research article point mutations in the folp gene partly...

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2013 Article ID 367021 6 pageshttpdxdoiorg1011552013367021

Research ArticlePoint Mutations in the folP Gene Partly Explain SulfonamideResistance of Streptococcus mutans

W Buwembo1 S Aery2 C M Rwenyonyi3 G Swedberg2 and F Kironde4

1 Department of Anatomy Makerere University PO Box 7072 Kampala Uganda2Department of Medical Biochemistry and Microbiology Uppsala University Husargaten 3Building D7 Level 3 PO Box 582 SE-75123 Uppsala Sweden

3Department of Dentistry Makerere University PO Box 7072 Kampala Uganda4Department of Biochemistry Makerere University PO Box 7072 Kampala Uganda

Correspondence should be addressed to F Kironde kirondestarcomcoug

Received 5 November 2012 Accepted 24 January 2013

Academic Editor Marco Gobbetti

Copyright copy 2013 W Buwembo et al This 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

Cotrimoxazole inhibits dhfr and dhps and reportedly selects for drug resistance in pathogens Here Streptococcus mutans isolateswere obtained from saliva ofHIVAIDS patients taking cotrimoxazole prophylaxis inUgandaThe isolates were tested for resistanceto cotrimoxazole and their folP DNA (which encodes sulfonamide-targeted enzyme dhps) cloned in pUC19 A set of recombinantplasmids carrying different point mutations in cloned folP were separately transformed into folP-deficient Escherichia coli Usingsulfonamide-containing media we assessed the growth of folP-deficient bacteria harbouring plasmids with differing folP pointmutations Interestingly cloned folP with three mutations (A37V N172D R193Q) derived from Streptococcus mutans 8 conferredsubstantial resistance against sulfonamide to folP-deficient bacteria Indeed change of any of the three residues (A37V N172D andR193Q) in plasmid-encoded folP diminished the bacterial resistance to sulfonamide while removal of all three mutations abolishedthe resistance In contrast plasmids carrying four other mutations (A46V E80K Q122H and S146G) in folP did not similarlyconfer any sulfonamide resistance to folP-knockout bacteria Nevertheless sulfonamide resistance (MIC= 50 120583M) of folP-knockoutbacteria transformed with plasmid-encoded folP was much less than the resistance (MIC = 4mM) expressed by chromosomally-encoded folP Therefore folP point mutations only partially explain bacterial resistance to sulfonamide

1 Introduction

Streptococcus mutans are commensal bacteria found in theoral cavity [1] These bacteria which belong to the ViridansStreptococci Group (VSG) cause dental caries and infre-quently give rise to extra oral infections like subacute bac-terial endocarditis [1 2] Although dental caries is not usuallytreated by antibiotics the VSG have attracted interest due totheir potential to act as reservoirs of resistance to antibioticdeterminants [3] Additionally in individuals taking antibi-otics as prophylaxis resistance of commensals to antibi-otic determinants could be selected [4] and transferred topathogenic organisms [5] such as Streptococcus pneumoniaewhich kills over 1000000 children worldwide every year [4]

Cotrimoxazole (SXT) is a combination drug (sul-famethoxazole plus trimethoprim) that is commonly used

as prophylaxis in HIVAIDS patients [6] Sulfamethoxa-zole is a long-acting sulphonamide In addition to wideusage as prophylaxis SXT is also a highly prescribed drugespecially in Sub-Saharan Africa due to its low cost andeasy availability Sub-Saharan Africa is reputed for highantibiotic abuse [7] In Uganda SXT is not only highlyprescribed in dental practice [8] but also selected for mul-tiple antibiotic resistance in Streptococcus mutans amongHIVAIDS patients [7] Despite these findings data onthe mechanisms of SXT resistance in commensal bacteriasuch as Streptococcus mutans is still scanty In order tobetter understand the mechanism of Streptococcus mutansresistance to SXT we characterised the S mutans folP genethat encodes dihydropteroate synthase the target enzymeof sulfonamides [9] Previously we reported [7] that folP genefrom the highly sulfonamide resistant S mutans isolate 797

2 International Journal of Microbiology

did not confer sulfonamide resistance to E coli folP knockoutbacteria and that sequencing of the folA gene of trimethoprim(TMP) resistant isolates did not reveal any mutations How-ever the folP gene is very polymorphic [7] and at least one ofthe variants of folP confers high sulfonamide resistance to Ecoli folP knockout cells In the current study we report site-directed mutagenesis experiments in which we altered pointmutations in S mutans folP gene inserting the mutagenizedfolP DNA in pUC19 plasmids which were then transformedinto folP deficientE coliC600 cells By assessing the growth ofthe transformant E coli Delta folP cells on media containingdifferent levels of sulfamethoxazole the influence of individ-ual amino acids on sulfonamide resistance in the folP geneknockout bacteria was determined

2 Methods

The mechanism of resistance to sulfamethoxazole (SMX) inS mutans was characterised as summarized in Figure 1

21 Bacterial Strains and Plasmids The bacterial strains usedin this study (Table 1) were previously isolated [7] fromoral specimens of HIVAIDS patients taking cotrimoxazoleas prophylaxis in Kampala Uganda The cloning vectorspJet12blunt (Fermentas Lithuania) and pUC19 [12] wereused E coli top ten cells (Invitrogen USA) and E coli recip-ient strain C600 ΔfolP [13] which is a folP knockout strainwere used in the transformation experiments Streptococcuspneumoniae ATCC 49619 was used as a susceptible controlwhen determining MICs

22 Susceptibility Testing Minimal inhibitory concentrations(MICs) were determined by the 119864-test method (AB BiodiskSweden) following the manufacturerrsquos recommendations Alltests were done on Iso-Sensitest Agar (ISA Oxoid UK)Plates were incubated at 37∘C in 5 CO

2for 24 h For

determination of sulfonamide resistance conferred by clonedfolP genes E coli C600 bacteria were grown in Iso-SensitestBroth (ISB Oxoid UK) to a cell density of 108mL dilutedto 104mL and plated on ISA plates containing varyingconcentrations of sulfathiazole (Sigma Aldrich USA)

23 DNA Extraction Isolates of Streptococcus mutans wereincubated at 37∘C for 12 h on Iso-Sensitest Agar Bacterialcolonies were re-suspended in brain heart infusion broth(BioMerieux France) and incubated at 37∘C for 24 h in anatmosphere of 5 carbon dioxide Chromosomal bacterialDNA from the cultured broth was then extracted using theWizard Genomic DNA Purification Kit (Promega USA)

24 Cloning The PCR primer sequences used were basedon the published sequence of the folP gene of StreptococcusmutansUA159 [10] (Table 1) FolP gene amplificationwas per-formed in 50-120583L volumes containing 05 120583M of each primer100 120583M of the four deoxyribonucleoside triphosphates 5units of DNA polymerase (Pfu Fermentas) 2120583L of template

for studying bacterial resistance tosulfamethoxazole (SMX)

Extracted Streptococcus genomicDNA folP DNA cloned into pJET

mutation patterns in the folP gene were used

Recombinant pJET transformedinto E coli top ten cells

replicated by culture and extracted

folP DNA recloned in pUC 19Recombinant pUC 19

mutagenized to produce orremove specific folP mutations

Recombinant pUC 19 carryingdifferent mutations in folP

transformed into ΔfolP E coli C60013

Reconstituted ΔfolP E coli C600grown at different sulfonamide

concentrations to determine MICs

DNA of recombinant pUC 19

determine and confirm the plasmidand insert nucleotide sequences

carrying folP mutations sequenced to

797 (10) have been described previously8Streptococcus isolates 8 797 135 7 477 and

Isolates 8 797 135 7 and 477 with different

Figure 1 Flow chart showing characterization of folP gene Plasmidscarrying folP gene were transformed in folP gene knockout bacteriato determine the effect of different mutations in plasmid encodedfolP on bacterial resistance to sulfonamides

DNA (50ndash500 ng) preparation and 1X reaction buffer (PfuFermentas) containing 2mM MgSO

4 Amplification reac-

tions were performedwith the Eppendorf mastercycler gradi-ent thermocycler (Eppendorf Germany) using the followingprogram heating at 94∘C for 2min followed by 25 cyclesconsisting of a denaturation at 94∘C for 30 s annealing at50∘C for 30 s and an extension at 72∘C for 15min This wasfollowed by a final extension of 72∘C for 5min and a holdingstep at 16∘C

The PCR products were cleaned by the PCR Cleanup kit(Omega USA) and then used for cloning into the pJet vectorusing the blunt end pJET cloning kit protocol (FermentasLithuania) The ligated products were introduced into E colitop ten cells by heat-shock CaCl

2transformation method

Plasmids were prepared from the transformants usingthe plasmid preparation kit (Omega USA) and prepared forsequencing

25 Sequence Analysis For sequencing the plasmids theBigDye Terminator labelled cycle-sequencing kit (AppliedBiosystems) and anABI prism 310Genetic Analyzer (AppliedBiosystems) were used The results of the folP gene sequenceanalysis were compared with database sequences of Strepto-coccus mutans UA159 [10] and NN2025 [11] using the BLASTprogramme at NCBI [14]

International Journal of Microbiology 3

Table 1 Characteristics of bacterial isolates used in the current study the respective genes and susceptibility to Cotrimoxazole (STX)sulfamethoxazole (SMX) and trimethoprim (TMP) as determined by 119864-test

Isolate Accession number of folP genenucleotide sequence used

Mutations in the folPgene STX Sulfonamide

susceptibilityTrimethoprimsusceptibility

Mutations in thefolA gene

S mutans 8Not yet submitted to genebanks but previouslypublished [7]

A37V N172Dand R193Qlowast gt32 120583gmL gt1024120583gmL

(gt4 mM) gt32120583gmL None

S mutans 797 HE5995331 A46V E80K Q122Hand S146Glowastlowast 05 120583gmL gt1024120583gmL

(gt4mM) 2 120583gmL None

S mutans 135 Not yet submitted butpreviously published [7]

A63S W174LK L175Fand M189Ilowastlowast 8120583gmL gt1024120583gmL


S sobrinus 7 HE 5995351 None gt32 120583gmL gt1024120583gmL(gt4mM) gt32120583gmL None

S downei 477 Similar to ZP 077252571 None 0125 120583gmL Not done Not done Not donelowastMutations as compared to UA159 [10] lowastlowastMutations as compared to NN2025 [11]

Table 2 Primers used for cloning and site-directed mutagenesis

Primer name Nucleotide sequenceMutans DHPSph 51015840-GAT CGA TCG CAT GCA CAT CAT AAC TAG GGA GCA AGC-31015840

mutansDHPSBam 51015840-GAT GGA TCG GAT CCA AAA TAATCT TAT CCA TAA CAC CCT CA-31015840

dhpssfwph 51015840-AAC CTA CTG CAT GCA TAA GAA TCA G-31015840

dhpssreveco 51015840-ATT GTA GGA ATT CTT CTA GAA AGA TCC-31015840

downeifolpfw 51015840-GCA TGC CAA AGA CAG GAA TTG CTG AC-31015840

Downeifolprevps 51015840-CTG CAG CCA CAA AAA TTT GCC CCA GAC-31015840

Primers for changing specific amino acids in isolate 797DHPS46AVfw 51015840-TGA AGC CAT GTT AGT AGC AGG AGC GGC TA-31015840

DHPS46AVrev 51015840-TAG CCG CTC CTG CTA CTA ACA TGG CTT CA-31015840

DHPS80aEKfw 51015840-TCG TTC CAA TTG TTA AAG CTA TTA GCG AA-31015840

DHPS80aEKrev 51015840-TTC GCT AAT AGC TTT AAC AAT TGG AAC GA-31015840

DHPS122QHfw 51015840-CTT TAT GAT GGG CAC ATG TTT CAA TTA GC-31015840

DHPS122QHrev 51015840-GCT AAT TGA AAC ATG TGC CCA TCA TAA AG-31015840

DHPS146SGfw 51015840-GTG AAG AAG TTT ATG GCA ATG TAA CAG AA-31015840

DHPS146SGrev 51015840-TTC TGT TAC ATT GCC ATA AAC TTC TTC AC-31015840

Primers for changing specific amino acids in isolate 8V37Afw 51015840-AAC CAA TCG ATC AGG CTC TAA AAC AGG TTG A-31015840

V37Arev 51015840-TCA ACC TGT TTT AGA GCC TGA TCG ATT GTT T-31015840

D172Nfw 51015840-GGA GTT AAA AAA GAA AAT ATT TGG CTT GAT C-3D172Nrev 51015840-GAT CAA GCC AAA TAT TTT CTT TTT TAA CTC C-31015840

Q193Rfw 51015840-ACA TGG AAC TTC TAC GAG GCT TAG CGG AGG T-3Q193Rrev 51015840-ACC TCC GCT AAG CCT CGT AGA AGT TCC ATG T-31015840

26 Site-Directed Mutagenesis Mutagenesis was carried outusing a 50120583L reactionmix (Fermentas Lithuania) containing1X Pfu buffer with MgSO

4 25 units Pfu DNA Polymerase

(Fermentas Lithuania) 01mM dNTPs 10ndash100 ng of tem-plate DNA inserted in pUC19 and 1120583M of each primer(Table 2) The PCR program started with a heating step at95∘C for 30 s followed by 18 cycles consisting of a denatura-tion step of 95∘C for 30 s annealing of 50∘C for 30 s and anextension of 68∘C for 7min

Site-directed mutagenesis products (17120583L of the PCRproduct) were digested with 5 units of the restriction enzyme

Dpn1 (Fermentas Lithuania) in Buffer Tango to removeunchanged DNA The mixture was incubated at 37∘C for 4 hbefore incubating at 80∘C for 20min to inactivate Dpn1

27 Transformation Ten 120583L of the digested mutagenesisproduct were transformed into CaCl

2-treated folP knockout

E coli competent cells Recombinant plasmids were preparedfrom part of a single transformant bacterial colony usingthe plasmid miniprep kit (Omega USA) The plasmids werethen sequenced as described above to confirm that site-directed mutagenesis had occurred Bacteria from the same


transformant colony were then tested for growth at differentSMX concentrations

3 Results

The results of transformation of S mutans folP gene intofolP gene knockout E coli are shown in Figure 2 As previ-ously reported [7] S mutans isolate 797 carries four pointmutations (A46V E80K Q122H and S146G) in folP geneas compared to the control strain NN2025 [11] but harboursone such mutation (S146G) if compared to control strainUA159 [12] (Table 1) Two other isolates with DHPS sequencediffering from both reference strains were cloned using thesame conditions as for 797Thesewere isolate 8 (A37VN172Dand R193Q) and isolate 135 (A63SW174L L175F andM189I)Of these only the cloned folP gene from isolate 8 conferredsulfonamide resistance to the E coli C600ΔfolP recipient Inthe present paper the above-mentioned four point muta-tions (A46V E80K Q122H and S146G) and those in Smutans isolate 8 (A37V N172D and R193Q) (Table 1) weresuccessfully altered or removed from folP gene by site-directed mutagenesis Nonmutagenized chromosomal DNAfrom isolates S sobrinus 7 and S downei 477 was individuallyinserted into pUC19 as well Effects of the different folP geneconstructs on resistance to sulphonamide were then investi-gated by transforming the recombinant pUC 19 plasmids infolP knockout E coli C600 and assessing the growth of trans-formant bacteria on media containing different concentra-tions of sulfamethoxazole (see Figure 1) Interestingly plas-mids harbouring triple mutant folP of S mutans isolate8 (bearing the mutations A37V N172D and R193Q) con-ferred (to folP knockout E coli) intermediate level resistance(MIC 50 120583M) against sulfonamide (bar A in Figure 2) eventhough this is not equal to 4mM the MIC arising from thechromosomal folP gene in the natural isolate S mutans 8(Table 1) In addition altering any of the three polymorphicamino acids (A37V N172D and R193Q) back to the UA159sequence and transforming the double mutant folP gene intothe E coli Delta-folP cells produced reconstituted knock-out E coli cells of lower level (MIC 30120583M) resistance tosulphonamide (Figure 2 bars B C D) while reversing twoamino acidmutations (Figure 2 bar E) or all three amino acidmutations (Figure 2 bar F) towild-type and transforming folPknockout E coli likewise yielded transformant clones withlow resistance to sulphonamide (MIC 20120583M) On the otherhand as previously reported [7] cloned folP gene from 797did not confer resistance of folP deficient E coli C600 tosulphonamide Moreover changing the DNA encoding fouramino acid mutations (A46V E80K Q122H and S146G) offolP in S mutans isolate 797 and transforming the mutantDNA in folP knockout E coli to comply with either NN2025or UA159 sequences (Figure 2 bar G) did not change suscep-tibility of folP knockout bacteria to sulphonamide Controlsconsisting of folP knockout E coli transformed with pUC 19plasmids encoding mutant folP from S mutans isolate 135(Figure 2 bar H) wild-type folP from S sobrinus 7 (Figure 2bar I) or wild-type folP from S downei 477 produced mini-mal or reduced resistance

A37

V +

N172

D +

R193

Qlowast

37

A +

N172

D +

R193

Qlowast

A37

V +172

N +

R193

Qlowast

A37

V +

N172

D +193

Rlowast

37

A +172

N +

R193

Qlowast

37

A +172

N +193

Rlowast

A46

V +

E80

K +

Q122

H +

S14

6G

A63

S +

W174

LK

+ L175

F +

M189

I

60

40

20

0

MIC

(120583M

)

A

B C D

E F G H I

J

folP mutation genotype

lowastlowastlowast

lowastlowast

S d

owne

i 477

wild

-type

folP

gen

e

S so

brin

us 7

wild

-type

folP

gen

e

Figure 2 Comparing sulfamethoxazole minimal inhibitory con-centrations (MICs) for folP knockout E coli cells transformedwith pUC19 plasmid carrying differing chromosomal folP genes ofstreptococci To determine the effect plasmid encoded mutant folPhas on the sulfamethoxazole resistance of transformed C600ΔfolPE coli bacteria growths (in SMX containing media) were comparedfor folP-deficient E coli transformed with pUC19 carrying triple-mutant folP (residues 37 172 and 193 bar A) double-mutant folP(bars B C and D) single-mutant folP (residue 193 bar E) and wild-type folP (bar F) from S mutans isolate 8 The sulfamethoxazoleresistance of transformed folP deficient cells was notably increasedby transformation with plasmid encoding triple-mutated folP (wild-type folPMIC = 20 120583M triple-mutant folPMIC = 50 120583M) Note theMIC (sulfamethoxazole) for chromosome-encoded folP in Smutansisolate 8 was 4mM (see Table 1) Controls comprising C600ΔfolP Ecoli transformed with pUC19 encoding either mutant folP from Smutans isolates 797 (bar G) and 135 (bar H) or wild-type folP fromS sobrinus isolate 7 (bar I) or S downei isolate 477 (bar J) showedbasal or less sulfamethoxazole resistance (MICs = 20ndash30120583M) lowast Smutans isolate 8 mutant folP lowastlowast S mutans isolate 797 mutant folPlowastlowastlowast S mutans isolate 135 mutant folP

4 Discussion

In the present study we examined Ugandan Streptococcusmutans isolates from HIVAIDS patients who were takingcotrimoxazole as prophylaxis [7] The isolates were found tohave different point mutations in the folP gene in relation towild-type sequences found in databases It should be notedthat these same isolates lacked anymutations in the folA gene

Resistance to sulfonamides in gram positive bacteria hasbeen shown to be due to mutations in the folP gene thatrender the encoded dihydropteroate synthase insensitive to


the drug [15] In the corresponding gene ofPlasmodiummorepoint mutation combinations were previously found to beassociated with higher resistance rates against sulfadoxine inP falciparum [16] In the present study we assessed the influ-ence of different combinations of mutations in S mutans folPby performing mutagenesis experiments to remove muta-tions in the folP gene and transforming the changedDNA intofolP knockout E coli cells which were subsequently tested forgrowth in the presence of varying levels of sulfamethoxazoleWe found that the cloned folP gene from isolate 8 conferredsubstantial sulphonamide resistance to folP knockout E coli(MIC 50 120583M) (Figure 2) but not to the level observed forthe natural isolate S mutans 8 (MIC 4mM) Changes inany of the three divergent amino acids (residues 37 172 and193) of folP reduced the level of resistance to sulfonamideand the removal of all three polymorphisms totally abolishedthe resistance In contrast no combination of the mutationsin folP from isolate 797 (A46V E80K Q122H and S146G)led to differences in susceptibility of folP knockout bacteriato sulfonamide In addition we found that isolates 135 7and 477 with different mutation patterns in folP grew to thesame resistance level (MIC 20120583M) as isolate 797This findingcorroborates the previous report [7] that the cloned folP genefrom 797 does not confer sulfonamide resistance to folP-geneknockoutE coli cells However that the cloned folP gene fromisolate S mutans 8 did not confer equally high sulphonamideresistance to folP knockout E coli as shown by the naturalisolate Smutans 8 (MIC 4mM) suggests that there is anothermechanism of resistance to sulfonamide other than the pointmutations One possibility may be that DHPS synthesisand expression are increased as was found in Streptococcusagalactiae [17] or that there may be point mutations in otherfolate pathway genes We could not rule out other causesof resistance to sulfonamide in Streptococcus mutans sincesequencing the folA gene of Smutans 8 and flanking regionsincluding the promoter did not reveal any mutations (resultsnot shown) Further experiments including whole genomesequencing of S mutans 8 and other sulfonamide resistantstrains may enhance the understanding of sulfa resistance inStreptococci

5 Conclusions

Point mutations are one of the explanations for the mecha-nism of resistance to sulfonamide in Streptococcus mutansHowever cloned folP gene did not confer full resistance tofolP knockout cells compared to the original isolate 797 aresult which does not rule out other possible mechanisms forthe resistance to sulfonamides

Conflict of Interests

The authors report no conflict of interests

Authorsrsquo Contribution

W Buwembo carried out the preparation of bacterial iso-lates and polymerase chain reaction tests participated in

the microbiological antibiotic resistance tests cloning andsequencing analyses and drafted the paper S Aery partici-pated in the mutagenesis experiments G Swedberg designedthe primers and participated in cloning and transformationexperiments F Kironde C M Rwenyonyi and G Swedbergconceived the study participated in its design and coordina-tion and helped in writing the paper All authors read andapproved the final paper

Acknowledgments

The authors are grateful to GS project students namely MaryMacRitchie Soheila Rajabi Marcus Andersson and FredrikJonsson who assisted in this work We sincerely thank thepatients who donated clinical specimens research associatesand administrators of TASO clinic This work was financiallysupported by the Swedish Agency for Research Cooperationwith Developing Countries All the authors are responsiblefor the content and writing of the paper

References

[1] W J Loesche ldquoRole of streptococcus mutans in human dentaldecayrdquoMicrobiological Reviews vol 50 no 4 pp 353ndash380 1986

[2] R Facklam ldquoWhat happened to the streptococci overview oftaxonomic and nomenclature changesrdquo Clinical MicrobiologyReviews vol 15 no 4 pp 613ndash630 2002

[3] A Bryskier ldquoViridans group streptococci a reservoir of resis-tant bacteria in oral cavitiesrdquo Clinical Microbiology and Infec-tion vol 8 no 2 pp 65ndash69 2002

[4] M Wilen W Buwembo H Sendagire F Kironde and GSwedberg ldquoCotrimoxazole resistance of Streptococcus pneu-moniae and commensal streptococci from Kampala UgandardquoScandinavian Journal of Infectious Diseases vol 41 no 2 pp113ndash121 2009

[5] P Echave J Bille C Audet I Talla B Vaudaux and M GehrildquoPercentage bacterial etiology and antibiotic susceptibility ofacute respiratory infection and pneumonia among children inrural Senegalrdquo Journal of Tropical Pediatrics vol 49 no 1 pp28ndash32 2003

[6] A Sosa ldquoWho issues guidelines on use of cotrimoxazole pro-phylaxisrdquo 2006 httpwwwwhointhivpubguidelinesctxenindexhtml

[7] B William C M Rwenyonyi G Swedberg and F KirondeldquoCotrimoxazole prophylaxis specifically selects for cotrimoxa-zole resistance in streptococcus mutans and Streptococcus sobri-nus with varied polymorphisms in the target genes folA andfolPrdquo International Journal of Microbiology vol 2012 Article ID916129 10 pages 2012

[8] A Kamulegeya B William and C M Rwenyonyi ldquoKnowledgeand antibiotics prescription pattern among ugandan oral healthcare providers a cross-sectional surveyrdquo Journal of DentalResearch Dental Clinics Dental Prospects vol 5 no 2 pp 61ndash66 2011 httpdentistrytbzmedacirjoddd

[9] O Skold ldquoResistance to trimethoprim and sulfonamidesrdquoVeterinary Research vol 32 no 3-4 pp 261ndash273 2001

[10] D Ajdic W M McShan R E McLaughlin et al ldquoGenomesequence of streptococcus mutans UA159 a cariogenic dentalpathogenrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 99 no 22 pp 14434ndash144392002


[11] F Maruyama M Kobata K Kurokawa et al ldquoComparativegenomic analyses of streptococcus mutans provide insightsinto chromosomal shuffling and species-specific contentrdquo BMCGenomics vol 10 article 358 2009

[12] C Yanisch-Perron J Vieira and J Messing ldquoImproved M13phage cloning vectors and host strains nucleotide sequences ofthe M13mp18 and pUC19 vectorsrdquo Gene vol 33 no 1 pp 103ndash119 1985

[13] C Fermer and G Swedberg ldquoAdaptation to sulfonamide resis-tance in Neisseria meningitidis may have required compen-satory changes to retain enzyme function kinetic analysis ofdihydropteroate synthases from N meningitidis expressed in aknockout mutant of Escherichia colirdquo Journal of Bacteriologyvol 179 no 3 pp 831ndash837 1997

[14] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[15] O Skold ldquoSulfonamides and trimethoprimrdquo Expert review ofanti-infective therapy vol 8 no 1 pp 1ndash6 2010

[16] S Sridaran S K McClintock L M Syphard K M Herman JW Barnwell and V Udhayakumar ldquoAnti-folAte drug resistancein Africa meta-analysis of reported dihydrofolAte reductase(dhfr) and dihydropteroate synthase (dhps) mutant genotypefrequencies in African Plasmodium falciparum parasite popu-lationsrdquoMalaria Journal vol 9 no 1 article 247 2010

[17] M Brochet E Couve M Zouine C Poyart and P Glaser ldquoAnaturally occurring gene amplification leading to sulfonamideand trimethoprim resistance in Streptococcus agalactiaerdquo Jour-nal of Bacteriology vol 190 no 2 pp 672ndash680 2008

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did not confer sulfonamide resistance to E coli folP knockoutbacteria and that sequencing of the folA gene of trimethoprim(TMP) resistant isolates did not reveal any mutations How-ever the folP gene is very polymorphic [7] and at least one ofthe variants of folP confers high sulfonamide resistance to Ecoli folP knockout cells In the current study we report site-directed mutagenesis experiments in which we altered pointmutations in S mutans folP gene inserting the mutagenizedfolP DNA in pUC19 plasmids which were then transformedinto folP deficientE coliC600 cells By assessing the growth ofthe transformant E coli Delta folP cells on media containingdifferent levels of sulfamethoxazole the influence of individ-ual amino acids on sulfonamide resistance in the folP geneknockout bacteria was determined

2 Methods

The mechanism of resistance to sulfamethoxazole (SMX) inS mutans was characterised as summarized in Figure 1

21 Bacterial Strains and Plasmids The bacterial strains usedin this study (Table 1) were previously isolated [7] fromoral specimens of HIVAIDS patients taking cotrimoxazoleas prophylaxis in Kampala Uganda The cloning vectorspJet12blunt (Fermentas Lithuania) and pUC19 [12] wereused E coli top ten cells (Invitrogen USA) and E coli recip-ient strain C600 ΔfolP [13] which is a folP knockout strainwere used in the transformation experiments Streptococcuspneumoniae ATCC 49619 was used as a susceptible controlwhen determining MICs

22 Susceptibility Testing Minimal inhibitory concentrations(MICs) were determined by the 119864-test method (AB BiodiskSweden) following the manufacturerrsquos recommendations Alltests were done on Iso-Sensitest Agar (ISA Oxoid UK)Plates were incubated at 37∘C in 5 CO

2for 24 h For

determination of sulfonamide resistance conferred by clonedfolP genes E coli C600 bacteria were grown in Iso-SensitestBroth (ISB Oxoid UK) to a cell density of 108mL dilutedto 104mL and plated on ISA plates containing varyingconcentrations of sulfathiazole (Sigma Aldrich USA)

23 DNA Extraction Isolates of Streptococcus mutans wereincubated at 37∘C for 12 h on Iso-Sensitest Agar Bacterialcolonies were re-suspended in brain heart infusion broth(BioMerieux France) and incubated at 37∘C for 24 h in anatmosphere of 5 carbon dioxide Chromosomal bacterialDNA from the cultured broth was then extracted using theWizard Genomic DNA Purification Kit (Promega USA)

24 Cloning The PCR primer sequences used were basedon the published sequence of the folP gene of StreptococcusmutansUA159 [10] (Table 1) FolP gene amplificationwas per-formed in 50-120583L volumes containing 05 120583M of each primer100 120583M of the four deoxyribonucleoside triphosphates 5units of DNA polymerase (Pfu Fermentas) 2120583L of template

for studying bacterial resistance tosulfamethoxazole (SMX)

Extracted Streptococcus genomicDNA folP DNA cloned into pJET

mutation patterns in the folP gene were used

Recombinant pJET transformedinto E coli top ten cells

replicated by culture and extracted

folP DNA recloned in pUC 19Recombinant pUC 19

mutagenized to produce orremove specific folP mutations

Recombinant pUC 19 carryingdifferent mutations in folP

transformed into ΔfolP E coli C60013

Reconstituted ΔfolP E coli C600grown at different sulfonamide

concentrations to determine MICs

DNA of recombinant pUC 19

determine and confirm the plasmidand insert nucleotide sequences

carrying folP mutations sequenced to

797 (10) have been described previously8Streptococcus isolates 8 797 135 7 477 and

Isolates 8 797 135 7 and 477 with different

Figure 1 Flow chart showing characterization of folP gene Plasmidscarrying folP gene were transformed in folP gene knockout bacteriato determine the effect of different mutations in plasmid encodedfolP on bacterial resistance to sulfonamides

DNA (50ndash500 ng) preparation and 1X reaction buffer (PfuFermentas) containing 2mM MgSO

4 Amplification reac-

tions were performedwith the Eppendorf mastercycler gradi-ent thermocycler (Eppendorf Germany) using the followingprogram heating at 94∘C for 2min followed by 25 cyclesconsisting of a denaturation at 94∘C for 30 s annealing at50∘C for 30 s and an extension at 72∘C for 15min This wasfollowed by a final extension of 72∘C for 5min and a holdingstep at 16∘C

The PCR products were cleaned by the PCR Cleanup kit(Omega USA) and then used for cloning into the pJet vectorusing the blunt end pJET cloning kit protocol (FermentasLithuania) The ligated products were introduced into E colitop ten cells by heat-shock CaCl

2transformation method

Plasmids were prepared from the transformants usingthe plasmid preparation kit (Omega USA) and prepared forsequencing

25 Sequence Analysis For sequencing the plasmids theBigDye Terminator labelled cycle-sequencing kit (AppliedBiosystems) and anABI prism 310Genetic Analyzer (AppliedBiosystems) were used The results of the folP gene sequenceanalysis were compared with database sequences of Strepto-coccus mutans UA159 [10] and NN2025 [11] using the BLASTprogramme at NCBI [14]














































3 Results


A37

V +

N172

D +

R193

Qlowast

37

A +

N172

D +

R193

Qlowast

A37

V +172

N +

R193

Qlowast

A37

V +

N172

D +193

Rlowast

37

A +172

N +

R193

Qlowast

37

A +172

N +193

Rlowast

A46

V +

E80

K +

Q122

H +

S14

6G

A63

S +

W174

LK

+ L175

F +

M189

I

60

40

20

0

MIC

(120583M

)

A

B C D

E F G H I

J


lowastlowastlowast

lowastlowast

S d

owne

i 477

wild

-type

folP

gen

e

S so

brin

us 7

wild

-type

folP

gen

e


4 Discussion





5 Conclusions







Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































3 Results


A37

V +

N172

D +

R193

Qlowast

37

A +

N172

D +

R193

Qlowast

A37

V +172

N +

R193

Qlowast

A37

V +

N172

D +193

Rlowast

37

A +172

N +

R193

Qlowast

37

A +172

N +193

Rlowast

A46

V +

E80

K +

Q122

H +

S14

6G

A63

S +

W174

LK

+ L175

F +

M189

I

60

40

20

0

MIC

(120583M

)

A

B C D

E F G H I

J


lowastlowastlowast

lowastlowast

S d

owne

i 477

wild

-type

folP

gen

e

S so

brin

us 7

wild

-type

folP

gen

e


4 Discussion





5 Conclusions







Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions







Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article point mutations in the folp gene partly...

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