caused by B. pseudomallei (Ellis and Titball, 1999).
A person could be infected easily by B. pseudomallei through simple means of ingestion of contaminated materials, open cuts or wounds, abrasions or even through inoculation of infected needles during laboratory experiments by researcher themselves (White, 2003). Once infected with B. pseudomallei, it may stay dormant in the host body for years without showing any obvious physical or bodily symptoms (Mays et al., 1975). Examples of reported cases include cases of American soldiers who returned from the Vietnam War, only to start showing symptoms of melioidosis after 30 years. The mortality rate of melioidosis is high and relapses are not uncommon as the disease can affect almost every organ in the host body (Sanford, 1995). Up till now, there is still no vaccine for the prevention of B. pseudomallei infection and an infected person is likely to suffer from either acute septicaemia (blood poisoning) or pneumonia (lung infections) (Deshazer et al., 1998, Reckseidler et al., 2001 and Perry et al., 1995). Wiersinga et al. (2006) and Tiangpitayakorn et al. (1997) also reported that this septicaemic form of melioidosis involves a rapid onset and death is inevitable usually during the first 12 to 24 hours. The disease that was caused by this bacterium was also found to be endemic in Thailand (Chao et al., 1989; Vuddhakul et al., 1999; Leelarasamee 2000), Malaysia (Puthucheary et al., 1992), Singapore (Chan et. al., 1985; Yap et al., 1995; Lim et al., 1997) and northern Australia (Cheng et al., 2008). Besides that, sporadic cases were also reported in Southern Taiwan (Shih et al., 2008), North, Central and South America (Inglis et al., 2006), India (Saravu et al., 2008), Africa, the Caribbean, and the Middle East (Dance, 2000).
Therefore, the incidence of melioidosis should not be taken lightly as it could spiral out of control as it can affect anyone. Furthermore B. pseudomallei has been classified as being a potential bio-agent to be used in future bioterrorism attacks.
Melioidosis in animals
Melioidosis affects animals as much as human. Although there is still no incidence of melioidosis being transmitted from animals to humans, precautions have to be observed at all times. For example, face masks and gloves should be worn during handling of infected animals. Meanwhile, farmers should also be prudent and not hesitant to cull infected live stock though loss is expected as it may cause serious loss of human lives later on.
Treatment of melioidosis
Treatment of melioidosis is challenging due to the fact that B. pseudomallei itself is resistant to certain antibiotics such as gentamicin and streptomycin (Chaowagul et al., 2000). On top of that, it has also been reported that B. pseudomallei could survive in soil and water for many years and, therefore, must have a certain adaptive mechanism which allows them to continue to survive in harsh and stressful environments (Pumirat et al., 2009). Proteomics studies conducted by Pumirat et al. (2009), showed the changes in protein secretions in B. pseudomallei under high salt (sodium chloride) concentration stress. It was reported that bacteria induced with a high salt environment had a 19-fold increase in a beta-lactamase-like protein which render it resistant to beta-lactam antibiotics, and hence, increases its survival rate.
Despite this, treatment of melioidosis is not impossible and the percentage of recovery increases with early detection and proper diagnosis. There were many cases of patients being successfully treated with various types of antibiotics and these include penicillins and cephalosporins (Leelarasamee and Bovornkitt, 1989; Cheng and Currie, 2005). Generally, the treatment would be conducted in two stages; the antibiotic being administered intravenously in multiple doses until the steady state of drugs in blood level is achieved. This would then followed by giving the patients drugs orally to prevent further recurrence or relapse (Cheng and Currie, 2005).
Current studies supporting successful treatments of melioidosis include a study conducted by Simpson et al. (1999), whereby ceftazidime or imipenem were administered intravenously, after the onset of the disease. Nevertheless, the easiest route of administration, the oral route, is still lacking data to support its effectiveness. Despite this, there are evidence in mice studies showing that animals administered with co-trimoxazole as pre-exposure and post-exposure prophylaxis had longer survival rate, compared to the control group (without administration of antibiotics) and those administered with amoxicillin or clavulanic acid (Suppiah et al., 2007).
Virulence determinants of Burkholderia pseudomallei
The huge magnitude of the disease caused by this pathogen may imply that this bacterium utilizes a broad range of virulence factors as means of survival in the infection of animals. It is therefore essential to investigate the roles of virulence factors to understand the pathogenesis of B. pseudomallei. To date, the virulence factors in this fastidious bacterium are currently understudied compared to other pathogenic bacteria. Elucidation of these virulence determinants in B. pseudomallei is currently the main focus of study in the scientific community. A better understanding of these virulence factors can form a rational basis in producing novel vaccines and therapies in curing melioidosis. The completion of B. pseudomallei K96243 genome sequencing by Sanger Institute, genomic sequencing analysis using bioinformatics and the development of genetic tools had facilitated identification and isolation of putative virulence factors (Holden et al., 2004; Reckseidler-Zenteno et al., 2003). Genetic techniques that were used in identifying genes associated with virulence factors include Tn5 transposon mutagenesis (DeShazer et al., 1997; Burtnick, et al., 2001), counter-selection markers that facilitate allelic exchange (Moore et al., 1999; Brown et al., 2004; Burtnick and Woods, 1999, Lopez et al., 2009) and signature-tagged mutagenesis using animal infection models (Atkins et al., 2002, Moore et al., 2004). Apart from the reported genetic manipulation methods, subtractive hybridization was also introduced in search for novel virulence factors (Brown and Beacham, 2000; Reckseidler et al., 2001).
In order to study the virulence factors in B. pseudomallei, animal models had been developed. These animal models include BALB/c mice (Leakey et al., 1998; Hoppe et al., 1999; Liu et al., 2002; Jeddeloh et al., 2003;), Syrian hamster (Brett et al., 1997), diabetic rats (Woods et al., 1993), guinea pigs (DeShazer et al., 1998), pigs (Najdenski et al., 2004) and Caenorhabditis elegans (O' Quinn et al., 2001; Gan et al., 2002).
The diabetic rat model using intraperitonal route of infection was devised following the correlation of diabetes mellitus and melioidosis (Woods et al., 1993). This model had been used by many researchers to study a number of putative B. pseudomallei virulence factors with some success (Sexton et al., 1994; DeShazer et al., 1997; Jones et al., 1997; DeShazer et al., 1998). DeShazer et al. (1998) also utilized guinea pig as an animal model by using intraperitonal route of infection to comparing the lipopolysaccharide mutants with the wild type B. pseudomallei 1026b strain. However, there is no subsequent use of guinea pigs in any literature reported for the study of virulence determinants. Brett et al. (1997) had reported that Syrian hamster were highly susceptible to B. pseudomallei when the animal was infected via intraperitonal route with an LD50 10 CFU. Subsequent studies had reported the use of Syrian hamster to study the putative virulence factors (DeShazer et al., 1997; Jones et al., 1997; DeShazer et al., 1998; DeShazer et al., 1999; Moore et al., 1999; Reckseidler et al., 2001; Ulrich et al., 2004, Warawa and Woods, 2005).
With the employment of genetic tools and animal studies, a number of virulence determinants were identified. The identified virulence factors that are involved in the pathogenesis of B. pseudomallei include capsule (Ahmed et al., 1999; Reckseidler-Zenteno et al., 2005), lipopolysaccharide (LPS) (DeShazer et al., 1998; Burtnick and Woods, 1999), flagella (DeShazer et al., 1997; Chua et al., 2003; Boonbumrung et al., 2006; Chuaygud et al., 2008), Type III secretion systems (Bsa) (Stevens et al., 2002, 2003; Burtnick et al., 2008) and quorum sensing (Valade et al., 2004; Song et al., 2005; Lumjiaktase et al., 2006). Other virulence determinants that are also been studied but play minor roles in pathogenesis include pili (Essex-Lopresti et al., 2005; Brown et al., 2002; Boddey et al., 2006), efflux pumps (Chan and Chua, 2005), a siderophore (Loprasert et al., 2000), exoproducts (DeShazer et al., 1999; Gautheir et al., 2000) and morphotype switching (Chantratita et al., 2007). Exoproducts are secreted factors which are proteases, lipases, lecithinases and haemolysins (Ashdown and Koehler, 1990).
Burkholderia pseudomallei capsular polysaccharide
The polysaccharide capsule is the structure that encapsulates and 'surrounds' the outer-most layer of the bacterium. It interacts with the existing environment and therefore, plays an important role in protecting the bacterium from harsh conditions as well as to give rise to causing virulence in other living beings.
B. pseudomallei extracellular polysaccharide has been classified into 2 major categories which are the capsular polysaccharides (CPS), whereby the polysaccharide is closely in contact with the bacteria cell and the other, slime polysaccharides, which is loosely associated with the cell respectively (Whitfield, 1995). It is difficult to differentiate between both types since CPS may sometimes resemble the slime polysaccharides when they are being 'released' from the particular cell (Whitfield, 1995). Moreover, CPS also contains lipopolysaccharides (LPS) which are similar to other types of polysaccharides found on the surface of the cell (Whitfield, 1995).
Roles of bacterial capsule in Burkholderia pseudomallei
Bacterial capsule in each pathogenic bacterium has its own function(s). Although the role of capsular polysaccharide in B. pseudomallei is still not satisfyingly clear, the capsule basically helps the bacteria in terms of survival as well as to cause virulence.
Previous studies have shown that capsular polysaccharide plays a vital role in virulence by using in vivo animal models such as hamsters and mice. Virulence studies conducted by Reckseidler et al. (2001) and Atkins et al. (2002) showed that mutants in the capsule operon genes wcbB (N-acetylglucosaminyltransferase) and wcbE (mannosyltransferase) were attenuated more than 105 fold in their virulence factors when the mutants were exposed to hamsters and mouse models intraperitoneally and intraveneously. In addition to that, there were also studies that utilize signature tagged mutagenesis to identify 12 genes from the capsule operon that were critical in causing diseases and virulence in mice (Cuccui et al., 2007). Inactivation of the genes wcbB, wcbC and wcbN resulted in reduced survival of these mutants in mice (Cuccui et al., 2007). More recently, Wikraiphat et al. (2009) did a comparative in vivo and in vitro analysis to determine the effects of 3 different types of B. pseudomallei mutants (without lipopolysacccharide, without capsule and without flagelin) in causing virulence in BALB/c mice and in both human polymorphonuclear cells (PMNs) and macrophages (M?s). The lipopolysaccharide and capsule mutants, which were generated through molecular means, were reported to demonstrate a significant decrease in virulence in both mice models as well as in the presence of human PMNs and M?s, suggesting that both lipopolysaccharide and capsule are highly associated with the virulence of the bacterium (Wikraiphat et al., 2009).
On the other hand, there were also studies which indicated the importance of capsular polysaccharide in protecting the B. pseudomallei from host serum cidal activity and opsonophagocytosis by reducing levels of complement C3 deposition (Reckseidler-Zenteno et al., 1995). Another similar study was conducted by Wikraiphat et al. (2009) in which the acapsular B. pseudomallei mutant was exposed to human PMNs and M?s. The experiment showed a decreased level in bacteria residual numbers as compared to the wild type (with capsule), suggesting that without the presence of capsule, the bacteria is more susceptible to intracellular killing by the host's defence systems. Besides that, Wikraiphat et al. (2009) also reported that the presence of the B. pseudomallei capsule provided a mediated resistance towards histatin and lactoferrin, suggesting that the capsule itself is important for the resistance towards certain antimicrobial peptides.
On top of that, B. pseudomallei capsular polysaccharide mutant has also been identified as a possible candidate for vaccine therapy (Sarkar-Tyson et al., 2007). Mice that were vaccinated with mutants in wcbH gene had higher levels of survival with 70% of mice that survived at day 35 as compared to mice that were vaccinated with wild type strain with 40% survivors (Sarkar-Tyson et al., 2007). Thus, this suggested that immunization with killed capsular polysaccharide mutant strains may confer immunity against B. pseudomallei.
Other suggested functions of the bacterial capsule which have yet to be determined in B. pseudomallei include its involvement in the prevention of desiccation, adherence for colonization and resistance to specific host immunity (Roberts, 1996).
Genetic organization of capsular polysaccharide gene cluster of B. pseudomallei
According to the B. pseudomallei genomic sequence, the genes that are responsible for the synthesis and export of capsular polysaccharide in B. pseudomallei are located at the sites 3327179 bp and 3359841 bp of the Chromosome 1 of B. pseudomallei K96243 (Holden et al., 2004). There are 22 genes all together in this cluster (Figure 2.1). The functions of these genes were annotated by Sanger Institute as shown in table 2.1 (Holden et al., 2004). The capsular polysaccharide gene cluster was shown to have homology to the capsular clusters of other bacteria like Escherichia coli, Neisseria meningitidis and Haemophilus influenzae (Reckseidler-Zenteno et al., 2009) This gene cluster however, does not have genes products that show homology to E. coli KpsF and KpsU capsule proteins (Reckseidler-Zenteno et al., 2001) . Furthermore, the genetic organization of B. pseudomallei capsule gene cluster is different from other capsule gene clusters of E. coli, N. meningitidis and H. influenzae as it lacks two transport regions flanking a single biosynthetic region (Reckseidler-Zenteno et al., 2001) (Figure 2.2). Furthermore, the biosynthetic genes are not organized in a continuous transcriptional unit as the genes wcbB and wcbP are separated from the other biosynthetic genes (Figure 2.2). In addition, the E. coli kpsC gene is normally located next to kpsS gene in E. coli capsule gene cluster and this is not the case with the B. pseudomallei wcbA and wcbO genes (Reckseidler et al., 2001) (Figure 2.2).
The genes wcbB, wcbE and wcbH encode for proteins that have high homology to glycosyltransferases from a various species of bacteria (Table 2.1). As the capsule is a homopolymer of mannoheptopyranosyl residues, these genes are most likely to be responsible in the biosynthesis of polysaccharide. Glycosyltransferases functions to catalyze the sequential transfer of sugar residues from nucleotide precursors to the membrane-bound acceptor (Rocchetta et al., 1998).The wcbB gene encodes for a protein that has high percentage of homology to a glycosyltransferase, WbpX from Pseudomonas aeruginosa (Reckseidler-Zenteno et al., 2001). The WcbB protein is important for transferring mannose residues in the capsule synthesis. Reckseidler-Zenteno et al. (2001) and Atkins et al. (2002) demonstrated loss of capsule when ?wcbB mutant was generated. This was further confirmed by Cuccui et al. (2007) by disrupting the wcbB gene using signature-tagged transposon mutagenesis system. Furthermore, the ?wcbE and ?wcbH mutants that were constructed by Reckseidler-Zenteno et al. (2001) did not produce any capsule. The wcbH gene product showed similarity with glycosyl transferase of Caulobacter crescentus with 27.02% identity (Holden et al., 2004). Owing to the findings mentioned, it can be deduced that the genes wcbB, wcbE and wcbH play important roles in the production of capsular polysaccharide.
The genes wcbA and wcbO predicts proteins that show homology to the E. coli proteins KpsC and KpsS respectively. These genes are involved in exporting and synthesizing of capsular polysaccharide in these organisms (Frosch et al., 1993; Roberts 1996). The wcbA gene, together with wcbO gene encode proteins that are homology to the KpsC and KpsS proteins of E. coli and LipA and LipB proteins of N. meningitidis, respectively (Reckseidler-Zenteno et al., 2001). The wcbA mutant was generated by allelic exchange. The wcbA mutant did not produce any polysaccharide and demonstrated attenuated virulence in Syrian hamster model (Reckseidler-Zenteno et al., 2001). Thus, the role of wcbA gene was confirmed to be the gene responsible for production of polysaccharide. The wcbC gene encodes the protein that are homology to KpsD, a periplasmic protein that is involve in exporting capsular proteins in E. coli (Frosh et al., 1992; Kroll et al., 1990; Wunder et al., 1994). ?wcbC mutant was constructed by inserting the trimethoprim cassette into the gene (Reckseidler-Zenteno et al., 2001). It was found that the ?wcbC mutant did not show any decrease in virulence in the hamster model and capsule is still detected (Reckseidler-Zenteno et al., 2001). However, these findings contradicted the findings by Cuccui et al. (2007). According to Cucui et al.s' findings, the amount of capsular polysaccharide had reduced even though the capsular polysaccharide is still present in ?wcbC mutant. These findings were observed using immunoflourescence microscope. Furthermore, ?wcbC mutant was attenuated in BALB/c mice (Cuccui et al., 2007). Therefore, wcbC gene is important for producing capsules.
Based on the annotations provided by the Sanger Institute, the wcbD, wzm and wzt2 genes are putative ATP-binding cassette (ABC) transporters of capsular polysaccharide (Holden et al., 2004). Furthermore, these genes were homology to proteins that transport capsular polysaccharide (Frosch et al., 1991; Kroll et al., 1990; Rosenow et al., 1995). Based on the information by Sanger Institute, the wzm and wzt2 gene products are homologous to KpsM and KpsT proteins of E. coli, CtrC and CtrD proteins of Neisseria meningitidis and BexA and BexB of Haemophilus influenza (Reckseidler-Zenteno et al., 2001). From the genome sequence of B. pseudomallei K96243, the termination codon of wzm gene overlaps the initiation codon of wzt2 gene (Reckseidler-Zenteno et al., 2001). Thus, it can be deduced that these two genes are translationally coupled. The E. coli capsular genes kpsM and kpsT are organized into a single transcriptional unit and these two genes are translationally coupled (Nsahlai, 2001). Although the role of wzm gene in the production of capsular polysaccharide was not studied in B. pseudomallei, this capsular gene was well studied in Escherichia coli and Mannheimia haemolytica (Nsahlai, 2001; Mckerral, 2001). Loss of capsular polysaccharide was observed in E. coli mutant in kpsM was generated (Nsahlai, 2001). The capsule of E. coli was fully restored when Mckerral (2001) performed a complementation study of kpsM in E. coli by transforming a plasmid containing M. haemolytica A1 capsular polysaccharide wzm gene. In addition, Cuccui et al. (2007) had proven that ?wzm and ?wzt2 were attenuated in BALB/c mice. Furthermore, Reckseidler-Zenteno et al. (2001) demonstrated that the ?wzt2 mutant did not produce capsular polysaccharide. Thus, it can be suggested that both wzm and wzt2 genes are important for producing capsular polysaccharide.
Apart from the genes mentioned, Reckseidler-Zenteno et al. (2001) had proven that the wcbP gene was involved in producing capsule. The wcbP gene product shares homology to the YooP protein of M. tuberculosis (Reckseidler-Zenteno et al., 2001). However, YooP protein was annotated as a putative oxidoreductase (Cole et al., 1998). As for wcbT gene, its gene product has high similarity with an Acyl-CoA tranferase from Rhizobium meliloti (Reckseidler-Zenteno et al., 2001). No capsule was observed when this gene was disrupted using allelic exchange approach. Hence, wcbT gene plays important role in capsule production (Reckseidler-Zenteno et al., 2001).
The wcbS gene is most likely to be responsible for producing capsule in B. pseudomallei as the role of this gene has already demonstrated to play a role in capsule production in B. mallei. Furthermore, the percentage homology between these two capsule genes is more than 99% (DeShazer et al., 2001). As for wcbF, wcbG, wcbI, wcbJ wcbQ and wcbR genes, the gene products of these genes are involved in capsular polysaccharide biosynthesis (Holden et al., 2004). In addition, wcbK and wcbL genes are annotated by Sanger Institute as putative GDP sugar epimerase/dehyratase protein and putative sugar kinase respectively (Holden et al., 2004). As for wcbM and wcbN genes, they are annotated as putative D-glycero-d-mannoheptose 1-phosphate guanosyltransferase and putative D-glycero-d-mannohptose 1,7-biosphosphate phosphatase (Holden et al., 2004).
Figure 2.1: Genetic organization of B. pseudomallei K96243 capsular polysaccharide cluster located at the sites 3327179 bp and 3359841 bp of the Chromosome 1 of B. pseudomallei K96243. This capsule cluster is approximately 30kb in size. Genes are not drawn to scale. (Adapted from Reckseidler-Zenteno et al., 2001; Cuccui et al., 2007)
Figure 2.2: Genetic organization of B. pseudomallei, E. coli K5, H. influenzae type b, and N. meningitidis group B capsule gene clusters. Large boxes show the conserved regions while the smaller boxes depict the specific genes that are located within the cluster. The hatched boxes in E. coli K5 capsule gene cluster are the intergenic gaps. Only selected B. pseudomallei capsule genes were shown in this diagram as this is just a comparison among the capsule gene clusters. Single copy of H. influenzae capsule gene cluster is depicted in this diagram. The genes and regions are not drawn to scale. (Adapted from Roberts, 1996)
Genetic tools for studying virulence determinants
Various virulence-associated factors such as bacterial toxins, siderospore and lipopolysaccharide were successfully identified by screening related secreted macromolecules using biochemical purification and immuno detection techniques (Quinn et al., 1997). However, these virulence factors can only be identified by using assays that have already determined, like cytotoxicity and enzymatic acitivities. Furthermore, the genes that are responsible for these virulence-associated factors are not known. Thus, there is a need for genetic tools to not only study the genes that encode for the virulence factors but also able to characterize the virulence factors.
By publishing the annotated genomic DNA of a B pseudomallei strain, a Thai clinical isolate K96243 in 2004 and together with the other 20 genome sequences listed in National Center for Biotechnology website with three strains fully annotated (strains 1106a, 1710a and 668), many putative virulence factors can be identified (Holden et al., 2004; Natalie et al., 2009). This information in turn, this has led to the publication of a list of bioinformatics analysis (Harland et al., 2007; Lim et al., 2007). Furthermore, a number of researchers have published papers on genomic and proteomic studies (Jitsurong et al., 2003; Ou et al., 2005; Rodrigues et al., 2006; Harding et al., 2007; Thongboonkerd et al., 2007; Wongtrakoongate et al., 2007; Osiriphun et al, 2009). The data obtained from these large-scaled studies may aid scientists to having a better understanding of the complex pathways of B. pseudomallei that permit infection, invasion and persistence.
To determine the functions of the putative virulence factors, various genetic techniques have been employed in studying genes by constructing mutants. Several methods had been used and among them are transposon Tn5 mutagenesis system, allelic exchange and insertional methods. Apart from these methods, DNA microarray system, subtractive hybridization techniques and in vivo gene expression technologies (IVET) have been used to identify the bacterial genes that are important for infection.
Transposon mutagenesis system is widely used by scientists to construct mutants by disrupting the genes in the genome randomly. By using this system, a large library of mutants can be obtained easily and loss of specific function and phenotypes related to virulence are screened. This method of screening for virulence factors had led to the identification of genes involved in B. pseudomallei motility (DeShazer et al., 1997), antimicrobial resistance (Burtnick and Woods, 1999; Moore et al., 2004), intracellular life cycle (Pilatz et al., 2006), capsular polysaccharide (Reckseidler et al., 2001), two-component regulatory system that is involved in invasion of eukaryotic cells and heavy metal resistance (Jones et al., 1997), type II O-antigenic polysaccharide (DeShazer et al., 1998) and type II general secretory pathway (GSP) gene cluster (DeShazer et al., 1999). Furthermore, Taweechaisupapong et al. (2005) used transposon mutagenesis system to study the correlation between biofilm formation and virulence. A derivative to transposon mutagenesis had been devised by combining the random insertional mutagenesis with in vivo negative selection in an animal. This derivative method is known as signature-tagged mutagenesis system (STM). In this system, attenuated mutants that died in the animal host can be identified from a mixture of mutants using a variable sequence tag. Cuccui et al. (2007) and Atkins et al. (2002) had utilized this method in studying the genes that are involved in capsular polysaccharide genes, DNA replication and repair, a putative oxidoreductase and a lipoprotein that is essential for intercellular spreading.
Apart from transposon mutagenesis system, allelic exchange technique is also widely used for generating mutants. Allelic exchange, also known as gene replacement or targeted mutagenesis inactivates the function of a particular gene by either deleting the gene itself or replacing the desired gene with an antibiotic resistance cassette.
There are two possibilities exist in performing allelic exchange. This includes allelic exchange using a linear vector and allelic exchange using a circular vector. When a linear fragment is used for mutagenesis of the gene, the DNA sequences of the ends of the fragment have to be homologous to the chromosome (Thongdee et al., 2008). Furthermore, a selection marker has to be incorporated into the linear fragment in order to select for transformants after performing transformation. Thongdee et al. (2008) had demonstrated that some B. pseudomallei strains were able to accept linear double stranded DNA from the environment via natural transformation and integrate the linear fragment into the genome.
An alternative method in performing allelic exchange is to use a circular vector. In this technique, DNA sequences using the regions that are homology to the flanking regions of the gene to be mutagenized were designed and cloned into a non-replicative plasmid. Single and double cross-over mutants can be constructed via homologous recombination depending on the plasmid. Single cross-over mutants are generated by inserting the whole non-replicative plasmid into the gene of interest and thus disrupting the function of that particular gene. Many scientists have generated single cross-over mutants in B. pseudomallei to study polysaccharide gene clusters (Sarkar-Tyson et al., 2007), stationary growth phase sigma factor sS (RpoS) (Subsin et al., 2003), type III translocator protein (BipB) (Suparak et al., 2005), two phospholipase C enzymes (PLC-1 and PLC2) (Korbsrisate et al., 2007), LuxRI AHL-dependent QS system called BpsRI (Lumjiaktase et al., 2006), and alternative sigma factor sE (RpoE) (Korbsrisate et al., 2005). Apart from that, several genes in B. pseudomallei had been mutagenized using counter selection markers that facilitate double cross over. The virulence factors that were studied include flagella (Chua et al., 2003), type III secretion systems (Stevens et al., 2004) and type IV pilus (Boddey et al., 2006).
On top of that, molecular analysis like DNA microarray analysis was also used to study the genes in B. pseudomallei as well as to compare the genomes of B. pseudomallei, B. mallei, and B. thailandensis (Moore et al., 2004; Ong et al., 2004; Kim et al., 2005). Ou et al. (2005) utilized microarray technology to study the molecular mechanisms that leads to phenotypic variability of B. pseudomallei isolates by comparing the genomes, transcriptomes, and proteomes of two natural isolates. Furthermore, microarray technology was used to study iron regulation in both B. pseudomallei and B. mallei as well as investigating B. pseudomallei infection in a hamster model (Tuanyok et al., 2005, 2006). Using DNA microarray, Alice et al. (2006) had conducted a transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in B. pseudomallei.
Besides the genetic tools mentioned above, other methods like subtractive hybridization techniques and in vivo gene expression technologies (IVET) were also utilized for investigating the roles of virulence factors. For instance, Reckseidler-Zenteno et al. (2001) used subtractive hybridization technique to study the function of capsular polysaccharide of B. pseudomallei. With the advent of IVET, Shalom et al. (2007) had successfully located a type VI secretion system locus in the genomic DNA of B. pseudomallei. Apart from diverse array of genetic tools for gene manipulation and analysis, novel allelic exchange vectors were developed specifically to study the B. pseudomallei genes that associated with virulence factors efficiently (Choi et al., 2008; Barrett et al., 2008; Rholl et al., 2008; Hamad et al., 2009; Lopez et al., 2009; Norris et al., 2009).
With the genetic tools mentioned above and emerging of novel allelic exchange vectors, the scientific community has started to understand the molecular and cellular basis of pathogenesis. Nevertheless, important questions like how B. pseudomallei attaches, invades and survives within epithelial and phagocytic cells remain unanswered.
Objective of thesis
Although the genes in the B. pseudomallei capsular polysaccharide gene cluster have been identified (Reckseidler-Zenteno et al., 2001; Cuccui et al., 2007), the functions of capsular polysaccharide is still not clear. Furthermore, the wzm gene that will be investigated in this project is not satisfyingly studied in previous studies (Reckseidler-Zenteno et al., 2001; Cuccui et al., 2007). Hence, to achieve these objectives, a markerless and in-frame deletion ?wzm mutant is constructed and studied using a few in vitro studies that are desiccation survival assay, biofilm formation assay, bacterial aggregation assay and osmotic stress assay. Transmission electron microscopy and scanning electron microscopy will be utilized to observe the loss of capsule in B. pseudomallei. Furthermore, the mutant as well as the wild type will be subjected to tests that deal with the response of these strains with acidic and oxidative conditions.
Hence, the objectives of this thesis are summarized as follows:
- Construct a unmarked and in-frame deletion, acapsular mutant strain of B. pseudomallei UKMS-01 using allelic exchange
- Characterize this acapsular mutant to confirm the loss of capsular polysaccharide
- Investigate the role of capsular polysaccharide with various in vitro assays
Luria Bertani (LB) medium and agar
The contents to prepare a 1 L medium were 10 g of bacto-tryptone, 5 g of bacto-yeast extract and 5 g of NaCl as described by Sambrook et al. (1989). The contents of LB were then dissolved with 1 L of deionized water. The pH of the medium was adjusted to pH 7.0. As for LB agar, 15 g of bacteriological agar was added into LB before autoclaving.
Blomfield medium and agar
The contents to prepare a 1 L medium were 10 g of bacto-tryptone and 5 g of bacto-yeast extract as described by Blomfield et al. (1991). The contents of the medium were then dissolved with 1 L of deionized water. The pH of the medium was adjusted to pH 7.0. As for Blomfield agar, 15 g of bacteriological agar was added into medium before autoclaving.
M9 minimal medium
To prepare 1 L of M9 medium, 6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl and 1 g NH4Cl were added and topped up with 1L of distilled water. This was then autoclaved at 121oC for 15 minutes. Subsequently, 2 mL of 1M MgSO4 and 0.1 mL of 1M CaCl2 that were autoclaved separately were added. In addition, 20 mL of 20% (w/v) glucose solution was added after being filter sterilized.
General molecular biology methods
Restriction enzyme digestion of DNA
All restriction enzymes that were used in this study were purchased from Promega, New England Biolabs and Fermentas. Reaction mixtures for digestion using restriction enzymes were done as recommended by the manufacturers. Generally, digestion were performed using a final volume of 20 L or 100 L and comprised 1x endonuclease buffer, 1- 3 L of each restriction enzymes, appropriate volumes of DNA and sterile distilled water. BSA was added at a concentration of 1x when it was required. All reactions were performed for 16 hours at 37oC.
Ligation of DNA
Generally, the contents of ligation reaction were 1 unit of T4 DNA ligase, 1x ligase buffer (Amersham Biosciences, Sweden), sterile distilled water, vector and insert DNA. The concentration of the vector and insert ratios that were used in this study were normally 1:1 or 1:3 ratio. Reactions were performed at 16oC over night.
Electrophoresis was run at 100 V with 0.7% (w/v) agarose and 0.5X TAE running buffer (Appendix A ). Subsequently, the agarose gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained with water before observing the gel under UV light. All agarose gel photographs were taken using Molecular Imager Gel Doc XR (Bio-Rad Laboratories, USA).
E. coli strain JM109 was used for transformation. Transformation via heat shock method was performed as described by Cohen et al. (1972). A single colony of E. coli strain JM109 was inoculated into 5 mL LB. The culture was then incubated for overnight (~16 hours) at 37oC with 200 rpm agitation. Approximately 200 L of overnight culture was transferred into 20 mL of fresh LB and incubated at 37oC with 200 rpm agitation until the mid log phase of the bacterial growth was reached (OD600 = 0.5). The culture was incubated on ice for 5 minutes. The culture was centrifuged at 4000x g for 10 minutes and supernatant was removed. Cell pellet was resuspended with 500 L of cold 1x TSS (Appendix B ). Approximately 100 L of resuspended culture was transferred to 5 tubes.
Approximately 5 L of ligation mixture was transferred into 100 L of competent cells and the contents were mixed by flicking the tube. The mixture was then incubated on ice for 30 minutes and heat shocked at 42oC for 2 minutes. The tubes containing the mixture were put on ice for 5 minutes. After that, 600 L of LB was added into each tube. The tubes were incubated at 37oC with 200 rpm agitation for 45 minutes. The tubes were centrifuged for 1 minute at 12,000 g and this was followed by removing the supernatant but leaving approximately 200 L of supernatant inside the tube. The cell pellets were resuspended with the remaining supernatant. Approximately 100 L of resuspended culture was plated on LB agar supplemented with the appropriate antibiotics. All plates were incubated at 37oC for 16 hours.
Burkholderia pseudomallei genomic DNA extraction
Genomic DNA extraction of B. pseudomallei strain UKMS-01 was isolated as described by Cutting and vander Horn (1990) but with slight modifications. A single colony of UKMS-01 was inoculated into 50 mL of LB and cultured at 37oC overnight with 200 rpm agitation. The culture was harvested by centrifugation at 4000 g for 10 minutes. The cell pellet was then resuspended with Lysis Solution (50 mM EDTA, 0.1M NaCl, pH7.5). The resuspended cell was then centrifuged again at 4000 g for 10 minutes. The cell pellet was then resuspended with 4 mL of Lysis Solution that contained 10 mg/mL lysozyme. The cell mixture was then incubated at 37oC for 1 hour to allow cell lysis to occur. Subsequently, 0.3 mL of 20% N-lauroylsarcosine was added to the cell mixture and was then incubated at 37oC for 10 minutes.
Genomic DNA was then purified from cell lysis by subjecting the cell mixture to phenol:chloroform (1:1) for three times. Finally, the cell mixture was then subjected to chloroform:isoamyl alcohol (24:1) for four times. The aqueous layer containing genomic DNA was then transferred to a new tube and precipitated with 0.1 volumes of 3M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold absolute ethanol. The tube was incubated for 20 minutes at -20oC to precipitate DNA. It was then subjected to centrifugation at 12 000 g to pellet the DNA. The pellet was washed with 70% ethanol and left to air dry. The genomic DNA was dissolved with 500 L of deionized water and kept at -20oC.
Plasmid DNA extraction
Plasmid DNA extraction was performed using Wizard Plus SV Minipreps DNA Purification System (Promega Corp, USA) according to manufacturer's instructions (Appendix C).
Polymerase Chain Reaction
PCR amplification was done in total volume of 20 l of PCR reaction mixture which consisted of 10 pmol/primer, 1 g template DNA, 2.5 mM dNTPs, 1.5 - 3.0 mM MgCl2, depending on template and primers, 0.5 units of Taq DNA polymerase and 1x PCR buffer. PCR reactions mixtures were performed using PCR amplification kits from Intron (Korea) or Invitrogen (USA). The reaction mixture was then placed into a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, USA).
PCR programs were specific for every reaction performed but it comprised the following: 94oC initial denaturation for 2 minutes, 94oC denaturation for 30 seconds, optimum annealing temperature of the pair of primers for 30 seconds, 72oC extension time depending on the size of PCR fragment synthesized and the denaturation, annealing and extension steps were repeated for 30 cycles. This is followed by 72oC final extension for 5 minutes.
PCR product purification
PCR products were excised from agarose gel and purified using Megaquick Spin (Intron, Korea). The procedure was done as recommended by the manufacturer (Appendix D).
Automated DNA sequencing was done by Macrogen (Korea) or 1stBase Laboratories (Malaysia). The plasmid pCW110 was sequenced using primers NQCAT, NQREV, Plasmid-SEQ-F, Plasmid-SEQ-R, wzm-ApaI-R and wzm-ApaI-F to check the sequence of the insert that was cloned into pDM4. DNA sequences were analysed using BioEdit program and BlastN program through NCBI.
Construction of wzm gene deletion construct, pCW110
Cloning methods were as described previously by Sambrook (1989).Upstream and downstream regions of wzm gene that were approximately 1 kb in sizes were amplified using primers wzm-Aout-F, wzm-ApaI-R, wzm-ApaI-F and wzm-Bout-R as listed in table 3.2. The annealing temperatures for wzm-Aout-F and wzm-ApaI-R were 55oC while the annealing temperatures for primer pair wzm-ApaI-F and wzm-Bout-R were 60oC. Subsequently, the PCR products were digested using ApaI , ligated together and reamplified using wzm-Aout-F and wzm-Bout-R primers with the annealing temperature of 55oC . The 2500 bp fragment was then subcloned into pGEMT-Easy (Promega, USA) (Appendix E) and thus generating pCW-easy. The ?wzm allele was then digested with SpeI and XbaI and cloned into pDM4 to produce pCW110.
Construction of unmarked and in-frame deletion of wzm in B. pseudomallei UKMS-01
Unmarked and in-frame wzm deletion mutant was constructed by performing allelic exchange and sacB counter-selection on sucrose media as described previously (Blomfield et al., 1991; Milton et al. 1996; Edwards et al., 1998; Logue et al., 2009; Hamad et al., 2009).
The deletion construct, pCW110 was conjugatively mobilized from the recombinant E. coli S17-1 ? pir into B. pseudomallei UKMS-01. To perform biparental-mating, 8:1 ratio of overnight culture of recombinant E. coli S17-1 ? pir and overnight culture of B. pseudomallei UKMS-01 was spotted on a LB plate supplemented with 10mM MgSO4. The plate was then incubated overnight at 37oC. The following day, the growth was then scrapped with an inoculating loop and resuspended with 1 ml of 10mM MgSO4. Approximately 100 l aliquots were plated on Blomfield agar supplemented with 200 g/ml chloramphremnicol and 50 g/ml gentamycin.
Excision of the plasmid by a second homologous recombination event that results in either wild type or deletion was selected on Blomfield agar supplemented with 10% sucrose by plating overnight cultures grown at 37oC at 10-1, 10-2, 10-3, 10-4 and 10-5 dilutions. After 48 hours incubation, the colonies formed were checked for chloramphenicol sensitivity. The ?wzm mutant was identified by PCR verification using primers wzmUSDS-SCR-F and wzmUSDS-SCR-R. The annealing temperatures for these two primers were 60oC. The expected size of the PCR product of mutant was 2724 bp while the expected size for wild type was 3420 bp. Both PCR products of mutant and wild type were cloned into pGEMT-easy before sequencing the resulting plasmids, pGEM-deletion and pGEM-wild type using primers M13-F(-20), M13-R(-20) and SCR-F.
Furthermore, the ?wzm mutant was PCR verified by checking the absence of wzm gene using the primer sets; wzm-SCR-F and wzm-SCR-R (616 bp product). To verify whether the mutant was not a merodiploid, the ?wzm mutant was identified by the absence of chloramphrenicol resistance gene from pDM4 plasmid using primers Cat-SCR-F and Cat-SCR-R (905 bp product). The annealing temperatures for primers sets wzm-SCR-F and wzm-SCR-R and Cat-SCR-F and Cat-SCR-R were 50oC.
Phenotypic characterization of acapsular mutant strain (wzm gene) of Burkholderia pseudomallei
Growth curve of B. pseudomallei UKMS-01 and CW-01
Both strains B. pseudomallei UKMS-01 and CW-01 were grown overnight in LB broth with shaking at 180 rpm at 37oC. The following day, fresh LB medium or M9 medium supplemented with 20% glucose were inoculated with overnight cultures at 1:100 dilutions (500 l to 50 ml media). The cultures were incubated at 180 rpm at 37oC and cultures were sampled at various time intervals to measure the OD at 600 nm with U-1900 UV/Vis spectrophotometer 200V (Hitachi, Japan). The OD600nm for each strain was performed in triplicates. The average OD600nm values were used to construct growth curves.
Scanning electron microscopy
Scanning electron microscopy was performed as described by Glauert (1980). Briefly, bacteria were grown to exponential phase and centrifuged at 2,000 x g for 15 minutes. Subsequently, the supernatant was discarded and the pellet was resuspended with McDowell- Trump fixative prepared in 0.1M phosphate buffer (pH 7.2) for at least 2 hours. The resuspended sample was again centrifuged at 2,000 x g for 15 minutes and supernatant was discarded. This was followed by resuspending the pellet in 0.1M phosphate buffer. This step was repeated twice. The resuspended sample was again centrifuged at 2,000 x g for 15 minutes. The supernatant was again discarded and the pellet was resuspended in 1% Osmium tetroxide prepared in the phosphate buffer above for 1 hour. Subsequently the sample was washed two times with distilled water. The resuspended sample was again centrifuged and dehydrated with 50% ethanol for 10 minutes, 75% ethanol for 10 minutes, 95% ethanol for 10 minutes, 100% ethanol for 20 minutes and Hexamethyldisilazane (HMDS) for 10 minutes. Subsequently, the HMDS was decanted from the tube and the tube containing the cells was left in a dessicator to air-dry at room temperature. The dried cells were then mounted onto a SEM specimen stub with a double-sided sticky tape. Prior to viewing in a scanning electron microscope, the specimen was coated with gold, gold/palladium, chromium or carbon.
Biofilm formation assay
This assay was performed as described by Loprasert et al.(2002) but with slight modifications. Bacteria were grown overnight in LB broth at 37oC with shaking at 200 rpm. Prior to biofilm formation assay, non-sterile 96-well polyvinyl chloride (PVC) culture plates were sterilized with 70% (w/v) ethanol and air-dried in a laminar flow cabinet. Approximately 100 l of LB broth was then added to the well followed by 1 l of overnight cultures. Wells were covered and incubated without shaking for 18 hours at 37oC. Then, 1 l from each well was transferred into triplicate wells containing 100 l fresh LB broth or M9 medium (supplemented with 0.5% [w/v] casamino acids) and plates were incubated without shaking at 27oC and 37oC for 18 hours. The medium that was inside the well was carefully removed and the wells were stained with 150 l 1% (w/v) crystal violet for 30 minutes at room temperature. The stain was removed and the wells were washed twice with 175 l sterile deionised water. Crystal violet stain was solubilised by the addition of 175 l DMSO to each well. The absorbance was measured at 595 nm in Bio Rad Model 680 microplate reader (Bio-Rad Laboratories, USA). The OD reading was adjusted by subtracting the OD of wells that contained the media but no bacteria (negative control), following the addition of DMSO, from the overall OD of wells that had bacteria. Each strain was studied in triplicates.
Bacterial cell aggregation assay
This method was performed as described by Loprasert et al.(2002) but with slight modifications. Bacteria were grown for 16 hours with shaking in LB broth at 37oC. Sterile 55 mm Petri dishes containing 3 mL LB broth or M9 medium supplemented with 0.5% (w/v) casamino acids was inoculated 1:1000 with overnight-grown bacteria and incubated without shaking at both 27oC and 37oC for 18 hours. The possible formation of "clumps" in the petri dish was then observed.
Desiccation survival assay
To study the role of capsular polysaccharide in protecting bacteria from desiccation, the bacteria were tested for their survival under conditions of dehydration as described by Ophir and Gutnick (1994). Bacterial cells were grown on LB medium at 37oC with shaking at 180 rpm. 10 l of over night culture was spotted on Millipore filters which were placed on LB plates and incubated at 37oC for 18 hours before desiccation. Subsequently, the filters were removed from the LB plates and placed in empty sterile petri dish plates for 0, 1, 2, 3 and 3 hours at 37oC. The filters were then resuspended with 5 ml of sterile water. A serial dilution of 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 of resuspended cells were performed. The colony forming units (CFU) were measured by plating 100 l resuspended on LB plates. The survival rate was thendetermined by using the following formula: VC before drying/ VCafter drying x 100.
Sensitivity to oxidative stress
To test susceptibility of bacteria to oxidative stress, disc inhibition assays were performed as described by Bauer et al. (1966). A single colony of mutant or wild type strain was streaked on LB agar plate and incubated at 37oC overnight. Subsequently, 4 or 5 isolated colonies were transferred into a 5 ml 0.5% sterile saline solution until the turbidity of the cell suspension reached the same density as the 0.5 McFarland standard. Subsequently, a sterile cotton swab was then dipped into the cell suspension and spread the cells onto the LB agar plates. The plates were left to dry for 5 minutes. Then, 6 mm paper discs containing 10 l of 0%, 2.5%, 5.0%, 10%, 15%, 20%, 25% and 30% H2O2 were placed on the lawn culture and the LB agar plates were incubated in 37oC overnight. The zone of inhibition was then measured the following day. This oxidative susceptibility test was performed in triplicates.
Osmotic Stress assay
To determine whether the capsular mutant strain is sensitive to osmotic stress, the method was performed as described by Subsin et al. (2003). Cells were grown overnight in LB at 37oC with shaking. This was followed by washing and resuspending the bacterial cells in M9 medium supplemented with 4M NaCl. The resuspended cells were incubated at 37oC with aeration during which time aliquots were taken at 5, 24 and 48 hours and the number of CFU was determined by plating dilutions on agar plates. The viability was expressed as percentage of CFU, with the maximum number of CFU being given a value of 100%.
Sensitivity to low pH
To determine the effects of acidic conditions on capsular polysaccharide, both mutant and wild type strains were spread on LB agar plates which were adjusted to pH 5 and pH 7 as a control. Serial dilution of overnight cultures at 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 was performed and spread on LB agar plates. Cell viability was determined by making viable counts. The viability was expressed as percentage of CFU, with the maximum number of CFU being given a value of 100%.