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Effect of Smoking on the Oral Micro-flora

Vishalkumar V. K, Sreedevi N, Angelica M, Priti Talwar
Abstract: It is a well-known fact that cigarette smoking (CS) is directly associated with cardiovascular diseases, chronic bronchitis, and various types of cancers worldwide. In this study, two parameters were taken in consideration. Firstly, we have focused on how cigarette smoking is affecting the oral micro-flora. Second parameter was to figure out if cigarette smoking has any change in alpha-amylase activity. Human mouth harbors complex, abundant, and highly diverse micro-flora, the comparative study was conducted to check the bacterial colonization between smokers (test samples) and nonsmokers (control). Thirty male volunteers from VIT University (15 smoker and 15 nonsmokers) ranging between 20 to 30 years of age were taken in this study. For this, salivary samples were taken from the volunteers both smoker and nonsmoker. The salivary samples taken were spread plated on nutrient agar media and comparative study was done on bacterial colonization, which showed that the test samples showed maximum bacterial colonies when compared to control. The second parameter, which was considered was alpha-amylase activity. Alpha amylase is one such protein enzyme present in human saliva, which hydrolyses alpha bonds of larger alpha-linked polysaccharides. These polysaccharides are mainly present in glycogen and starch, which on breakdown yields glucose, maltose, and dextrin, which makes the digestion process easier. We are currently focusing on estimation of amylase activity between smokers and nonsmokers for which salivary samples were taken from the same volunteers. It could be possible that the activity of amylase in smokers could quite be different from those in nonsmokers.
Cigarette smoking is one of the leading preventable causes of cardiovascular diseases, chronic bronchitis, and various types of cancers and finally leading to death worldwide. All most every organ of the body and overall health of a person is affected by cigarette smoking. Death rate is around 480,000 each year in United States, which is found to be one in five deaths. Apart from all these facts about smoking, there are more than 700 microbial species found in the oral cavity of which over 50% have been detected and cultivated. There were 2 main purposes of our study: (i) our first attempt was to find out on how cigarette smoking was affecting the oral micro-flora, and (ii) to figure out if cigarette smoking has any change in alpha-amylase activity, which is one of the prominent and digestive enzymes in human saliva. The micro-flora present in human mouth shows complex, abundant, and highly diverse in nature and keeping that in mind we had conducted a comparative study to check bacterial colonization between smokers (test samples) and nonsmokers (control). For this study, 30 volunteers participated; all of them gave their informed consent before the study. Thirty male volunteers from VIT University (15 smoker and 15 nonsmokers) ranging between 20 to 30 years of age participated in this study. Saliva was collected from these volunteers for our study purpose. All the volunteers were asked about their detailed smoking history. For nonsmokers, volunteers with no history of any kind of smoking activity are considered. For smoker, volunteers with the following criteria were considered while taking salivary samples:
(i) Number of cigarettes or beedies consumed daily [1]
(ii) Frequency of smoking [1].
(iii) Number of years of smoking[1].
Our first parameter was, (i) bacterial colonization, saliva was taken from the volunteers both from smokers and nonsmokers and using spread plate method, salivary samples were spread plated on the nutrient agar medium. Comparative studies were done by comparing the samples of both the groups (smokers and nonsmokers).
For second parameter was, (ii) estimation of alpha-amylase activity. Alpha amylase is one of the major enzyme present in human saliva, which helps in partial digestion of the food in mouth by hydrolyzing the alpha bonds of the polysaccharides, which are alpha-linked. These polysaccharides are major components of glycogen and starch, which on breakdown yields glucose, maltose, and dextrin, which makes the digestion process easier. To explore more about the estimation of amylase activity between smokers and nonsmokers for which same salivary samples were taken. It could be possible that the activity of amylase in smokers could quite be different from those in nonsmokers.
A. Volunteers selection
Thirty males comprising of fifteen smokers and fifteen nonsmokers all in the age group ranging from 20-25 years were selected. The subjects were randomly selected on basis of their smoking history. We took precaution that all those thirty volunteers who had participated were not on any antibiotic medication during last 6 months.
B. Sample Collection
Participants were asked to rinse their mouth with water 10 min prior to saliva collection. Approximately 3 ml of saliva was collected from donor into the sterile glass vial according to the method described by Navazesh 1993 (cited by Rohleder et al 2008).
C. Bacterial Colonization
Serial dilution was performed with the samples collected with distilled water. Serial dilutions (1/10000) of the samples were plated on NA medium inside the laminar air flow hood by spread plate method. In the spread plate, 0.5 ml of inoculum was spread over the surface of the agar. The plates were turned upside down and place in incubated at 37ºC for 24 hours.
The total colony forming unit (CFU) determined after incubating the spread plate for 24 hours by using Colony Counter.
The number of colonies for each petri dish culture was determined with the formula:
CFU/ mL = CFU/plate x dilution factor x 1/aliquot
D. Salivary Amylase Activity
Amylase activity was estimated using standard protocol from Lab Manual (VIT University, 2013). For the experimental use saliva was diluted in distilled water (1:10). For preparation of test solution by taking 1 ml of phosphate buffer (0.050 M, pH 7.1) and 1 ml of diluted salivary amylase enzyme. With the help of test blank, which contains 1ml starch and 2ml of phosphate buffer incubate for 15 min at 30ºC. The amount of maltose released is estimated by DNS method. Series of standards using maltose from 200 mg to 1000 mg concentration is prepared.
The absorbance at 510 nm was measured and a standard graph was plotted. With the help of standard the amount of maltose breakdown by the enzyme amylase was estimated, and this gives the enzyme activity.
E. Enzyme activity =
F. Statistical analysis:
Mean has been calculated for representing the entire data with single value which gives more convenient result. Also standard deviation has been calculated for show the dispersion among all sample variables. Student t test have been use to know the significance difference between smoker and non-smoker salivary amylase activity.
The Microsoft Word and Excel were used to generate graphs, tables, and so forth.
Bacterial Colonization:
The 30 volunteers from VIT University participated in our study in which 15 were smoker and remaining 15 were nonsmoker (control). We diluted salivary samples and inoculated in petri plates and incubated for 24 hours (as described above). After the incubation period it was observed that colonies formed in smoker samples were higher in number when compared to non-smoker sample.
Colony numbers
Table 1: Number of participant falling in different class of colony number

Figure 1: number of colonies formed by smoker and non-smoker sample (DF 10:10000)
The mean value for smoker’s colony number was 49.83±6.54 which was higher when compare to nonsmoker’s colony number which was 40.5±5.68. This clearly indicates that smoking influence mouth micro-flora and also the factor responsible for more dense colonization (figure 1).
Amylase activity:
The salivary amylase collected from all participants was analyzed with standard protocol and calculated the activity of amylase per unit. The sample was immediately analyzed to reduce the error causing in calculating amylase activity.
The amylase activity calculated from standard maltose graph showed significant difference between smokers and nonsmokers salivary amylase activity.

Figure 2: Showing difference between salivary amylase activities in smoker and non-smoker
The salivary amylase activity seems to be higher in smokers when compare to nonsmoker. The mean value for smoker amylase activity was 0.33±0.15 IU, and when it was compared to nonsmokers it was just 0.045±0.05 IU.
Non- smoker
Table 2: Statistical analysis of amylase activity (n=30)
The percentage of amylase activity showing the calculated value for student t test at 5% is higher, (calculated value[15.5]> table value [2.04]) which indicated that there was significant difference in amylase activity between smoker and non-smoker.
At the global level smoking is the major public health hazard. This is just not harming the individual alone but also to other people who are surrounding them. Smoking has diverse effect on human body like mouth, cardiovascular, liver, lungs and so on.
This study is an explorative attempt to know the effect of smoking on mouth microbe’s health and activity of salivary amylase with different parameters.
The comparative data obtained from smokers and nonsmokers volunteers showed significant difference in microbial colony number and also the amylase activity. To minimize the effect of all other possible factors volunteers belonging to same sex and same age group had been taken in consideration.
The 24 hours incubation at 37ºC for all sample showed diverse result. In smokers petri plates CFUs/ml was higher when compared to nonsmokers petri plates (Table 1). The highest colony number was found to be 59 in smoker’s plate (test plate) and the lowest was 32 in nonsmoker’s petri plate (figure 1).
The immediate analysis of salivary sample obtained from all 30 volunteers showed high amylase activity in smokers sample (test sample) and low amylase activity in nonsmoker sample (Figure 2). The mean value for amylase activity in smoker’s sample (control sample) was found to be 0.33±0.15 IU and non-smoker’s sample was just 0.045±0.05 IU (Table 2).
The findings of given study emphasizes that smoker volunteers had effected on both amylase activity of salivary amylase as well as health of mouth micro-biota. So the best way to prevent this is by educating people about it and if possible by including these issues in public health education.
[1] Maddipati Sreedevi[1], Alampalli Ramesh, and Chini Dwarakanath Periodontal Status in Smokers and Nonsmokers: A Clinical, Microbiological, and Histopathological Study
[2] G. Calsina, J.-M. Ram´ on, and J.-J. Echeverr´Ä±a, “Effects of smoking on periodontal tissues,” Journal of Clinical Periodontology, vol. 29, no. 8, pp. 771–776, 2002.
[3] J. Haber, J.Wattles,M. Crowley, R.Mandell, K. Joshipura, and R. L. Kent, “Evidence for cigarette smoking as a major risk factor for periodontitis,” Journal of Periodontology, vol. 64, no. 1, pp. 16–23, 1993.
[4] Jørn A. Aas,1,2* Bruce J. Paster,1,3 Lauren N. Stokes,1 Ingar Olsen,2 and Floyd E. Dewhirst1,3 Defining the Normal Bacterial Flora of the Oral Cavity, Vol. 43, No. 11.
[5] D. Weiner1, Y. Levy2, E.V. Khankin3, A.Z. Rezick11. Inhibition of salivary amylase activity by cigarette smoke aldehydes.s

Answer to the Chicken and Egg Question

Chicken vs. Egg
The question that drives everyone insane: which one came first, the chicken or the egg? One always ends up at the same place while thinking over again and again. This is a life cycle of chicken: from egg to chick then hen or rooster. ‘A circle has no beginning’, said by dotty the witch from Harry Potter. It is a no win-win situation, it depends on ones perception and theories. I think it is impossible to answer this question; they both need each other for its success. I think the chicken and the egg exist at the same time; one cannot exist without another. This is a scientific answer I can give but from the viewpoint of a religious person, it will be a chicken that comes first. According to the bible, God created the creature first then come to the egg (Genesis 1:21).
Here is a little information on how the chicken is formed. In all living thing, their DNA revolves around their environment because of the natural selection[1]. In the case of chicken, DNA from the male sperm cell and a female ovum meet and combine to form a zygote[2]. This cell then will be divided again for numerous times to form a complete cell of an animal. In the animal, every cell contains the same DNA and its come from zygote.
Evolutionist claimed that egg came first. They said that birds evolved from reptiles over millions of year so the reptiles laid the egg that hatched as a chicken. The chicken egg should not always contain chicken eggshell and chicken fetus together. It is not necessary to have chicken eggshell and chicken fetus to create a chicken. An egg should be classified by the species that lay it rather than what species it contains.
The complicated part is the eggshell because it contains any genetic material. Well, the obvious parts of the determined chicken egg will the chicken fetus. The chicken eggshell is made of calcium so we can say that the chicken egg consists of two distinct parts, the chicken eggshell and the chicken fetus.
The question is who generates the eggshell? The fetus has nothing to do with fertilizing the eggshell, if the fetus generates the eggshell then it could be classified as a single entity but there exist unfertilized eggs. So it is proven that the fetus has nothing to do with generating the eggshell. The eggshell is fertilized by the hen only when it is mate with the roosters; see even here it is proven that there would be no fertilized egg unless the hen and the roosters mate so it also means the egg cannot exist without the chicken and the other way round.
Some of the scientists said that some animal which is not a chicken laid an egg. The fetus inside the egg undergoes a minor evolutionary change that became a hen. This hen then laid the first chicken egg which was a rooster. Then that rooster mated with some animal which was not a chicken and laid an egg which is not a chicken egg since the rooster’s DNA could not affect the egg. Then the egg hatched which became a hen who then laid the first chicken egg.
According to NPR’s Robert Krulwich and the majority of scientists stated that long time ago there was a chicken-like bird. It was called a proto-chicken. So this is how it goes, proto-hen laid an egg and proto-rooster fertilized it but when the genes combine in a new way, resulting in mutation[3] that make the baby different from the parents. It became a new species called chicken. In the end, two birds which were not a chicken created a chicken egg. She was trying to say that the egg comes first then the chicken. (Breyer)
There is another article that agrees that egg comes first. Modern scientist found out that non- chicken birds can be mix with the chicken, only with a similar physical traits example like horse and donkey and produces mule which cannot produce fertile offspring. They found evidence that the chicken belongs to Galliformes order. This theory was possible because domestic chicken appeared, scientifically known as Gallus Gallus domesticus[4]. So again it is saying that there were the different genetics of different kinds or some call it as variety within species. As this diversity evolved from parents to offspring, eventually non-chicken bird laid an egg containing a chicken. So likely, it is saying the Gallus domesticus egg came first.
Jean- Baptiste Lamark, a French naturalist, he believes that one species developed into another as it changes – evolution. He also believes that the way species change was by developing a feature or different traits and passing on to the future generation. He uses giraffe as an example; he thought that it had come from a smaller antelope that stretched its neck, legs and tongue to feed on trees. These stretch features were then passed on to the offspring and when they stretched to reach higher their necks, legs and tongue became longer and longer. This theory was known as the theory of acquired characteristics. (Riley)
Phylogenetic tree:

According to Evolutionary theory, 300 years ago, humans had a common ancestor with the chicken but recently in evolutionary time like in this diagram, we are more close to the mouse than the chicken. Researcher finds out that long-supposed evolutionary is based on the genetic information. You will notice that the diagram focused on one kind of common ancestor we had, the vertebrate[5] species and it was made based on tyrosinase[6].
The new knowledge that has been discovered can affect our society for example like religion, to some people religion play a big role in their life. According to bible and most of the religion books, it is believed that God create the creature and science has proven them wrong most of the time so it give them a second thought and people’s believe started to fall apart resulting in confusion whether to believe their religion or science.
On the other hand, it helps the world to progress, without discovering new species and evolution of course scientist and society will not progress. It plays a big role in medical field, we can make medicine out of some species, and this give us advantage and to the scientist and researcher too. Discovering new species mean we must find a way to live with it, this give a society a headache, because the world is developing so which mean deforestation, we need more space for our rapid population, the home to millions of species, so the solution to this might be artificial environment example like bridge connected from one small forest to another, so the animal can cross to another place without interrupting our society. This will be very useful to migrating animal such as Zebra, Elephants and Caribou, this type of animal move long distance for searching food and mating. (Hunter)
(1,196 Words)
Bibliography: MLA
Online Resources
“Finally Answered! Which Came First, the Chicken or the Egg?” MNN. N.p., n.d. Web. 16 Feb. 2014.
“Which Came First, the Chicken or the Egg?” HowStuffWorks., 01 Apr. 2000. Web. 17 Feb. 2014.
“Zygote Definition – Google Search.” Zygote Definition – Google Search. N.p., n.d. Web. 17 Feb. 2014.
“Understanding Humanity’s Infatuation With Chickens.” Chicken Evolution And Its Role In Humanity. N.p., n.d. Web. 25 Feb. 2014
Bryner, Jeanna. “Which Came First? Eggs Before Chickens, Scientists Now Say.”LiveScience. TechMedia Network, 14 Nov. 2008. Web. 25 Feb. 2014.
“Fertile Chicken Eggs | Chicken Egg Incubators.” Facts about Fertilized Chicken Eggs :. N.p., n.d. Web. 25 Feb. 2014.
“Comparison Places Humans Closer to Chickens than Mice.” Bible Creation Lessons. N.p., n.d. Web. 26 Feb. 2014. Nature Publishing Group, n.d. Web. 24 Feb. 2014.
Journal Article
“Journal Article.” Perspectives on the Knowledge-Based Society. An Introduction to the Special Issue — Economics E-Journal. N.p., n.d. Web. 26 Feb. 2014.
Hunter, Philip. EMBO Reports. Rep. no. PMCID: PMC1852758. EMBO Rep, 8 Apr. 2007. Web. 26 Feb. 2014
Riley, Peter D. Cambridge Checkpoint. 2nd ed. London: Hodder Education, 2011. Print
Pickering, Ron. Complete Biology For Cambridge IGCSE. 2nd ed. Singapore: Oxford UP, 2006. Print.
[1] The greater chance of passing on genes by the best adapted organism.
[2] The cell resulting from the union of an ovum and a spermatozoon
[3] Is a change in a gene or chromosome. (Pickering)
[4] Scientific name for domestic chicken.
[5] An animal with backbone.
[6] A copper-containing oxidoreductase enzyme found in plant and animal tissues that catalyzes the oxidation of tyrosine into melanin and other pigments. (Dictionary)