Research Article Differential Bacteriostatic Effects of...

Hindawi Publishing CorporationISRN ToxicologyVolume 2013 Article ID 415070 6 pageshttpdxdoiorg1011552013415070

Research ArticleDifferential Bacteriostatic Effects of Sucralose onVarious Species of Environmental Bacteria

Arthur Omran1 Ronald Baker2 and Charles Coughlin1

1 Department of Biology University of North Florida Jacksonville FL 32224 USA2 Florida Department of Health Bureau of Public Health Laboratories Jacksonville FL 32202 USA

Correspondence should be addressed to Charles Coughlin cbcoughlunfedu

Received 22 July 2013 Accepted 28 August 2013

Academic Editors C C Yang and B Zhou

Copyright copy 2013 Arthur Omran et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sucralose was developed as a low-cost artificial sweetener that is nonmetabolizable and can withstand changes in pH andtemperature It is not degraded by the wastewater treatment process and thus has been found in waste water estuaries rivers and theGulf Stream Since themolecule canwithstand heat acidification andmicrobial degradation it is accumulating in the environmentThe highest concentration of environmental sucralose detected to date is 300 ngL Our lab has isolated six bacterial species fromareas that have been exposed to sucraloseWe then cultured these isolates in the presence of sucralose looking for potential sucralosemetabolism or growth acceleration Instead we found something very interesting bacteriostatic effects exhibited on all six isolatesThis inhibition was directly proportional to the concentration of sucralose exposureThe efficiency of the growth inhibition seemedto be species specific with various concentrations inhibiting each organism differently

1 Introduction

An unexpected contaminant in our aquatic and costal envi-ronments is artificial sweeteners [1] Due to the human inabil-ity tometabolize them they are passed on to the environmentvia human excrement Naturally the highest concentration ofartificial sweetener contaminants is in waste water treatmentplantsrsquo reservoirs Artificial sweeteners such as saccharinand cyclamates are detected in lower concentrations and arefound 90 degraded by the wastewater treatment processSucralose however is found in higher concentrations and isminimally degraded [2] Degradation only occurs to a limitedextent during hydrolysis ozonation and microbial processesindicating that breakdown of sucralose will likely be slow andincomplete leading to accumulation of sucralose in surfacewaters [3]This indicates that conventional waste water treat-ment is ineffective at degrading sucralose [2] Fromwastewa-ter facilities the pollutants are dumped into public waterwaysand sucralose has been detected in rivers in North Carolinain the Gulf Stream and even in the waters of the Florida Keys[4] Also for the first time scientists are detecting sucralose inthe USA inland surface waters [2] Artificial sweeteners havebeen considered contaminants by environmental scientists

for a short time because artificial sweeteners are water con-taminants that are highly specific to wastewater [1]

Most artificial sweeteners are either partially or complete-ly broken down due to the waste water treatment processusing high temperatures changes in pH and constant filtra-tion It would seem that the exception is sucralose due to itsability to withstand drastic pH and temperature changes itis also small enough to pass through the filtration processassociated with the treatment Hence sucralose is being con-tinuously released into our environment in ever increasingconcentrations and due to the human consumersrsquo inabilityto metabolize this artificial sweetener we are dumping itinto our water ways and it is collecting over time [4] Astime passes sucralose will spread to other aquatic and costalecosystems increasing concentration [3]Thepersistent qual-ities of sucralose may lead to chronic low-dose exposure withlargely unknown consequences for human and environmen-tal health [3] It is also unknownwhat this increasing concen-tration of sucralose is doing to environmental microbes

Sucralose may be acting on microbes by inhibitinggrowth Studies of human oral and gut bacteria have shownan inhibition of bacterial growth in the presence of sucralose[5] The same may be true for environmental microbes In

2 ISRN Toxicology

Table 1 A list of environmental sample sites

Sample number Location Sample type GPS coordinate northwest1 Lake Oneida Water and soil 30266912 minus815133472 The Rudder Club Dock (St Johns River) Water and soil 30193071 minus816912663 Duval County Dock (St Johns River) Surface water 30165346 minus816455594 St Johns Parkway Dock (St Johns River) Surface water 30045679 minus816671925 Clay County WasteWater Facility Nutrient poor wastewater 30093079 minus817645246 St Johns County Waste Facility Purified wastewater product 30106153 minus816256937 Guana River Road (Estuary) Water and soil 30 0110158402304ndash81 1910158404221

Table 2 Isolate identity and morphology

Organism identity based on 16s rRNA gene Gram character Shape Colony morphologyMicrobacterium sp U 13 Gram+ Cocci Greyish pale filamentous flat with filiform marginsStenotrophomonas sp I 61 Gramminus Cocci Yellowish white circular umbonate form with entire marginsRhizobium borbori Gramminus Cocci Grey circular convex form with entire marginsCitrobacter murlinlae Gramminus Basili Bright white umbonate form with entire marginsEnsifer arboris Gramminus Basili Dull white rhizoid form with filiform marginsStreptomyces badius Gram+ Basili Bright white filamentous form with filiform margins

one study the incorporation of 126mmolL sucralose intoglucose agar medium caused total inhibition of growth ofStreptococcus sobrinus 6715-17 Streptococcus sanguis 10904Streptococcus sanguis Challis Streptococcus salivarius andActinomyces viscosusWVU627 [5] In a related study ratswereinfected with Streptococcus sobrinus and given sucrose waterdiet and they developed caries lesionsThen another group ofrats was given the same bacteria but sucralosewater instead ofsugar waterThose rats had a drastic decrease in caries lesionsin their teeth demonstrating that oral bacteria cannot growon the artificial sweetener hence causing less damage provingthat sucralose is noncariogenic [6] These are good examplesof sucralose inhibiting bacterial growth however there havebeenminimal studies on the inhibitory effects of sucralose onenvironmental microbes

Sucralose is increasing in concentration in ourwaterwaysand it has been shown in previous studies to be harmful tooral and gut bacteriaWe propose that sucralose can negative-ly affect environmental bacteria

2 Methods and Materials

21 Collection of Bacteria Water samples and soil samplesfrom various test sites around Jacksonville Florida (Table 1)were collected aseptically using autoclaved collection flasksand jars The samples were then used for microbial isolation

22 Isolation of Bacteria Fluid and soil extracted from sam-ples were serially diluted with sterile 089NaCl solutionthen spread plated on Tryptic Soy Agar (TSA) (Difco Labora-tories Michigan USA) plates infused with 80mM sucraloseand incubated at 327∘C for 48 h Bacteria were isolated intopure cultures on subsequent TSA slants based on colonymor-phology (Table 2) Isolates of each bacterium were incubated(327∘C) for one day for microbial analysis

23 Microbial Analysis Individual isolates were extractedfrom pure culture and gram stained The isolates were thenanalyzed using basic light microscopy to identify individualgram characters and cellular morphology (Table 2) Once alist of the isolates with their microbial characters was formedthey were screened for sucralose metabolism

24 Sucralose Metabolism Inspection In order to inspectorganismal growth in the presence of sucralose 01mL ofisolated cell cultures was diluted with 29mL of 089 NaClsolutionThese samples were streak plated ontoM9 agar con-taining glucose (Technova Nova Scotia Canada) (positivecontrol) M9 agar containing sucrose (positive control) M9agar containing sucralose and glucose (experimental) M9agar containing sucralose (experimental) and M9 agar con-taining no sugars (negative control) Isolates which exhibitedgrowth on theM9 agar containing sucralose and glucose wereselected for further experimentation (Table 3) This is due tothe possibility that they may be resistant to or metabolizingsucralose which was inspected during the growth testingexperiment The selected isolates were then identified via 16SrRNA sequencing

25 Genomic DNA Extraction and PCR of 16S rRNA GeneGenomic DNA was extracted from each of the selectedbacterial isolates using the Ultraclean Microbial DNA Iso-lation Kit in accordance with the manufacturer protocols(MO BIO Laboratories California USA) The 16S rRNAgene was amplified using the bacterial consensus primers8F (51015840AGTTGATCCTGGCTCAG 31015840) and 1492R (51015840 ACC-TTGTTACGACTT 31015840) The long polymerase chain reactions(PCR) consisted of 417 120583L dH

2O 50120583L 10xTaq buffer 15 120583L

50mMMgCl2 05 120583L 10 120583M forward primer 10 120583M reverse

primer 04 120583L 25mM dNTPs 04 120583L 5U120583L Taq polymeraseand 1120583L genomic DNA in a final volume of 50 120583L DNA

ISRN Toxicology 3

Table 3 Initial CFU counts for isolate selection

Organism identity basedon 16s rRNA gene

CFU count on M9agar with glucose Sucrose Glucose and

80mM sucralose Sucralose Nosugars

Microbacterium sp U 13 281 201 45 0 0Stenotrophomonas spI 61 233 80 40 0 0

Rhizobium borbori 171 126 32 0 0Citrobacter murlinlae 262 99 60 0 0Ensifer arboris 285 140 63 0 0Streptomyces badius 294 133 88 0 0

amplificationwas performedwith the following thermocyclerregime 2min at 98∘C followed by 33 cycles of 98∘C for 30 s45∘C for 60 s 72∘C for 90 s and a single step at 72∘C for 10minShort PCR amplification consisted of 50120583L reactions withanalogous reagentsconcentrations to the long PCR usingthe additional primers 760R (51015840 CTACCAGGGTATCTAAT31015840) and 790F (51015840 ATTAGATACCCTGGTAG 31015840) with thefollowing thermocycler settings 25 cycles of 98∘C for 30 s44∘C for 45 s and 72∘C for 90 s

26 PCR Cleanup Cycle Sequencing and Ethanol Precipita-tion The short PCR products were cleaned up using theQIAquick PCR Purification Kit following the manufacturerprotocols (Qiagen California USA)The four primers afore-mentioned were employed for cycle sequencing on a CEQ8000 Genetic Analysis System (Beckman Coulter CaliforniaUSA) using 1 120583L GenomeLab DTCS Quick Start Master Mix2120583L primer 2 120583L DNA and 7 120583L dH

2O Cycle sequencing

consisted of 33 cycles at 96∘C for 30 s 37ndash47∘C for 15 s and60∘C for 4min Sequencing reactions were performed usingeach of the amplification primers and internal primers so thateach fragmentwas sequenced in both the forward and reversedirections Products were cleaned and precipitated accordingto the manufacturer specifications (Beckman Coulter Cali-fornia USA)

27 Identification of Bacterial Species The obtained sequen-ces were compared to other sequences using the BLASTfunction through the NCBI website (httpwwwncbinlmgovBLAST) Sequences were determined 99 certain thatthe isolates were not new species Isolates were then identifiedto the level of species (Table 2)

28 GrowthTurbidity Testing Individual isolates were thencultured in Tryptic Soy Broth (TSB) media (Difco Labora-tories Michigan USA) and incubated at 25∘F The controlgroup consisted of 5mL of TSB and the experimental groupsincluded 5mL TSB with 05mL of 10 20 30 and 40sucralose added respectively Turbidity of the cultures wasmeasured over the next 9 days at the same time each day usinga Sequoia Turner Ultraviolet Light Spectrophotometer set to620 nm wavelength (Figure 1)

29 Sucralose Metabolism Validation Individual isolateswere cultured in M9 Broth media (Technova Nova Scotia

002040608

1

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)

Average bacterial sucralose metabolism growth curve

M9 glucoseM9 sucraloseM9 no carbon

minus02

Figure 1 Composite growth curves for bacteria cultured in M9media with various carbon sources

Canada) and incubated at 25∘C The control group consistedof 5mL ofM9 broth with no carbon source the experimentalgroup included a 5mL ofM9 broth with sucralose as the onlycarbon source Turbidity of the cultures was measured overthe next 9 days at the same time each day using a SequoiaTurner Ultraviolet light Spectrophotometer set to 620 nmwavelength (Figure 2)

210 Disk Diffusion Assay and Determination of the Type ofInhibition Each bacterial isolate was spread plated into alawn of confluent growth onto TSA media Filter disks wereprepared by whole punching out Whatman Grade Num-ber 2V filter paper and impregnating the disks with 16Msucralose The antibiotic sensor disk was placed onto the sur-face of the media 3 disks per petri dish These samples wereincubated overnight at 25∘C Diameters of the zones of inhi-bition were measured and recorded The zones of inhibitionwere then swabbed and used to inoculate new TSA mediaThese re-culture plates were incubated over night at 25∘CThere-culture plates were then inspected for any growth

3 Results and Discussion

The glucose100mM sucralose M9 agar completely inhibitedgrowth for 22 of the 28 isolates (results not shown) Theisolates that were chosen for gene sequencing and furtherexperimentation all were able to withstand the M9 sucralose

4 ISRN Toxicology

Table 4 Disk diffusion assay data and zone of inhibitions are indicated Regrowth from inhibited zones was tested regrowth indicated abacteriostatic inhibition not bactericidal

Isolate Regrowth Diameters of inhibition (mm) Averageinhibition

Microbacterium sp U 13 yes 112 11 15 1 105 1 112Stenotrophomonas sp I 61 yes 2 21 23 15 16 19 19Rhizobium borbori yes 11 08 11 115 13 1 107Citrobacter murlinlae yes 13 1 09 08 1 1 1Ensifer arboris yes 09 09 08 07 09 1 0866Streptomyces badius yes 05 06 07 07 05 1 0667

00102030405060708

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Time (days)

Ensifer arboris

Control1117 mM8375mM

5578mM278mM

Figure 2 Growth curves for bacterial isolate Ensifer arboris undervarying concentrations of sucralose

and glucose agar (Table 3) Of the 28 isolates extracted fromenvironmental samples only 6 had growth on the sucra-loseglucose media They were identified as the bacteriaMicrobacterium sp U13 Stenotrophomonas sp I 61 Rhizobi-um borbori Citrobacter murlinlae Ensifer arboris and Strep-tomyces badius

These 6 isolates had fewer CFUs on the sucraloseglucosemedia than they had on the positive control groups of sucroseand glucose and had no growth on the sucralose onlyM9 agar(Table 3) These organisms were not metabolizing sucralose(Table 3 Figure 1)

Six unique bacterial isolates were obtained of which fourwere Gramminus and 2 Gram+ (Table 2) The isolates were sub-cultured in triplicate in the presence of 278mM 5578mM8375mM and 1117mM sucralose to elucidate effects ofsucralose on bacterial growth with controls consisting of iso-lates emended with an additional volume of growth medium

A growth curve showed a decrease in growth with thosestrains receiving sucralose addition compared to the control(Figure 1) An ANOVA indicated a significant (119875 lt 0001)difference between control groups and experimental strainsamended with 8375mM and 1117mM sucralose with exper-imental strains showing decreased growth rate (Figures 23 4 5 6 and 7) These results indicate that the addition ofsucralose is a growth inhibitor formultiple strains of bacteria

0005

01015

02025

03035

04

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)

Stenotrophomonas sp I 61

Control

8375mM

5578mM278mM1117mM

Figure 3 Growth curves for bacterial isolate Stenotrophomonas spI 61 under varying concentrations of sucralose

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

0

02

04

06

08

1

12

14

Abso

rban

ce (n

m)

Rhizobium borbori

Figure 4 Growth curves for bacterial isolate Rhizobium borboriunder varying concentrations of sucralose

We observed that there was no statistically significant(119875 gt 099) difference between control groups and 257mMfor our isolates (Figures 2 3 4 5 6 and 7) The additionof low concentrations of sucralose (257 nm and less) had nosignificant effect on growth whereas higher levels (8375mMand 1117mM) reduced growth drastically and possibly ledto cell death At 1117mM significant inhibition of total cell

ISRN Toxicology 5

002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

Microbacterium sp U 13

Figure 5 Growth curves for bacterial isolate Microbacterium spU13 under varying concentrations of sucralose

002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)

Citrobacter murlinlae

Control

8375mM

5578mM278mM1117mM

Figure 6 Growth curves for bacterial isolate Citrobacter murlinlaeunder varying concentrations of sucralose

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

002040608

112141618

Abso

rban

ce (n

m)

Streptomyces badius

Figure 7 Growth curves for bacterial isolate Streptomyces badiusunder varying concentrations of sucralose

culture growth (Figures 3 4 6 and 7) Of those 6 bacterialisolates not completely inhibited by sucralose two showedsignificantly decreased growth (119875 lt 005) response inthe presence of 5578mM sucralose (Figures 2 and 3) Thenegative effect that sucralose had on their growth rates wasdirectly proportional to the concentration of sucralose addedto the growth media On average the 278mM and 5578mMsucralose treatments did not significantly inhibit the growthrates of these isolates or minimally inhibit the isolates intheir growth rates The 8375mM and 1117mM treatmentsdid have a rather drastic inhibitory effect on bacterial growthacross the board The inhibition exhibited on all isolates wassimilar to that of previous studies in which sucralose causestotal growth inhibition of oral bacteria in lab mice [5 7]

Disk diffusion assay data indicated differential growthinhibition as well Regrowth from inhibited zones was testedregrowth indicated a bacteriostatic inhibition for each bacte-rial isolate This means that sucralose is a bacteriostatic agent(Table 4) This study is limited in its scope as it is an inquiryof pure scienceThis is due to current environmental concen-trations being in the 300 ngL and below range The researchis meant to investigate the possibility of growth inhibitionoccurring in environmental isolates in pure culture

4 Conclusions

The current environmental concentrations of sucralose(300 ngL in waste water and less in fresh waters) do notseem to have any effect on bacterial growth Sucralose ishowever increasing in its concentration due to its inability tobe degraded by pH and temperature changes [2] and itsnonmetabolism by microbes (Figure 2) Sucralose would athigher concentrations potentially 5578mM hurt the bacte-rial community This type of contamination would take avery long time to accumulate however it is troubling becausethe bacterial community is the basis for the health of manyecosystems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper The authors ofthis paper do not have a direct financial relation with thecommercial identitiesmentioned in the paper thatmight leadto a conflict of interests nor have they received funding fromany third party

Acknowledgments

Special thanks are to Dr Paul D Fiorella of the FloridaDepartment of Health for help with 16S sequencing andfor guidance with species identification Also thanks due toDr Janice Swenson for guidance with the inhibition typedetermination

References

[1] A Lillicrap K Langford andK E Tollefsen ldquoBioconcentrationof the intense sweetener sucralose in a multitrophic battery of

6 ISRN Toxicology

aquatic organismsrdquo Environmental Toxicology and Chemistryvol 30 no 3 pp 673ndash681 2011

[2] C I Torres S Ramakrishna C-A Chiu K G Nelson P West-erhoff and R Krajmalnik-Brown ldquoFate of sucralose duringwastewater treatmentrdquo Environmental Engineering Science vol28 no 5 pp 325ndash331 2011

[3] L SohKAConnors BWBrooks and J Zimmerman ldquoFate ofsucralose through environmental and water treatment process-es and impact on plant indicator speciesrdquoEnvironmental Scienceamp Technology vol 45 no 4 pp 1363ndash1369 2011

[4] R Mead J Morgan G Avery Jr et al ldquoOccurrence of the artifi-cial sweetener sucralose in coastal and marine waters of theUnited Statesrdquo Marine Chemistry vol 116 no 1ndash4 pp 13ndash172009

[5] D A Young and W H Bowen ldquoThe influence of sucralose onbacterial metabolismrdquo Journal of Dental Research vol 69 no 8pp 1480ndash1484 1990

[6] W H Bowen and S K Pearson ldquoThe effects of sucralose xyli-tol and sorbitol on remineralization of caries lesions in ratsrdquoJournal of Dental Research vol 71 no 5 pp 1166ndash1168 1992

[7] W H Bowen D A Young and S K Pearson ldquoThe effects ofsucralose on coronal and root-surface cariesrdquo Journal of DentalResearch vol 69 no 8 pp 1485ndash1487 1990

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2 ISRN Toxicology

Table 1 A list of environmental sample sites

Sample number Location Sample type GPS coordinate northwest1 Lake Oneida Water and soil 30266912 minus815133472 The Rudder Club Dock (St Johns River) Water and soil 30193071 minus816912663 Duval County Dock (St Johns River) Surface water 30165346 minus816455594 St Johns Parkway Dock (St Johns River) Surface water 30045679 minus816671925 Clay County WasteWater Facility Nutrient poor wastewater 30093079 minus817645246 St Johns County Waste Facility Purified wastewater product 30106153 minus816256937 Guana River Road (Estuary) Water and soil 30 0110158402304ndash81 1910158404221

Table 2 Isolate identity and morphology

Organism identity based on 16s rRNA gene Gram character Shape Colony morphologyMicrobacterium sp U 13 Gram+ Cocci Greyish pale filamentous flat with filiform marginsStenotrophomonas sp I 61 Gramminus Cocci Yellowish white circular umbonate form with entire marginsRhizobium borbori Gramminus Cocci Grey circular convex form with entire marginsCitrobacter murlinlae Gramminus Basili Bright white umbonate form with entire marginsEnsifer arboris Gramminus Basili Dull white rhizoid form with filiform marginsStreptomyces badius Gram+ Basili Bright white filamentous form with filiform margins

one study the incorporation of 126mmolL sucralose intoglucose agar medium caused total inhibition of growth ofStreptococcus sobrinus 6715-17 Streptococcus sanguis 10904Streptococcus sanguis Challis Streptococcus salivarius andActinomyces viscosusWVU627 [5] In a related study ratswereinfected with Streptococcus sobrinus and given sucrose waterdiet and they developed caries lesionsThen another group ofrats was given the same bacteria but sucralosewater instead ofsugar waterThose rats had a drastic decrease in caries lesionsin their teeth demonstrating that oral bacteria cannot growon the artificial sweetener hence causing less damage provingthat sucralose is noncariogenic [6] These are good examplesof sucralose inhibiting bacterial growth however there havebeenminimal studies on the inhibitory effects of sucralose onenvironmental microbes

Sucralose is increasing in concentration in ourwaterwaysand it has been shown in previous studies to be harmful tooral and gut bacteriaWe propose that sucralose can negative-ly affect environmental bacteria

2 Methods and Materials

21 Collection of Bacteria Water samples and soil samplesfrom various test sites around Jacksonville Florida (Table 1)were collected aseptically using autoclaved collection flasksand jars The samples were then used for microbial isolation

22 Isolation of Bacteria Fluid and soil extracted from sam-ples were serially diluted with sterile 089NaCl solutionthen spread plated on Tryptic Soy Agar (TSA) (Difco Labora-tories Michigan USA) plates infused with 80mM sucraloseand incubated at 327∘C for 48 h Bacteria were isolated intopure cultures on subsequent TSA slants based on colonymor-phology (Table 2) Isolates of each bacterium were incubated(327∘C) for one day for microbial analysis

23 Microbial Analysis Individual isolates were extractedfrom pure culture and gram stained The isolates were thenanalyzed using basic light microscopy to identify individualgram characters and cellular morphology (Table 2) Once alist of the isolates with their microbial characters was formedthey were screened for sucralose metabolism

24 Sucralose Metabolism Inspection In order to inspectorganismal growth in the presence of sucralose 01mL ofisolated cell cultures was diluted with 29mL of 089 NaClsolutionThese samples were streak plated ontoM9 agar con-taining glucose (Technova Nova Scotia Canada) (positivecontrol) M9 agar containing sucrose (positive control) M9agar containing sucralose and glucose (experimental) M9agar containing sucralose (experimental) and M9 agar con-taining no sugars (negative control) Isolates which exhibitedgrowth on theM9 agar containing sucralose and glucose wereselected for further experimentation (Table 3) This is due tothe possibility that they may be resistant to or metabolizingsucralose which was inspected during the growth testingexperiment The selected isolates were then identified via 16SrRNA sequencing

25 Genomic DNA Extraction and PCR of 16S rRNA GeneGenomic DNA was extracted from each of the selectedbacterial isolates using the Ultraclean Microbial DNA Iso-lation Kit in accordance with the manufacturer protocols(MO BIO Laboratories California USA) The 16S rRNAgene was amplified using the bacterial consensus primers8F (51015840AGTTGATCCTGGCTCAG 31015840) and 1492R (51015840 ACC-TTGTTACGACTT 31015840) The long polymerase chain reactions(PCR) consisted of 417 120583L dH

2O 50120583L 10xTaq buffer 15 120583L

50mMMgCl2 05 120583L 10 120583M forward primer 10 120583M reverse

primer 04 120583L 25mM dNTPs 04 120583L 5U120583L Taq polymeraseand 1120583L genomic DNA in a final volume of 50 120583L DNA

ISRN Toxicology 3









2O Cycle sequencing





002040608

1

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)



minus02






4 ISRN Toxicology




00102030405060708

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Time (days)

Ensifer arboris


5578mM278mM






0005

01015

02025

03035

04

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

0

02

04

06

08

1

12

14

Abso

rban

ce (n

m)

Rhizobium borbori



ISRN Toxicology 5

002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM



002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

002040608

112141618

Abso

rban

ce (n

m)

Streptomyces badius




4 Conclusions




Acknowledgments


References


6 ISRN Toxicology












Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 3









2O Cycle sequencing





002040608

1

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)



minus02






4 ISRN Toxicology




00102030405060708

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Time (days)

Ensifer arboris


5578mM278mM






0005

01015

02025

03035

04

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

0

02

04

06

08

1

12

14

Abso

rban

ce (n

m)

Rhizobium borbori



ISRN Toxicology 5

002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM



002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

002040608

112141618

Abso

rban

ce (n

m)

Streptomyces badius




4 Conclusions




Acknowledgments


References


6 ISRN Toxicology












Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

4 ISRN Toxicology




00102030405060708

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Time (days)

Ensifer arboris


5578mM278mM






0005

01015

02025

03035

04

1 2 3 4 5 6 7 8 9Ab

sorb

ance

(nm

)

Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

0

02

04

06

08

1

12

14

Abso

rban

ce (n

m)

Rhizobium borbori



ISRN Toxicology 5

002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM



002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

002040608

112141618

Abso

rban

ce (n

m)

Streptomyces badius




4 Conclusions




Acknowledgments


References


6 ISRN Toxicology












Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 5

002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM



002040608

1121416

Abso

rban

ce (n

m)

1 2 3 4 5 6 7 8 9Time (days)


Control

8375mM

5578mM278mM1117mM


1 2 3 4 5 6 7 8 9Time (days)

Control

8375mM

5578mM278mM1117mM

002040608

112141618

Abso

rban

ce (n

m)

Streptomyces badius




4 Conclusions




Acknowledgments


References


6 ISRN Toxicology












Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

6 ISRN Toxicology












Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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