Is lactate an undiscovered pneumococcal virulence factor?

Abstract
Streptococcus pneumoniae is Gram-positive alpha haemolytic bacteria that commonly found in the nosphrynax of elderly people and young children, it causes approximately 2 million deaths mostly children under age of 5 year and people over 60 years of age. Most important diseases caused by S. pneumoniae are including pneumonaie, meningitis and bacteraemia. The pathogens can be transmitted through contact. Streptococcus pneumoniae obtains its energy mainly carbohydrates through fermentation process. However, in some situations where there are limited sugars or in the presence of galactose the homolactic fermentation is shifted to mixed fermentation in which in addition to lactate, ethanol, formate and acetate are formed. In this study, the role of lactate (lactic acid) and formate (formic acid) in bacterial competition and cytotoxicy was investigated. We hypothesised that lactic acid and formic acid are able to contribute to the virulence of streptococcus pneumoniae. Bacteria were grown on either BHI or BAB. The killing assay was done by exposing various acids on S.pneumoniae as control then lactic acid producing bacteria and non-acid producing bacteria was tested with these acids. Growth assay experiment was done followed by cytotoxicity test using A549 epithelial cells incubated for 24h. The effect of lactic acid for killing assay was significant. Similar effect was seen when lactic acid was exposed to A549 cells. However, a hydrochloric acid acid was unable to inhibit the growth of bacteria. This study concludes that lactic acid produced by Streptococcus pneumoniae is a potential virulence factor and may contribute to Streptococcus invasive disease.
Chapter 1 Introduction
1.1 The biology of Streptococcus pneumoniae
Streptococcus pneumoniae is a normal inhabitant of human nasopharynx, and it is a member of lactic acid family that gets its energy mainly by the process of fermentation. It is Gram positive, and catalase negative. Under light microscope S. pneumoniae can be seen in pairs and short chains. In blood agar they can be seen as ?-haemolytic.
S. pneumoniae is a fastidious facultative anaerobe that requires highly nutritious medium for growth. It grows in Brain Heart Infusion (BHI) media, as well as Blood Agar Base (BAB). This bacterium also grows in chemically defined medium that contains nutrients, such as vitamins, glucose, amino acids (Table 1) and pyruvate. However, the most easily observable characteristic of S. pneumoniae is its sensitivity to optochin (ethylhydrocupreine). This makes the pneumococcus distinguishable from other alpha haemolytic streptococcus. Like other Gram positive bacteria S. pneuminae possess three major surface layer that can be distinguishable: cell wall, plasma membrane and capsule (Alonsodevelasco, E. et, al 1995). There are more than 90 serotypes of S.pneumoniae based on their capsular polysaccharides coats.
1.2 The Genome:
The complete genome sequence of a type 4 isolate of S. pneumoniae comprises a single circular chromosome of 2,160,837 base pairs (bp) about 40% of G+C content(Tettelin et al., 2001). This genome contains 2236 predicted coding regions; of these genes around 64% are assigned a biological role (Tettelin et al., 2001). It also contains 73 noncoding RNA genes that include four rRNA operons. Moreover, S. pneumoniae has a high capacity for DNA uptake (Hoskins, J. et al 2001).
The pneumococcal genomes contain a considerable number of insertion elements such as transposon remnants and repeat sequences. The large number of insertion elements in the genome indicates that the pneumococcal genome is exposed to common inter and intra-genomic events. ( Lanie.J.A.,et, al. 2006)
1.3 The diseases caused by S. pneumoniae and their epidemiology:
The diseases caused by the pneumococcus is life threatening and include pneumonia, meningitis, bacteraemia and septicaemia. Additionally, it also causes otitis media, sinusitis, osteomyelitis, and peritonitis. The microorganism is also responsible for endocarditis, and septic arthritis (Kilian, 2007). The diseases caused by S.pneumoniae are results from either direct extension from the nasopharynx or by invasion and haematogenous spread.
Pneumonia is a very important cause of mortality and morbidity amongst elderly people.
( Nagaoka, S. et al..). Despite the availability of pneumococcal vaccine this microorganism still pose a great challenge to any attempt to eradicate or limit the spread of the disease because rising antibiotic resistance and limitations of vaccines. The pneumococcal infections are responsible for more than 1.6 million deaths each year worldwide (WHO, 2008). The highest incident of this disease occurs in children under the age of 5 year and in the elderly. Also very high incident in patients with predisposing conditions such as asplenia and those who are immune-compromised are reported.
1.4 The pneumococcal virulence determinants
1.4.1 Capsule: S.pneumoniae possess polysaccharide capsule which is considered as the most important virulence factor, because unencapsulated pneumococcus is almost harmless while the encapsulated bacteria from the same species cause disease (Alonsodevelasco,et,al 1995). It has been found that encapsulated strains are approximately 105 more virulent than unencapsulated strains (Alonsodevelasco,et,al 1995) I general, the vast majority of Streptococcus serotypes are unable to produce potential virulence (Lysenko, et al. 2010). The survival of the serotype in the blood stream and ability to cause invasive disease are mainly determined by the chemical structure of capsule polysaccharide and thicknes of capsule (Alonsodevelasco,et al. 1995).
1.4.2 Protein virulence determinants: Recent studies discovered that there are proteins that also contribute to virulence. They include, but are not limited to, hyaluronate lyase,(berry, M. et al., 1994), pneumolysin (Paton, J.c., et al 1986), neuraminidases (Elizabeth A. et., al 2002), galactosidases (Terra et al., 2010) and pyruvate formate lyase (Yesilkaya et al., 2009).
1.4.2a Hyaluronate lyase: Hyaluronate lyase degrades the hyaluronan, which is a hyaluronic acid derivative and its one of the most important polysaccharide component of animals, into disaccharide as a final product (Songlin, et al., 2000). Study carried by Berry et al. (1994), suggests that hyaluronidase plays vital role in migration of streptococci between tissues, in particular translocation from the lungs to vascular system. The other way in which hyaluronidase contributes the streptococcal pathogenesis is by allowing huge number of microorganisms to host tissue for colonization (Berry et al. (1994).
1.4.2b Pneumolysin: This is a membrane damaging toxin which inhibits neutrophil chemotaxis, phagocytosis and respiratory burst (Greenwood, D. et, al 2007). The sulfhydryl-activated cytolysin toxin functions by binding to cholesterol in host cell membranes. (Paton, J.c., et al 1986). It also damages blood vessels in the lungs and therefore, causes bleeding into air spaces. Moreover, pneumolysin leads to the activation of the classical complement pathway and the depletion of serum opsonic activity (Lock, R.A., 1988)
1.4.2c Neuraminidases: This enzyme is able to cleave N-acetylneuraminic acid from glycoproteins, such as mucin, oligosaccharides, and glycolipids on host cell surfaces. S. pneumoniae expresses several distinct neuraminidases. Studies carried out by Elizabeth A. et., al (2002) has indicated that neuraminidase activity might promote the colonization by decreasing the viscosity of mucus(Tong et al, 2000). The two neuraminidases (NanA and NanB) are part of virulence factors that cause disease (Tong et al, 2000). Although there are three forms of neuraminidases, NanA, NanB and NanC , the most abundant neuraminidase and probably the most important one is the Neuraminidase A(NanA ). Almost all the S. pneumoniae that were investigated has shown to have neuraminidase activity (Anirban et al., 2010). These investigations showed that NanA contributed to the colonisation of pneumococcus in the nasopharynax and also the development of otitis media (Anirban et al., 2010).
1.4.2d Galactosidases: Galactosidase is an important enzyme, that catalysis the hydrolysis of galactose from oligosaccharides (Jeong et al.,2009; Terra et al., 2010), These enzyme can be found in most mucosal microorganisms and they exist in different forms specific for individual galactosidic bonds. The size of the galactosidase depends on the type of the organisms. Nevertheless, most prokaryotic galactosidases are large proteins. Regarding the galactosidases virulence contribution in streptococci pneumoniae is not yet fully understood. However, study carried out by (Terra et al., 2010) exclusively showed that galactosidase is hugely important in mucindegradation. This study also investigated the role of galactosidase in pneumococcal virulence and eventually achieved that galactosidase is essential for survival in the nasopharynx (Terra et al., 2010)
1.4.2e Pyruvate formate lyase (PFL): PFL is a metabolic enzyme that is responsible for the conversion of pyruvate into formate and acetyl CoA under anaerobic or microaerobic conditions. This enzyme is produced in inactive form and posttranslationally activated by pyruvate formate- lyase activating enzyme (Leppanen, et al., 1999).
Pyruvate formate lyase (PFL) activity mediates mixed acid fermentation. Monosaccharides, such as galactose converts a considerable percentage of pyruvate to acetyl-CoA in both microaerobic and anaerobic conditions of glycerol. Study carried by (Yesilkaya et al., 2009) indicates that PFL/PFl-AE is essential for in vivo fitness of the pneumococcus. The study concluded that lack of PLF is able to influence alteration of lipid composition in cell membrane and reduction of pneumococcus virulence.
Despite considerable efforts, it is still not known completely how pneumococcus causes disease in its host. Therefore the study of S.pneumoniae virulence determinants is an important approach to developing new therapies such as vaccines and antibiotics.. Recent studies are showing that the pneumococcal fermentative metabolism is an important contributor to pneumococcal virulence.
1.5 The pneumococcal fermentative metabolism
The Lactic acid bacteria (LAB), one of which is the pneumococcus, receive its energy from fermentative breakdown of carbohydrates. These group of bacteria maintains fermentative metabolism regardless of presence of oxygen (Yesilkaya,et, al,2009). The pneumococcus undergoes fermentative metabolism, because pneumococcus lacks genes (approximately 18 genes) that are essential for respiration. The process that these bacteria undergoes is a classical pathway known as Embden-Meyerhof pathway which activates the breakdown of carbohydrate and eventually results the production of pyruvate, NADH and two moles of ATP (Yesilkaya, et al 2009).
Figure 1: Schematic representation of the lactate pathway in lactic acid bacteria. LDH, lactate dehydrogenase, PFL, pyruvate formate lyase, iPFL, inactive pyruvate formate lyase, PFL-AE, pyruvate formate activating enzyme, PDH, pyruvate dehydrogenase, POX, pyruvate oxidase, ADH, alcohol dehydrogenase, ACK, acetate kinase, PTA, phosphotransacetylase. (Taken from Yesilkaya, 2009)
NAD+ regeneration occurs through lactate dehydrogenase catalysed conversion of pyruvate to lactate. In some cases, in particular where there is limited sugars or in the presence of galactose the homolactic fermentation is shifted to mixed fermentation in which in addition to lactate, ethanol, formate and acetate are formed. The mixed- acid fermentation is mediated by PFL in anaerobiosis or microaerobiosis. Aerobically, pyruvate dehyrogenase complex (PDHC) contributes to the transformation of pyruvate but S. pneumoniae lacks genes for PDHC (Yesilkaya, et al 2009).
The process of shifting from hololatic fermentation to mixed-acid product formation is mainly explained by the allosteric modulation of the enzyme such as lactate dehydrogenase and pyruvate formate lyse which compete for pyruvate. Fructose-1,6-diphosphate (FDP) is an essential activator of LDH. In L. lactis glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) are strong inhibitors of PFL. The importance of fermentative and metabolic by product of streptococcus pneumonia was investigated in this study. The lactic acid and formic acid plays a major role in contributing virulence factors by killing other microbiota found in the nasophrynax and lungs. Similarly, these acids contribute the inflammations, since it has been reported that this acids cause both inflammation and ulceration. An experiment carried out by (Sakurazawa & Ohkusa, 2005) showed that organic acids could induce apoptosis and hence this cytotoxicity can contribute to the pathogenesis of ulcers (Sakurazawa & Ohkusa, 2005).
1.5.1 The Bacterial completion and the role of different bacterial products in other microorganisms
To colonise a new habitat emerging bacteria have to compete with previously colonised microorganisms, the competition determination depends on the number of bacterial populations that colonise a particular region of the host. Various ecological factors contribute to the colonisation of bacteria in the host, including the availability of natural resources. These resources, such as nutrients and spaces are limited in nasophrynax (Margolis et al. 2010). The established bacteria can produce toxins and harmful substances to inhibit the colonisation of incoming bacteria. Similarly, the host immune response plays crucial role in determining the colonisation of the bacteria.
A study investigating the role of hydrogen peroxide in the human nasopharynx showed that it is capable of eliminating various bacterial species in the respiratory tract (David,2003). Streptococcus pneumoniae and Haemophilus influenzae, are co-inhabitants in the upper respiratory and both cause life threatening disease. However, these pathogens compete in space and nutrients. Production of toxic chemicals is part of the space competition. Hydrogen peroxide produced by S.pneumoniae acts as an antimicrobial agent to eliminate growth of other bacteria. Indeed, a David (2003) showed that hydrogen peroxide produced by S. pneumoniae caused rapid killing of Haemophilus influenzae. Interestingly, exogenous catalyse exposure has exhibited safeguarding of H. influenzae and no killing activity of hydrogen peroxide was observed. This suggests that hydrogen peroxide may be responsible for this bactericidal activity. Moreover, S. pneumoniae that was unable to produce hydrogen peroxide did not exhibit killing effect for H. influenzae. Furthermore, other respiratory pathogens were affected by the hydrogen peroxide produced by S. pneumoniae since these chemicals killed other respiratory tract pathogens such as Moraxella catarrhalis and Neisseria meningitidis (David,2003). Production of hydrogen peroxide by S. pneumoniae also possess cytotoxic affects on host cells and tissue (Weiser, et al 2003) The mechanisms in which S. pneumoniae survive with endogenous hydrogen peroxide concentrations that are able to kill other species are not understood.
1.5.2 Haemophilia influenzae: Haemophilia Influenza is a Gram negative coccobacillus, rod shape nonmotile and non spore forming bacterium. It is facultative anaerobe fastidious bacteria that were identified in 1892. The chromosome sequence of Haemophilus nfluenza 1.83Mb was the first completed genome sequence (Tatusov, et al., 1996). Apparently, Haemophilia influenza is an obligate parasite and a resident of the upper respiratory system of humans (Tatusov, et al., 1996). Haemophilia influenza consists of encapsulated, and non encapsulated strains. The type B strain is recognised as the most virulent strains, since it cause the majority of the Haemophilia influenza invasive diseases. This bacteria is a leading cause of death children and amongst elderly people. It also causes number of different life-threatening diseases. Although, the type b strain is important in H. influenza invasive disease, there are other encapsulated trains such as serotype A, which is very similar to that of type B. Encapsulated strains have the ability to cause an important invasive disease such as meningitis. H. influenza strain can cause mucosal infections, including otitis media, conjunctivitis, sinusitis, bronchitis, and pneumonia.
Staphylococcus aureus
Staphylococcus aureus is a Gram positive spherical (coccus ) that resembles and arranged in grape-like cluster (Greenwood, D., 2006). S aureus form hemolytic on blood agar, the organism is facultative anaerobes and opportunistic pathogen bacteria. The organism is non-sporing and non-motile, and is able to grow both with and without oxygen (facultatively anaerobic), and catalase-positive. Staphylococcus aureus causes wide range of diseases, ranging from superficial lesions to life-threatening septicaemia. (Charlier,C, 2008). The skin is the best ecological niche for S. aureus. These organisms usually found in upper respiratory tract as microbiota and are common in animals. Healthy individuals carry the organism in nasophyranx and hands as well.
1.5.3 Streptococcus suis
The bacteria used in this study include Streptococcus suis, which is a Gram- positive bacteria, mainly a pathogen of pigs but also found in other animals such as goat, sheep and cattle. Recently, this microorganism was isolated from various animals, such as horses cats and dogs. The organism is carried in the nasopharynx of pigs and mainly transferred from pigs to human where there are physical contacts to pigs or during the consumption of pig meat (Barbara, 2006 p494). S. suis can cause various severe and life treating infections such as meningitis, bacteraemia, septicaemia, arthritis and bronchitis. Approximately 2000 incidences have been discovered in areas where pig products are used namely Netherlands and Denmark. Although, there are no human S.suis infection outbreaks, but there are several incidences reported in china. Serotype 2 and serotype 5 are the most dominant pathogenic serotypes that cause illness. In this study S. suis has been used merely because it is a member of the lactic acid producing bacteria. Although S.suis is zoonotic bacterial pathogen and mainly found in pigs and other animals it also isolated from humans. Streptococcus suis is similar to other Lactic acid Bacteria since it is a member of LAB. It causes similar diseases which may elicit the exact mechanism of diseases caused by Streptococcus pnemoniae. For instance, both streptococcus pneumonia and Streptococcus suis causes meningitis and pneumonia via similar mechanisms.
1.5.4AIMS AND OBJECTIVES
The aim of this project was to investigate whether the final metabolic product of Streptococcus pneumoniae contributes to pneumococcal virulence. This study concentrated on the effect of lactic acids on bacteria and compares the effect of formic acid with hydrochloric acid on streptococci pneumonia and other co-existent bacteria. Likewise, this study also focuses the impact of these acids on epithelial cells. To investigate these hypotheses several bacteria that are naturally found in the human nasopharynx were used as well as lactic acid producing bacteria and non lactic acid producing bacteria.
1.5.5 HYPOTHESIS
Organic acids produced by S. pneumoniae as a result of fermentative metabolism, lactic acid and formic acid, are able to kill or inhibit the other colonising bacteria in the nasophynax
Organic acids have an adverse effect on respiratory cells and they contribute to inflammation.
Chapter 2
2. Methods
2.1 Bacterial strains used:
Streptococcus pneumoniae serotype 2 strain D39 was mainly used in this experiment. The bacterial strains used in this study are listed in Table 2. In addition, some other bacterial strains were used that include Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus influenzae. The total of 8 different strains that were employed in this study, all of these strains were obtained from Dr Hasan Yesilkaya, the University of Leicester, and stock strains were prepared from them in glycerol, 50µl of aliquot and stored at -80?C for future experiments.
2.1.1 Bacterial media preparation:
The solid culture of bacterial strains was done in Blood Ager Base (BAB) supplemented with 5% defibrinated horse blood or in Luria Bertani agar. To prepare BAB, sixteen gram of BAB powder was mixed with 400 ml distilled water and autoclaved at 121 °C for 15 min. Once the medium was cooled at room temperature, 5% horse blood was added and mixed, and approximately 20 ml was poured into each petri dish. The reason why blood BAB medium was used was to increases the growth of these fastidious organisms. BAB also allows the detection of the haemolytic activity. The agar surfaces were dried before inoculation.
2.1.2 Bacterial growth and measurement of Optical density (OD500)
Stock cultures of Streptococcus pneumoniae strain, the wild type D39 was prepared by growing them in 10 ml BHI at 370 C under microaerophilic condition to optical density reached OD500 0.4-0.5 at. Then the cultures were centrifuged at 3500 rpm for 10 min in AllegraTM X-22, centrifuge (Beckman Coulter, CA, USA). inoculation were centrifuged at 1500g for 15minutes and supernatant was discarded, thereafter, the pallet was re-suspended in 2ml BHI serum broth is composed 80%of v/v BHI broth and 20% v/v filtered foetal calf serum. 0.5ml of the re-suspended pellet was transferred to 1.5ml eppendorf tubes and stored into -80c for future use.
2.1.3 Spreading and streaking of bacterial cells:
A frozen aliquot of bacteria were thawed, and 20µl of this bacteria were transferred on to petri plates and immediately streaked with a flame sterilized spreader. The objective of this process is to obtain an even distribution of cells over the surface of the plate. To avoid any contamination plates were kept on close to flame or closed and plates were sent to overnight incubator.
2.1.4 The broth cultures used is Brain hearth infusion (BHI) broth.
To prepare BHI 8 grams of BHI was mixed with 200ml of distilled water and autoclaved at 121 C for 15 minutes. After autoclave the colour of BHI medium appeared amber. The reason we used this medium is because it is reach in nutrients for bacteria and it is good to utilise it for the cultivation of many bacteria such as S. pneumoniae, and H. influenzae. Regarding the growth of the Staphylococcus aerus and Pseudomonas aeroginosa Luiria Bertoni in agar was prepared, As shown in figure 1.
Unlike other bacteria employed in this project, Luria bertoni Agar was plated and dried 20µl of bacteria dropped on each plate and streaked using the flame sterilized spreader. Because these bacteria grow anaerobic condition we incubated at 37?C without co2 jar or jar with co2.
2.1.5 Luiria Bertoni broth
The Luiria Bertoni is one of the most important medium used in laboratory, because it contains nutrition that microorganisms required to maintain life. 400ml of Luiria Bertoni was prepared to use for killing experiment of both Staphylococcus aerus and Pseudomonas aeruginosa. 1 g of Tryptone, 2 g of yeast extract (LP0021), 2 of NaCl and 6 g of Agar (Bacteriogical Agar, Oxid Ltd Bensingstone, Hampshire England) was dissolved in 400ml of distilled water and finally the mixture was sent to autoclave at 121 ?C for 15min.
2.2.1 Chemical defined medium (CDM) preparation
To prepare 10ml of chemically defined media (CDM), 8.7ml of basal solution was transferred to universal tube; 200µl of glucose was added to the solution subsequently. The following essential nutrition was added to the universal tube, 100 µl of nitrogenous base, 100 µl micronutrients, 100µl vitamins 40µl choline and 10µl pyruvate.
2.2.2 Gram staining
To determine the morphological properties of bacteria, such as the shape, and to determine whether it is a Gram negative or positive, the Gram staining procedure was followed. A loop of bacteria was collected from plate culture and dispersed onto clean microscope slide using 20 µ l sterile PBS. The bacterial growth was removed by passing slide through the hot Bunsen flame. The slide was treated with crystal violet for about 2 minutes with excess. Similarly, a large quantity of Iodine was poured on the slide for 2minutes, Acetone was also poured and using water the slides were washed. Thereafter, Safranin was poured excessively for another two minutes and the slides were blotted and dried. The slide was viewed under the microscope, for the first time magnification of 10x is used. However, in later stage the magnification was adjusted to 100x. To minimise the uncertainty between air and light scattering the microscope immersion oil was used.
2.2.3 Preparing salt solutions
To rule out the possibilities of effect of salts, various salt solutions were prepared. 50ml, of 50mM 100mM 300mm and 500mM of Sodium lactate, sodium chloride and sodium formate were dissolved in water. For example the molecular weight of sodium chloride is 58.44g to find the concentration of 50mM we calculated like this (Concentration of 50mM =( 58.44g/mol)*(50mol)/1000=2.922.
The solutions were filter sterilized using a 0.2 µm acrodise syringe filter (Pall Corporation, MI, and USA) and the salt solutions were stored at room temperature for immediate use and the rest was stored at -20?C for future usage.
2.2.4 Determination of organic and in organic salt susceptibility test
The solution of sodium chloride, sodium lactate or sodium formate were exposed to Streptococci pneumoniae D39 strain, and the final concentration of these salts was 500 mM. The control (without salts) was added 195µl of CDM and 5µl of bacteria. The other wells was added a solution containing salts at various concentrations and each well was put 195µl of the mixture. The cultures were incubated at 37 ?C in flat bottomed microtitter 96 wells for 2 h. After an incubation period,180µl Phospate buffered saline (PBS) was added to the empty wells to dilute the incubated samples by transferring 20µl of the incubated samples to next well. The dried blood agar plates were divided into six segments and 60 µl of the sample was put to each segment, plates were put near to the flame when dried plates were placed in CO2 jar, plates were inverted and placed in overnight incubator. Next day plates were collected and counted the colonies on the plates. Data and figures explaining these results are presented in result and discussion sections.
2.2.5 The impact of Organic/Inorganic acids on cell culture viability using different concentrations.
To investigate the effect of the Lactic acid, Formic acid and Hydrochloric acid on pneumococcal growth, the bacteria were grown by providing all nutrition that they require to maintain live. However, Lactic acid solution in different concentrations was exposed to bacteria, but prior to this, the level of pH was initially measured. The initial pH of chemically defined media (CDM) was 6.5 and then subsequent measurements of pH was done by adding lactic acid, formic acid or Hydrochloric acid to the solution of CDM in drop wise.
The composition of solution in which bacteria were grown contained 10ml of chemically defined medium (CDM) as explained in section (2.1.3). In these studies three different experiments with a series of Lactic acid, Formic acid and hydrochloric acid in different concentrations was done. The concentration was brought up to 500mM, and 1moler. The pH of the solution with acids was constantly measured and recorded. In the situation of Lactic acid the rate of pH dropped from 6.4 to 5.8 when used various amount of lactic acids. However, several consecutive measurement of pH for both formic acid and HCl acid was made. Nevertheless, the rate of the pH stayed roughly the same as lactic acid. The experiment of acids were carried same as salts in above (2.1.3) To kill the bacteria pure acids such as lactic acid, formic acid and hydrochloric acids should be utilized and tested on both streptococcus pneumaniae and other bacteria employed in this project.
2.3. The Lactic acid and its effect on S. pneumonoiae, D39 strain
To determine the impact of lactic acid on bacterial strains, it’s important to calculate the amount of lactic acid needed for, to bring up the volume into 200µl. 1Molar was calculated by 1.010 of lactic acid was added to 8.990 of distilled water, this makes the amount of Lactic acid solution into 10ml. This amount of solution was divided into approximately 20 tubes and eventually the mixture was stored in -20C. Using microtitre plate, 10µl of D39 strain and proportional amount of PBS were added to bring up the volume in to 200ul in total. The sample that contains lactic acid, CDM and the frozen aliquot was incubated for 2 hours at 37°C. Thereafter, 60µl of solution was transferred to previously labelled agar plate and the plates were covered until they become dry and co2 jar was used, the plates were sent to the incubator for overnight incubation at 37°C. Next morning plates were viewed to count the bacteria in the marked area, the most concentrated plates had the greatest numbers of bacteria, and the resulted were recorded. While the most diluted plates showed decrease or lysis of bacteria Fig4. The experiments were repeated at least twice for using only CDM but different concentration of lactic acids.
2.3.1 The effect of lactic acid on growth of other bacteria
In this chapter other bacteria were used to test the effect of lactic acid on other species that are found naturally in nasophyrax that might compete with the natural resources. These species are include Heamophilus influenza, Staphylococci aerus, Streptococci Suis, Group B bacteria, Streptococci Agalactiae and Pseudomonas aeruginosa. The killing effect of lactic acid on a S. pneumoniae D39 strain was tested. Furthermore, various concentrations of lactic acid were exposed to all the above bacteria. Although, a different bacterial species were utilised to test the capability of lactic acid the growth medium for the assay was chemically defined medium (CDM).
2.3.2Bacterial growth studies
The bacterial strains were cultivated in chemically defined medium prepared as described in Section 2.1.3. The growth medium was composed of 180 µl of chemically defined medium added with 20 µl of lactic acid formic acid or hydrochloric acid, prepared as described in section 2.1.5, to bring to the final concentration of 5, 10, 25, 40 and 50 mM. The growth studies were done using flat-bottomed microtitre plates (Nunc, Roskilde, Denmark). 5 µl (5X106) of stock frozen bacteria that (prepared as described in Section 2.1.1) was added to the sample. The samples were further diluted 180µl in phosphate-buffered saline (PBS) was added to the medium. Another 20 µl of phosphate-buffered saline was added to control cultures without lactic acid, formic acid or hydrochloric acids.
The microwell plate was then placed in spectrophotometric plate reader (Varioskan, Thermo-Electron Corporation, USA), set up to take absorbance every 30 minutes for 16 h at 500 nm at 370 C and shaking 3 sec before taking readings. Each sample was prepared in triplicate and repeated at least three times. Growth curves were obtained and the growth rate was calculated by using the slope of the curve from the exponential phase of the growth while growth yield was obtained by taking a highest optical density in the stationary phase.
2.3.3 Cells Culture Methods
The A549 cells (was generously obtained from Dr Hasan, from the University of Leicester) were cultured in RPMI-1640 (500ml) medium containing 1% (5ml) antibiotic [penicillin-streptomycin] and 10 % (50ml) Fetal Bovine Serum (FBS), and this was called ‘complete medium’.
This image was taken from www.a549.com
2.3.4Maintenance of Cells (A549 cells and Hep-2 cells)
To thaw the frozen cells, the cells were immediately placed into 370 C water bath. After thawing the cells, the vial was wiped with 70% ethanol and allowed to dry before opening. The thawed cells were then transferred into a sterile centrifuge tube containing 2 ml of warm complete medium; the cells were centrifuged for 10 min at 250 x g at room temperature. The supernatant was discarded and the cell pellet was suspended in 1 ml of complete medium and then transferred into 25 cm2 tissue culture flask with 15 ml of complete medium and incubated at 37 0C at 5 % CO2. Cells were checked and media was replaced every 2-3 days and 24 h prior to MTT assay.
2.3.5 Trypsinizing and Sub-culturing the Cells
Subculturing was done when the cells reach confluence. Old media was removed and the cells washed with 10 ml of phosphate-buffered Saline (PBS) t

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