Mechanism of Reduced Susceptibility to Fosfomycin …downloads.hindawi.com/journals/bmri/2017/5470241.pdf · Mechanism of Reduced Susceptibility to Fosfomycin in Escherichia coli

Research ArticleMechanism of Reduced Susceptibility to Fosfomycin inEscherichia coli Clinical Isolates

Yasuo Ohkoshi12 Toyotaka Sato1 Yuuki Suzuki1 Soh Yamamoto1 Tsukasa Shiraishi1

Noriko Ogasawara1 and Shin-ichi Yokota1

1Department of Microbiology Sapporo Medical University School of Medicine Chuo-ku Sapporo 060-8556 Japan2Department of Clinical Laboratory NTT East Sapporo Hospital Chuo-ku Sapporo 060-0061 Japan

Correspondence should be addressed to Shin-ichi Yokota syokotasapmedacjp

Received 15 July 2016 Revised 22 December 2016 Accepted 27 December 2016 Published 19 January 2017

Academic Editor Yun-Peng Chao

Copyright copy 2017 Yasuo Ohkoshi 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

In recent years multidrug resistance of Escherichia coli has become a serious problem However resistance to fosfomycin (FOM)has been low We screened E coli clinical isolates with reduced susceptibility to FOM and characterized molecular mechanismsof resistance and reduced susceptibility of these strains Ten strains showing reduced FOM susceptibility (MIC ge 8 120583gmL) in 211clinical isolates were found and examined Acquisition of genes encoding FOM-modifying enzyme genes (fos genes) andmutationsin murA that underlie high resistance to FOM were not observed We examined ability of FOM incorporation via glucose-6-phosphate (G6P) transporter and sn-glycerol-3-phosphate transporter In ten strains nine showed lack of growth onM9minimumsalt agar supplementedwithG6P Eight of the ten strains showed fluctuated induction byG6P of uhpT that encodes G6P transporterexpression Nucleotide sequences of the uhpT uhpA glpT ptsI and cyaA shared several deletions and amino acid mutations in thenine strains with lack of growth onG6P-supplementedM9 agar In conclusion reduction of uhpT function is largely responsible forthe reduced sensitivity to FOM in clinical isolates that have not acquired FOM-modifying genes or mutations in murA Howeverthere are a few strains whose mechanisms of reduced susceptibility to FOM are still unclear

1 Introduction

Escherichia coli is a causative agent of uncomplicated urinarytract infections in immunocompetent hosts and oppor-tunistic infections in immunocompromised hosts In recentyears fluoroquinolone-resistant andor extended spectrum120573-lactamase- (ESBL-) producing E coli strains have been fre-quently isolated from such patients [1ndash4] In addition theseE coli resistant strains occasionally show cross-resistance toaminoglycosides [5] Thus these multidrug-resistant E colihave an impact on the selection of therapeutically effectivedrugs

Because of this serious concern the use of fosfomycin(FOM) an antibiotic as a bacterial cell wall synthesis inhibitordeveloped 40 years ago has been reevaluated against drug-resistant bacteria especially E coli [6 7] Since FOM hasa unique mode of action that differed from other antibi-otics it is expected to display little cross-resistance to otherantimicrobial agents E coli is the most frequent causative

pathogenic bacterium of acute cystitis [8 9] For instancetrimethoprimsulfamethoxazole and FOMare recommendedas the first-line treatment in acute uncomplicated urinarytract infections according to the US guideline [10] On theother hand the guideline of the Japanese Association forInfectious Diseases and Japan Society for Chemotherapyrecommends fluoroquinolones as the first-line drug andFOM as the second-line drug [11]

FOM inhibits UDP-N-acetylglucosamine enolpyruvyltransferase (MurA) an enzyme involved in the synthesisof the essential peptidoglycan component N-acetylmuramicacid [12] FOM is incorporated into the bacterial cellsvia glucose-6-phosphate (G6P) transporter UhpT and sn-glycerol-3-phosphate (G3P) transporter GlpT [13 14] UhpTis upregulated by exogenously added G6P [15ndash17] It is thusrecommended that the addition of G6P to a growth mediumis used in the measurement of FOMMIC

High resistance to FOM primarily occurs by the acquisi-tion of glutathione S-transferase genes such as fosA fosA2 to

HindawiBioMed Research InternationalVolume 2017 Article ID 5470241 8 pageshttpsdoiorg10115520175470241

2 BioMed Research International

fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 found in var-ious bacteria [19 21ndash26] and mutation(s) in murA gene [1824 27 28] Furthermore mutations in the transporter genesuhpT and glpT and genes encoding proteins regulating uhpTexpression such as uhpA reduce the susceptibility to FOM[18 19 27ndash29] In addition expression levels of UhpT andGlpT are positively regulated by cyclic AMP (cAMP) [16 17]The levels of cAMP are controlled by phosphoenolpyruvate-protein phosphotransferase I encoded by ptsI and adenylcyclase encoded by cyaA andmutations in these genes resultin reduced susceptibility to FOM [29ndash31]

Nevertheless several surveillance studies report that therate of emergence of E coli isolates showing FOM resistanceor reduced FOM susceptibility has been markedly low [79 32] Although spontaneously mutational rate to acquireFOM resistance is high FOM resistance confers biologicalcosts such as reduced cell growth rate in Gram-negativebacteria [29 33] This indicates that FOM continues to bean effective agent against E coli infections However theoverall up-to-date status of FOM resistance needs to becontinuously surveyed and its molecular characteristics haveto be understood to prevent future emergence and increaseof multidrug-resistant E coli with FOM resistance in theclinic In this study we screened E coli clinical isolates fromJapan showing resistance or reduced susceptibility to FOMand identified the molecular mechanisms of their reducedsusceptibility

2 Materials and Methods

21 Bacterial Strains E coli clinical isolates (211 strains) werecollected in the years 2008-2009 as described previously [45]Thesewere identified and stocked in SapporoClinical Lab-oratory Inc (Sapporo Japan) These strains were collectedfrom a variety of clinical specimens in almost entire area ofHokkaido Prefecture Japan This study was approved by thereview boards of the relevant institutions The strains wereisolated from the following clinical specimens urine (119899 =87 410) catheter urine (119899 = 76 358) sputum (119899 =15 71) stool (119899 = 7 33) vaginal secretion (119899 = 628) pus (119899 = 3 14) aspiration tube (119899 = 3 14)drainage tube intravenous hyperalimentation catheter tuberhinorrhea (two strains from each type of specimen 09)ascites anal gland fluid decubitus injury site intestinal juicestoma PEG insertion site pharynx fluid and synovial fluid(one strain from each type of specimen 05) Identificationwas performed using the MicroScan WalkAway 96 system(Beckman Coulter Tokyo Japan) E coli strain ATCC25922was obtained from American Type Culture Collection (Man-assas VA)

22 Antibiotic Susceptibility FOM imipenem (IPM) andceftazidime (CAZ) were provided by Meiji Seika Pharma(Tokyo Japan) MSD (Tokyo Japan) and Glaxo SmithKline(Tokyo Japan) respectively Minimum inhibitory concentra-tion (MIC) was determined by broth microdilution methodor agar plate dilution method according to the recommen-dations of the Clinical and Laboratory Standards Institute(CLSI) with breakpoints according to CLSI guidelines [34]

Table 1 PCR and real-time RT-PCR primers used in this study

Primer Sequence (51015840-31015840) Reference

murA-fulla F AAACAGCAGACGGTCTATGG[18]R CCATGAGTTTATCGACAGAACG

glpT-full F GCGAGTCGCGAGTTTTCATTG[18]R GGCAAATATCCACTGGCACC

uhpT-full F TTTTTGAACGCCCAGACACC[18]R AGTCAGGGGCTATTTGATGG

uhpT-partial F ATGCTGGCTTTCTTAAACC[19]R TTATGCCACTGTCAACTGC

uhpA-full F GATCGCGGTGTTTTTTCAG[18]R GATACTCCACAGGCAAAACC

uhpA-partial F ATCACCGTTGCCCTTATAGA[19]R TCACCAGCCATCAAACAT

ptsI-full F GAAAGCGGTTGAACATCTGG[18]R TCCTTCTTGTCGTCGGAAAC

cyaA-full F AACCAGGCGCGAAAAGTGG[18]R ACCTTCTGGGATTTGCTGG

rpoD-qPCR F CAAGCCGTGGTCGGAAAA[20]R GGGCGCGATGCACTTCT

uhpT-qPCR F AAGCCGACCCTGGACCTT[20]R ACGGTTTGAACCACATTTTGC

aPrimers designated ldquofullrdquo were used for direct sequencing and ldquoqPCRrdquowereused for real-time RT-PCR

TheMIC of FOM was measured by the agar dilution methodin the absence of G6P in the presence of 25120583gmL G6P andin the presence of 25120583gmL G6P and 2mM cAMP

23 Measurements of Carbohydrate Phosphate TransporterActivity E coli cells were cultured for 24 h inMullerndashHintonbroth and harvested by centrifugation The cell pellet waswashed twice with saline E coli cell suspension with salinewas then used to inoculate to M9 minimum salt (BectonDickinson Franklin Lakes NJ) agar supplemented with 02G6P or 02 G3P [18] Cell growth was observed afterincubation for 24 h in the case of G6P and for 48 h in the caseof G3P

24 Genetic Analysis DNA was isolated using DNeasy Kit(Qiagen Hilden Germany) according to the manufacturerrsquosinstructions Polymerase chain reaction (PCR) was per-formed using KAPATaq Extra HotStart Ready Mix with dye(NIPPON Genetics Tokyo Japan) Serogroups [35 36] andphylogenetic groups [37] were determined by PCR Multilo-cus sequence typing (MLST) was determined according toTartof et al [38] Genes of fosA fosA3 fosC2 [22] fosB2fosC fosX [26] fosB [21] fosA34 [25] and fosKP96 [24]were detected by PCRusing the primers described previouslyGene of fosA5 was detected by PCR using primer set 51015840-ACTGAATCACCTGACCCTGG-31015840 and 51015840-CGCATAATG-GGTGTAGTCGC-31015840 Full nucleotide sequences of six genes(murA uhpT glpT uhpA ptsI and cyaA) were determinedby a combination of direct sequencing and primer walkingwith the respective PCR products PCR primer sequences aregiven in Table 1 The sequencing was performed with Big

BioMed Research International 3

Table 2 Antibiotic susceptibility and the presence of ESBL genes in Ecoli strains with FOMMIC ge 8120583gmL

StrainsMIC (120583gmL)

ESBLageneFosfomycin(ge128)b

Levofloxacin(ge4)b

Gentamicin(ge8)b

Imipenem(ge2)b

Ceftazidime(ge8)b

SRE257 1024lowast le0125 1 0125 0125 mdashSRE91 128 32lowast 4 025 0125 mdashSRE49 128 16lowast 2 0125 025 mdashSRE54 64 ge64lowast ge64lowast 0125 2 CTX-M14SRE237 64 le0125 4 0125 0125 mdashSRE29 32 16lowast ge64lowast 0125 0125 mdashSRE252 32 05 2 0125 0125 mdashSRE280 32 le0125 8lowast 05 0125 mdashSRE18 16 32lowast 2 0125 2 CTX-M2SRE253 8 le0125 4 05 0125 mdashaESBL extended spectrum 120573-lactamasebBreakpoints (120583gmL) are according to CLSIlowastResistantIntermediate

Dye Terminator Kit version 31 and 3730xI DNA analyzer(Applied Biosystems Carlsbad CA) at Hokkaido SystemScience (Sapporo Japan)

25 Real-Time Reverse-Transcription (RT) PCR E coli cellswere grown for 24 h in Luria-Bertani (LB) broth and thecells were harvested and washed twice with M9 minimumsalt solution The suspended cells were used to inoculateto M9 minimum salt solution with or without 02 G6Psupplementation and incubated for 30min at 37∘C RNAwas isolated from the cells using RNeasy Plus Mini Kit(Qiagen) according to themanufacturerrsquos instructions cDNAwas prepared from the RNA using SuperScript III FirstStrand Synthesis Kit (Invitrogen Carlsbad CA) and randomhexamer oligonucleotide primers mRNA levels of uhpT andrpoD were quantified using QuantiFast SYBR Green PCRMastermix (Qiagen) by LightCycler LC480 (Roche BaselSwitzerland) with the cDNA as a template PCR primers weregiven in Table 1 Levels of uhpT transcript were calculated by2minusΔΔct method and data were normalized to the levels of thehouse-keeping gene rpoDmRNA

3 Results

31 Antibiotic Susceptibility Antibiotic susceptibility was ex-amined for 211 E coli clinical isolates MICs were determinedby broth microdilution method for LVX GEN IPM andCAZ and by agar plate dilution method for FOM (Table 2)The distribution of FOM MIC was shown in Figure 1 Threestrains (14) were not susceptible including resistant andintermediate Furthermore seven strains (3) with elevatedFOM MIC (ge8120583gmL le64 120583gmL) were observed out ofnormal distribution of the susceptible strains FOM MICsof these ten strains were not affected by the presence orabsence of G6P By contrast other susceptible strains (MICle 2 120583gmL) showed an increase of FOM MICs in theabsence of G6P (Table 3) Susceptibility to other antibiotics

012

8289

18

0 1 1 3 2 2 0 0 1 00

25

50

75

100N

umbe

r of s

trai

ns

MIC (120583gmL)

S I R0125

025

05 1 2 4 8

16

32

64

128

256

512

1024

2048

N = 211

Figure 1 Distribution of fosfomycinMICs of the E coli clinical iso-lates The breakpoint is according to CLSI guideline S susceptibleI intermediate and R resistant

was examined for strains with reduced susceptibility toFOM (Table 2) LVX-resistant strains comprised 50 andGEN-resistant strains comprised 30 of these strains IPM-resistant and CAZ-resistant strains were not observed how-ever 20 strains shared CTX-M-type ESBL genesThe occur-rence of antimicrobial nonsusceptibility was not significantlyhigh compared to total strains examined (data not shown)Genotypes (ie phylogenetic groups and MLST) and O-serogroups of these strains were variable (Table 3)

32 Analysis of Genes Associated with FOM Sensitivity Weexamined molecular mechanisms underlying the reductionof FOM sensitivity (MIC ge 8 120583gmL) First genes encodingFOM-modifying enzymes were investigated None of thefollowing were detected in the strains examined fosA fosA2to fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 (datanot shown) Next nucleotide sequences of murA uhpTuhpA glpT ptsI and cyaA genes were determined (Table 3)No murA coding sequence mutations that would result in


Table3Ch

aracteris

ticso

fEcolistrains

used

inthes

tudy

Strain

Specim

enSero-

grou

p

Phylo-

genetic

grou

pMLS

T

MIC

a

(120583gmL)

Growth

onM9agar

supp

lemented

with

uhpT

expressio

nindu

cedby

G6P

b

Aminoacid

resid

uealternations

inproteins

encodedby

glpT

ptsI

cyaA

murA

uhpA

and

uhpT

genesc

G6Pminus

G6P

+G6P

+cA

MP+

G3P

G6P

cyaA

glpT

murA

ptsI

uhpA

uhpT

SRE2

57Urin

eO1

B295

1024

1024

1024

+minus

124

mdashmdash

mdashVa

l399Leu

163sim

188

deletio

nmdash

SRE9

1Aspira

tion

tube

O1

D64

8128

128

32minus

minus378

mdash155sim

158

deletio

nPh

e176Leu

mdashmdash

Thr3Ala

mdash

SRE4

9Urin

eO25bH4

B2131

128

128

128

+minus

NT

His7

16Leu

mdashmdash

Lys410Arg

ND

ND

SRE5

4Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

Ala44

3Thr

Gly452A

spmdash

mdash

SRE2

37Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

mdashmdash

mdashSR

E29

Urin

eO25bH4

B2131

3232

32+

+122622

mdashmdash

mdashmdash

mdashmdash

SRE2

52Urin

eO25a

D73

3232

4minus

minus368

ND

Ile171Th

rmdash

Lys145Asn

mdashmdash

SRE2

80Ascites

O12

D1486

3232

32+

minus056

Ser142As

nmdash

mdashmdash

mdashmdash

SRE18

Urin

eND

D405

1616

16+

minus222

mdashmdash

mdashmdash

Met1Ile

mdashSR

E253

Urin

eO18

B295

88

8minus

minus19809

His7

16Leu

mdashmdash

mdashmdash

mdashSR

E40

Decub

itus

O25a

D501

3205

NT

++

28405

SRE4

1Ca

theter

urine

O1

D64

88

05

NT

++

32856

SRE110

Catheter

urine

O25bH4

B2131

805

NT

++

19002

SRE2

05Urin

eND

A131

805

NT

++

73419

SRE2

27Pu

sO1

B295

805

NT

++

2472

8SR

E30

Urin

eO1

D64

88

025

NT

++

7190

8AT

CC25922

NT

NT

3205

NT

++

30869

a FOM

MICsw

ered

etermined

inthep

resence(+)

orabsence(minus)o

fglucose-6-pho

sphate(G

6P)a

ndorc

AMP

b Ecolicellswereincub

ated

inM9minim

umsaltsolutio

nin

thep

resenceo

rabsence

ofG6PTh

euhpTmRN

Alevelswered

etermined

byreal-timeR

T-PC

Randthed

ataweren

ormalized

torpoD

mRN

Alevels

Indu

ctionof

uhpT

expressio

nby

G6P

was

calculated

bydividing

theu

hpTmRN

Alevelinthep

resenceo

fG6P

bytheu

hpTmRN

Alevelinthea

bsence

ofG6P

c Aminoacid

mutations

foun

don

lyin

strains

with

redu

cedFO

Msusceptib

ility(M

ICge8120583

gmL)

comparedwith

strains

with

FOM

MIClt1120583

gmL

NDnot

detectedN

Tno

ttested


Growth in the presence of G6P +minus

P lt 0001

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(a)

Fosfomycin MIC (120583gml)

P lt 0005

ge8 le4

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(b)

Figure 2 Induced expression levels of uhpT expression by the addition of G6P Nine clinical isolates with reduced susceptibility to FOM sixsusceptible clinical isolates and one standard strain are included Data used to generate this figure are given in Table 2 Statistical significancewas determined byMannndashWhitney test (a)Comparison of strains not grown (minus) or strains grown (+) inM9minimumsalt solution containingG6P (b) Comparison of strains with FOMMIC ge 8 120583gmL or le4 120583gmL

changes of amino acid residues in MurA were observed Aresistant strain (SRE257) and an intermediate-resistant strain(SRE91) had mutations in the genes that would result indeletions of a part of amino acid residues in UhpA andGlpT respectively In another intermediate-resistant strain(SRE49) uhpA and uhpT were not detected by PCR ampli-fication with two distinct primer pairs (ldquofullrdquo and ldquopartialrdquo inTable 1) In one strain with MIC 32 120583gmL (SRE252) cyaAwas not detectable by PCR All strains with reduced FOMMIC (ge8120583gmL) except one (SRE29) had several mutationsin one or more genes leading to amino acid deletion or pointmutation(s) of amino acid residues compared with othersusceptible strains

33 Function and Expression of Carbohydrate PhosphateTransporters To determine the activity of UhpT and GlpTwe examined cell growth in M9 minimum salt solutionsupplementedwithG6P orG3PNine of ten strainswith FOMMIC ge 8120583gmL did not grow within 24 h on G6P-containingM9 minimum salt agar On the other hand only three often strains did not grow in the presence of G3P (Table 3)and the two strains (SRE91 and SRE252) showed reducedMIC to FOM by addition of cAMP This suggested that thesetwo strains shared insufficient intracellular concentration ofcAMP for full expression of GlpT andor UhpTThese resultssuggested that FOM incorporation through the UhpT systemis involved to a greater extent in the reduction of E coli FOMcompared to the GlpT system

Expression of UhpT is induced by G6P [15ndash17] Wetherefore determined uhpT gene induction by G6P usingquantitative RT-PCR analysis (Table 3 and Figure 2) In

strains with FOM MIC lt 1 120583gmL uhpT expression wasstrongly induced (190- to 730-fold) by G6P By contrast ineight of ten strains with FOM MIC ge 8120583gmL the uhpTinduction was markedly lower (056- to 38-fold) or no uhpTsignal was amplified by PCR Of the remaining two strainswe observed a high induction (200-fold) of uhpT expressionby G6P in SRE253 strain however the strain did not grow onG6P-containing M9 minimum agar High induction (1200-fold) of uhpT by G6P was observed in SRE29 strain and thestrain grew on G6P-containing M9 minimum agar howeverit showed reduced susceptibility to FOM (MIC 32 120583gmL)These results indicated that G6P-dependent growth lacked inmost strains with reduced susceptibility to FOM because oflack of uhpT or a markedly reduced uhpT induction by G6PHowever the changes in uhpT expression and UhpT activitydid not account for the reduced FOM susceptibility of a fewstrains (Figure 2)

4 Discussion

The frequency of FOM resistance has been recognized to below And it has been expected that the frequency of cross-resistance of FOMand other antibiotics is very low because ofa unique mode of action However several reports examinedFOM resistance in ESBL-producing E coli [19 23 24 28] Areport indicates that FOM-resistant and intermediate E coliare more frequently resistant to other types of antimicrobialsthan FOM-susceptible strains [28] These reports suggestthe importance for surveillance of FOM-resistant E coliparticularly for focus on their cross-resistance of FOM inmultidrug resistance


In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes

Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis

In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed

reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation

Competing Interests

The authors declare no competing interests

Acknowledgments

The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation

References

[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten

[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009

[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012

[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012

[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014

[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009

[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010

[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007

[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015

[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015


[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)

[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995

[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974

[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004

[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971

[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995

[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002

[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010

[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010

[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014

[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997

[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012

[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012

[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013

[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014

[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015

[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015

[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015

[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003

[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976

[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003

[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012

[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012

[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012

[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007

[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010

[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000

[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005


[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 found in var-ious bacteria [19 21ndash26] and mutation(s) in murA gene [1824 27 28] Furthermore mutations in the transporter genesuhpT and glpT and genes encoding proteins regulating uhpTexpression such as uhpA reduce the susceptibility to FOM[18 19 27ndash29] In addition expression levels of UhpT andGlpT are positively regulated by cyclic AMP (cAMP) [16 17]The levels of cAMP are controlled by phosphoenolpyruvate-protein phosphotransferase I encoded by ptsI and adenylcyclase encoded by cyaA andmutations in these genes resultin reduced susceptibility to FOM [29ndash31]

Nevertheless several surveillance studies report that therate of emergence of E coli isolates showing FOM resistanceor reduced FOM susceptibility has been markedly low [79 32] Although spontaneously mutational rate to acquireFOM resistance is high FOM resistance confers biologicalcosts such as reduced cell growth rate in Gram-negativebacteria [29 33] This indicates that FOM continues to bean effective agent against E coli infections However theoverall up-to-date status of FOM resistance needs to becontinuously surveyed and its molecular characteristics haveto be understood to prevent future emergence and increaseof multidrug-resistant E coli with FOM resistance in theclinic In this study we screened E coli clinical isolates fromJapan showing resistance or reduced susceptibility to FOMand identified the molecular mechanisms of their reducedsusceptibility

2 Materials and Methods

21 Bacterial Strains E coli clinical isolates (211 strains) werecollected in the years 2008-2009 as described previously [45]Thesewere identified and stocked in SapporoClinical Lab-oratory Inc (Sapporo Japan) These strains were collectedfrom a variety of clinical specimens in almost entire area ofHokkaido Prefecture Japan This study was approved by thereview boards of the relevant institutions The strains wereisolated from the following clinical specimens urine (119899 =87 410) catheter urine (119899 = 76 358) sputum (119899 =15 71) stool (119899 = 7 33) vaginal secretion (119899 = 628) pus (119899 = 3 14) aspiration tube (119899 = 3 14)drainage tube intravenous hyperalimentation catheter tuberhinorrhea (two strains from each type of specimen 09)ascites anal gland fluid decubitus injury site intestinal juicestoma PEG insertion site pharynx fluid and synovial fluid(one strain from each type of specimen 05) Identificationwas performed using the MicroScan WalkAway 96 system(Beckman Coulter Tokyo Japan) E coli strain ATCC25922was obtained from American Type Culture Collection (Man-assas VA)

22 Antibiotic Susceptibility FOM imipenem (IPM) andceftazidime (CAZ) were provided by Meiji Seika Pharma(Tokyo Japan) MSD (Tokyo Japan) and Glaxo SmithKline(Tokyo Japan) respectively Minimum inhibitory concentra-tion (MIC) was determined by broth microdilution methodor agar plate dilution method according to the recommen-dations of the Clinical and Laboratory Standards Institute(CLSI) with breakpoints according to CLSI guidelines [34]

Table 1 PCR and real-time RT-PCR primers used in this study

Primer Sequence (51015840-31015840) Reference

murA-fulla F AAACAGCAGACGGTCTATGG[18]R CCATGAGTTTATCGACAGAACG

glpT-full F GCGAGTCGCGAGTTTTCATTG[18]R GGCAAATATCCACTGGCACC

uhpT-full F TTTTTGAACGCCCAGACACC[18]R AGTCAGGGGCTATTTGATGG

uhpT-partial F ATGCTGGCTTTCTTAAACC[19]R TTATGCCACTGTCAACTGC

uhpA-full F GATCGCGGTGTTTTTTCAG[18]R GATACTCCACAGGCAAAACC

uhpA-partial F ATCACCGTTGCCCTTATAGA[19]R TCACCAGCCATCAAACAT

ptsI-full F GAAAGCGGTTGAACATCTGG[18]R TCCTTCTTGTCGTCGGAAAC

cyaA-full F AACCAGGCGCGAAAAGTGG[18]R ACCTTCTGGGATTTGCTGG

rpoD-qPCR F CAAGCCGTGGTCGGAAAA[20]R GGGCGCGATGCACTTCT

uhpT-qPCR F AAGCCGACCCTGGACCTT[20]R ACGGTTTGAACCACATTTTGC

aPrimers designated ldquofullrdquo were used for direct sequencing and ldquoqPCRrdquowereused for real-time RT-PCR

TheMIC of FOM was measured by the agar dilution methodin the absence of G6P in the presence of 25120583gmL G6P andin the presence of 25120583gmL G6P and 2mM cAMP

23 Measurements of Carbohydrate Phosphate TransporterActivity E coli cells were cultured for 24 h inMullerndashHintonbroth and harvested by centrifugation The cell pellet waswashed twice with saline E coli cell suspension with salinewas then used to inoculate to M9 minimum salt (BectonDickinson Franklin Lakes NJ) agar supplemented with 02G6P or 02 G3P [18] Cell growth was observed afterincubation for 24 h in the case of G6P and for 48 h in the caseof G3P

24 Genetic Analysis DNA was isolated using DNeasy Kit(Qiagen Hilden Germany) according to the manufacturerrsquosinstructions Polymerase chain reaction (PCR) was per-formed using KAPATaq Extra HotStart Ready Mix with dye(NIPPON Genetics Tokyo Japan) Serogroups [35 36] andphylogenetic groups [37] were determined by PCR Multilo-cus sequence typing (MLST) was determined according toTartof et al [38] Genes of fosA fosA3 fosC2 [22] fosB2fosC fosX [26] fosB [21] fosA34 [25] and fosKP96 [24]were detected by PCRusing the primers described previouslyGene of fosA5 was detected by PCR using primer set 51015840-ACTGAATCACCTGACCCTGG-31015840 and 51015840-CGCATAATG-GGTGTAGTCGC-31015840 Full nucleotide sequences of six genes(murA uhpT glpT uhpA ptsI and cyaA) were determinedby a combination of direct sequencing and primer walkingwith the respective PCR products PCR primer sequences aregiven in Table 1 The sequencing was performed with Big





Levofloxacin(ge4)b

Gentamicin(ge8)b

Imipenem(ge2)b

Ceftazidime(ge8)b




3 Results


012

8289

18

0 1 1 3 2 2 0 0 1 00

25

50

75

100N

umbe

r of s

trai

ns

MIC (120583gmL)

S I R0125

025

05 1 2 4 8

16

32

64

128

256

512

1024

2048

N = 211





Table3Ch

aracteris

ticso

fEcolistrains

used

inthes

tudy

Strain

Specim

enSero-

grou

p

Phylo-

genetic

grou

pMLS

T

MIC

a

(120583gmL)

Growth

onM9agar

supp

lemented

with

uhpT

expressio

nindu

cedby

G6P

b

Aminoacid

resid

uealternations

inproteins

encodedby

glpT

ptsI

cyaA

murA

uhpA

and

uhpT

genesc

G6Pminus

G6P

+G6P

+cA

MP+

G3P

G6P

cyaA

glpT

murA

ptsI

uhpA

uhpT

SRE2

57Urin

eO1

B295

1024

1024

1024

+minus

124

mdashmdash

mdashVa

l399Leu

163sim

188

deletio

nmdash

SRE9

1Aspira

tion

tube

O1

D64

8128

128

32minus

minus378

mdash155sim

158

deletio

nPh

e176Leu

mdashmdash

Thr3Ala

mdash

SRE4

9Urin

eO25bH4

B2131

128

128

128

+minus

NT

His7

16Leu

mdashmdash

Lys410Arg

ND

ND

SRE5

4Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

Ala44

3Thr

Gly452A

spmdash

mdash

SRE2

37Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

mdashmdash

mdashSR

E29

Urin

eO25bH4

B2131

3232

32+

+122622

mdashmdash

mdashmdash

mdashmdash

SRE2

52Urin

eO25a

D73

3232

4minus

minus368

ND

Ile171Th

rmdash

Lys145Asn

mdashmdash

SRE2

80Ascites

O12

D1486

3232

32+

minus056

Ser142As

nmdash

mdashmdash

mdashmdash

SRE18

Urin

eND

D405

1616

16+

minus222

mdashmdash

mdashmdash

Met1Ile

mdashSR

E253

Urin

eO18

B295

88

8minus

minus19809

His7

16Leu

mdashmdash

mdashmdash

mdashSR

E40

Decub

itus

O25a

D501

3205

NT

++

28405

SRE4

1Ca

theter

urine

O1

D64

88

05

NT

++

32856

SRE110

Catheter

urine

O25bH4

B2131

805

NT

++

19002

SRE2

05Urin

eND

A131

805

NT

++

73419

SRE2

27Pu

sO1

B295

805

NT

++

2472

8SR

E30

Urin

eO1

D64

88

025

NT

++

7190

8AT

CC25922

NT

NT

3205

NT

++

30869

a FOM

MICsw

ered

etermined

inthep

resence(+)

orabsence(minus)o

fglucose-6-pho

sphate(G

6P)a

ndorc

AMP


ated

inM9minim

umsaltsolutio

nin

thep

resenceo

rabsence

ofG6PTh

euhpTmRN

Alevelswered

etermined

byreal-timeR

T-PC

Randthed

ataweren

ormalized

torpoD

mRN

Alevels

Indu

ctionof

uhpT

expressio

nby

G6P

was

calculated

bydividing

theu

hpTmRN

Alevelinthep

resenceo

fG6P

bytheu

hpTmRN

Alevelinthea

bsence

ofG6P

c Aminoacid

mutations

foun

don

lyin

strains

with

redu

cedFO

Msusceptib

ility(M

ICge8120583

gmL)

comparedwith

strains

with

FOM

MIClt1120583

gmL

NDnot

detectedN

Tno

ttested



P lt 0001

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(a)


P lt 0005

ge8 le4

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(b)






4 Discussion







Competing Interests


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Levofloxacin(ge4)b

Gentamicin(ge8)b

Imipenem(ge2)b

Ceftazidime(ge8)b




3 Results


012

8289

18

0 1 1 3 2 2 0 0 1 00

25

50

75

100N

umbe

r of s

trai

ns

MIC (120583gmL)

S I R0125

025

05 1 2 4 8

16

32

64

128

256

512

1024

2048

N = 211





Table3Ch

aracteris

ticso

fEcolistrains

used

inthes

tudy

Strain

Specim

enSero-

grou

p

Phylo-

genetic

grou

pMLS

T

MIC

a

(120583gmL)

Growth

onM9agar

supp

lemented

with

uhpT

expressio

nindu

cedby

G6P

b

Aminoacid

resid

uealternations

inproteins

encodedby

glpT

ptsI

cyaA

murA

uhpA

and

uhpT

genesc

G6Pminus

G6P

+G6P

+cA

MP+

G3P

G6P

cyaA

glpT

murA

ptsI

uhpA

uhpT

SRE2

57Urin

eO1

B295

1024

1024

1024

+minus

124

mdashmdash

mdashVa

l399Leu

163sim

188

deletio

nmdash

SRE9

1Aspira

tion

tube

O1

D64

8128

128

32minus

minus378

mdash155sim

158

deletio

nPh

e176Leu

mdashmdash

Thr3Ala

mdash

SRE4

9Urin

eO25bH4

B2131

128

128

128

+minus

NT

His7

16Leu

mdashmdash

Lys410Arg

ND

ND

SRE5

4Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

Ala44

3Thr

Gly452A

spmdash

mdash

SRE2

37Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

mdashmdash

mdashSR

E29

Urin

eO25bH4

B2131

3232

32+

+122622

mdashmdash

mdashmdash

mdashmdash

SRE2

52Urin

eO25a

D73

3232

4minus

minus368

ND

Ile171Th

rmdash

Lys145Asn

mdashmdash

SRE2

80Ascites

O12

D1486

3232

32+

minus056

Ser142As

nmdash

mdashmdash

mdashmdash

SRE18

Urin

eND

D405

1616

16+

minus222

mdashmdash

mdashmdash

Met1Ile

mdashSR

E253

Urin

eO18

B295

88

8minus

minus19809

His7

16Leu

mdashmdash

mdashmdash

mdashSR

E40

Decub

itus

O25a

D501

3205

NT

++

28405

SRE4

1Ca

theter

urine

O1

D64

88

05

NT

++

32856

SRE110

Catheter

urine

O25bH4

B2131

805

NT

++

19002

SRE2

05Urin

eND

A131

805

NT

++

73419

SRE2

27Pu

sO1

B295

805

NT

++

2472

8SR

E30

Urin

eO1

D64

88

025

NT

++

7190

8AT

CC25922

NT

NT

3205

NT

++

30869

a FOM

MICsw

ered

etermined

inthep

resence(+)

orabsence(minus)o

fglucose-6-pho

sphate(G

6P)a

ndorc

AMP


ated

inM9minim

umsaltsolutio

nin

thep

resenceo

rabsence

ofG6PTh

euhpTmRN

Alevelswered

etermined

byreal-timeR

T-PC

Randthed

ataweren

ormalized

torpoD

mRN

Alevels

Indu

ctionof

uhpT

expressio

nby

G6P

was

calculated

bydividing

theu

hpTmRN

Alevelinthep

resenceo

fG6P

bytheu

hpTmRN

Alevelinthea

bsence

ofG6P

c Aminoacid

mutations

foun

don

lyin

strains

with

redu

cedFO

Msusceptib

ility(M

ICge8120583

gmL)

comparedwith

strains

with

FOM

MIClt1120583

gmL

NDnot

detectedN

Tno

ttested



P lt 0001

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(a)


P lt 0005

ge8 le4

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(b)






4 Discussion







Competing Interests


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Ch

aracteris

ticso

fEcolistrains

used

inthes

tudy

Strain

Specim

enSero-

grou

p

Phylo-

genetic

grou

pMLS

T

MIC

a

(120583gmL)

Growth

onM9agar

supp

lemented

with

uhpT

expressio

nindu

cedby

G6P

b

Aminoacid

resid

uealternations

inproteins

encodedby

glpT

ptsI

cyaA

murA

uhpA

and

uhpT

genesc

G6Pminus

G6P

+G6P

+cA

MP+

G3P

G6P

cyaA

glpT

murA

ptsI

uhpA

uhpT

SRE2

57Urin

eO1

B295

1024

1024

1024

+minus

124

mdashmdash

mdashVa

l399Leu

163sim

188

deletio

nmdash

SRE9

1Aspira

tion

tube

O1

D64

8128

128

32minus

minus378

mdash155sim

158

deletio

nPh

e176Leu

mdashmdash

Thr3Ala

mdash

SRE4

9Urin

eO25bH4

B2131

128

128

128

+minus

NT

His7

16Leu

mdashmdash

Lys410Arg

ND

ND

SRE5

4Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

Ala44

3Thr

Gly452A

spmdash

mdash

SRE2

37Urin

eO25bH4

B2131

6464

64+

minus15

9His7

16Leu

mdashmdash

mdashmdash

mdashSR

E29

Urin

eO25bH4

B2131

3232

32+

+122622

mdashmdash

mdashmdash

mdashmdash

SRE2

52Urin

eO25a

D73

3232

4minus

minus368

ND

Ile171Th

rmdash

Lys145Asn

mdashmdash

SRE2

80Ascites

O12

D1486

3232

32+

minus056

Ser142As

nmdash

mdashmdash

mdashmdash

SRE18

Urin

eND

D405

1616

16+

minus222

mdashmdash

mdashmdash

Met1Ile

mdashSR

E253

Urin

eO18

B295

88

8minus

minus19809

His7

16Leu

mdashmdash

mdashmdash

mdashSR

E40

Decub

itus

O25a

D501

3205

NT

++

28405

SRE4

1Ca

theter

urine

O1

D64

88

05

NT

++

32856

SRE110

Catheter

urine

O25bH4

B2131

805

NT

++

19002

SRE2

05Urin

eND

A131

805

NT

++

73419

SRE2

27Pu

sO1

B295

805

NT

++

2472

8SR

E30

Urin

eO1

D64

88

025

NT

++

7190

8AT

CC25922

NT

NT

3205

NT

++

30869

a FOM

MICsw

ered

etermined

inthep

resence(+)

orabsence(minus)o

fglucose-6-pho

sphate(G

6P)a

ndorc

AMP


ated

inM9minim

umsaltsolutio

nin

thep

resenceo

rabsence

ofG6PTh

euhpTmRN

Alevelswered

etermined

byreal-timeR

T-PC

Randthed

ataweren

ormalized

torpoD

mRN

Alevels

Indu

ctionof

uhpT

expressio

nby

G6P

was

calculated

bydividing

theu

hpTmRN

Alevelinthep

resenceo

fG6P

bytheu

hpTmRN

Alevelinthea

bsence

ofG6P

c Aminoacid

mutations

foun

don

lyin

strains

with

redu

cedFO

Msusceptib

ility(M

ICge8120583

gmL)

comparedwith

strains

with

FOM

MIClt1120583

gmL

NDnot

detectedN

Tno

ttested



P lt 0001

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(a)


P lt 0005

ge8 le4

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(b)






4 Discussion







Competing Interests


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



P lt 0001

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(a)


P lt 0005

ge8 le4

01

1

10

100

1000

10000

Fold

indu

ctio

n of

uhpT

mRN

A ex

pres

sion

(b)






4 Discussion







Competing Interests


Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Competing Interests


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










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Mechanism of Reduced Susceptibility to Fosfomycin …downloads.hindawi.com/journals/bmri/2017/5470241.pdf · Mechanism of Reduced Susceptibility to Fosfomycin in Escherichia coli