Research ArticleKinetics of Targeted Phage Rescue in a Mouse Model ofSystemic Escherichia coli K1

Gyoumlrgy Schneider 1 Nikolett Szentes2 Marianna Horvaacuteth1 Aacutegnes Dorn1 Alysia Cox3

Gaacutebor Nagy1 Zsolt Doffkay4 Gergely Maroacuteti5 Gaacutebor Raacutekhely46 and Tamaacutes Kovaacutecs 3

1Department of Medical Microbiology and Immunology University of Pecs Medical School Hungary2Department of Pharmacology and Pharmacotherapy University of Pecs Medical School Hungary3Department of Biotechnology Nanophagetherapy Center Enviroinvest Corporation Pecs Hungary4Department of Biotechnology University of Szeged Hungary5Institute of Biochemistry Biological Research Centre Hungarian Academy of Sciences Szeged Hungary6Institute of Biophysics Biological Research Center Hungarian Academy of Sciences Szeged Hungary

Correspondence should be addressed to Gyorgy Schneider gyorgyschneideraokptehu

Received 16 January 2018 Accepted 3 June 2018 Published 11 July 2018

Academic Editor Phillip E Klebba

Copyright copy 2018 Gyorgy Schneider et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Escherichia (E) coli K1 strains remain common causative agents of neonatal sepsis and meningitis We have isolated a lyticbacteriophage (ΦIK1) against E coli strain IHE3034 and tested its specificity in vitro as well as distribution and protective efficacyin vivo The phage was shown to be specific to the K1 capsular polysaccharide In the lethal murine model a high level ofprotection was afforded by the phage with strict kinetics A single dose of 1 x 108 phage particles administered 10 and 60 minutesfollowing the bacterial challenge elicited 100 and 95 survival respectively No mice could be rescued if phage administrationoccurred 3 hours postinfection Tissue distribution surveys in the surviving mice revealed that the spleen was the primary organin which accumulation of active ΦIK1 phages could be detected two weeks after phage administration These results suggest thatbacteriophages have potential as therapeutic agents in the control of systemic infections

1 Introduction

Thenumber of bacterial septicemia cases has been on the riseworldwide affecting more than a million Americans [1] ofwhom between 28 and 50 die [2]

After entering the body survival and outcome of thehematogenous spread partially depends on the fitness andvirulence factors of the invader Certain factors enablebacteria to enter the central nervous system (CNS) andcause meningitis The K1 serotype of Escherichia (E) coliis the leading causative agent of Gram-negative bacterialmeningitis with significant mortality and morbidity in new-borns worldwide [3] Its pathogenesis and pathophysiologyhave been investigated mostly using two E coli K1 isolatesoriginally isolated from the cerebrospinal fluid (CSF) ofneonates with sepsis and meningitis [4] Serotypes of these

strains (RS218 and IHE3034) are identical O18K1H7 [4]TheK1 capsule identical to the capsule of Neisseria meningitidesB is thought to be one of the most important virulencefactors as it supports survival in the host [5] No vaccinationis available for this capsule type because of the molecularmimicry between the serogroup B capsule and the tissueantigen of neuron cell adhesion molecule [6]

Capsule is not only a virulence associated factor but adominant outer surface cellular structure that is an ideal tar-get for bacteriophages These viruses can infect and multiplyinside the bacterial host and due to their specificity theycan be used to reduce target bacteria Recently phage therapyhas regained general interest as resistance to antibiotics hasbecome a serious problem [7] and phage therapy has beenfound to be effective in treating many bacterial infectionsRecent animal experiments have demonstrated the potential

HindawiBioMed Research InternationalVolume 2018 Article ID 7569645 8 pageshttpsdoiorg10115520187569645

2 BioMed Research International

of phages as alternative therapeutic agents in various infec-tion models ranging from topical wound and burn infections[8ndash10] pneumonia [11] and lung- [12] and intra-abdominal[13 14] bacteremic infections

In this work we isolated a lytic bacteriophage specificfor the K1 capsule of the newborn meningitis E coli strainIHE3034 and demonstrated its therapeutic efficacy in aprogressive intravenous mouse model We demonstratedthat intravenous administration of phages could effectivelycontrol progressive infections evoked by high bacterial cellnumber

2 Materials and Methods

21 Bacterial Strains and Culture Conditions The newbornmeningitis E coli (NMEC) strain IHE3034 (O18acK1H7phylogroup B2 sequence type ST95) was used throughoutthe study It was originally isolated by 1976 in Finland [4] andkindly provided by the authors

22 Bacteriophage Isolation Bacteriophages against theNMEC strain IHE3034 were isolated from raw sewage waterharvested from the Tettye Forrashaz Treatment Plant locatedin Pecs-Pellerd Hungary Briefly 5 ml sewage and 1 ml ONculture of IHE3034 were added to 50 ml Luria-Bertani broth(LB) medium (Oxoid USA) and incubated at 37∘C and150 rpm for 24 h The following day 15 ml of the infectedLB-sewage mixture was harvested and centrifuged at 8000 xg for 5 min to pellet cells and debris From the supernatant980 120583l was transferred to a 15 ml reagent tube and 20 120583l ofchloroform (Sigma USA) was added and left at 4∘C aftershaking After ON incubation the sample was centrifuged(8000 x g for 5 min) and a 100x serial dilution series (102x104x 106x 108x) was prepared for plaque forming unit (PFU)determination Ten 120583l from each dilution step was added to4 ml of molten top agar (04 agar) tempered to 54∘C andpoured onto LB agar plates (15 agar) previously layeredwith lawns of logarithmic phase (OD

600=08) culture of

IHE3034 Plates were incubated ON at 37∘C Phages werestored at 4∘C tested before use and diluted accordingly

23 Selection of Bacteriophages Specific for the K1 CapsulendashK1Mutant Construction Phage plaques from the top agar plateswere individually isolated and propagated in the presenceof the wild-type IHE3034 NMEC strain Tests of the phagesuspensionswere performed on the IHE3034wild-type strainand effective phage plaques were picked up propagated onIHE3034 K1 and counterselected on the lawn of its isogeniccapsule mutant (ΔK1)

The capsule mutant was gained by using the Datsenkoand Wanner method [15] based on the application oflambda Red recombinase expressing helper plasmidpKD46 Briefly pKD46 was first electroporated intothe wild-type IHE3034 and maintained at 30∘C inthe presence of Ampicillin (100120583g mlminus1) PCRs wereperformed by using the primer pairs IHE R2 pKDfw51015840-TGGGTTTATTATGGGGGGAACACAACAAACTGC-CAACATAATATATATTAAATTTCAAGTCAATAT-CTTTTGAATTTTAAGTGTAGGCTGGAGCTGCTTC

and IHE R2-pKDrev 5rsquo-TTTATCTTAACAAAGAGG-GGCAAGGTAAATTTTATAAAAATGTTACGG-AAGCCATTGCTGATTACAAAAAAGACCTAT-AGCATATGAATATCCTCCTTAGTTCCTATTCC from thetemplate plasmid pKD3 containing the cat cassette (encodingfor the chloramphenicol resistance) The PCR product waselectroporated into the freshly made competent cells ofIHE3034 containing the L-arabinose (gt10 mmol) inducedlambda Red recombinase encoded on the pKD64 helperplasmid Selection of the mutants was carried out in thepresence of chloramphenicol Elimination of Region2 ofthe capsule locus was tested by PCR by using the primerskpsT IHEell 51015840-CATGGCCCGTTGGATTGGC and KpsSIHEell 51015840-CAGTACGGCGGGGATCTCT Loss of the K1capsule was phenotypically confirmed by latex agglutination(Thermo Fisher Scientific USA) and designated as IHE3034ΔK1

Phages specific for K1 were purified three times on theIHE3034 strain by using the standard procedure described bySambrook et al [16]

24 Propagation and Purification of Phage Strains High-titer phage stocks for in vivo experimentation and sequenceanalysis were propagated and amplified in the IHE3034 hostbacterium strain by standard procedures [17] For large-scalephage preparations for animal experiments the propagatedphage and bacterium suspensions were centrifuged (2500 x g5 min) and the supernatants were filtered through a 022 120583mfilter (Sarstedt Germany) centrifuged (18000 x g 30 min)and resuspended in 01M phosphate-buffered saline (PBS)Plaque forming units (PFUs) were determined and expressedin PFUmlminus1 and stocks were stored at 4∘CThese stocks wereused for DNA isolation and subsequent sequencing

25 Electron Microscopic Analysis Morphology of phageswas investigated by transmission electron microscopy as pre-viously described [17] Briefly one drop of phage suspensionwas placed on copper gridswith carbon-coated Formvar filmsand negatively stained with 2 ammonium molybdate (pH68) for 15 min The samples were examined in a Jeol 1010transmission electron microscope (JEOL Inc Peabody MAUSA) operating at 80 kV

26 Pyrosequencing and Bioinformatics Phage nucleic acidwas isolated using the High Pure Viral Nucleic Acid Kit(Roche Applied Science Mannheim Germany) according tomanufacturerrsquos instructions The complete genome of ΦIK1was sequenced by the shotgun full-sequencing strategy usingthe GS Junior+platform (Roche Diagnostics GmbH Ger-many) The mean coverage of ΦIK1 was 1380 The assemblyof the sequence was performed with Geneious 8 softwareReads were mapped against the ΦIK1 genome sequence andthe coverage was investigated for detection of direct repeatsOpen reading frames (ORFs) were predicted using RAST[18]

27 In Vitro Activity Analysis of E coli IHE3034 Treated withΦIK1 In vitro activity was assessed to provide a quantitativeanalysis of the efficacy of the phage bactericidal activity in

BioMed Research International 3

the presence of different phage-bacterium ratios These testswere performed in a 96-well tissue culture plate Briefly ONculture of IHE3034 was set to an OD

600of 1 (sim2 x 108 CFU

mlminus1) and the initial ΦIK1 bacteriophage suspension was setto sim2 x 108 PFU mlminus1)

The 96-well tissue culture plate was partitioned on fourparallel sections Each section contained 3 columns (1-3 4-6 7-9 10-12) filled with 106 105 104 and 103 CFU mlminus1bacterial suspensions (180120583l) respectively Rows ldquoArdquo and ldquoHrdquocontained sterile growthmediumas a negative growth control(Figure 2) All other rows contained 20120583l of bacteriophagesuspensions from the phage dilutions of 106 105 104 103and 102 The final ΦIK1 phage concentrations were 10 timesdiluted (105 104 103 102 and 101 PFUs mlminus1)

By using this checker board testing the following phagebacteriummultiplicity of infection (MOI) values were tested1001 101 11 110 1100 11000 110000 1100000 The assaywas run for 24 h at 37∘CBacterial growthwas registered every5 min by measuring the OD of each well with a multimodemicroplate reader (BioTek Synergy HT)The experiment wasperformed three times

28 Mutation Rate Determination of the Receptor of ΦIK1IHE3034 suspension (1 x 108)was evenly spread on the surfaceof an LB agar plate and 10 120583l of the ΦIK1 suspension (1 x108 PFU mLminus1) was added dropwise The area of the clearedlytic zone was measured and emerged resistant colonies weredetermined Determination was performed three times

29 LethalDose of IHE3034 in the Intravenous InfectionMouseModel For in vivo tests 6-7-week-old female BALBc (19-21g) and NMRI (20-22 g) mice were purchased from CharlesRiver (Germany) Animals were cared for in accordance withthe guidelines of the European Federation for LaboratoryAnimal Science Associations (FELASA) and all procedurescare and handling of the animals were approved by theAnimal Welfare Committee of University of Pecs

For infection log-phase bacteria grown in LB broth werewashed in PBS and the optical density was set to OD

600=05

(sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFU mLminus1)OD600

=5 (sim1 x 109 CFUmLminus1) and OD600

=10 (sim2 x 109 CFUmLminus1) Groups of 10 mice received intravenous doses of 100120583l from the above suspensions giving a final amount of sim1 x107 sim2 x 107 sim1 x 108 and sim2 x 108 bacterial cells respectivelyDeath and general conditions were recorded for the following7 days and LD50 value for IHE3034 was determined on bothmouse strains

210 Therapeutic Efficacy of ΦIK1 in the Intravenous MouseModel This study was performed in strict accordance withthe FELASA (Federation of European Laboratory AnimalScience Associations) guidelines and recommendations Theanimal experiments were approved by the ethical committeeof the University of Pecs (Permit Number BA022000-202011) Altogether 6 groups (6mice per group) of 6-7-week-old female BALBcmice (19-21 g) were used Five groupswereintravenously infected with 01 mL OD

600=5 (1 x 108 CFU

mouse) IHE3034 E coli NMEC strain One group served

Figure 1 Morphology of ΦIK1 shows typical characteristics ofPodoviridae Bar represents 100nm

as a negative or bacteriophage control (GPC) receiving01 ml bacteriophage suspension (1 x 108 PFU mlminus1) Thebacterial control group (GBC) received only 01 ml OD

600=5

bacteria (1 x 108 CFU mouse) while 01 ml bacteriophagesuspensions (1 x 108 PFU mouse) were administered to allother groups 10 min (G10M) 1 h (G1H) 2 h (G2H) and3 h (G3H) postinfection General conditions and survivalrates of the mice were permanently monitored for two weeksExperiments were performed twice

211 Assessing the Titers of ΦIK1 in Different Organs Twoweeks after phage therapy treatments all mice were sacrificedand the concentration of active bacteriophages in the bloodbrain and spleenwas determinedOrganswere homogenizedin 25 ml PBS with a blade stirrer for 5 seconds Tissuesuspensions were centrifuged (8000 x g for 5 min) andchloroformed as described above Ten 120583l from these and the100x diluted tissue suspensions were added to a bacteriallawn incubated at 37∘C and PFUs were determined thefollowing day Detection limit of the test was 100 phageparticles in 1 ml blood and 250 phage particles in the brainand spleen

3 Results

31 Characterization of E coli IHE3034 Bacteriophages Iso-lated from Sewage Water Ten bacteriophages specific for theK1 capsule were purified from the local municipal sewagetreatment facility of Pecs-Pellerd Comparison of their DNArestriction profiles and recognition patterns on an E colistrain collection has suggested that the 10 isolated phageswere the same Onewas chosen for further in vitro and in vivostudies and called ΦIK1 Transmission electron microscopyanalysis of ΦIK1 indicates the typical characteristics ofPodoviridae with a head diameter of sim80 nm (Figure 1)

32 K1 Mutant Production from IHE3034 and MutationRate Determination No clearing zone was observed on thecapsule deficient isogenic mutant strain IHE3034 ΔK1 when10120583l of ΦIK1 suspension was added to a bacteria-coated agarplate However it did cause a clear plaque in the presence ofthe wild-type IHE3034 E coli K1 strain

4 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12

A

LB LB LB LB LB LB LB LB LB LB LB LB

B

IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034C

110 110 110 11 11 11 101 101 101 1001 1001 1001D

1100 1100 1100 110 110 110 11 11 11 101 101 101E

11000 11000 11000 1100 1100 1100 110 110 110 11 11 11F

110000 110000 110000 11000 11000 11000 1100 1100 1100 110 110 110G

1100000 1100000 1100000 110000 110000 110000 11000 11000 11000 1100 1100 1100H

LB LB LB LB LB LB LB LB LB LB LB LB

Figure 2 In vitro kinetics of theΦIK1-IHE3034 interaction in different MOIs Lines A (medium control) B (bacterium growth control) andH (medium control) are control fields In rows C D E F and G the final concentration of phages are 105 104 103 102 and 101 respectivelyIn columns 1-3 4-6 7-9 and 10-12 bacterial end concentrations were 106 105 104 and 103 respectively Accordingly ratios are presented PFUCFU

The mutation rate of the receptor of ΦIK1 was 41 x 104The K1 agglutination assay revealed that 100 (3434) of thetested colonies resistant to ΦIK1 lost the K1 capsule

33 Sequence Analysis Sequence analysis of ΦIK1 revealedthat the genome size of ΦIK1 was 44246 bps with 94-96 sequence similarity to 7 previously sequenced E colibacteriophages also belonging to Podoviridae Altogether 58ORFs of ΦIK1 were identified The sequence of ΦIK1 wasdeposited in the Genbank under the accession numberKY435490

34 In Vitro Activity Analysis The 24 h in vitro time killanalysis revealed that ΦIK1 could hinder the proliferationof IHE3034 This inhibitory effect depended on the starting

CFU of IHE3034 (Figure 2) If an MOI with a phage bac-terium ratio of 110 (105106) was applied (see row C columns1-3) IHE3034 could overcome the attack of bacteriophageand by the end of the 24th hour the CFU number equaledthe positive controls (only bacterium no phage row B) Incontrast ΦIK1 could successfully clear IHE3034 if at least a11 bacteriophage bacterium ratio was applied (MOIs 104104D7-9 103103 E10-12)

At a bacterial concentration of 105 CFU mlminus1 the lyticeffect of ΦIK1 was not significant as IHE3034 culture couldavoid the inhibitory effect ofΦIK1 if the phage was diluted to104 103 102 and 101 PFUmlminus1 (D4-6 E4-6 F4-6 G4-6)Thistendency changed if a 104 CFU mLminus1 bacterium suspensionwas tested in the presence of 10x 100x and 1000x less phage

BioMed Research International 5

0 6 12 18 24 30 36 42 48 54 60 660

20

40

60

80

100

Hours post challenge

Perc

ent s

urvi

val

GBCG3HG2H

G1HG10MGPC

p=00021

p=044

plt00001

Figure 3 In the phage rescue experiment survival of IHE3034infected BALBc mice strongly depended on the elapsed timebetween the injection of bacterium suspension (1 x 108 CFUmouse)and ΦIK1 (1 x 108 PFU mouse) Phage administration occurred 10min (G10M) 1 h (G1H) 2 h (G2H) and 3 h (G3H) postinfectionwhile the IHE3034 and the ΦIK1 suspensions were used as positive(GBC) and negative (GPC) controls respectively The obtainedsurvival curves were statistically compared by the logrank (Matel-Cox) test using the GraphPad Prism 60 software

particles (MOI 110 1100 and 11000 see wells E7-9 F7-9 andG7-9 respectively) In these cases the growth of bacteria waseffectively hindered by ΦIK1 This was also observed if thebacterial cell number was further decreased to 103 CFUmlminus1

This threshold at which IHE3034 could grow at aninoculum of 105 (Figure 2 column 4-6) is in accordance withthe mutation rate of ΦIK1 receptor (sim104)

35 IntravenousMouse SepsisModel of IHE 3034 Twomousestrains were used to establish the systemic infection mousemodel for IHE3034 BALBc mice were more sensitive toIHE3034 as intravenous administration of 01 ml cell suspen-sion (OD

600=10) caused 100 mortality in 24 h (SFigure 1)

Most of the mice survived if 01 ml suspensions of OD600

=05(1 x 107 CFU) or OD

600=1 (2 x 107 CFU) bacterial cells

were injected intravenously Administration of 01 ml fromOD600

=5 (1 x 108 CFUs mouse) caused the death of all micein 48 h

In contrast no complete mortality could be achievedin any of the NMRI mice groups treated with the abovesuspensions (SFigure 1)

36 Phage Rescue of the IHE3034 Rapid Sepsis by ΦIK1 Thebactericidal activity of ΦIK1 observed in vitro could be cor-roborated in the rapid sepsis mouse model The restrainingeffect strongly depended on the time between infection andphage administration

All 12 positive control mice (GBC) infected with 1 x108 CFU IHE3034 bacterium suspension died within 48 h(Figure 3) No effect was observed when phage suspension

only (1 x 108 PFU mouse) was administered to healthymice (groupof phage control GPC) Bacteriophages exerted a100 curing effect if administered within 60 min of bacterialinfection saving the life of all infected mice

Efficacy of phage treatment was drastically altered ifadministration of ΦIK1 followed bacterial challenge by 2 h(G2H) In this case survival rate at 24 h was 916 (1112)but it drastically decreased to 333 (412) by the 48th hour(Figure 3) Two mice survived from this group after the 60thhour and eventually recovered after 7-10 days No rescueeffect of ΦIK1 was detected with a 0 survival rate at 48 hpostinfection if phage was administered 3 h after bacterialinfection

37 Reisolation of Persisting ΦIK1 Phages from DifferentOrgans Two weeks after bacterial challenge and subsequentphage administrations surviving mice were sacrificed andactive ΦIK1 phage particles were isolated from the bloodspleen and brain (Figure 4 x-axis) The spleen was the onlyorgan in which active phage particles could be detected ina significant amount totalling lt35000 PFU spleen Thenumber of detectable phage particles was more limited in thebrain (lt1750 PFU brain) and blood (lt1250 120583lminus1) (Figure 4)In the phage control group (GPC) no phage particles (012)were detected in the brain and a limited number of phages(lt1000) were found in the blood of two sacrificed animals(212)

4 Discussion

Bacterial sepsis can progress if an aetiological agent entersthe body and its hematogenous spread occursThis conditionstrikes more than a million Americans [1] and has a high(28-50) mortality [2] Due to the emergence of multidrugresistance (MDR) among the aetiological agents it is increas-ingly obvious that new treatment options are needed [10]Theapplication of targeted bacteriophages could be a promisingapproach

Counterselection of isolated lytic phageswith the isogenicreceptor mutant bacteria is an effective method to isolatetargeted phages for a well-determined receptor For thispurpose we have constructed and used the IHE3034 ΔK1strain and isolatedΦIK1 targeting one of the most importantvirulence factors of the newborn meningitis-causing E colistrain IHE3034 [19 20] We have chosen the K1 capsuleas a target since it not only hinders opsonization [21] butalso contributes to adhesion promoting invasion through theblood-brain barrier (BBB) [19 22]

Although the exact timing of the pathomechanism inIHE3034 in mice is not known the 100 efficacy of theΦIK1treatment could be achieved if the phages were administered1 h after bacterial challenge (G10M and G1H Figure 3)Thereare several possible reasons for the inefficacy of phage rescuewhen performed after the first hour of infection (G2H andG3H) including the presence of phage resistant colonies inthe inoculum However their frequency was quite high (104mlminus1) so resistant cells would have been incapable of evokingthe pathogenic process This is supported by the results ofthe in vitro experiments where ΦIK1 controlled IHE3034

6 BioMed Research International

Figure 4 Detection of active phages in the surviving mice two weeks after bacterial challenge and phage rescue Results are subdividedaccording to the organs (X axis) In each subsection (1-12) the summarized results of the GPC G10MG1H andG2H are presented as columnsG3H and GBC were not drafted as no mice survived in G3H while no phage was administered to GBC (STable 1)

at 11000 MOI (Figure 2) but only when the bacterial cellnumber was below 104 CFU (the mutation rate)

Still enough K1 expressing cells were in the blood of theexperimental animals to take over a fatal pathogenic processIn our model system IHE3034 could only trigger a rapidlyescalating infection if the bacterium number was fairly high(1 x 108 CFU mouse) This is in accordance with otherexperimental setups performed with Enterococcus faecium as109 [13] Staphylococcus aureus as 108 [23] and Pseudomonasaeruginosa as 107 [14] Compared to these experiments the100 efficacy (G1H) of the phage rescue in relation of ΦIK1and IHE3034 (11 MOI) is comparable to results of otherstudies where 100 survival of the challenged mice werereported ifsim13 (3x1081x109) 101 (109108)MOIswere appliedand administration of phages occurred 45 min and 1 h afterbacterial challenges [13 23] Amore striking rescue effect wasreported by Wang et al [14] where the 1100 MOI still hada 100 protective effect if the isolated Pseudomonas-specificphage was administered up to 1 h after the infection Thiscould be due to the fact that differences in the bacteriumspecies used E faecalis S aureus P aeruginosa and NME coli possess different pathomechanisms This stronglyinfluences the availability of the infectious agent in the host

Bacteria that are capable of intracellular survival suchas IHE3034 [20] can hide from bacteriophages and by thiscan assure their survival and lead to pathogenesis E faeciumis able to survive and proliferate in murine macrophages[24] and there is also a mounting evidence that S aureushas the potential to internalize and survive within hostcells [25] In contrast P aeruginosa is thought to be anextracellular bacterium and therefore it is more vulnerableto bacteriophages Intracellular bacteria are vulnerable untilthey reach adhere to and invade certain cells protectingthemselves from the extracellular phages This may explainwhy phage rescue experiments were more effective withextracellular P aeruginosa

This process could be influenced by the tissue penetrationpotential of bacteriophages The presence of ΦIK1 in thespleens of animals in the phage control group (GPC) and thesurviving animals in groups G10M G1H and G2H indicates

that the phage could survive in the spleen for at least 2 weeksuntil the mice were sacrificed

Lack of ΦIK1 in the brains of the GPC group suggeststhat this phage cannot penetrate the BBB under normalconditions However phage was detected in the brains ofgroups G10M and G1H (Figure 4) We hypothesize thatin group (G1H) ΦIK1 phages could also exert their lyticeffects in the brain contributing to rescue At least twodifferent mechanisms could be considered for this processIt is known that upon inflammation the BBB can becomepermeable facilitating the transfer of macromolecules andviruses [26] Our study contradicts this in the case of ΦIK1as administration of 108 heat-killed IHE3034 cells 1 h priorto the intravenous phage administration did not result inthe detection of active phage particles in the brains ofexperimental animals when sacrifice occurred 1 3 or 7 daysafter administration (unpublished data)

Based on this observation we postulate that the alreadyphage-infected IHE3034 cells crossed the BBB into the braintissue

Phages are alternative therapeutics against multidrugresistant bacteria but there are still questions about theirpractical use especially in systemic infections Besides theimmunological aspects a good understanding of phage-bacteria interactions and phage survival in the body isrequired Our results demonstrate that in accordance withprevious findings phages can not only combat bacteriaalready present in the circulatory system but also survivein the spleen afterwards Future work must be dedicated tothose factors that influence this and possibly enhance thetherapeutic potential of phages

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

BioMed Research International 7

Acknowledgments

Rozsa Lajko is acknowledged for excellent technical assis-tance and Balazs Ambrusitcs and Tettye Forrashaz Kft areacknowledged for access to sewage samples The project wasfunded by AOK-KA-20163 These supports do not lead toany conflicts of interest The present scientific contributionis dedicated to the 650th anniversary of the foundation of theUniversity of Pecs Hungary

Supplementary Materials

Supplementary figure 1 concentration-dependent sen-sitivity of the BALBc (A) and CD1 (B) mice for theIHE3034 E coli K1 meningitis strain on a time scale Ten-ten mice were intravenously injected with 100 120583L doses ofOD600

=05 (sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFUmLminus1) OD

600=5 (sim1 x 109 CFU mLminus1) and OD

600=10 (sim

2 x 109 CFU mLminus1) bacterial suspensions getting a finalamount of sim1 x 107 sim2 x 107 sim1 x 108 and sim2 x 108bacterial cells respectively Death rates are represented inboth mice strains Supplementary Table 1 detection ofactive phages particles in the different organs of the sur-vived mice two weeks after bacterial challenge and phagerescue administration (Supplementary Table for Figure 4)(Supplementary Materials)

References

[1] M J Hall S N Williams C J DeFrances and A GolosinskiyldquoInpatient care for septicemia or sepsis a challenge for patientsand hospitalsrdquo NCHS Data Brief no 62 pp 1ndash8 2011

[2] K AWood andDCAngus ldquoPharmacoeconomic implicationsof new therapies in sepsisrdquo PharmacoEconomics vol 22 no 14pp 895ndash906 2004

[3] T Barichello V S Dagostim J S Generoso et al ldquoNeonatalEscherichia coli K1 meningitis causes learning and memoryimpairments in adulthoodrdquo Journal of Neuroimmunology vol272 no 1-2 pp 35ndash41 2014

[4] T K Korhonen M V Valtonen J Parkkinen et al ldquoSerotypeshemolysin production and receptor recognition of Escherichiacoli strains associated with neonatal sepsis and meningitisrdquoInfection and Immunity vol 48 pp 486ndash491 1985

[5] J A Hoffman C Wass M F Stins and K S Kim ldquoThe capsulesupports survival but not traversal of Escherichia coli K1 acrossthe blood-brain barrierrdquo Infection and Immunity vol 67 no 7pp 3566ndash3570 1999

[6] M Muhlenhoff M Eckhardt and R Gerardy-SchahnldquoPolysialic acid Three-dimensional structure biosynthesis andfunctionrdquo Current Opinion in Structural Biology vol 8 no 5pp 558ndash564 1998

[7] P Hyman and S T Abedon ldquoBacteriophage host range andbacterial resistancerdquo Advances in Applied Microbiology vol 70pp 217ndash248 2010

[8] C S McVay M Velasquez and J A Fralick ldquoPhage therapyof Pseudomonas aeruginosa infection in a mouse burn woundmodelrdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 1934ndash1938 2007

[9] S Kumari K Harjai and S Chhibber ldquoEfficacy of bacterio-phage treatment in murine burn wound infection induced by

Klebsiella pneumoniaerdquo Journal ofMicrobiology and Biotechnol-ogy vol 19 no 6 pp 622ndash628 2009

[10] J M Regeimbal A C Jacobs B W Corey et al ldquoPersonalizedtherapeutic cocktail of wild environmental phages rescues micefrom acinetobacter baumannii wound infectionsrdquo Antimicro-bial Agents and Chemotherapy vol 60 no 10 pp 5806ndash58162016

[11] S Chhibber S Kaur and S Kumari ldquoTherapeutic potentialof bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in micerdquo Journal of Medical Micro-biology vol 57 no 12 pp 1508ndash1513 2008

[12] I Takemura-Uchiyama J Uchiyama M Osanai et al ldquoExper-imental phage therapy against lethal lung-derived septicemiacaused by Staphylococcus aureus in micerdquo Microbes and Infec-tion vol 16 no 6 pp 512ndash517 2014

[13] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol70 no 1 pp 204ndash210 2002

[14] J Wang B Hu M Xu et al ldquoUse of bacteriophage in thetreatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosardquo International Journal ofMolecular Medicine vol 17 pp 309ndash317 2006

[15] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[16] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Col Spring Harbor Press Cold SpringHarbor NY USA 2nd edition 1989

[17] I Toth D Svab B Balint M Brown-Jaque and G MarotildquoComparative analysis of the Shiga toxin converting bacterio-phage first detected in Shigella sonneirdquo Infection Genetics andEvolution vol 37 pp 150ndash157 2016

[18] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 p 75 2008

[19] J R Johnson P Delavari and T OrsquoBryan ldquoEscherichia coli O18K1H7 isolates from patients with acute cystitis and neonatalmeningitis exhibit common phylogenetic origins and virulencefactor profilesrdquo The Journal of Infectious Diseases vol 183 no10 pp 425ndash434 2001

[20] K J Kim S J Elliott F Di CelloM F Stins andK S Kim ldquoTheK1 capsule modulates trafficking of E coli-containing vacuolesand enhances intracellular bacterial survival in human brainmicrovascular endothelial cellsrdquo Cellular Microbiology vol 5no 4 pp 245ndash252 2003

[21] M Moulin-Schouleur C Schouler P Tailliez et al ldquoCommonvirulence factors and genetic relationships between O18K1H7Escherichia coli isolates of human and avian originrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3484ndash3492 2006

[22] K S Kim H Itabashi P Gemski J Sadoff R L Warren andA S Cross ldquoThe K1 capsule is the critical determinant in thedevelopment of Escherichia coli meningitis in the ratrdquo TheJournal of Clinical Investigation vol 90 no 3 pp 897ndash905 1992

[23] R Capparelli M Parlato G Borriello P Salvatore and DIannelli ldquoExperimental phage therapy against Staphylococcusaureus in micerdquo Antimicrobial Agents and Chemotherapy vol51 no 8 pp 2765ndash2773 2007

[24] F Lebreton W van Schaik M Sanguinetti et al ldquoAsrR Isan Oxidative Stress Sensing Regulator Modulating Enterococ-cus faecium Opportunistic Traits Antimicrobial Resistance

BioMed Research International 3

the presence of different phage-bacterium ratios These testswere performed in a 96-well tissue culture plate Briefly ONculture of IHE3034 was set to an OD

600of 1 (sim2 x 108 CFU

mlminus1) and the initial ΦIK1 bacteriophage suspension was setto sim2 x 108 PFU mlminus1)

The 96-well tissue culture plate was partitioned on fourparallel sections Each section contained 3 columns (1-3 4-6 7-9 10-12) filled with 106 105 104 and 103 CFU mlminus1bacterial suspensions (180120583l) respectively Rows ldquoArdquo and ldquoHrdquocontained sterile growthmediumas a negative growth control(Figure 2) All other rows contained 20120583l of bacteriophagesuspensions from the phage dilutions of 106 105 104 103and 102 The final ΦIK1 phage concentrations were 10 timesdiluted (105 104 103 102 and 101 PFUs mlminus1)

By using this checker board testing the following phagebacteriummultiplicity of infection (MOI) values were tested1001 101 11 110 1100 11000 110000 1100000 The assaywas run for 24 h at 37∘CBacterial growthwas registered every5 min by measuring the OD of each well with a multimodemicroplate reader (BioTek Synergy HT)The experiment wasperformed three times

28 Mutation Rate Determination of the Receptor of ΦIK1IHE3034 suspension (1 x 108)was evenly spread on the surfaceof an LB agar plate and 10 120583l of the ΦIK1 suspension (1 x108 PFU mLminus1) was added dropwise The area of the clearedlytic zone was measured and emerged resistant colonies weredetermined Determination was performed three times

29 LethalDose of IHE3034 in the Intravenous InfectionMouseModel For in vivo tests 6-7-week-old female BALBc (19-21g) and NMRI (20-22 g) mice were purchased from CharlesRiver (Germany) Animals were cared for in accordance withthe guidelines of the European Federation for LaboratoryAnimal Science Associations (FELASA) and all procedurescare and handling of the animals were approved by theAnimal Welfare Committee of University of Pecs

For infection log-phase bacteria grown in LB broth werewashed in PBS and the optical density was set to OD

600=05

(sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFU mLminus1)OD600

=5 (sim1 x 109 CFUmLminus1) and OD600

=10 (sim2 x 109 CFUmLminus1) Groups of 10 mice received intravenous doses of 100120583l from the above suspensions giving a final amount of sim1 x107 sim2 x 107 sim1 x 108 and sim2 x 108 bacterial cells respectivelyDeath and general conditions were recorded for the following7 days and LD50 value for IHE3034 was determined on bothmouse strains

210 Therapeutic Efficacy of ΦIK1 in the Intravenous MouseModel This study was performed in strict accordance withthe FELASA (Federation of European Laboratory AnimalScience Associations) guidelines and recommendations Theanimal experiments were approved by the ethical committeeof the University of Pecs (Permit Number BA022000-202011) Altogether 6 groups (6mice per group) of 6-7-week-old female BALBcmice (19-21 g) were used Five groupswereintravenously infected with 01 mL OD

600=5 (1 x 108 CFU

mouse) IHE3034 E coli NMEC strain One group served

Figure 1 Morphology of ΦIK1 shows typical characteristics ofPodoviridae Bar represents 100nm

as a negative or bacteriophage control (GPC) receiving01 ml bacteriophage suspension (1 x 108 PFU mlminus1) Thebacterial control group (GBC) received only 01 ml OD

600=5

bacteria (1 x 108 CFU mouse) while 01 ml bacteriophagesuspensions (1 x 108 PFU mouse) were administered to allother groups 10 min (G10M) 1 h (G1H) 2 h (G2H) and3 h (G3H) postinfection General conditions and survivalrates of the mice were permanently monitored for two weeksExperiments were performed twice

211 Assessing the Titers of ΦIK1 in Different Organs Twoweeks after phage therapy treatments all mice were sacrificedand the concentration of active bacteriophages in the bloodbrain and spleenwas determinedOrganswere homogenizedin 25 ml PBS with a blade stirrer for 5 seconds Tissuesuspensions were centrifuged (8000 x g for 5 min) andchloroformed as described above Ten 120583l from these and the100x diluted tissue suspensions were added to a bacteriallawn incubated at 37∘C and PFUs were determined thefollowing day Detection limit of the test was 100 phageparticles in 1 ml blood and 250 phage particles in the brainand spleen

3 Results

31 Characterization of E coli IHE3034 Bacteriophages Iso-lated from Sewage Water Ten bacteriophages specific for theK1 capsule were purified from the local municipal sewagetreatment facility of Pecs-Pellerd Comparison of their DNArestriction profiles and recognition patterns on an E colistrain collection has suggested that the 10 isolated phageswere the same Onewas chosen for further in vitro and in vivostudies and called ΦIK1 Transmission electron microscopyanalysis of ΦIK1 indicates the typical characteristics ofPodoviridae with a head diameter of sim80 nm (Figure 1)

32 K1 Mutant Production from IHE3034 and MutationRate Determination No clearing zone was observed on thecapsule deficient isogenic mutant strain IHE3034 ΔK1 when10120583l of ΦIK1 suspension was added to a bacteria-coated agarplate However it did cause a clear plaque in the presence ofthe wild-type IHE3034 E coli K1 strain

4 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12

A

LB LB LB LB LB LB LB LB LB LB LB LB

B

IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034C

110 110 110 11 11 11 101 101 101 1001 1001 1001D

1100 1100 1100 110 110 110 11 11 11 101 101 101E

11000 11000 11000 1100 1100 1100 110 110 110 11 11 11F

110000 110000 110000 11000 11000 11000 1100 1100 1100 110 110 110G

1100000 1100000 1100000 110000 110000 110000 11000 11000 11000 1100 1100 1100H

LB LB LB LB LB LB LB LB LB LB LB LB

Figure 2 In vitro kinetics of theΦIK1-IHE3034 interaction in different MOIs Lines A (medium control) B (bacterium growth control) andH (medium control) are control fields In rows C D E F and G the final concentration of phages are 105 104 103 102 and 101 respectivelyIn columns 1-3 4-6 7-9 and 10-12 bacterial end concentrations were 106 105 104 and 103 respectively Accordingly ratios are presented PFUCFU

The mutation rate of the receptor of ΦIK1 was 41 x 104The K1 agglutination assay revealed that 100 (3434) of thetested colonies resistant to ΦIK1 lost the K1 capsule

33 Sequence Analysis Sequence analysis of ΦIK1 revealedthat the genome size of ΦIK1 was 44246 bps with 94-96 sequence similarity to 7 previously sequenced E colibacteriophages also belonging to Podoviridae Altogether 58ORFs of ΦIK1 were identified The sequence of ΦIK1 wasdeposited in the Genbank under the accession numberKY435490

34 In Vitro Activity Analysis The 24 h in vitro time killanalysis revealed that ΦIK1 could hinder the proliferationof IHE3034 This inhibitory effect depended on the starting

CFU of IHE3034 (Figure 2) If an MOI with a phage bac-terium ratio of 110 (105106) was applied (see row C columns1-3) IHE3034 could overcome the attack of bacteriophageand by the end of the 24th hour the CFU number equaledthe positive controls (only bacterium no phage row B) Incontrast ΦIK1 could successfully clear IHE3034 if at least a11 bacteriophage bacterium ratio was applied (MOIs 104104D7-9 103103 E10-12)

At a bacterial concentration of 105 CFU mlminus1 the lyticeffect of ΦIK1 was not significant as IHE3034 culture couldavoid the inhibitory effect ofΦIK1 if the phage was diluted to104 103 102 and 101 PFUmlminus1 (D4-6 E4-6 F4-6 G4-6)Thistendency changed if a 104 CFU mLminus1 bacterium suspensionwas tested in the presence of 10x 100x and 1000x less phage

BioMed Research International 5

0 6 12 18 24 30 36 42 48 54 60 660

20

40

60

80

100

Hours post challenge

Perc

ent s

urvi

val

GBCG3HG2H

G1HG10MGPC

p=00021

p=044

plt00001

Figure 3 In the phage rescue experiment survival of IHE3034infected BALBc mice strongly depended on the elapsed timebetween the injection of bacterium suspension (1 x 108 CFUmouse)and ΦIK1 (1 x 108 PFU mouse) Phage administration occurred 10min (G10M) 1 h (G1H) 2 h (G2H) and 3 h (G3H) postinfectionwhile the IHE3034 and the ΦIK1 suspensions were used as positive(GBC) and negative (GPC) controls respectively The obtainedsurvival curves were statistically compared by the logrank (Matel-Cox) test using the GraphPad Prism 60 software

particles (MOI 110 1100 and 11000 see wells E7-9 F7-9 andG7-9 respectively) In these cases the growth of bacteria waseffectively hindered by ΦIK1 This was also observed if thebacterial cell number was further decreased to 103 CFUmlminus1

This threshold at which IHE3034 could grow at aninoculum of 105 (Figure 2 column 4-6) is in accordance withthe mutation rate of ΦIK1 receptor (sim104)

35 IntravenousMouse SepsisModel of IHE 3034 Twomousestrains were used to establish the systemic infection mousemodel for IHE3034 BALBc mice were more sensitive toIHE3034 as intravenous administration of 01 ml cell suspen-sion (OD

600=10) caused 100 mortality in 24 h (SFigure 1)

Most of the mice survived if 01 ml suspensions of OD600

=05(1 x 107 CFU) or OD

600=1 (2 x 107 CFU) bacterial cells

were injected intravenously Administration of 01 ml fromOD600

=5 (1 x 108 CFUs mouse) caused the death of all micein 48 h

In contrast no complete mortality could be achievedin any of the NMRI mice groups treated with the abovesuspensions (SFigure 1)

36 Phage Rescue of the IHE3034 Rapid Sepsis by ΦIK1 Thebactericidal activity of ΦIK1 observed in vitro could be cor-roborated in the rapid sepsis mouse model The restrainingeffect strongly depended on the time between infection andphage administration

All 12 positive control mice (GBC) infected with 1 x108 CFU IHE3034 bacterium suspension died within 48 h(Figure 3) No effect was observed when phage suspension

only (1 x 108 PFU mouse) was administered to healthymice (groupof phage control GPC) Bacteriophages exerted a100 curing effect if administered within 60 min of bacterialinfection saving the life of all infected mice

Efficacy of phage treatment was drastically altered ifadministration of ΦIK1 followed bacterial challenge by 2 h(G2H) In this case survival rate at 24 h was 916 (1112)but it drastically decreased to 333 (412) by the 48th hour(Figure 3) Two mice survived from this group after the 60thhour and eventually recovered after 7-10 days No rescueeffect of ΦIK1 was detected with a 0 survival rate at 48 hpostinfection if phage was administered 3 h after bacterialinfection

37 Reisolation of Persisting ΦIK1 Phages from DifferentOrgans Two weeks after bacterial challenge and subsequentphage administrations surviving mice were sacrificed andactive ΦIK1 phage particles were isolated from the bloodspleen and brain (Figure 4 x-axis) The spleen was the onlyorgan in which active phage particles could be detected ina significant amount totalling lt35000 PFU spleen Thenumber of detectable phage particles was more limited in thebrain (lt1750 PFU brain) and blood (lt1250 120583lminus1) (Figure 4)In the phage control group (GPC) no phage particles (012)were detected in the brain and a limited number of phages(lt1000) were found in the blood of two sacrificed animals(212)

4 Discussion

Bacterial sepsis can progress if an aetiological agent entersthe body and its hematogenous spread occursThis conditionstrikes more than a million Americans [1] and has a high(28-50) mortality [2] Due to the emergence of multidrugresistance (MDR) among the aetiological agents it is increas-ingly obvious that new treatment options are needed [10]Theapplication of targeted bacteriophages could be a promisingapproach

Counterselection of isolated lytic phageswith the isogenicreceptor mutant bacteria is an effective method to isolatetargeted phages for a well-determined receptor For thispurpose we have constructed and used the IHE3034 ΔK1strain and isolatedΦIK1 targeting one of the most importantvirulence factors of the newborn meningitis-causing E colistrain IHE3034 [19 20] We have chosen the K1 capsuleas a target since it not only hinders opsonization [21] butalso contributes to adhesion promoting invasion through theblood-brain barrier (BBB) [19 22]

Although the exact timing of the pathomechanism inIHE3034 in mice is not known the 100 efficacy of theΦIK1treatment could be achieved if the phages were administered1 h after bacterial challenge (G10M and G1H Figure 3)Thereare several possible reasons for the inefficacy of phage rescuewhen performed after the first hour of infection (G2H andG3H) including the presence of phage resistant colonies inthe inoculum However their frequency was quite high (104mlminus1) so resistant cells would have been incapable of evokingthe pathogenic process This is supported by the results ofthe in vitro experiments where ΦIK1 controlled IHE3034

6 BioMed Research International

Figure 4 Detection of active phages in the surviving mice two weeks after bacterial challenge and phage rescue Results are subdividedaccording to the organs (X axis) In each subsection (1-12) the summarized results of the GPC G10MG1H andG2H are presented as columnsG3H and GBC were not drafted as no mice survived in G3H while no phage was administered to GBC (STable 1)

at 11000 MOI (Figure 2) but only when the bacterial cellnumber was below 104 CFU (the mutation rate)

Still enough K1 expressing cells were in the blood of theexperimental animals to take over a fatal pathogenic processIn our model system IHE3034 could only trigger a rapidlyescalating infection if the bacterium number was fairly high(1 x 108 CFU mouse) This is in accordance with otherexperimental setups performed with Enterococcus faecium as109 [13] Staphylococcus aureus as 108 [23] and Pseudomonasaeruginosa as 107 [14] Compared to these experiments the100 efficacy (G1H) of the phage rescue in relation of ΦIK1and IHE3034 (11 MOI) is comparable to results of otherstudies where 100 survival of the challenged mice werereported ifsim13 (3x1081x109) 101 (109108)MOIswere appliedand administration of phages occurred 45 min and 1 h afterbacterial challenges [13 23] Amore striking rescue effect wasreported by Wang et al [14] where the 1100 MOI still hada 100 protective effect if the isolated Pseudomonas-specificphage was administered up to 1 h after the infection Thiscould be due to the fact that differences in the bacteriumspecies used E faecalis S aureus P aeruginosa and NME coli possess different pathomechanisms This stronglyinfluences the availability of the infectious agent in the host

Bacteria that are capable of intracellular survival suchas IHE3034 [20] can hide from bacteriophages and by thiscan assure their survival and lead to pathogenesis E faeciumis able to survive and proliferate in murine macrophages[24] and there is also a mounting evidence that S aureushas the potential to internalize and survive within hostcells [25] In contrast P aeruginosa is thought to be anextracellular bacterium and therefore it is more vulnerableto bacteriophages Intracellular bacteria are vulnerable untilthey reach adhere to and invade certain cells protectingthemselves from the extracellular phages This may explainwhy phage rescue experiments were more effective withextracellular P aeruginosa

This process could be influenced by the tissue penetrationpotential of bacteriophages The presence of ΦIK1 in thespleens of animals in the phage control group (GPC) and thesurviving animals in groups G10M G1H and G2H indicates

that the phage could survive in the spleen for at least 2 weeksuntil the mice were sacrificed

Lack of ΦIK1 in the brains of the GPC group suggeststhat this phage cannot penetrate the BBB under normalconditions However phage was detected in the brains ofgroups G10M and G1H (Figure 4) We hypothesize thatin group (G1H) ΦIK1 phages could also exert their lyticeffects in the brain contributing to rescue At least twodifferent mechanisms could be considered for this processIt is known that upon inflammation the BBB can becomepermeable facilitating the transfer of macromolecules andviruses [26] Our study contradicts this in the case of ΦIK1as administration of 108 heat-killed IHE3034 cells 1 h priorto the intravenous phage administration did not result inthe detection of active phage particles in the brains ofexperimental animals when sacrifice occurred 1 3 or 7 daysafter administration (unpublished data)

Based on this observation we postulate that the alreadyphage-infected IHE3034 cells crossed the BBB into the braintissue

Phages are alternative therapeutics against multidrugresistant bacteria but there are still questions about theirpractical use especially in systemic infections Besides theimmunological aspects a good understanding of phage-bacteria interactions and phage survival in the body isrequired Our results demonstrate that in accordance withprevious findings phages can not only combat bacteriaalready present in the circulatory system but also survivein the spleen afterwards Future work must be dedicated tothose factors that influence this and possibly enhance thetherapeutic potential of phages

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

BioMed Research International 7

Acknowledgments

Rozsa Lajko is acknowledged for excellent technical assis-tance and Balazs Ambrusitcs and Tettye Forrashaz Kft areacknowledged for access to sewage samples The project wasfunded by AOK-KA-20163 These supports do not lead toany conflicts of interest The present scientific contributionis dedicated to the 650th anniversary of the foundation of theUniversity of Pecs Hungary

Supplementary Materials

Supplementary figure 1 concentration-dependent sen-sitivity of the BALBc (A) and CD1 (B) mice for theIHE3034 E coli K1 meningitis strain on a time scale Ten-ten mice were intravenously injected with 100 120583L doses ofOD600

=05 (sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFUmLminus1) OD

600=5 (sim1 x 109 CFU mLminus1) and OD

600=10 (sim

2 x 109 CFU mLminus1) bacterial suspensions getting a finalamount of sim1 x 107 sim2 x 107 sim1 x 108 and sim2 x 108bacterial cells respectively Death rates are represented inboth mice strains Supplementary Table 1 detection ofactive phages particles in the different organs of the sur-vived mice two weeks after bacterial challenge and phagerescue administration (Supplementary Table for Figure 4)(Supplementary Materials)

References

[1] M J Hall S N Williams C J DeFrances and A GolosinskiyldquoInpatient care for septicemia or sepsis a challenge for patientsand hospitalsrdquo NCHS Data Brief no 62 pp 1ndash8 2011

[2] K AWood andDCAngus ldquoPharmacoeconomic implicationsof new therapies in sepsisrdquo PharmacoEconomics vol 22 no 14pp 895ndash906 2004

[3] T Barichello V S Dagostim J S Generoso et al ldquoNeonatalEscherichia coli K1 meningitis causes learning and memoryimpairments in adulthoodrdquo Journal of Neuroimmunology vol272 no 1-2 pp 35ndash41 2014

[4] T K Korhonen M V Valtonen J Parkkinen et al ldquoSerotypeshemolysin production and receptor recognition of Escherichiacoli strains associated with neonatal sepsis and meningitisrdquoInfection and Immunity vol 48 pp 486ndash491 1985

[5] J A Hoffman C Wass M F Stins and K S Kim ldquoThe capsulesupports survival but not traversal of Escherichia coli K1 acrossthe blood-brain barrierrdquo Infection and Immunity vol 67 no 7pp 3566ndash3570 1999

[6] M Muhlenhoff M Eckhardt and R Gerardy-SchahnldquoPolysialic acid Three-dimensional structure biosynthesis andfunctionrdquo Current Opinion in Structural Biology vol 8 no 5pp 558ndash564 1998

[7] P Hyman and S T Abedon ldquoBacteriophage host range andbacterial resistancerdquo Advances in Applied Microbiology vol 70pp 217ndash248 2010

[8] C S McVay M Velasquez and J A Fralick ldquoPhage therapyof Pseudomonas aeruginosa infection in a mouse burn woundmodelrdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 1934ndash1938 2007

[9] S Kumari K Harjai and S Chhibber ldquoEfficacy of bacterio-phage treatment in murine burn wound infection induced by

Klebsiella pneumoniaerdquo Journal ofMicrobiology and Biotechnol-ogy vol 19 no 6 pp 622ndash628 2009

[10] J M Regeimbal A C Jacobs B W Corey et al ldquoPersonalizedtherapeutic cocktail of wild environmental phages rescues micefrom acinetobacter baumannii wound infectionsrdquo Antimicro-bial Agents and Chemotherapy vol 60 no 10 pp 5806ndash58162016

[11] S Chhibber S Kaur and S Kumari ldquoTherapeutic potentialof bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in micerdquo Journal of Medical Micro-biology vol 57 no 12 pp 1508ndash1513 2008

[12] I Takemura-Uchiyama J Uchiyama M Osanai et al ldquoExper-imental phage therapy against lethal lung-derived septicemiacaused by Staphylococcus aureus in micerdquo Microbes and Infec-tion vol 16 no 6 pp 512ndash517 2014

[13] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol70 no 1 pp 204ndash210 2002

[14] J Wang B Hu M Xu et al ldquoUse of bacteriophage in thetreatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosardquo International Journal ofMolecular Medicine vol 17 pp 309ndash317 2006

[15] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[16] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Col Spring Harbor Press Cold SpringHarbor NY USA 2nd edition 1989

[17] I Toth D Svab B Balint M Brown-Jaque and G MarotildquoComparative analysis of the Shiga toxin converting bacterio-phage first detected in Shigella sonneirdquo Infection Genetics andEvolution vol 37 pp 150ndash157 2016

[18] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 p 75 2008

[19] J R Johnson P Delavari and T OrsquoBryan ldquoEscherichia coli O18K1H7 isolates from patients with acute cystitis and neonatalmeningitis exhibit common phylogenetic origins and virulencefactor profilesrdquo The Journal of Infectious Diseases vol 183 no10 pp 425ndash434 2001

[20] K J Kim S J Elliott F Di CelloM F Stins andK S Kim ldquoTheK1 capsule modulates trafficking of E coli-containing vacuolesand enhances intracellular bacterial survival in human brainmicrovascular endothelial cellsrdquo Cellular Microbiology vol 5no 4 pp 245ndash252 2003

[21] M Moulin-Schouleur C Schouler P Tailliez et al ldquoCommonvirulence factors and genetic relationships between O18K1H7Escherichia coli isolates of human and avian originrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3484ndash3492 2006

[22] K S Kim H Itabashi P Gemski J Sadoff R L Warren andA S Cross ldquoThe K1 capsule is the critical determinant in thedevelopment of Escherichia coli meningitis in the ratrdquo TheJournal of Clinical Investigation vol 90 no 3 pp 897ndash905 1992

[23] R Capparelli M Parlato G Borriello P Salvatore and DIannelli ldquoExperimental phage therapy against Staphylococcusaureus in micerdquo Antimicrobial Agents and Chemotherapy vol51 no 8 pp 2765ndash2773 2007

[24] F Lebreton W van Schaik M Sanguinetti et al ldquoAsrR Isan Oxidative Stress Sensing Regulator Modulating Enterococ-cus faecium Opportunistic Traits Antimicrobial Resistance

4 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12

A

LB LB LB LB LB LB LB LB LB LB LB LB

B

IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034 IHE3034C

110 110 110 11 11 11 101 101 101 1001 1001 1001D

1100 1100 1100 110 110 110 11 11 11 101 101 101E

11000 11000 11000 1100 1100 1100 110 110 110 11 11 11F

110000 110000 110000 11000 11000 11000 1100 1100 1100 110 110 110G

1100000 1100000 1100000 110000 110000 110000 11000 11000 11000 1100 1100 1100H

LB LB LB LB LB LB LB LB LB LB LB LB

Figure 2 In vitro kinetics of theΦIK1-IHE3034 interaction in different MOIs Lines A (medium control) B (bacterium growth control) andH (medium control) are control fields In rows C D E F and G the final concentration of phages are 105 104 103 102 and 101 respectivelyIn columns 1-3 4-6 7-9 and 10-12 bacterial end concentrations were 106 105 104 and 103 respectively Accordingly ratios are presented PFUCFU

The mutation rate of the receptor of ΦIK1 was 41 x 104The K1 agglutination assay revealed that 100 (3434) of thetested colonies resistant to ΦIK1 lost the K1 capsule

33 Sequence Analysis Sequence analysis of ΦIK1 revealedthat the genome size of ΦIK1 was 44246 bps with 94-96 sequence similarity to 7 previously sequenced E colibacteriophages also belonging to Podoviridae Altogether 58ORFs of ΦIK1 were identified The sequence of ΦIK1 wasdeposited in the Genbank under the accession numberKY435490

34 In Vitro Activity Analysis The 24 h in vitro time killanalysis revealed that ΦIK1 could hinder the proliferationof IHE3034 This inhibitory effect depended on the starting

CFU of IHE3034 (Figure 2) If an MOI with a phage bac-terium ratio of 110 (105106) was applied (see row C columns1-3) IHE3034 could overcome the attack of bacteriophageand by the end of the 24th hour the CFU number equaledthe positive controls (only bacterium no phage row B) Incontrast ΦIK1 could successfully clear IHE3034 if at least a11 bacteriophage bacterium ratio was applied (MOIs 104104D7-9 103103 E10-12)

At a bacterial concentration of 105 CFU mlminus1 the lyticeffect of ΦIK1 was not significant as IHE3034 culture couldavoid the inhibitory effect ofΦIK1 if the phage was diluted to104 103 102 and 101 PFUmlminus1 (D4-6 E4-6 F4-6 G4-6)Thistendency changed if a 104 CFU mLminus1 bacterium suspensionwas tested in the presence of 10x 100x and 1000x less phage

BioMed Research International 5

0 6 12 18 24 30 36 42 48 54 60 660

20

40

60

80

100

Hours post challenge

Perc

ent s

urvi

val

GBCG3HG2H

G1HG10MGPC

p=00021

p=044

plt00001

Figure 3 In the phage rescue experiment survival of IHE3034infected BALBc mice strongly depended on the elapsed timebetween the injection of bacterium suspension (1 x 108 CFUmouse)and ΦIK1 (1 x 108 PFU mouse) Phage administration occurred 10min (G10M) 1 h (G1H) 2 h (G2H) and 3 h (G3H) postinfectionwhile the IHE3034 and the ΦIK1 suspensions were used as positive(GBC) and negative (GPC) controls respectively The obtainedsurvival curves were statistically compared by the logrank (Matel-Cox) test using the GraphPad Prism 60 software

particles (MOI 110 1100 and 11000 see wells E7-9 F7-9 andG7-9 respectively) In these cases the growth of bacteria waseffectively hindered by ΦIK1 This was also observed if thebacterial cell number was further decreased to 103 CFUmlminus1

This threshold at which IHE3034 could grow at aninoculum of 105 (Figure 2 column 4-6) is in accordance withthe mutation rate of ΦIK1 receptor (sim104)

35 IntravenousMouse SepsisModel of IHE 3034 Twomousestrains were used to establish the systemic infection mousemodel for IHE3034 BALBc mice were more sensitive toIHE3034 as intravenous administration of 01 ml cell suspen-sion (OD

600=10) caused 100 mortality in 24 h (SFigure 1)

Most of the mice survived if 01 ml suspensions of OD600

=05(1 x 107 CFU) or OD

600=1 (2 x 107 CFU) bacterial cells

were injected intravenously Administration of 01 ml fromOD600

=5 (1 x 108 CFUs mouse) caused the death of all micein 48 h

In contrast no complete mortality could be achievedin any of the NMRI mice groups treated with the abovesuspensions (SFigure 1)

36 Phage Rescue of the IHE3034 Rapid Sepsis by ΦIK1 Thebactericidal activity of ΦIK1 observed in vitro could be cor-roborated in the rapid sepsis mouse model The restrainingeffect strongly depended on the time between infection andphage administration

All 12 positive control mice (GBC) infected with 1 x108 CFU IHE3034 bacterium suspension died within 48 h(Figure 3) No effect was observed when phage suspension

only (1 x 108 PFU mouse) was administered to healthymice (groupof phage control GPC) Bacteriophages exerted a100 curing effect if administered within 60 min of bacterialinfection saving the life of all infected mice

Efficacy of phage treatment was drastically altered ifadministration of ΦIK1 followed bacterial challenge by 2 h(G2H) In this case survival rate at 24 h was 916 (1112)but it drastically decreased to 333 (412) by the 48th hour(Figure 3) Two mice survived from this group after the 60thhour and eventually recovered after 7-10 days No rescueeffect of ΦIK1 was detected with a 0 survival rate at 48 hpostinfection if phage was administered 3 h after bacterialinfection

37 Reisolation of Persisting ΦIK1 Phages from DifferentOrgans Two weeks after bacterial challenge and subsequentphage administrations surviving mice were sacrificed andactive ΦIK1 phage particles were isolated from the bloodspleen and brain (Figure 4 x-axis) The spleen was the onlyorgan in which active phage particles could be detected ina significant amount totalling lt35000 PFU spleen Thenumber of detectable phage particles was more limited in thebrain (lt1750 PFU brain) and blood (lt1250 120583lminus1) (Figure 4)In the phage control group (GPC) no phage particles (012)were detected in the brain and a limited number of phages(lt1000) were found in the blood of two sacrificed animals(212)

4 Discussion

Bacterial sepsis can progress if an aetiological agent entersthe body and its hematogenous spread occursThis conditionstrikes more than a million Americans [1] and has a high(28-50) mortality [2] Due to the emergence of multidrugresistance (MDR) among the aetiological agents it is increas-ingly obvious that new treatment options are needed [10]Theapplication of targeted bacteriophages could be a promisingapproach

Counterselection of isolated lytic phageswith the isogenicreceptor mutant bacteria is an effective method to isolatetargeted phages for a well-determined receptor For thispurpose we have constructed and used the IHE3034 ΔK1strain and isolatedΦIK1 targeting one of the most importantvirulence factors of the newborn meningitis-causing E colistrain IHE3034 [19 20] We have chosen the K1 capsuleas a target since it not only hinders opsonization [21] butalso contributes to adhesion promoting invasion through theblood-brain barrier (BBB) [19 22]

Although the exact timing of the pathomechanism inIHE3034 in mice is not known the 100 efficacy of theΦIK1treatment could be achieved if the phages were administered1 h after bacterial challenge (G10M and G1H Figure 3)Thereare several possible reasons for the inefficacy of phage rescuewhen performed after the first hour of infection (G2H andG3H) including the presence of phage resistant colonies inthe inoculum However their frequency was quite high (104mlminus1) so resistant cells would have been incapable of evokingthe pathogenic process This is supported by the results ofthe in vitro experiments where ΦIK1 controlled IHE3034

6 BioMed Research International

Figure 4 Detection of active phages in the surviving mice two weeks after bacterial challenge and phage rescue Results are subdividedaccording to the organs (X axis) In each subsection (1-12) the summarized results of the GPC G10MG1H andG2H are presented as columnsG3H and GBC were not drafted as no mice survived in G3H while no phage was administered to GBC (STable 1)

at 11000 MOI (Figure 2) but only when the bacterial cellnumber was below 104 CFU (the mutation rate)

Still enough K1 expressing cells were in the blood of theexperimental animals to take over a fatal pathogenic processIn our model system IHE3034 could only trigger a rapidlyescalating infection if the bacterium number was fairly high(1 x 108 CFU mouse) This is in accordance with otherexperimental setups performed with Enterococcus faecium as109 [13] Staphylococcus aureus as 108 [23] and Pseudomonasaeruginosa as 107 [14] Compared to these experiments the100 efficacy (G1H) of the phage rescue in relation of ΦIK1and IHE3034 (11 MOI) is comparable to results of otherstudies where 100 survival of the challenged mice werereported ifsim13 (3x1081x109) 101 (109108)MOIswere appliedand administration of phages occurred 45 min and 1 h afterbacterial challenges [13 23] Amore striking rescue effect wasreported by Wang et al [14] where the 1100 MOI still hada 100 protective effect if the isolated Pseudomonas-specificphage was administered up to 1 h after the infection Thiscould be due to the fact that differences in the bacteriumspecies used E faecalis S aureus P aeruginosa and NME coli possess different pathomechanisms This stronglyinfluences the availability of the infectious agent in the host

Bacteria that are capable of intracellular survival suchas IHE3034 [20] can hide from bacteriophages and by thiscan assure their survival and lead to pathogenesis E faeciumis able to survive and proliferate in murine macrophages[24] and there is also a mounting evidence that S aureushas the potential to internalize and survive within hostcells [25] In contrast P aeruginosa is thought to be anextracellular bacterium and therefore it is more vulnerableto bacteriophages Intracellular bacteria are vulnerable untilthey reach adhere to and invade certain cells protectingthemselves from the extracellular phages This may explainwhy phage rescue experiments were more effective withextracellular P aeruginosa

This process could be influenced by the tissue penetrationpotential of bacteriophages The presence of ΦIK1 in thespleens of animals in the phage control group (GPC) and thesurviving animals in groups G10M G1H and G2H indicates

that the phage could survive in the spleen for at least 2 weeksuntil the mice were sacrificed

Lack of ΦIK1 in the brains of the GPC group suggeststhat this phage cannot penetrate the BBB under normalconditions However phage was detected in the brains ofgroups G10M and G1H (Figure 4) We hypothesize thatin group (G1H) ΦIK1 phages could also exert their lyticeffects in the brain contributing to rescue At least twodifferent mechanisms could be considered for this processIt is known that upon inflammation the BBB can becomepermeable facilitating the transfer of macromolecules andviruses [26] Our study contradicts this in the case of ΦIK1as administration of 108 heat-killed IHE3034 cells 1 h priorto the intravenous phage administration did not result inthe detection of active phage particles in the brains ofexperimental animals when sacrifice occurred 1 3 or 7 daysafter administration (unpublished data)

Based on this observation we postulate that the alreadyphage-infected IHE3034 cells crossed the BBB into the braintissue

Phages are alternative therapeutics against multidrugresistant bacteria but there are still questions about theirpractical use especially in systemic infections Besides theimmunological aspects a good understanding of phage-bacteria interactions and phage survival in the body isrequired Our results demonstrate that in accordance withprevious findings phages can not only combat bacteriaalready present in the circulatory system but also survivein the spleen afterwards Future work must be dedicated tothose factors that influence this and possibly enhance thetherapeutic potential of phages

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

BioMed Research International 7

Acknowledgments

Rozsa Lajko is acknowledged for excellent technical assis-tance and Balazs Ambrusitcs and Tettye Forrashaz Kft areacknowledged for access to sewage samples The project wasfunded by AOK-KA-20163 These supports do not lead toany conflicts of interest The present scientific contributionis dedicated to the 650th anniversary of the foundation of theUniversity of Pecs Hungary

Supplementary Materials

Supplementary figure 1 concentration-dependent sen-sitivity of the BALBc (A) and CD1 (B) mice for theIHE3034 E coli K1 meningitis strain on a time scale Ten-ten mice were intravenously injected with 100 120583L doses ofOD600

=05 (sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFUmLminus1) OD

600=5 (sim1 x 109 CFU mLminus1) and OD

600=10 (sim

2 x 109 CFU mLminus1) bacterial suspensions getting a finalamount of sim1 x 107 sim2 x 107 sim1 x 108 and sim2 x 108bacterial cells respectively Death rates are represented inboth mice strains Supplementary Table 1 detection ofactive phages particles in the different organs of the sur-vived mice two weeks after bacterial challenge and phagerescue administration (Supplementary Table for Figure 4)(Supplementary Materials)

References

[1] M J Hall S N Williams C J DeFrances and A GolosinskiyldquoInpatient care for septicemia or sepsis a challenge for patientsand hospitalsrdquo NCHS Data Brief no 62 pp 1ndash8 2011

[2] K AWood andDCAngus ldquoPharmacoeconomic implicationsof new therapies in sepsisrdquo PharmacoEconomics vol 22 no 14pp 895ndash906 2004

[3] T Barichello V S Dagostim J S Generoso et al ldquoNeonatalEscherichia coli K1 meningitis causes learning and memoryimpairments in adulthoodrdquo Journal of Neuroimmunology vol272 no 1-2 pp 35ndash41 2014

[4] T K Korhonen M V Valtonen J Parkkinen et al ldquoSerotypeshemolysin production and receptor recognition of Escherichiacoli strains associated with neonatal sepsis and meningitisrdquoInfection and Immunity vol 48 pp 486ndash491 1985

[5] J A Hoffman C Wass M F Stins and K S Kim ldquoThe capsulesupports survival but not traversal of Escherichia coli K1 acrossthe blood-brain barrierrdquo Infection and Immunity vol 67 no 7pp 3566ndash3570 1999

[6] M Muhlenhoff M Eckhardt and R Gerardy-SchahnldquoPolysialic acid Three-dimensional structure biosynthesis andfunctionrdquo Current Opinion in Structural Biology vol 8 no 5pp 558ndash564 1998

[7] P Hyman and S T Abedon ldquoBacteriophage host range andbacterial resistancerdquo Advances in Applied Microbiology vol 70pp 217ndash248 2010

[8] C S McVay M Velasquez and J A Fralick ldquoPhage therapyof Pseudomonas aeruginosa infection in a mouse burn woundmodelrdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 1934ndash1938 2007

[9] S Kumari K Harjai and S Chhibber ldquoEfficacy of bacterio-phage treatment in murine burn wound infection induced by

Klebsiella pneumoniaerdquo Journal ofMicrobiology and Biotechnol-ogy vol 19 no 6 pp 622ndash628 2009

[10] J M Regeimbal A C Jacobs B W Corey et al ldquoPersonalizedtherapeutic cocktail of wild environmental phages rescues micefrom acinetobacter baumannii wound infectionsrdquo Antimicro-bial Agents and Chemotherapy vol 60 no 10 pp 5806ndash58162016

[11] S Chhibber S Kaur and S Kumari ldquoTherapeutic potentialof bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in micerdquo Journal of Medical Micro-biology vol 57 no 12 pp 1508ndash1513 2008

[12] I Takemura-Uchiyama J Uchiyama M Osanai et al ldquoExper-imental phage therapy against lethal lung-derived septicemiacaused by Staphylococcus aureus in micerdquo Microbes and Infec-tion vol 16 no 6 pp 512ndash517 2014

[13] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol70 no 1 pp 204ndash210 2002

[14] J Wang B Hu M Xu et al ldquoUse of bacteriophage in thetreatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosardquo International Journal ofMolecular Medicine vol 17 pp 309ndash317 2006

[15] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[16] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Col Spring Harbor Press Cold SpringHarbor NY USA 2nd edition 1989

[17] I Toth D Svab B Balint M Brown-Jaque and G MarotildquoComparative analysis of the Shiga toxin converting bacterio-phage first detected in Shigella sonneirdquo Infection Genetics andEvolution vol 37 pp 150ndash157 2016

[18] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 p 75 2008

[19] J R Johnson P Delavari and T OrsquoBryan ldquoEscherichia coli O18K1H7 isolates from patients with acute cystitis and neonatalmeningitis exhibit common phylogenetic origins and virulencefactor profilesrdquo The Journal of Infectious Diseases vol 183 no10 pp 425ndash434 2001

[20] K J Kim S J Elliott F Di CelloM F Stins andK S Kim ldquoTheK1 capsule modulates trafficking of E coli-containing vacuolesand enhances intracellular bacterial survival in human brainmicrovascular endothelial cellsrdquo Cellular Microbiology vol 5no 4 pp 245ndash252 2003

[21] M Moulin-Schouleur C Schouler P Tailliez et al ldquoCommonvirulence factors and genetic relationships between O18K1H7Escherichia coli isolates of human and avian originrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3484ndash3492 2006

[22] K S Kim H Itabashi P Gemski J Sadoff R L Warren andA S Cross ldquoThe K1 capsule is the critical determinant in thedevelopment of Escherichia coli meningitis in the ratrdquo TheJournal of Clinical Investigation vol 90 no 3 pp 897ndash905 1992

[23] R Capparelli M Parlato G Borriello P Salvatore and DIannelli ldquoExperimental phage therapy against Staphylococcusaureus in micerdquo Antimicrobial Agents and Chemotherapy vol51 no 8 pp 2765ndash2773 2007

[24] F Lebreton W van Schaik M Sanguinetti et al ldquoAsrR Isan Oxidative Stress Sensing Regulator Modulating Enterococ-cus faecium Opportunistic Traits Antimicrobial Resistance

BioMed Research International 5

0 6 12 18 24 30 36 42 48 54 60 660

20

40

60

80

100

Hours post challenge

Perc

ent s

urvi

val

GBCG3HG2H

G1HG10MGPC

p=00021

p=044

plt00001

Figure 3 In the phage rescue experiment survival of IHE3034infected BALBc mice strongly depended on the elapsed timebetween the injection of bacterium suspension (1 x 108 CFUmouse)and ΦIK1 (1 x 108 PFU mouse) Phage administration occurred 10min (G10M) 1 h (G1H) 2 h (G2H) and 3 h (G3H) postinfectionwhile the IHE3034 and the ΦIK1 suspensions were used as positive(GBC) and negative (GPC) controls respectively The obtainedsurvival curves were statistically compared by the logrank (Matel-Cox) test using the GraphPad Prism 60 software

particles (MOI 110 1100 and 11000 see wells E7-9 F7-9 andG7-9 respectively) In these cases the growth of bacteria waseffectively hindered by ΦIK1 This was also observed if thebacterial cell number was further decreased to 103 CFUmlminus1

This threshold at which IHE3034 could grow at aninoculum of 105 (Figure 2 column 4-6) is in accordance withthe mutation rate of ΦIK1 receptor (sim104)

35 IntravenousMouse SepsisModel of IHE 3034 Twomousestrains were used to establish the systemic infection mousemodel for IHE3034 BALBc mice were more sensitive toIHE3034 as intravenous administration of 01 ml cell suspen-sion (OD

600=10) caused 100 mortality in 24 h (SFigure 1)

Most of the mice survived if 01 ml suspensions of OD600

=05(1 x 107 CFU) or OD

600=1 (2 x 107 CFU) bacterial cells

were injected intravenously Administration of 01 ml fromOD600

=5 (1 x 108 CFUs mouse) caused the death of all micein 48 h

In contrast no complete mortality could be achievedin any of the NMRI mice groups treated with the abovesuspensions (SFigure 1)

36 Phage Rescue of the IHE3034 Rapid Sepsis by ΦIK1 Thebactericidal activity of ΦIK1 observed in vitro could be cor-roborated in the rapid sepsis mouse model The restrainingeffect strongly depended on the time between infection andphage administration

All 12 positive control mice (GBC) infected with 1 x108 CFU IHE3034 bacterium suspension died within 48 h(Figure 3) No effect was observed when phage suspension

only (1 x 108 PFU mouse) was administered to healthymice (groupof phage control GPC) Bacteriophages exerted a100 curing effect if administered within 60 min of bacterialinfection saving the life of all infected mice

Efficacy of phage treatment was drastically altered ifadministration of ΦIK1 followed bacterial challenge by 2 h(G2H) In this case survival rate at 24 h was 916 (1112)but it drastically decreased to 333 (412) by the 48th hour(Figure 3) Two mice survived from this group after the 60thhour and eventually recovered after 7-10 days No rescueeffect of ΦIK1 was detected with a 0 survival rate at 48 hpostinfection if phage was administered 3 h after bacterialinfection

37 Reisolation of Persisting ΦIK1 Phages from DifferentOrgans Two weeks after bacterial challenge and subsequentphage administrations surviving mice were sacrificed andactive ΦIK1 phage particles were isolated from the bloodspleen and brain (Figure 4 x-axis) The spleen was the onlyorgan in which active phage particles could be detected ina significant amount totalling lt35000 PFU spleen Thenumber of detectable phage particles was more limited in thebrain (lt1750 PFU brain) and blood (lt1250 120583lminus1) (Figure 4)In the phage control group (GPC) no phage particles (012)were detected in the brain and a limited number of phages(lt1000) were found in the blood of two sacrificed animals(212)

4 Discussion

Bacterial sepsis can progress if an aetiological agent entersthe body and its hematogenous spread occursThis conditionstrikes more than a million Americans [1] and has a high(28-50) mortality [2] Due to the emergence of multidrugresistance (MDR) among the aetiological agents it is increas-ingly obvious that new treatment options are needed [10]Theapplication of targeted bacteriophages could be a promisingapproach

Counterselection of isolated lytic phageswith the isogenicreceptor mutant bacteria is an effective method to isolatetargeted phages for a well-determined receptor For thispurpose we have constructed and used the IHE3034 ΔK1strain and isolatedΦIK1 targeting one of the most importantvirulence factors of the newborn meningitis-causing E colistrain IHE3034 [19 20] We have chosen the K1 capsuleas a target since it not only hinders opsonization [21] butalso contributes to adhesion promoting invasion through theblood-brain barrier (BBB) [19 22]

Although the exact timing of the pathomechanism inIHE3034 in mice is not known the 100 efficacy of theΦIK1treatment could be achieved if the phages were administered1 h after bacterial challenge (G10M and G1H Figure 3)Thereare several possible reasons for the inefficacy of phage rescuewhen performed after the first hour of infection (G2H andG3H) including the presence of phage resistant colonies inthe inoculum However their frequency was quite high (104mlminus1) so resistant cells would have been incapable of evokingthe pathogenic process This is supported by the results ofthe in vitro experiments where ΦIK1 controlled IHE3034

6 BioMed Research International

Figure 4 Detection of active phages in the surviving mice two weeks after bacterial challenge and phage rescue Results are subdividedaccording to the organs (X axis) In each subsection (1-12) the summarized results of the GPC G10MG1H andG2H are presented as columnsG3H and GBC were not drafted as no mice survived in G3H while no phage was administered to GBC (STable 1)

at 11000 MOI (Figure 2) but only when the bacterial cellnumber was below 104 CFU (the mutation rate)

Still enough K1 expressing cells were in the blood of theexperimental animals to take over a fatal pathogenic processIn our model system IHE3034 could only trigger a rapidlyescalating infection if the bacterium number was fairly high(1 x 108 CFU mouse) This is in accordance with otherexperimental setups performed with Enterococcus faecium as109 [13] Staphylococcus aureus as 108 [23] and Pseudomonasaeruginosa as 107 [14] Compared to these experiments the100 efficacy (G1H) of the phage rescue in relation of ΦIK1and IHE3034 (11 MOI) is comparable to results of otherstudies where 100 survival of the challenged mice werereported ifsim13 (3x1081x109) 101 (109108)MOIswere appliedand administration of phages occurred 45 min and 1 h afterbacterial challenges [13 23] Amore striking rescue effect wasreported by Wang et al [14] where the 1100 MOI still hada 100 protective effect if the isolated Pseudomonas-specificphage was administered up to 1 h after the infection Thiscould be due to the fact that differences in the bacteriumspecies used E faecalis S aureus P aeruginosa and NME coli possess different pathomechanisms This stronglyinfluences the availability of the infectious agent in the host

Bacteria that are capable of intracellular survival suchas IHE3034 [20] can hide from bacteriophages and by thiscan assure their survival and lead to pathogenesis E faeciumis able to survive and proliferate in murine macrophages[24] and there is also a mounting evidence that S aureushas the potential to internalize and survive within hostcells [25] In contrast P aeruginosa is thought to be anextracellular bacterium and therefore it is more vulnerableto bacteriophages Intracellular bacteria are vulnerable untilthey reach adhere to and invade certain cells protectingthemselves from the extracellular phages This may explainwhy phage rescue experiments were more effective withextracellular P aeruginosa

This process could be influenced by the tissue penetrationpotential of bacteriophages The presence of ΦIK1 in thespleens of animals in the phage control group (GPC) and thesurviving animals in groups G10M G1H and G2H indicates

that the phage could survive in the spleen for at least 2 weeksuntil the mice were sacrificed

Lack of ΦIK1 in the brains of the GPC group suggeststhat this phage cannot penetrate the BBB under normalconditions However phage was detected in the brains ofgroups G10M and G1H (Figure 4) We hypothesize thatin group (G1H) ΦIK1 phages could also exert their lyticeffects in the brain contributing to rescue At least twodifferent mechanisms could be considered for this processIt is known that upon inflammation the BBB can becomepermeable facilitating the transfer of macromolecules andviruses [26] Our study contradicts this in the case of ΦIK1as administration of 108 heat-killed IHE3034 cells 1 h priorto the intravenous phage administration did not result inthe detection of active phage particles in the brains ofexperimental animals when sacrifice occurred 1 3 or 7 daysafter administration (unpublished data)

Based on this observation we postulate that the alreadyphage-infected IHE3034 cells crossed the BBB into the braintissue

Phages are alternative therapeutics against multidrugresistant bacteria but there are still questions about theirpractical use especially in systemic infections Besides theimmunological aspects a good understanding of phage-bacteria interactions and phage survival in the body isrequired Our results demonstrate that in accordance withprevious findings phages can not only combat bacteriaalready present in the circulatory system but also survivein the spleen afterwards Future work must be dedicated tothose factors that influence this and possibly enhance thetherapeutic potential of phages

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

BioMed Research International 7

Acknowledgments

Rozsa Lajko is acknowledged for excellent technical assis-tance and Balazs Ambrusitcs and Tettye Forrashaz Kft areacknowledged for access to sewage samples The project wasfunded by AOK-KA-20163 These supports do not lead toany conflicts of interest The present scientific contributionis dedicated to the 650th anniversary of the foundation of theUniversity of Pecs Hungary

Supplementary Materials

Supplementary figure 1 concentration-dependent sen-sitivity of the BALBc (A) and CD1 (B) mice for theIHE3034 E coli K1 meningitis strain on a time scale Ten-ten mice were intravenously injected with 100 120583L doses ofOD600

=05 (sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFUmLminus1) OD

600=5 (sim1 x 109 CFU mLminus1) and OD

600=10 (sim

2 x 109 CFU mLminus1) bacterial suspensions getting a finalamount of sim1 x 107 sim2 x 107 sim1 x 108 and sim2 x 108bacterial cells respectively Death rates are represented inboth mice strains Supplementary Table 1 detection ofactive phages particles in the different organs of the sur-vived mice two weeks after bacterial challenge and phagerescue administration (Supplementary Table for Figure 4)(Supplementary Materials)

References

[1] M J Hall S N Williams C J DeFrances and A GolosinskiyldquoInpatient care for septicemia or sepsis a challenge for patientsand hospitalsrdquo NCHS Data Brief no 62 pp 1ndash8 2011

[2] K AWood andDCAngus ldquoPharmacoeconomic implicationsof new therapies in sepsisrdquo PharmacoEconomics vol 22 no 14pp 895ndash906 2004

[3] T Barichello V S Dagostim J S Generoso et al ldquoNeonatalEscherichia coli K1 meningitis causes learning and memoryimpairments in adulthoodrdquo Journal of Neuroimmunology vol272 no 1-2 pp 35ndash41 2014

[4] T K Korhonen M V Valtonen J Parkkinen et al ldquoSerotypeshemolysin production and receptor recognition of Escherichiacoli strains associated with neonatal sepsis and meningitisrdquoInfection and Immunity vol 48 pp 486ndash491 1985

[5] J A Hoffman C Wass M F Stins and K S Kim ldquoThe capsulesupports survival but not traversal of Escherichia coli K1 acrossthe blood-brain barrierrdquo Infection and Immunity vol 67 no 7pp 3566ndash3570 1999

[6] M Muhlenhoff M Eckhardt and R Gerardy-SchahnldquoPolysialic acid Three-dimensional structure biosynthesis andfunctionrdquo Current Opinion in Structural Biology vol 8 no 5pp 558ndash564 1998

[7] P Hyman and S T Abedon ldquoBacteriophage host range andbacterial resistancerdquo Advances in Applied Microbiology vol 70pp 217ndash248 2010

[8] C S McVay M Velasquez and J A Fralick ldquoPhage therapyof Pseudomonas aeruginosa infection in a mouse burn woundmodelrdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 1934ndash1938 2007

[9] S Kumari K Harjai and S Chhibber ldquoEfficacy of bacterio-phage treatment in murine burn wound infection induced by

Klebsiella pneumoniaerdquo Journal ofMicrobiology and Biotechnol-ogy vol 19 no 6 pp 622ndash628 2009

[10] J M Regeimbal A C Jacobs B W Corey et al ldquoPersonalizedtherapeutic cocktail of wild environmental phages rescues micefrom acinetobacter baumannii wound infectionsrdquo Antimicro-bial Agents and Chemotherapy vol 60 no 10 pp 5806ndash58162016

[11] S Chhibber S Kaur and S Kumari ldquoTherapeutic potentialof bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in micerdquo Journal of Medical Micro-biology vol 57 no 12 pp 1508ndash1513 2008

[12] I Takemura-Uchiyama J Uchiyama M Osanai et al ldquoExper-imental phage therapy against lethal lung-derived septicemiacaused by Staphylococcus aureus in micerdquo Microbes and Infec-tion vol 16 no 6 pp 512ndash517 2014

[13] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol70 no 1 pp 204ndash210 2002

[14] J Wang B Hu M Xu et al ldquoUse of bacteriophage in thetreatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosardquo International Journal ofMolecular Medicine vol 17 pp 309ndash317 2006

[15] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[16] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Col Spring Harbor Press Cold SpringHarbor NY USA 2nd edition 1989

[17] I Toth D Svab B Balint M Brown-Jaque and G MarotildquoComparative analysis of the Shiga toxin converting bacterio-phage first detected in Shigella sonneirdquo Infection Genetics andEvolution vol 37 pp 150ndash157 2016

[18] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 p 75 2008

[19] J R Johnson P Delavari and T OrsquoBryan ldquoEscherichia coli O18K1H7 isolates from patients with acute cystitis and neonatalmeningitis exhibit common phylogenetic origins and virulencefactor profilesrdquo The Journal of Infectious Diseases vol 183 no10 pp 425ndash434 2001

[20] K J Kim S J Elliott F Di CelloM F Stins andK S Kim ldquoTheK1 capsule modulates trafficking of E coli-containing vacuolesand enhances intracellular bacterial survival in human brainmicrovascular endothelial cellsrdquo Cellular Microbiology vol 5no 4 pp 245ndash252 2003

[21] M Moulin-Schouleur C Schouler P Tailliez et al ldquoCommonvirulence factors and genetic relationships between O18K1H7Escherichia coli isolates of human and avian originrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3484ndash3492 2006

[22] K S Kim H Itabashi P Gemski J Sadoff R L Warren andA S Cross ldquoThe K1 capsule is the critical determinant in thedevelopment of Escherichia coli meningitis in the ratrdquo TheJournal of Clinical Investigation vol 90 no 3 pp 897ndash905 1992

[23] R Capparelli M Parlato G Borriello P Salvatore and DIannelli ldquoExperimental phage therapy against Staphylococcusaureus in micerdquo Antimicrobial Agents and Chemotherapy vol51 no 8 pp 2765ndash2773 2007

[24] F Lebreton W van Schaik M Sanguinetti et al ldquoAsrR Isan Oxidative Stress Sensing Regulator Modulating Enterococ-cus faecium Opportunistic Traits Antimicrobial Resistance

6 BioMed Research International

Figure 4 Detection of active phages in the surviving mice two weeks after bacterial challenge and phage rescue Results are subdividedaccording to the organs (X axis) In each subsection (1-12) the summarized results of the GPC G10MG1H andG2H are presented as columnsG3H and GBC were not drafted as no mice survived in G3H while no phage was administered to GBC (STable 1)

at 11000 MOI (Figure 2) but only when the bacterial cellnumber was below 104 CFU (the mutation rate)

Still enough K1 expressing cells were in the blood of theexperimental animals to take over a fatal pathogenic processIn our model system IHE3034 could only trigger a rapidlyescalating infection if the bacterium number was fairly high(1 x 108 CFU mouse) This is in accordance with otherexperimental setups performed with Enterococcus faecium as109 [13] Staphylococcus aureus as 108 [23] and Pseudomonasaeruginosa as 107 [14] Compared to these experiments the100 efficacy (G1H) of the phage rescue in relation of ΦIK1and IHE3034 (11 MOI) is comparable to results of otherstudies where 100 survival of the challenged mice werereported ifsim13 (3x1081x109) 101 (109108)MOIswere appliedand administration of phages occurred 45 min and 1 h afterbacterial challenges [13 23] Amore striking rescue effect wasreported by Wang et al [14] where the 1100 MOI still hada 100 protective effect if the isolated Pseudomonas-specificphage was administered up to 1 h after the infection Thiscould be due to the fact that differences in the bacteriumspecies used E faecalis S aureus P aeruginosa and NME coli possess different pathomechanisms This stronglyinfluences the availability of the infectious agent in the host

Bacteria that are capable of intracellular survival suchas IHE3034 [20] can hide from bacteriophages and by thiscan assure their survival and lead to pathogenesis E faeciumis able to survive and proliferate in murine macrophages[24] and there is also a mounting evidence that S aureushas the potential to internalize and survive within hostcells [25] In contrast P aeruginosa is thought to be anextracellular bacterium and therefore it is more vulnerableto bacteriophages Intracellular bacteria are vulnerable untilthey reach adhere to and invade certain cells protectingthemselves from the extracellular phages This may explainwhy phage rescue experiments were more effective withextracellular P aeruginosa

This process could be influenced by the tissue penetrationpotential of bacteriophages The presence of ΦIK1 in thespleens of animals in the phage control group (GPC) and thesurviving animals in groups G10M G1H and G2H indicates

that the phage could survive in the spleen for at least 2 weeksuntil the mice were sacrificed

Lack of ΦIK1 in the brains of the GPC group suggeststhat this phage cannot penetrate the BBB under normalconditions However phage was detected in the brains ofgroups G10M and G1H (Figure 4) We hypothesize thatin group (G1H) ΦIK1 phages could also exert their lyticeffects in the brain contributing to rescue At least twodifferent mechanisms could be considered for this processIt is known that upon inflammation the BBB can becomepermeable facilitating the transfer of macromolecules andviruses [26] Our study contradicts this in the case of ΦIK1as administration of 108 heat-killed IHE3034 cells 1 h priorto the intravenous phage administration did not result inthe detection of active phage particles in the brains ofexperimental animals when sacrifice occurred 1 3 or 7 daysafter administration (unpublished data)

Based on this observation we postulate that the alreadyphage-infected IHE3034 cells crossed the BBB into the braintissue

Phages are alternative therapeutics against multidrugresistant bacteria but there are still questions about theirpractical use especially in systemic infections Besides theimmunological aspects a good understanding of phage-bacteria interactions and phage survival in the body isrequired Our results demonstrate that in accordance withprevious findings phages can not only combat bacteriaalready present in the circulatory system but also survivein the spleen afterwards Future work must be dedicated tothose factors that influence this and possibly enhance thetherapeutic potential of phages

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

BioMed Research International 7

Acknowledgments

Rozsa Lajko is acknowledged for excellent technical assis-tance and Balazs Ambrusitcs and Tettye Forrashaz Kft areacknowledged for access to sewage samples The project wasfunded by AOK-KA-20163 These supports do not lead toany conflicts of interest The present scientific contributionis dedicated to the 650th anniversary of the foundation of theUniversity of Pecs Hungary

Supplementary Materials

Supplementary figure 1 concentration-dependent sen-sitivity of the BALBc (A) and CD1 (B) mice for theIHE3034 E coli K1 meningitis strain on a time scale Ten-ten mice were intravenously injected with 100 120583L doses ofOD600

=05 (sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFUmLminus1) OD

600=5 (sim1 x 109 CFU mLminus1) and OD

600=10 (sim

2 x 109 CFU mLminus1) bacterial suspensions getting a finalamount of sim1 x 107 sim2 x 107 sim1 x 108 and sim2 x 108bacterial cells respectively Death rates are represented inboth mice strains Supplementary Table 1 detection ofactive phages particles in the different organs of the sur-vived mice two weeks after bacterial challenge and phagerescue administration (Supplementary Table for Figure 4)(Supplementary Materials)

References

[1] M J Hall S N Williams C J DeFrances and A GolosinskiyldquoInpatient care for septicemia or sepsis a challenge for patientsand hospitalsrdquo NCHS Data Brief no 62 pp 1ndash8 2011

[2] K AWood andDCAngus ldquoPharmacoeconomic implicationsof new therapies in sepsisrdquo PharmacoEconomics vol 22 no 14pp 895ndash906 2004

[3] T Barichello V S Dagostim J S Generoso et al ldquoNeonatalEscherichia coli K1 meningitis causes learning and memoryimpairments in adulthoodrdquo Journal of Neuroimmunology vol272 no 1-2 pp 35ndash41 2014

[4] T K Korhonen M V Valtonen J Parkkinen et al ldquoSerotypeshemolysin production and receptor recognition of Escherichiacoli strains associated with neonatal sepsis and meningitisrdquoInfection and Immunity vol 48 pp 486ndash491 1985

[5] J A Hoffman C Wass M F Stins and K S Kim ldquoThe capsulesupports survival but not traversal of Escherichia coli K1 acrossthe blood-brain barrierrdquo Infection and Immunity vol 67 no 7pp 3566ndash3570 1999

[6] M Muhlenhoff M Eckhardt and R Gerardy-SchahnldquoPolysialic acid Three-dimensional structure biosynthesis andfunctionrdquo Current Opinion in Structural Biology vol 8 no 5pp 558ndash564 1998

[7] P Hyman and S T Abedon ldquoBacteriophage host range andbacterial resistancerdquo Advances in Applied Microbiology vol 70pp 217ndash248 2010

[8] C S McVay M Velasquez and J A Fralick ldquoPhage therapyof Pseudomonas aeruginosa infection in a mouse burn woundmodelrdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 1934ndash1938 2007

[9] S Kumari K Harjai and S Chhibber ldquoEfficacy of bacterio-phage treatment in murine burn wound infection induced by

Klebsiella pneumoniaerdquo Journal ofMicrobiology and Biotechnol-ogy vol 19 no 6 pp 622ndash628 2009

[10] J M Regeimbal A C Jacobs B W Corey et al ldquoPersonalizedtherapeutic cocktail of wild environmental phages rescues micefrom acinetobacter baumannii wound infectionsrdquo Antimicro-bial Agents and Chemotherapy vol 60 no 10 pp 5806ndash58162016

[11] S Chhibber S Kaur and S Kumari ldquoTherapeutic potentialof bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in micerdquo Journal of Medical Micro-biology vol 57 no 12 pp 1508ndash1513 2008

[12] I Takemura-Uchiyama J Uchiyama M Osanai et al ldquoExper-imental phage therapy against lethal lung-derived septicemiacaused by Staphylococcus aureus in micerdquo Microbes and Infec-tion vol 16 no 6 pp 512ndash517 2014

[13] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol70 no 1 pp 204ndash210 2002

[14] J Wang B Hu M Xu et al ldquoUse of bacteriophage in thetreatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosardquo International Journal ofMolecular Medicine vol 17 pp 309ndash317 2006

[15] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[16] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Col Spring Harbor Press Cold SpringHarbor NY USA 2nd edition 1989

[17] I Toth D Svab B Balint M Brown-Jaque and G MarotildquoComparative analysis of the Shiga toxin converting bacterio-phage first detected in Shigella sonneirdquo Infection Genetics andEvolution vol 37 pp 150ndash157 2016

[18] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 p 75 2008

[19] J R Johnson P Delavari and T OrsquoBryan ldquoEscherichia coli O18K1H7 isolates from patients with acute cystitis and neonatalmeningitis exhibit common phylogenetic origins and virulencefactor profilesrdquo The Journal of Infectious Diseases vol 183 no10 pp 425ndash434 2001

[20] K J Kim S J Elliott F Di CelloM F Stins andK S Kim ldquoTheK1 capsule modulates trafficking of E coli-containing vacuolesand enhances intracellular bacterial survival in human brainmicrovascular endothelial cellsrdquo Cellular Microbiology vol 5no 4 pp 245ndash252 2003

[21] M Moulin-Schouleur C Schouler P Tailliez et al ldquoCommonvirulence factors and genetic relationships between O18K1H7Escherichia coli isolates of human and avian originrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3484ndash3492 2006

[22] K S Kim H Itabashi P Gemski J Sadoff R L Warren andA S Cross ldquoThe K1 capsule is the critical determinant in thedevelopment of Escherichia coli meningitis in the ratrdquo TheJournal of Clinical Investigation vol 90 no 3 pp 897ndash905 1992

[23] R Capparelli M Parlato G Borriello P Salvatore and DIannelli ldquoExperimental phage therapy against Staphylococcusaureus in micerdquo Antimicrobial Agents and Chemotherapy vol51 no 8 pp 2765ndash2773 2007

[24] F Lebreton W van Schaik M Sanguinetti et al ldquoAsrR Isan Oxidative Stress Sensing Regulator Modulating Enterococ-cus faecium Opportunistic Traits Antimicrobial Resistance

BioMed Research International 7

Acknowledgments

Rozsa Lajko is acknowledged for excellent technical assis-tance and Balazs Ambrusitcs and Tettye Forrashaz Kft areacknowledged for access to sewage samples The project wasfunded by AOK-KA-20163 These supports do not lead toany conflicts of interest The present scientific contributionis dedicated to the 650th anniversary of the foundation of theUniversity of Pecs Hungary

Supplementary Materials

Supplementary figure 1 concentration-dependent sen-sitivity of the BALBc (A) and CD1 (B) mice for theIHE3034 E coli K1 meningitis strain on a time scale Ten-ten mice were intravenously injected with 100 120583L doses ofOD600

=05 (sim1 x 108 CFU mLminus1) OD600

=1 (sim2 x 108 CFUmLminus1) OD

600=5 (sim1 x 109 CFU mLminus1) and OD

600=10 (sim

2 x 109 CFU mLminus1) bacterial suspensions getting a finalamount of sim1 x 107 sim2 x 107 sim1 x 108 and sim2 x 108bacterial cells respectively Death rates are represented inboth mice strains Supplementary Table 1 detection ofactive phages particles in the different organs of the sur-vived mice two weeks after bacterial challenge and phagerescue administration (Supplementary Table for Figure 4)(Supplementary Materials)

References

[1] M J Hall S N Williams C J DeFrances and A GolosinskiyldquoInpatient care for septicemia or sepsis a challenge for patientsand hospitalsrdquo NCHS Data Brief no 62 pp 1ndash8 2011

[2] K AWood andDCAngus ldquoPharmacoeconomic implicationsof new therapies in sepsisrdquo PharmacoEconomics vol 22 no 14pp 895ndash906 2004

[3] T Barichello V S Dagostim J S Generoso et al ldquoNeonatalEscherichia coli K1 meningitis causes learning and memoryimpairments in adulthoodrdquo Journal of Neuroimmunology vol272 no 1-2 pp 35ndash41 2014

[4] T K Korhonen M V Valtonen J Parkkinen et al ldquoSerotypeshemolysin production and receptor recognition of Escherichiacoli strains associated with neonatal sepsis and meningitisrdquoInfection and Immunity vol 48 pp 486ndash491 1985

[5] J A Hoffman C Wass M F Stins and K S Kim ldquoThe capsulesupports survival but not traversal of Escherichia coli K1 acrossthe blood-brain barrierrdquo Infection and Immunity vol 67 no 7pp 3566ndash3570 1999

[6] M Muhlenhoff M Eckhardt and R Gerardy-SchahnldquoPolysialic acid Three-dimensional structure biosynthesis andfunctionrdquo Current Opinion in Structural Biology vol 8 no 5pp 558ndash564 1998

[7] P Hyman and S T Abedon ldquoBacteriophage host range andbacterial resistancerdquo Advances in Applied Microbiology vol 70pp 217ndash248 2010

[8] C S McVay M Velasquez and J A Fralick ldquoPhage therapyof Pseudomonas aeruginosa infection in a mouse burn woundmodelrdquo Antimicrobial Agents and Chemotherapy vol 51 no 6pp 1934ndash1938 2007

[9] S Kumari K Harjai and S Chhibber ldquoEfficacy of bacterio-phage treatment in murine burn wound infection induced by

Klebsiella pneumoniaerdquo Journal ofMicrobiology and Biotechnol-ogy vol 19 no 6 pp 622ndash628 2009

[10] J M Regeimbal A C Jacobs B W Corey et al ldquoPersonalizedtherapeutic cocktail of wild environmental phages rescues micefrom acinetobacter baumannii wound infectionsrdquo Antimicro-bial Agents and Chemotherapy vol 60 no 10 pp 5806ndash58162016

[11] S Chhibber S Kaur and S Kumari ldquoTherapeutic potentialof bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in micerdquo Journal of Medical Micro-biology vol 57 no 12 pp 1508ndash1513 2008

[12] I Takemura-Uchiyama J Uchiyama M Osanai et al ldquoExper-imental phage therapy against lethal lung-derived septicemiacaused by Staphylococcus aureus in micerdquo Microbes and Infec-tion vol 16 no 6 pp 512ndash517 2014

[13] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol70 no 1 pp 204ndash210 2002

[14] J Wang B Hu M Xu et al ldquoUse of bacteriophage in thetreatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosardquo International Journal ofMolecular Medicine vol 17 pp 309ndash317 2006

[15] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[16] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Col Spring Harbor Press Cold SpringHarbor NY USA 2nd edition 1989

[17] I Toth D Svab B Balint M Brown-Jaque and G MarotildquoComparative analysis of the Shiga toxin converting bacterio-phage first detected in Shigella sonneirdquo Infection Genetics andEvolution vol 37 pp 150ndash157 2016

[18] R K Aziz D Bartels A Best et al ldquoThe RAST Server rapidannotations using subsystems technologyrdquo BMCGenomics vol9 p 75 2008

[19] J R Johnson P Delavari and T OrsquoBryan ldquoEscherichia coli O18K1H7 isolates from patients with acute cystitis and neonatalmeningitis exhibit common phylogenetic origins and virulencefactor profilesrdquo The Journal of Infectious Diseases vol 183 no10 pp 425ndash434 2001

[20] K J Kim S J Elliott F Di CelloM F Stins andK S Kim ldquoTheK1 capsule modulates trafficking of E coli-containing vacuolesand enhances intracellular bacterial survival in human brainmicrovascular endothelial cellsrdquo Cellular Microbiology vol 5no 4 pp 245ndash252 2003

[21] M Moulin-Schouleur C Schouler P Tailliez et al ldquoCommonvirulence factors and genetic relationships between O18K1H7Escherichia coli isolates of human and avian originrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3484ndash3492 2006

[22] K S Kim H Itabashi P Gemski J Sadoff R L Warren andA S Cross ldquoThe K1 capsule is the critical determinant in thedevelopment of Escherichia coli meningitis in the ratrdquo TheJournal of Clinical Investigation vol 90 no 3 pp 897ndash905 1992

[23] R Capparelli M Parlato G Borriello P Salvatore and DIannelli ldquoExperimental phage therapy against Staphylococcusaureus in micerdquo Antimicrobial Agents and Chemotherapy vol51 no 8 pp 2765ndash2773 2007

[24] F Lebreton W van Schaik M Sanguinetti et al ldquoAsrR Isan Oxidative Stress Sensing Regulator Modulating Enterococ-cus faecium Opportunistic Traits Antimicrobial Resistance

8 BioMed Research International

and Pathogenicityrdquo PLoS Pathogens vol 8 no 8 Article IDe1002834 2012

[25] C Garzoni and W L Kelley ldquoStaphylococcus aureus newevidence for intracellular persistencerdquo Trends in Microbiologyvol 17 no 2 pp 59ndash65 2009

[26] A Varatharaj and I Galea ldquoThe blood-brain barrier in systemicinflammationrdquo Brain Behavior and Immunity vol 60 pp 1ndash122017

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom