The Antibiotic Resistance In Bacteria Biology Essay
In this experiment, bacterial civilizations were drawn from two different environments and compared to see if there was any difference in the sum of antibiotic immune bacteriums in each environment. The two environments chosen were the door grips of three bathrooms of West Holmes Hall and Intramural Sports East ( IM East ) . We hypothesized that there would be more antibiotic immune bacteriums coming from IM East than that of Holmes because there is a greater figure of people from more diverse locations that use the installations at IM East, and therefore there should be a greater diverseness of bacteriums contained in this country and a greater sum that would be immune.
The consequences of the chi-squared analysis were a p-value of 0.671 for Achromycin, 0.791 for Principen, and 0.823 for Kantrex. This suggests that there was no important difference in the sum of antibiotic immune bacteriums between West Holmes Hall and IM East. This contradicts our hypothesis that there would be more bacteriums in IM East. We chose to analyze one bacterium further because of its Kantrex opposition.
This bacterium was from Holmes bathroom 2, # 11 on the original run home base. The strain appeared to be Gram-negative as a consequence of a Gram-stain, but Gram-positive with the KOH, EMB agar, and MacConkey agar trials. We attempted to insulate a plasmid from the bacteriums to see if the opposition was as a consequence of familial information on a plasmid but were non successful.
This could intend that the opposition is coded in the bacterial chromosome, but it could besides intend that we merely were unable to insulate a plasmid from the bacterium. The consequences of the survey show that there are more factors act uponing the prevalence of antibiotic opposition in a community of bacteriums than merely the sum or diverseness of people that use the installations in that environment.As a consequence of the over-administration and abuse of antibiotics and antiseptics, bacterial opposition to antibiotics is a turning job. One of the prima causes of the spread of antibiotic opposition is due to the distribution of bacteriums that contain opposition cistrons. Bacterias that contain opposition cistrons can give them to other bacteriums in the population in a assortment of ways, doing potentially the whole population resistant to antibiotics. With their deadness to many types of drugs, antibiotic-resistant strains of bacteriums present a serious jeopardy to human wellness and the populace should be kept informed. Due to the considerable wellness hazards presented by antibiotic-resistant bacteriums, it is necessary to research where the happening of immune strains is greatest.
Antibiotic opposition is a turning job for several grounds that are largely related to the medical community overprescribing antibiotic medicine and patients non utilizing them decently. An illustration this is when antibiotics are prescribed to people that do non necessitate them for the unwellness that they have, such as a viral infection. Not merely will this non impact the illness that they have, but it will besides potentially let for bacteriums that are already populating in their organic structures that have the opposition to the drug and thrive. Besides, when prescribed with an antibiotic decently some people do non mind the waies and merely take some of the proper dosage.
Basically what this does is kill some of the bacteriums that are in their organic structure but non plenty for the immune system to complete off the balance of the bacterium. This allows for the Numberss of bacteriums to lift one time once more, with an addition in the sum of bacteriums that were immune ( Levy 1998 ) .Another job lending to the tendency of increasing antibiotic resistant bacterium is the popular antiseptics manus sanitizers and soaps that have antibiotics right in the expression. Hand sanitizers and antiseptics kill the bulk of bacteriums that are on surfaces. However, the bacteriums that do survive are by and large those that are predisposed to lasting in such conditions and their competition for resources has been eliminated ( Levy 1998 ) . This allows them to reproduce and turn quickly, finally making big sums of bacteriums that are besides predisposed to defying such actions against them. The inordinate usage of such merchandises is engendering a whole new set of bacteriums that are immune to interventions that normally would kill them. A end of this research is to happen out where such bacteriums are populating in the universe around us.
Another end of the research is to research the ways that bacteria really resist the antibiotics. There are several ways that bacteriums can inherit cistrons that code for the proteins doing them resistant. First would be chromosomal heritage, where coevalss of bacteriums are of course predisposed to holding immune features and they pass these characters to their offspring.
Another manner that bacteriums can give others this immune cistron is through extra familial fragments called plasmids. Plasmids contain Deoxyribonucleic acid that does non happen in the chromosome of the bacteriums, and the plasmid Deoxyribonucleic acid can straight give this information to others in the environment around it through a procedure called junction ( Levy 1998 ) .Due to abundant wet and the prevalence of cast and mold, bathrooms are normally thought of as some of the most bacteria-rich environments. A survey done in Nipponese families found that the sum of bacteriums found in bathrooms was 2nd in figure merely to those found in kitchens ( Ojima et al. 2002 ) . It is because of these findings that the inside door grips of two different bathrooms were swabbed for bugs.
Then, the opposition and the assortment of bacteriums from each location were analyzed and compared. Testing for antibiotic opposition in different environments may supply information on where antibiotic immune strains of bacteriums are most likely to happen. Hopefully, this will let a greater apprehension of how antibiotic immune strains of bacteriums get into the environment in which they live.
Public bathrooms are topographic points where a assortment of different people enter each twenty-four hours, each with differing hygienic wonts. The people that visit these public toilets bring a many bacteriums with them. The most common topographic points to happen antibiotic immune bacteriums on a college campus, harmonizing to one survey, were in cafeterias, bathrooms, and computing machines ( Shanks, 2009 ) . Bathrooms were an easy and accessible topographic point to swob for this experiment, and this seemed like a sensible topographic point to seek for antibiotic immune strains of bacteriums.Although bugs thrive in dark moist topographic points, they are non needfully the premier location for bacteriums ; alternatively most bacteriums are found on surfaces with high degrees of tactile traffic ( Ojima 2002 ) . As one of the most touched surfaces in any edifice, surpassing bathroom door grips were our proving environments. The two environments tested in this survey were public bathrooms in West Holmes Hall and public bathrooms in Intramural Sports East.
Although a old survey found no important difference between the figure of antibiotic-resistant bacteriums on surfaces in medical and domestic locations ( Blouin, 1998 ) , our hypothesis is that there will be more immune bacterium from the grips at the Intramural fittingness centre than at West Holmes. Our hypothesis is based on the thought that the greater diverseness of locations from which the visitants to the fittingness centre are coming agencies that there will be a greater diverseness of bacteriums brought in to the installations by those people.
Methods
Written by: Curtis Abell and Kris Kutskill
Bacteria aggregation and pouring home bases
Bacterias were collected by swobing the door-handles of three different bathrooms in West Holmes Hall utilizing a cotton swab dipped in phosphate-buffered saline. This solution allows for the bacteriums to be transferred from one surface to another without killing them. Two of these were work forces ‘s bathrooms and one was a adult females ‘s bathroom, and combined these are group 1 of the research. Samples of bacteriums were besides collected from Intramural ( IM ) Sports East in the same manner ; two work forces ‘s bathrooms and one adult females ‘s bathroom for experimental group 2. The environmental samples were placed onto a LB agar home base and incubated at 37A°C overnight.
Home plates of agar medium were made by making a solution incorporating 8g of bacto-agar, 12g LB pulverization, and 600mL of H2O. These solutions were autoclaved to guarantee that they were non contaminated by any other bugs from the environment. Six of the home bases were allowed to stay with merely the LB agar and were used for spot home bases, while three other groups of six home bases had antibiotics added to them. The three antibiotics used were Kantrexs, Principen, and Achromycin at a concentration of. Patch home bases were made by streaking a individual settlement of bacteriums onto an LB home base that had labeled with 16 different co-ordinates on it.
These settlements were chosen indiscriminately from the original swab home base and patched on to these home bases in one co-ordinate. These home bases were so transferred into the brooder for 20 hours at 37A°C.
Making Patch Home plates
A sum of 24 spot home bases were made to find antibiotic opposition in bacteriums to one or more of the antibiotics Principen, Kantrex, and Achromycin. An LB agar merely home base was besides used as the control when streaking.
The LB merely home base was streaked last to guarantee that bacteriums had still been on the cringle through the antibiotic home bases prior to streaking the LB home base. The home bases were marked as before with 16 quarter-circles fiting the maestro plates. A unfertile cringle was so used to grate up bacteriums from the maestro home base and so streak each antibiotic home base and the LB merely plate last without traveling back to the maestro home base or re-sterilizing the cringle. The home bases were patched matching to their appropriate Numberss. For illustration quadrant one on the maestro home base was streaked on quadrant one for the antibiotic and LB merely plates. Once all 24 home bases were streaked, the home bases were grouped harmonizing to environment, turned upside down, and incubated at 37oC for 24 hours.
Agarose gel cataphoresis
Rubber stoppers were placed on a gel caste that serves as the cast for the gel. A comb was put in the slots with the points confronting in to do the lanes in the gel. Then 0.4g ultrapure agarose and 40mL TBE were assorted in a 125mL Erlenmeyer flask. The mixture was microwaved for one minute. Once it cooled, 2AµL of ethidium bromide ( EtBr ) was added and poured into the gel caste. The gel box was so filled half of the manner with used TBE solution.
The finished gel was so placed in the gel box with the TBE and the lanes are ready to be loaded. The gels were loaded with 5 AµL of 1kb ladder for the control lane. The other lanes were loaded with 5 AµL of 6x lading dye and 1 AµL of the solution being tested. The gels were run under about 100 Vs of electricity for about 30-45 proceedingss ; until the ladder passed through two tierces of the gel.
Gram discoloration
For the Gram discoloration, a bacterium settlement was spread into 50AµL of H2O and so affixed with a fire onto a slide. The settlement was treated consecutive with crystal violet, I, ethyl alcohol and saffranine. The crystal violet dye was allowed to stand on the slide for about a minute before being rinsed.
Then the I was added for a minute and rinsed with H2O once more. Ethanol was applied until there was no more bluish dye dripping off of the slide. Finally the saffranine was added to the slide and was allowed to put on the slide for one minute and rinsed with H2O. Then the slide was observed through a compound microscope to denominate whether the bacteriums were Gram-positive or Gram-negative. If the settlements were pink, they would be considered Gram-negative. If the settlements appeared violet, they were recognized as Gram-positive.
KOH trial
Five AµL of 3 % KOH solution were placed on a slide in a bead. Then a loop full of bacterium from environmental settlements was introduced to the solution and assorted for about a minute.
Gram-negative cells will lyse and the solution thickens as it is pulled off from the slide. Gram-positive cells do non lyse and the solution incorporating the cells and KOH will non go gluey.
MacConkey Agar trial and Eosin Methylene Blue ( EMB ) trial
The MacConkey Agar trial, environmental samples of bacteriums were placed on MacConkey ‘s Agar medium and placed in an brooder for 24 hours at 37A°C. Merely Gram-negative bacteriums will turn on this medium.
Environmental samples of bacteriums were besides placed on EMB agar and incubated at 37A°C for 24 hours. Gram-negative bacteriums by and large grow on this agar entirely.
Plasmid isolation and agarose gel cataphoresis analyzation
Positive Red control plasmid from E. coli bacterium was purified utilizing Promega ‘s Wizard Plus Miniprep kit and protocol. The environmental bacteriums were harvested from the antibiotic run home base and plasmid DNA was purified from the bacteriums besides utilizing Promega ‘s Wizard Plus Miniprep kit and protocol ( Promega, 2009 ) . The optical density of the purified plasmid DNA was measured utilizing a spectrophotometer.
The figure of base braces in the plasmid DNA was determined by agarose gel cataphoresis when compared to a 1kb ladder solution. The values were compared to the ladder solution because the figure of base brace is known on the ladder as it goes through the agarose gel.
Restriction enzyme digestion
We intended to happen out more information about our Red control plasmid by digesting it with limitation enzymes. We used a solution of 1AµL of the limitation enzymes PST1 and Eco RV along with 2AµL of buffer 3 and 7AµL of nuclease-free H2O for our first trial. The gel cataphoresis for this process is determined in figure 7. For the 2nd limitation we used a solution similar to the first merely with Eco RI and PST1 limitation enzymes alternatively of PST1 and Eco RV.
The solutions were incubated for 20 hours at 37A°C. After incubation the solutions were used in the lanes of an agarose gel cataphoresis.
Consequences
Written by: Kris Kutskill and Curtis AbellFigure 1 shows the bacterium from both environments that were collected for farther survey. These home bases were the original swab home bases that were used to roll up settlements to be tested for antibiotic opposition. The bacteriums were counted from the home bases in figure 2 to see how many of the settlements from each environment had antibiotic opposition and statistical analysis was done on it. Table 1 shows that there were less antibiotic resistant bacteriums in the Intramural Sports East edifice bathrooms than those in West Holmes. The entire figure of bacteriums in IM East was 44 and the entire figure of antibiotic resistant strains in West Holmes was 33 ( table 1 ) . However, a chi-squared analysis revealed that the difference between the two environments was undistinguished.
The p-value for Kantrex was 0.823, Principen was 0.791, and Achromycin was 0.671 ( table 1 ) .The consequences of the cataphoresis on the agarose gel are that no plasmids occurred in our environmental specimens ( figure 3 ) .
However, figure 3 shows that the ruddy control plasmid had a positive consequence. Figure 4 shows the consequence of a Gram-stain trial on bacteriums from Holmes bathroom 2 # 11, and when compared with the control stain it appeared to be Gram-negative. However, the consequences of the KOH trial, EMB agar trial, and MacConkey agar trial ( figure 6 ) were negative, as in the bacterium in the trial appeared to be Gram-positive ( table 2 ) .The ruddy plasmid contains the information for the Principen opposition cistron, as shown by the consequences of a transmutation trial on E. coli in figure 5. The consequences of this trial show growing in E. coli bacteria that antecedently were non antibiotic resistant in home bases that contain the antibiotic Principen.
The pLitmus28i was a positive control in this trial, and H2O was used as a negative control. The negative control was to do certain that the solution was non contaminated and to see that the antibiotic in the agar was working. Bacteria added to the H2O control home bases were non transformed, and therefore they did non turn on the Principen home base.The consequences of the limitation digestion of the Red control plasmid are inconclusive because the ladder in the sample ran faster than normal ( figure 7 ) . When compared to the control group in the agarose gel, it is clear that the digestion of the plasmid occurred in the 2nd lane.
This lane had the limitation enzymes EcoRV and PST I, and the sets indicated are similar to our anticipation of there being three fragments with base brace lengths of 2860bp ( base brace ) , 1150bp, and 529bp. Lane 1 had a digest with enzymes Eco RI and PST 1 and had two fragments and had predicted base brace lengths of 1591 and 2984bp.
Discussion
Written by: Kris Kutskill and Curtis AbellWe found rather a few bacterial settlements on each of the door grips of the bathrooms which is consistent with the survey done on Nipponese families ( Ojima et al. 2002 ) . When we compared the sums of immune strains from each environment, found that there were more antibiotic immune strains of bacteriums in the bathrooms West Holmes Hall ( moderate tactile traffic ) when compared to the bathrooms in Intramural Sports East ( high traffic ) . However, the consequences of the chi-squared analysis on each of the antibiotic types revealed that there was non a important difference between the sums of antibiotic resistant strains in each environment. This was because the p-values of each of the trials were above 0.5.
These consequences are contrary to our original hypothesis that there would be more antibiotic resistant strains in IM East than in Holmes Hall because of the diverseness of locations that people that use the installations come from. This consequence could hold several possible accounts ; first it is possible that the installations are cleaned more frequently at IM East than in Holmes Hall as a consequence of the cognition that many people use the bathrooms each twenty-four hours. Additionally, the cleansing techniques could be more thorough at IM East when compared to those used at Holmes Hall. Finally, we may hold underestimated the sum of people that use the bathrooms in Holmes. There really could be more people utilizing the bathrooms at that place than at IM East. As a consequence of the premise that more people use the bathrooms at IM East from a greater diverseness of locations, we could hold overlooked the fact that many people could besides utilize the bathrooms in Holmes outside of the homogenous group contained within the residential college. In other words, many more people other than the people that live in the edifice of West Holmes could be utilizing the bathrooms during the twenty-four hours.
Another possible job with the survey was trying and plating. The original samples that were patched onto antibiotic home bases were discarded after an unusual sum of bacterium was turning on them. All of the home bases proving for antibiotic opposition had to be re-patched from our environmental spot plates ; this could hold altered the consequences. It is non clear whether the spot plates grew ab initio as a consequence of faulty home bases or if the bacteriums were immune.
We chose to analyze one strain of antibiotic resistant bacteriums that was obtained from the environment farther. These bacteriums were a strain found on Holmes bathroom 2, # 11 on the original spot home base. These bacteriums were studied farther as a consequence of its opposition to kanamycin. The bacterium appeared to be Gram-negative as a consequence of the Gram-stain trial, but seemed to demo features of Gram-positive bacteriums under the KOH, EMB, and MacConkey agar trials. It is ill-defined whether this strain is Gram-negative or positive as a consequence of this contradiction.
Colonies of Holmes 2 # 11 were white in colour on the original spot home bases, and the settlements formed little circles with clear borders. Under magnification ( during the Gram-stain trial ) the single bacteria appeared to hold cocci form.During the two different attempts at insulating a plasmid utilizing the mini-prep protocol and agarose gel cataphoresis, we were unable to insulate a plasmid from our environmental bacterium. This consequence could bespeak that the cistron for opposition to kanamycin is on the bacterial chromosome instead than on a plasmid. On the other manus, the plasmid that codes for the antibiotic opposition could hold existed, but we were merely unable to insulate it. If clip would hold permitted we could hold continued to seek to insulate a plasmid from the bacteriums, but the sum of clip allotted merely allowed for two trials.
As a consequence of non being able to insulate an environmental plasmid, we chose to utilize the control Red plasmid for farther usage in limitation digestion and transmutation. The consequences from the transmutation trial that was done on E. coli bacterium allowed us to deduce which antibiotic the control Red plasmid coded. This information was deduced by looking at the control pLitmus28i plates in this trial that were guaranteed to turn in the presence of Principen if the transmutation was successful.
The E. coli that were transformed with the Red plasmid were non antecedently immune to ampicillin, as shown by the negative control with merely H2O added to the cells, but they became immune to the antibiotic after the transmutation. Due to the fact that the bacterium became immune after the transmutation procedure, it was concluded that the Red plasmid contained the familial information to give the E. coli bacteria opposition to the antibiotic Principen.A cardinal thing that was learned from this experiment is that merely because a location is frequented by a more diverse group of people, it does non needfully intend that the sum of antibiotic immune strains of bacteriums will be higher. The figure of antibiotic immune bacterium is influenced by many factors, and a topographic point such as Holmes Hall that has the same people utilizing the bathroom installations could incorporate even more antibiotic resistant bacteriums than a topographic point that has many different people from a big assortment of topographic points. Antibiotic opposition could be created by a closely knit group of people every bit good as people from diverse locations.
Due to the decisions drawn from this experiment, it would be interesting to see the consequences of a similar experiment done in a different but similar state of affairs. It would be interesting to see if there is a greater assortment of bacteriums coming from the different environments. To understand the job even better, it would be necessary to happen out specifically which bacteriums were coming from these environments and see where the most bacteriums in these environments occurs. Bacteria live in other parts of the bathroom and non merely on the door grips. The most common topographic points to happen bacterium in bathrooms are in the sinks or on lavatory seats ( Shanks, 2009 ) .
More bacteriums may populate in such countries of the bathroom and may give a more accurate representation of the bathroom environment.
Mentions
Blouin, S. A. 1998. “ A comparing of antibiotic opposition in bacteriums found in medical and domestic environments. ” Bios.
69: 109-118.Levy, S.B.
1998. “ The Challenge of Antibiotic Resistance. ” Scientific American.
March 1998. 46-53.Ojima, M.
, Y. Toshima, E. Koya, K. Ara, H. Tokuda, S. Kawai, F. Kasuga, N. Ueda 2002.
“ Hygiene measures sing existent distributions of micro-organisms in Nipponese families. ” Journal of Applied Microbiology. 93 ( 5 ) : 800-809.
Promega Corporation. 2009. Wizard Plus SV Minipreps DNA Purification System. Retrieved ( 4/25/10 ) , from Promega Web Site: hypertext transfer protocol: //www.promega.
com/tbs/tb22 5/tb225
Shanks, C. R. and M. A. Peteroy-Kelly. 2009. “ Analysis of Antimicrobial Resistance in
Bacteria Found at Various Sites on Surfaces in an Urban University. ” Bios.
80 ( 3 ) : 105-113.
Tables and Figures
A ) B ) C )D ) E ) F )Figure 1. Bacterias collected from three locations within two different environments. The bacteriums were obtained from the door handles go forthing each bathroom. A-C are a samples from three different West Holmes bathrooms ( environmental group 1 ) . The bacterium from environmental group 1 are representative of moderate tactile traffic.
D-F are samples from three different bathrooms in IM Sports East ( environmental group 2 ) . Environment group 2 represents high tactile traffic.
a ) IM East # 2
B ) Holmes # 3
Figure 2. Antibiotic spot home bases from environmental samples. The left column are control LB merely plates, the 2nd column are plates incorporating LB agar and kanamycin antibiotic, the 3rd column contains home bases with LB agar and Achromycin, and the 4th column contains home bases with LB agar and Principen. Rows a, vitamin D, and e incorporate bacteriums from environment 2 ( IM East ) , and rows B, degree Celsius, and f incorporate bacteriums from environment 1 ( Holmes Hall ) .
degree Fahrenheit ) Holmes # 2
vitamin E ) IM East # 3
vitamin D ) IM East # 1
degree Celsius ) Holmes # 1
1 2 3 1 2 3 4 5 6 7 8a ) B )Figure 3. Agarose gel with Red plasmid control and Environmental mini-prep consequences. a ) An agarose gel with 1kb ladder in the left lane and Red plasmid control in lanes 2 and 3. B ) Agarose gel made by cataphoresis with 1kb ladder in the left lane and ruddy control plasmid in lane 2. Lanes 3 and 4 contained sample from environment Holmes bathroom 1 # 1. Lanes 5 and 6 contained Holmes bathroom 2 # 11, and lanes 7 and 8 had sample from IM East bathroom 1 # 12.a ) B )Figure 4.
Determination of the Gram individuality of environmental isolate Holmes 2 # 11. a ) Control Gram stain incorporating Gram positive and Gram negative bacteriums. The Gram positive Staphylococcus aureus bacterium ( stained purple ) appear in the upper centre of the exposure, while the Gram negative E. coli ( stained pink ) appear around the fringe. B ) Gram discoloration of bacteriums strain # 11 isolated from Holmes bathroom 2. Based on a comparing to the Gram positive and Gram negative controls, we concluded that Holmes 2 # 11 was Gram negative.
Cells from both a ) and B ) were observed with a compound microscope with the 100x aim ( 1000x magnification ) under oil submergence.3214765
Figure 5. Trial of transmutation using control Red plasmid on E. coli bacteriums.
These plates show a successful transmutation of E. coli bacteriums to hold antibiotic opposition to ampicillin after transforming them with the control Red plasmid. Plate 4 is a home base that had the transmutation, but is turning on an LB agar home base without any antibiotic.
Testing growing of the E. coli without transmutation ( adding merely H2O ) was used to demo that the antibiotic Principen was working ( 3 and 7 ) . The pLITMUS28i plates serve as a positive control for transmutation of the bacteriums ( 5 and 6 ) . We concluded from the positive pLITMUS28i trial that the Red plasmid contains the familial information for Principen opposition due to the growing of the E. coli on the home base 2 incorporating this antibiotic.
The bacterium did non turn on Kantrex after the transmutation ( 1 ) , so we concluded that the plasmid did non code for Kantrex opposition.Figure 5. These transmutation plates show a successful transmutation of E. coli bacteriums to hold antibiotic opposition to ampicillin after transforming them with the control Red plasmid.a ) B )Figure 6. Eosin Methylene Blue ( EMB ) agar and MacConkey agar trials on Holmes 2 # 11.
a ) The sample of bacteriums from Holmes 2 # 11 did non turn on the EMB agar home base after being incubated at 37A°C for 24 hours. Bacterias that are Gram-negative grow on such agar, so this trial would take us to reason that the bacteriums are Gram-positive. B ) Holmes 2 # 11 was streaked onto a home base incorporating MacConkey agar and incubated at 37A°C for 24 hours. The bacterium did non turn in this agar which would propose a consequence of Gram-positive bacteriums.1 2 3 4Figure 7. Restriction digestion of Red control plasmid utilizing Eco RV, Eco RI, and PST 1 limitation enzymes.
Lane 4 contains the 1kb ladder, and it ran highly fast in this case. It is unknown why it ran faster than usual. Lane 3 contains an undigested Red control plasmid. Lane 2 contains a Red control plasmid digested with EcoRV and PST I restriction enzymes. Lane 1 contains Red control plasmid digested with EcoRI and PST I restriction enzymes.
Table 1. Colony count of antibiotic immune strains of bacteriums
A
Holmes 1Holmes 2Holmes 3IM East 1IM East 2IM East 3Tetracycline534151Ampicillin263630Kanamycin31085111Entire101915121910X2 consequences:Tetracycline: p-value = 0.671Ampicillin: p-value = 0.791Kanamycins: p-value = 0.823
