research article evaluation of the efficacy of a...

Research ArticleEvaluation of the Efficacy of a Bacteriophage inthe Treatment of Pneumonia Induced by Multidrug ResistanceKlebsiella pneumoniae in Mice

Fang Cao12 Xitao Wang1 Linhui Wang1 Zhen Li1 Jian Che1 Lili Wang13

Xiaoyu Li13 Zhenhui Cao1 Jiancheng Zhang1 Liji Jin1 and Yongping Xu13

1School of Life Science and Biotechnology Dalian University of Technology Dalian 116024 China2Orthopedics Research Center Affiliated Zhongshan Hospital of Dalian University Dalian 116001 China3Ministry of Education Center for Food Safety of Animal Origin Dalian University of Technology Dalian 116620 China

Correspondence should be addressed to Yongping Xu 61901223qqcom

Received 31 October 2014 Revised 19 January 2015 Accepted 4 March 2015

Academic Editor Teresa M Coque

Copyright copy 2015 Fang Cao et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Multidrug-resistant Klebsiella pneumoniae (MRKP) has steadily grown beyond antibiotic control However a bacteriophage isconsidered to be a potential antibiotic alternative for treating bacterial infections In this study a lytic bacteriophage phage 1513was isolated using a clinical MRKP isolate KP 1513 as the host and was characterized It produced a clear plaque with a halo andwas classified as Siphoviridae It had a short latent period of 30 min a burst size of 264 and could inhibit KP 1513 growth in vitrowith a dose-dependent pattern Intranasal administration of a single dose of 2 times 109 PFUmouse 2 h after KP 1513 inoculation wasable to protect mice against lethal pneumonia In a sublethal pneumonia model phage-treated mice exhibited a lower level of Kpneumoniae burden in the lungs as compared to the untreated control These mice lost less body weight and exhibited lower levelsof inflammatory cytokines in their lungs Lung lesion conditions were obviously improved by phage therapy Therefore phage 1513has a great effect in vitro and in vivo which has potential to be used as an alternative to an antibiotic treatment of pneumonia thatis caused by the multidrug-resistant K pneumoniae

1 Introduction

Klebsiella pneumoniae (K pneumoniae) is one of the mostcommon gram-negative bacteria that is responsible forhospital-acquired bacterial infections including pneumo-nia bacteremia urinary tract infections liver abscess andwound infections in immune compromised patients [1 2]Pneumonia that is caused by K pneumoniae is usuallyassociated with a high mortality rate In fact despite thedevelopment of broad-spectrum antibiotics mortality ratesgreater than 50 have been reported in those infected withKlebsiella pneumonia [3] In addition treating Klebsiellapneumoniae has become more difficult due to the worldwideincrease in multidrug resistant strains leaving only limitedclinical treatment options [4ndash8] In effect about 80 ofthe nosocomial infections caused by K pneumoniae are dueto these multidrug-resistant strains the incidence of ESBLs

(extended-spectrum beta-lactamase) isolates ranges from 8to 44 [9 10]

Bacteriophages or phages are viruses that specificallyattack and kill their host bacteria a process that is regardedas a possible treatment method for combating bacterialinfectious diseases Bacteriophage treatment is particularlydesirable because of the side effects and inefficacy associatedwith antibiotics and the emergence of new antibiotic-resistantstrains The bacteriophages have garnered increasing atten-tion as an alternative to controlling bacterial infectious dis-eases with antibiotics since avoiding an antibiotic treatmentwould avoid the spread of multiresistant bacteria [11ndash14] Incomparisonwith antibiotics phages havemany advantageousqualities such as a high abundance host specificity rapidexponential replication and a decline in numbers when thetarget bacteria decrease Furthermore phage therapy hasalready been tested in a variety of bacterial species and has

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 752930 9 pageshttpdxdoiorg1011552015752930

2 BioMed Research International

accomplished some achievements [15ndash17] For exampleChhibber et al delivered phage via an intraperitoneal route totreat experimental lobar pneumonia that was induced by Kpneumoniae in mice and had a great efficacy [9] However anintraperitoneal administration of a phage would not be eitherpractical or efficient for treating pneumonia in humans In thepresent study we evaluated the therapeutic efficacy of phage1513 which was delivered intranasally in order to protectagainst pneumonia that is induced by a strain of multidrugresistant K pneumoniae

2 Materials and Methods

21 Bacterial Strain A K pneumoniae strain was isolatedfrom a patient with pneumonia at the Zhongshan HospitalAffiliated to Dalian University (Dalian China) and then wasdesignated KP 1513 KP 1513 was identified as a multipleantibiotics-resistant and ESBLs-producing strain by theWalkAway Plus System (Siemenrsquos Diagnostics) Stocks of the strainwere stored at minus80∘C in Luria-Bertani broth (yeast extract5 g tryptone 10 g and NaCl 10 g in 1 L of distilled water)containing 25 glycerol

22 Isolation Enrichment and Purification of BacteriophageKP 1513 was used as a host to isolate a lytic phage from thesewage samples from the Zhongshan Hospital Affiliated toDalian University Briefly 20mL of the sewage sample wascentrifuged at 5000 g for 5min at 4∘C to remove any debrisand soilThen the supernatant was pooled and supplementedwith CaCl

2to 1mmol Lminus1 TenmL of this supernatant was

combined with 10mL of 2 times LB broth and 2mL of bacteriasolution and was then incubated for 12 h at 37∘Cwith shaking(120 rpm) Then the enrichment broths were centrifuged at8000 g at 4∘C for 5min The supernatant was pooled andpassed through a 022120583m filter (Millipore USA) A double-layered agar method was used to confirm phage presencein the filtrate and the plaque forming unit (PFU) wascalculated Single plaques were subcultured three times forphage purification The suspension that contained specificphage was incubated with DNase I and RNase (1 120583gmLminus1Takara Japan) at 37∘C for 30min prior to adding NaCl(1mol Lminus1) and polyethylene glycol 8000 (PEG8000 Sigma)to precipitate the phage particles This phage was designatedphage 1513 and was resuspended in SM buffer (NaCl 58 gMgSO

4sdot7H2O2 g TrissdotCl 1mol Lminus1 and 2gelatin 5mL in 1 L

of distilled water pH 75) Then cesium chloride equilibriumultracentrifugation (150000 g for 24 h in a Beckman Ti50rotor)was conducted for further phage purificationThebandthat contained the phage was collected and dialyzed againsta TBS buffer (NaCl 8 g KCl 02 g and tris base 3 g in 1 Lof distilled water pH 74 with pure HCl) at 4∘C overnightThen phages were passed through a Detoxi-Gel endotoxinremoving gel (Pierce Biotechnology Rockford IL) to removeendotoxins from the phage preparation Finally the purifiedphages were stored at 4∘C until use

23 Characterization of Phage 1513 Transmission elec-tron microscopy was used to observe phage 1513 with

a JEM-1200EX electron microscope (JEOL Japan) after neg-ative staining by phosphotungstic acid

Thermal pH and chloroform stability tests were con-ducted Aliquots of phage suspensions with high titers (2 times108 PFUmLminus1) were incubated at 4∘C 25∘C 37∘C 50∘C 60∘Cand 70∘C (pH 7) for 60min or at 4∘C (pH 3ndash12) for 10 h ormixed with chloroform and incubated at 4∘C for 24 h Thephage titers were tested by the double-layer agar method

A one-step growth experiment was carried out by mod-ifying the method described by Ellis and Delbruck [18]Briefly the phage was added to broth culture of KP 1513 toa MOI (multiplicity of infection) of 01 The sample was thenincubated at 37∘C for 15min in order to promote phage-hostadsorptionThemixture was centrifuged at 12000 g at 4∘C for10min to remove any free phage particles The supernatantwas removed and the pellet was resuspended in LB brothThe above-mentioned centrifugation procedure was repeatedoncemoreThe pellet was resuspended in 100mLLBmediumand incubated at 37∘C while shaking Aliquots were removedat 10min intervals and the phage titer was determined byspot method

Genome sequencing was performed by Shenzhen Ge-nomics Institute (Shenzhen China) using the Illumina High-Throughput Sequencing Platform (Illumina HiSeq 2000) onDNA obtained by means of standard SDS-proteinase K pro-ceduresThe complete genome sequence of the phage 1513wasaccessible in GenBank (Accession number KP658157)

24 Bactericidal Effect of Phage 1513 In Vitro An aliquot ofovernight culture of KP 1513 was seeded into fresh LBmedium and incubated for 6 h at 37∘C with shaking(150 rpm)The bacteria were pelleted and resuspended in PBSto an OD

650of 06 (sim2 times 109 CFUmLminus1) Different phage 1513

concentrations (2 times 1010 2 times 109 and 2 times 108 PFUmLminus1) orPBS (control) were added to the inocula and then furtherincubated for 12 h at 37∘C with shaking (150 rpm) Bacterialgrowth was monitored by measuring the OD

600at a 30min

interval

25 Mice Seven-week-old female Swiss-Webster mice(weight sim25 g per mouse) were purchased from the DalianMedical University in Dalian China (SCXK 2008-0002)All procedures were conducted according to guidelinesestablished by the Ethics Committee of Dalian Medical Uni-versity

26 Mouse Lethal PneumoniaModel Groups of 10 mice wereanesthetized by an IP injection with ketamine (120mg kgminus1)and xylazine (12mg kgminus1) and inoculated with 20120583L inoculaof KP 1513 (2 times 108 CFUmouse) intranasally LD

50of the KP

1513 was 417 times 109 CFUmLminus1 intranasally in the previousstudy In the phage therapeutic experiments a single dose ofphage (2 times 109 2 times 108 and 2 times 107 PFUmouse) or PBS wasgiven intranasally 2 h after infection

27 Bacterial Burden Histopathology and Cytokine Assay ofLung Tissue Groups of 11 mice were inoculated intranasally

BioMed Research International 3

(a) (b)

Figure 1 Morphology of K pneumoniae phage 1513 (a) Electron micrograph of phage 1513 (b) Plaque morphologies of phage 1513

with 6 times 107 CFUmouse of KP 1513 phages (6 times 107 PFUmouse) or PBS were administered intranasally 2 h afterinfection At 24 h after infection five mice from each groupwere euthanized and the left lungs were removed and fixedby 10 formalin for at least 24 h at 4∘C and were then sentto the pathology department of the Zhongshan Hospital forthe haematoxylin-eosin (HE) stain Another 6 mice fromeach group were euthanized and weighed and their wholelungs were homogenized in sterile saline An aliquot of thehomogenate was frozen at minus80∘C for cytokine testing (TNF-120572 and IL-6) using the Mouse ELISA Kit (Nanjing JianchengChina) The remaining homogenate was diluted and platedonto Luria-Bertani agar in order to determine the colonyformation unit To detect the phage level present in thelung the homogenate was centrifuged at 4000 g to obtain thesupernatants The supernatants were filtered through a 022120583m pore-size filter prior to conducting the double-layeredagar method to calculate the number of phages by plaqueforming unit (PFU)

28 Statistical Analysis A log rank test was performed totest the significant difference between the survival curvesTwo-tailedMann-Whitney paired tests were used to compareweight loss bacterial quantifications bacteriophages andcytokines in the lung sample Error bars represent sem

3 Results

31 Isolation and Morphological Study of Phage 1513 A lyticphage against KP 1513 was isolated from the sewage samplesand then purified By transmission electron microscopy thisphage was identified as a virion particle that was approxi-mately 150 nm long with an icosahedral head (approximately48 plusmn 3 nm long) and a tail (approximately 100 plusmn 10 nm long)which indicates that this phage belongs to the Siphoviridaefamily (Figure 1) In addition when cultured with KP 1513

the phage formed clear plaques andwas surrounded by a largehalo

32 Characterization of Phage 1513 pH and temperaturestabilities are important for the therapeutic application of thephage in animals In this study phage 1513 was susceptiblewith strongly acid and alkaline pH (pH lt 3 or pH gt 10)and was relatively stable at pH between 6 and 9 at 4∘Cfor 10 h In addition phage 1513 was stable at 4∘C (forshort time storage) and 37∘C at pH 7 (condition in lung)(Figure 2) Furthermore phage 1513 was not chloroform-sensitive which is important since chloroform was involvedin the purification procedures

The one-step growth curve revealed that phage 1513 hada latent period of 30min and an average burst size of 264(Figure 3)These calculationswere based on the ratio ofmeanyield of phage particles liberated to the mean phage particlesthat infected the bacterial cells in the latent period whichindicates that phage 1513 can quickly infect the host andpropagate

The complete genome sequence of the phage indicatedthat the linear double strandedDNAofKlebsiellaphage 1513 is49462 bp in length with 5061 of G + C as well as 72 CDSswhich comprised about 8281 of the genome Moreover atandem repeat sequence (51015840-GGTTTCTACGGTTTCGAC-GGTTTCTAC GGTTTCT-31015840 34 bp) was observed by Tan-dem Repeats Finder without tRNA gene

33 Effect of Phage 1513 against K pneumoniae In Vitro Toassess the ability of the phage to lyse host bacteria in vitrowe monitored the host bacteriarsquos growth while being in thephagersquos presence After adding phage 1513 to KP 1513 culturethe optical density at 650 nm constantly rose for 2 h butit then dropped down by different extents according to theMOI (Figure 3) The differences between treatment groups


90

85

80

75

70

65

60

55

50

3 4 5 6 7 8 9 10 11 12

pH

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

10

8

6

4

2

0

0 10 20 30 40 50 60 70 80

Temperature (∘C)

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(b)

Figure 2 Stability of phage 1513 under different pH (a) or temperature (b)

0 20 40 60 80 1004

5

6

7

8

Time after infection (min)

Latent time

Burs

t siz

e

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

0 1 2 3 4 5 6 7 8 9 10 11 1200

02

04

06

08

10

12

14

Negative control

Time (h)

MOI = 10

MOI = 1

MOI = 01

Opt

ical

den

sity

at650

nm

(b)

Figure 3 (a) Phage 1513rsquos one-step growth curve was measured Bacteriophages showed a latent period of about 30min and a burst sizeof 264 PFUmLminus1 (b) Lytic ability of phage 1513 in vitro KP (2 times 109 CFUmLminus1) was infected with the phage at different multiplicities ofinfection (MOI) The optical density at 650 nm was determined at each timepoint Infection dose of phage none (998771) 2 times 1010 PFUmLminus1 (998787)2 times 109 PFUmLminus1 (e) and 2 times 108 PFUmLminus1 (◼)

gradually became negligible after 8 h incubation Since theoptical density of the phage-inoculated groups was muchlower than that of the control group we could concludethat phage 1513 was able to effectively lyse KP 1513 in vitro(Figure 3)

34 Phage 1513rsquos Effect on Lethal Pneumonia As shownin Figure 4 without treatment 100 of the mice (1010)

died after an intranasal inoculation of 2 times 108 CFU of KP1513mouse By contrast all phage 1513 doses that weredelivered intranasally 2 h after infection improved the sur-vival in a dose-dependent manner the highest dose (2 times109 PFUmouse) showed the greatest survival rate comparedto that of the control group (80 versus 0 119875 = 00002)Thesurvival rate was respectively 60 (610 119875 = 00018) and30 (310 119875 = 00058) in the treatment group of MOI = 1(2 times 108 PFUmouse) and MOI = 01 (2 times 107 PFUmouse)


0 12 24 36 48 60 720

20

40

60

80

100

PBS(h)

Surv

ival

()

MOI = 01

MOI = 1

MOI = 10

Figure 4 Efficiency of phage 1513 in vivo Survival curves ofthe infected mice treated with either PBS or phages at differentmultiplicities of infection (MOI) A group of 10micewere inoculatedvia the intranasal route with 2 times 108 CFUmouse K pneumoniaeVarious doses of phage 1513 were administrated intranasally 2 h afterinfection Survival curves were compared for significance with thelog-rank test of the results obtained from the following parametersfrom the phage 1513 (2 times 107 PFUmouse) treatment group versusthose from the control group (119875 = 00058) from the phage 1513(2 times 108 PFUmouse) treatment group versus those from the controlgroup (119875 = 00018) and from the phage 1513 (2 times 109 PFUmouse)treatment group versus those from the control group (119875 = 00002)

35 PhageTreatment Improved the LungDamage Thepathol-ogy of the infected lung tissues showed focal consolidationareas in the phage-treated mice whereas diffuse consolida-tion was observed in the PBS-treated mice Furthermorethe results of histopathologic sections of the infected lungtissues were analogous In the control group the pulmonaryalveoli exhibited an abnormal structure and the majority ofthe alveolar space was obliterated by inflammatory exudateand immune cell infiltrate which was accompanied withhemorrhage and consolidation In contrast the phage-treatedmicersquos lungs showed local discrete lesions and most of thetissue was healthy (Figure 5)

36 Phage Treatmentrsquos Effect on Body Weight Loss BacterialBurden and Cytokines Released in Lung In the phage-treatedgroup the micersquos weight loss rate (weight difference beforeinfection and 24 h after infectionweight before infection)was significantly lower In the homogenate of lungs thenumber of bacteria (log10 CFUgminus1) in phage group andcontrol group was respectively 616 plusmn 010 and 799 plusmn010 so the number of bacteria was approximately two logunits lower than that in the control group The number ofbacteriophage (log10 PFU gminus1) in phage group and controlgroup was respectively 648 plusmn 011 and 776 plusmn 011 so thenumber of phage was more than 10 times higher in thetreatment group (Figure 6) In addition cytokine levels in

the phage-treated mice were remarkably lower (TNF-120572 119875 =00159 IL-6 119875 = 00079) than those of the control mice(Figure 7)

4 Discussion

Hospital-acquired pneumonia that is caused by Klebsiellapneumoniae is always a threat as well as a fastidious publichuman health problem [19] Despite advances in antimicro-bial therapy the morbidity and mortality remain high andout of control Furthermore the emergence of multidrugresistance aggravates this situation An increasing num-ber of extended-spectrum-120573-lactamase-producing and KPC-type carbapenemases-producing K pneumoniae nosocomialisolates have been reported [4 7 20ndash22] Since antibi-otic treatment has associated restrictions and shortcomingsphage therapy is now more frequently being considered asa potential treatment and prevention for bacterial infections[23] In the present study we isolated a new bacteriophage(phage 1513) that could effectively control pneumonia that iscaused by a clinical multidrug resistance K pneumoniae invitro and in vivo

The in vitro study showed phage 1513rsquos effectivenessagainst K pneumoniae with a short latent time and a largeburst size In addition the phage possessed stability withinphysiological ranges of temperature and pH It formedplagues on 5 of 10 clinical Klebsiella strains tested notablyKP 1513 Plaques did not exist on Pseudomonas aeruginosa(ATCC27853) Staphylococcus aureus (Newman) and E coli(CGMCC 1797) It was consistent with previous documentsthat bacteriophage are highly specific and can only infect asingle species of bacterium usually a subset of strains withinthat species [13 24] Therefore phage 1513 has a narrowspectrum and is potentially active against the MRKP strains

However it can be found that an early trend of phageresistance emerged after 11 hours of incubation (Figure 3)Mutants that are resistant to phage infections are a criticalproblem for the application of phage therapy [25] How-ever some studies have demonstrated that the resistanceis partially due to receptor molecule variation which actsas virulence factor of pathogens As a result bacteria haveto attenuate their virulence in order to be resistant to thephage lysis [26] On the other hand a phage can alsomutate to adapt to the change of pathogens [27] Furtherinvestigations are required to determine whether the phage-resistant K pneumoniae cells that have emerged throughphage therapy have reduced the pathogenic ability Perhapsa cocktail of several phage strains will be necessary forcontrolling bacterial variation

Chhibber et al have demonstrated that a single intraperi-toneal administration of a high bacteriophage dose whichwas 100 times the infectious bacterial dose administeredimmediately after the intranasal infection has been shown torescue 100 of animals [9] As we know the delivery routehas a critical influence on phage therapy People rarely deliverintraperitoneally in clinical treatments however they takedrugs by nebulisation which is similar to intranasal admin-istration Compared with intranasal injection intraperi-toneally administering the phage ismore suitable for systemic


(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

KP + PBS KP + phage

Figure 5 Histopathology of KP-infected lung tissue from PBS- or phage 1513-treated mice A group of 10 mice were inoculated intranasallywith 6 times 107 CFU KPmouse Additionally phage 1513 was administered intranasally The mice were euthanized 24 h after infection and lungtissues were removed as described above (a) PBS without K pneumoniae (b) K pneumoniae treated with PBS (c) K pneumoniae treatedwith phage 1513 ((a1) (b1) and (c1) were amplified 40 times (a2) (b2) and (c2) were amplified 100 times and (a3) (b3) and (c3) wereamplified 200 times)

infections but not for local infections like pneumonia More-over if phages were delivered intraperitoneally they would bedetected and cleared outmore quickly by the immune systemIn this study we demonstrated that intranasal administrationcan also effectively treat pneumonia that is caused by K

pneumoniae in mice We gave a various dose of phage (2 times109 PFUmouse 2 times 108 PFUmouse 2 times 107 PFUmouse)2 h after infection and as a result the survival rate wasaffected This is probably because the increasing dose ofbacteriophages results inmore rapid bacterial killing thereby


5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)

Figure 6 Body weight loss bacterial burden and the phage number present in the lung after phage treatment of K pneumoniae infectionGroups of 6 mice were inoculated via the intranasal route with 6 times 107 CFUmouse K pneumoniae PBS or phage 1513 (6 times 107 PFUmouse)was administered intranasally 2 h after infectionThe numbers of bacteriophage (a) or bacteria (b) in the lung homogenates were determinedMice were weighed (c) Bacteria counts and weight loss rate were significantly lower in the lung homogenate from the mice in the phage-treated group than in the control group (119875 = 00022 and 119875 = 00087) Bacteriophage counts were significantly higher in the infected groupthan in the noninfected animals (119875 = 00022)

0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)

Figure 7 Cytokine assay of lung tissue after phage treatment of K pneumoniae infection Groups of 6 mice were inoculated via the intranasalroute with 6 times 107 CFUmouse K pneumoniae PBS or phage 1513 (6 times 107 PFUmouse) was administered intranasally 2 h after infectionThe mice were killed 24 h after infection and the inflammatory cytokines of the lung homogenate were collected and detected Cytokineconcentrations were remarkably lower in the phage-treated group than in the control group (a) TNF-120572 (119875 = 00159) (b) IL-6 (119875 = 00079)


increasing the survival rate [28] Besides phage 1513 cannotprovide significant protection 24 h prior to infection (data notshown) It is speculated that the phage has been eliminatedbefore it attached to the host and cracked it The clearancerate of the phage particles from body fluids by the reticu-loendothelial system is a critical parameter for phage therapy[26] It may be more effective to increase the dose of phageor give it less than 24 h before infection But phage 1513 hasobvious advantages on account of the significant effect in thetreatment experiment

In addition from Figure 6 we can see that bacterianumbers significantly decreased in the phage-treated micewhich suggests that the phage effectively killedK pneumoniaein vivo Consequently in the phage-treated mice lung lesionsand an inflammatory response were clearly less prominent incomparison to the controlThesewere all concordantwith thesurvival study resultsMoreover many reports have indicatedthat a cytokine storm results in lung injury and poor clinicaloutcomes [29 30] Therefore the phage may be beneficial tothe lung by decreasing the hostrsquos inflammatory response asdemonstrated by the levels of IL-6 and TNF-120572

In conclusion the results of this study suggest that a phagetreatment that is administered intranasally has great potentialfor treating pneumonia and other infections caused by Kpneumoniae The safety of phage 1513 and its activity againstbiofilm formation will be investigated in further study

Conflict of Interests

No conflict of interests is declared

Authorsrsquo Contribution

Fang Cao and Xitao Wang contributed equally to the studyand are co-first authors

Acknowledgments

Support for this work was provided by the National NaturalScience Foundation of China (31001053) the Special Fundsof the Central Colleges Basic Scientific Research OperatingExpenses (DUT13 JB04) and the National Public Science andTechnology Research Funds Projects of Ocean (201405003)The authors gratefully thankProfessor PhilipAThacker fromthe University of Saskatchewan for his valuable suggestionsand for revising the paper

References

[1] H Hackstein S Kranz A Lippitsch et al ldquoModulation of res-piratory dendritic cells during Klebsiella pneumonia infectionrdquoRespiratory Research vol 14 no 1 article 91 2013

[2] R Podschun and U Ullmann ldquoKlebsiella spp as nosoco-mial pathogens epidemiology taxonomy typing methods andpathogenicity factorsrdquoClinical Microbiology Reviews vol 11 no4 pp 589ndash603 1998

[3] S J Cryz ldquoProgress in immunization against Klebsiella infec-tionsrdquo European Journal of Clinical Microbiology vol 2 no 6pp 523ndash528 1983

[4] J M Zhu R J Jiang H S Kong et al ldquoEmergence of novelvariants of gyrA parC qnrS genes in multi-drug resistantKlebsiella caused pneumoniardquo Zhonghua Liu Xing Bing Xue ZaZhi vol 34 pp 61ndash66 2013

[5] D K Pomakova C-B Hsiao J M Beanan et al ldquoClinicaland phenotypic differences between classic and hyperviru-lent Klebsiella pneumonia an emerging and under-recognizedpathogenic variantrdquo European Journal of Clinical Microbiologyamp Infectious Diseases vol 31 no 6 pp 981ndash989 2012

[6] S Shoma M Kamruzzaman A N Ginn J R Iredell and SR Partridge ldquoCharacterization ofmultidrug-resistantKlebsiellapneumoniae from Australia carrying bla

119873119863119872minus1rdquo Diagnostic

Microbiology and Infectious Disease vol 78 no 1 pp 93ndash972014

[7] KDemirdag and SHosoglu ldquoEpidemiology and risk factors forESBL-producing Klebsiella pneumoniae a case control studyrdquoJournal of Infection in Developing Countries vol 4 no 11 pp717ndash722 2010

[8] E B Hirsch and V H Tam ldquoDetection and treatment optionsfor Klebsiella pneumoniae carbapenemases (KPCs) an emerg-ing cause of multidrug-resistant infectionrdquo Journal of Antimi-crobial Chemotherapy vol 65 no 6 pp 1119ndash1125 2010

[9] 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

[10] J J Hilliard J L Melton L Hall et al ldquoComparative effects ofcarbapenems on bacterial load and host immune response ina Klebsiella pneumoniaemurine pneumonia modelrdquo Antimicro-bial Agents and Chemotherapy vol 55 no 2 pp 836ndash844 2011

[11] F Pouillot M Chomton H Blois et al ldquoEfficacy of bacterio-phage therapy in experimental sepsis and meningitis caused bya clone O25b H4-ST131 Escherichia coli strain producing CTX-M-15rdquo Antimicrobial Agents and Chemotherapy vol 56 no 7pp 3568ndash3575 2012

[12] B Biswas S Adhya P Washart et al ldquoBacteriophage therapyrescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faeciumrdquo Infection and Immunity vol 70no 1 pp 204ndash210 2002

[13] J Gu X Liu Y Li et al ldquoAmethod for generation phage cocktailwith great therapeutic potentialrdquo PLoSONE vol 7 no 3 ArticleID e31698 2012

[14] 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

[15] E Kutter D de Vos G Gvasalia et al ldquoPhage therapy in clinicalpractice treatment of human infectionsrdquo Current Pharmaceuti-cal Biotechnology vol 11 no 1 pp 69ndash86 2010

[16] H Brussow ldquoWhat is needed for phage therapy to become areality in Western medicinerdquo Virology vol 434 no 2 pp 138ndash142 2012

[17] B K Chan S T Abedon and C Loc-Carrillo ldquoPhage cocktailsand the future of phage therapyrdquo FutureMicrobiology vol 8 no6 pp 769ndash783 2013

[18] E L Ellis andM Delbruck ldquoThe growth of bacteriophagerdquoTheJournal of General Physiology vol 22 pp 365ndash384 1939

[19] FOkada Y Ando KHonda et al ldquoAcuteKlebsiella pneumoniaepneumonia alone and with concurrent infection comparisonof clinical and thin-section CT findingsrdquo British Journal ofRadiology vol 83 no 994 pp 854ndash860 2010


[20] C H Lester S S Olsen L Jakobsen et al ldquoEmergence ofextended-spectrum 120573-lactamase (ESBL)-producing Klebsiellapneumoniae in Danish hospitals This is in part explained byspread of two CTX-M-15 clones withmultilocus sequence types15 and 16 in Zealandrdquo International Journal of AntimicrobialAgents vol 38 no 2 pp 180ndash182 2011

[21] A Adler E Khabra I Chmelnitsky et al ldquoDevelopment andvalidation of a multiplex PCR assay for identification of theepidemic ST-258512 KPC-producing Klebsiella pneumoniaeclonerdquoDiagnostic Microbiology amp Infectious Disease vol 78 no1 pp 12ndash15 2014

[22] S Ambretti P Gaibani F Caroli L Miragliotta and V SambrildquoA carbapenem-resistant Klebsiella pneumoniae isolate harbor-ingKPC-I from ItalyrdquoNewMicrobiologica vol 33 no 3 pp 281ndash282 2010

[23] XWittebole S deRoock and SMOpal ldquoAhistorical overviewof bacteriophage therapy as an alternative to antibiotics for thetreatment of bacterial pathogensrdquo Virulence vol 5 no 1 pp226ndash235 2014

[24] D R Harper and M C Enright ldquoBacteriophages for thetreatment of Pseudomonas aeruginosa infectionsrdquo Journal ofApplied Microbiology vol 111 no 1 pp 1ndash7 2011

[25] M Kim and S Ryu ldquoCharacterization of a T5-like coliphageSPC35 and differential development of resistance to SPC35in Salmonella enterica serovar typhimurium and Escherichiacolirdquo Applied and Environmental Microbiology vol 77 no 6 pp2042ndash2050 2011

[26] M Skurnik and E Strauch ldquoPhage therapy facts and fictionrdquoInternational Journal ofMedicalMicrobiology vol 296 no 1 pp5ndash14 2006

[27] S Matsuzaki M Yasuda H Nishikawa et al ldquoExperimentalprotection ofmice against lethal Staphylococcus aureus infectionby novel bacteriophage 120593MR11rdquo Journal of Infectious Diseasesvol 187 no 4 pp 613ndash624 2003

[28] E Morello E Saussereau D Maura M Huerre L Touquiand LDebarbieux ldquoPulmonary bacteriophage therapy onPseu-domonas aeruginosa cystic fibrosis strains First steps towardstreatment and preventionrdquo PLoS ONE vol 6 no 2 Article IDe16963 2011

[29] J Bordon S Aliberti R Fernandez-Botran et al ldquoUnderstand-ing the roles of cytokines and neutrophil activity and neutrophilapoptosis in the protective versus deleterious inflammatoryresponse in pneumoniardquo International Journal of InfectiousDiseases vol 17 no 2 pp e76ndashe83 2013

[30] M Zimecki J Artym M Kocięba B Weber-Dabrowska JBorysowski and A Garski ldquoEffects of prophylactic adminis-tration of bacteriophages to immunosuppressed mice infectedwith Staphylococcus aureusrdquo BMC Microbiology vol 9 article169 2009

Submit your manuscripts athttpwwwhindawicom

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Anatomy Research International

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Molecular Biology International

GenomicsInternational Journal of


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BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


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Virolog y

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Stem CellsInternational



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Microbiology


accomplished some achievements [15ndash17] For exampleChhibber et al delivered phage via an intraperitoneal route totreat experimental lobar pneumonia that was induced by Kpneumoniae in mice and had a great efficacy [9] However anintraperitoneal administration of a phage would not be eitherpractical or efficient for treating pneumonia in humans In thepresent study we evaluated the therapeutic efficacy of phage1513 which was delivered intranasally in order to protectagainst pneumonia that is induced by a strain of multidrugresistant K pneumoniae

2 Materials and Methods

21 Bacterial Strain A K pneumoniae strain was isolatedfrom a patient with pneumonia at the Zhongshan HospitalAffiliated to Dalian University (Dalian China) and then wasdesignated KP 1513 KP 1513 was identified as a multipleantibiotics-resistant and ESBLs-producing strain by theWalkAway Plus System (Siemenrsquos Diagnostics) Stocks of the strainwere stored at minus80∘C in Luria-Bertani broth (yeast extract5 g tryptone 10 g and NaCl 10 g in 1 L of distilled water)containing 25 glycerol

22 Isolation Enrichment and Purification of BacteriophageKP 1513 was used as a host to isolate a lytic phage from thesewage samples from the Zhongshan Hospital Affiliated toDalian University Briefly 20mL of the sewage sample wascentrifuged at 5000 g for 5min at 4∘C to remove any debrisand soilThen the supernatant was pooled and supplementedwith CaCl

2to 1mmol Lminus1 TenmL of this supernatant was

combined with 10mL of 2 times LB broth and 2mL of bacteriasolution and was then incubated for 12 h at 37∘Cwith shaking(120 rpm) Then the enrichment broths were centrifuged at8000 g at 4∘C for 5min The supernatant was pooled andpassed through a 022120583m filter (Millipore USA) A double-layered agar method was used to confirm phage presencein the filtrate and the plaque forming unit (PFU) wascalculated Single plaques were subcultured three times forphage purification The suspension that contained specificphage was incubated with DNase I and RNase (1 120583gmLminus1Takara Japan) at 37∘C for 30min prior to adding NaCl(1mol Lminus1) and polyethylene glycol 8000 (PEG8000 Sigma)to precipitate the phage particles This phage was designatedphage 1513 and was resuspended in SM buffer (NaCl 58 gMgSO

4sdot7H2O2 g TrissdotCl 1mol Lminus1 and 2gelatin 5mL in 1 L

of distilled water pH 75) Then cesium chloride equilibriumultracentrifugation (150000 g for 24 h in a Beckman Ti50rotor)was conducted for further phage purificationThebandthat contained the phage was collected and dialyzed againsta TBS buffer (NaCl 8 g KCl 02 g and tris base 3 g in 1 Lof distilled water pH 74 with pure HCl) at 4∘C overnightThen phages were passed through a Detoxi-Gel endotoxinremoving gel (Pierce Biotechnology Rockford IL) to removeendotoxins from the phage preparation Finally the purifiedphages were stored at 4∘C until use

23 Characterization of Phage 1513 Transmission elec-tron microscopy was used to observe phage 1513 with

a JEM-1200EX electron microscope (JEOL Japan) after neg-ative staining by phosphotungstic acid

Thermal pH and chloroform stability tests were con-ducted Aliquots of phage suspensions with high titers (2 times108 PFUmLminus1) were incubated at 4∘C 25∘C 37∘C 50∘C 60∘Cand 70∘C (pH 7) for 60min or at 4∘C (pH 3ndash12) for 10 h ormixed with chloroform and incubated at 4∘C for 24 h Thephage titers were tested by the double-layer agar method

A one-step growth experiment was carried out by mod-ifying the method described by Ellis and Delbruck [18]Briefly the phage was added to broth culture of KP 1513 toa MOI (multiplicity of infection) of 01 The sample was thenincubated at 37∘C for 15min in order to promote phage-hostadsorptionThemixture was centrifuged at 12000 g at 4∘C for10min to remove any free phage particles The supernatantwas removed and the pellet was resuspended in LB brothThe above-mentioned centrifugation procedure was repeatedoncemoreThe pellet was resuspended in 100mLLBmediumand incubated at 37∘C while shaking Aliquots were removedat 10min intervals and the phage titer was determined byspot method

Genome sequencing was performed by Shenzhen Ge-nomics Institute (Shenzhen China) using the Illumina High-Throughput Sequencing Platform (Illumina HiSeq 2000) onDNA obtained by means of standard SDS-proteinase K pro-ceduresThe complete genome sequence of the phage 1513wasaccessible in GenBank (Accession number KP658157)

24 Bactericidal Effect of Phage 1513 In Vitro An aliquot ofovernight culture of KP 1513 was seeded into fresh LBmedium and incubated for 6 h at 37∘C with shaking(150 rpm)The bacteria were pelleted and resuspended in PBSto an OD

650of 06 (sim2 times 109 CFUmLminus1) Different phage 1513

concentrations (2 times 1010 2 times 109 and 2 times 108 PFUmLminus1) orPBS (control) were added to the inocula and then furtherincubated for 12 h at 37∘C with shaking (150 rpm) Bacterialgrowth was monitored by measuring the OD

600at a 30min

interval

25 Mice Seven-week-old female Swiss-Webster mice(weight sim25 g per mouse) were purchased from the DalianMedical University in Dalian China (SCXK 2008-0002)All procedures were conducted according to guidelinesestablished by the Ethics Committee of Dalian Medical Uni-versity

26 Mouse Lethal PneumoniaModel Groups of 10 mice wereanesthetized by an IP injection with ketamine (120mg kgminus1)and xylazine (12mg kgminus1) and inoculated with 20120583L inoculaof KP 1513 (2 times 108 CFUmouse) intranasally LD

50of the KP

1513 was 417 times 109 CFUmLminus1 intranasally in the previousstudy In the phage therapeutic experiments a single dose ofphage (2 times 109 2 times 108 and 2 times 107 PFUmouse) or PBS wasgiven intranasally 2 h after infection

27 Bacterial Burden Histopathology and Cytokine Assay ofLung Tissue Groups of 11 mice were inoculated intranasally


(a) (b)




3 Results








90

85

80

75

70

65

60

55

50

3 4 5 6 7 8 9 10 11 12

pH

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

10

8

6

4

2

0

0 10 20 30 40 50 60 70 80

Temperature (∘C)

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(b)


0 20 40 60 80 1004

5

6

7

8


Latent time

Burs

t siz

e

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

0 1 2 3 4 5 6 7 8 9 10 11 1200

02

04

06

08

10

12

14

Negative control

Time (h)

MOI = 10

MOI = 1

MOI = 01

Opt

ical

den

sity

at650

nm

(b)






0 12 24 36 48 60 720

20

40

60

80

100

PBS(h)

Surv

ival

()

MOI = 01

MOI = 1

MOI = 10





4 Discussion






(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

KP + PBS KP + phage





5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)


0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)










Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)




3 Results








90

85

80

75

70

65

60

55

50

3 4 5 6 7 8 9 10 11 12

pH

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

10

8

6

4

2

0

0 10 20 30 40 50 60 70 80

Temperature (∘C)

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(b)


0 20 40 60 80 1004

5

6

7

8


Latent time

Burs

t siz

e

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

0 1 2 3 4 5 6 7 8 9 10 11 1200

02

04

06

08

10

12

14

Negative control

Time (h)

MOI = 10

MOI = 1

MOI = 01

Opt

ical

den

sity

at650

nm

(b)






0 12 24 36 48 60 720

20

40

60

80

100

PBS(h)

Surv

ival

()

MOI = 01

MOI = 1

MOI = 10





4 Discussion






(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

KP + PBS KP + phage





5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)


0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)










Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


90

85

80

75

70

65

60

55

50

3 4 5 6 7 8 9 10 11 12

pH

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

10

8

6

4

2

0

0 10 20 30 40 50 60 70 80

Temperature (∘C)

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(b)


0 20 40 60 80 1004

5

6

7

8


Latent time

Burs

t siz

e

Plaq

ue fo

rmin

g un

its (l

og10

PFU

mLminus

1)

(a)

0 1 2 3 4 5 6 7 8 9 10 11 1200

02

04

06

08

10

12

14

Negative control

Time (h)

MOI = 10

MOI = 1

MOI = 01

Opt

ical

den

sity

at650

nm

(b)






0 12 24 36 48 60 720

20

40

60

80

100

PBS(h)

Surv

ival

()

MOI = 01

MOI = 1

MOI = 10





4 Discussion






(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

KP + PBS KP + phage





5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)


0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)










Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 12 24 36 48 60 720

20

40

60

80

100

PBS(h)

Surv

ival

()

MOI = 01

MOI = 1

MOI = 10





4 Discussion






(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

KP + PBS KP + phage





5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)


0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)










Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

KP + PBS KP + phage





5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)


0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)










Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5

6

7

8

9

KP + phage

lowastlowast

+ phagePBS

PFU

g o

f lun

glo

g

(a)

5

6

7

8

9

KP + PBS KP + phage

lowastlowast

CFU

g o

f lun

glo

g

(b)

0

minus15

minus10

minus5

KP + PBS KP + phage

Wei

ght l

oss (

)

lowastlowast

(c)


0

5000

10000

15000

20000

KP + PBS KP + phage

TNF-120572

(pg

mL

of lu

ng h

omog

enat

e)

lowast

(a)

0

2000

4000

6000

8000

IL-6

(pg

mL

of lu

ng h

omog

enat

e)

KP + PBS KP + phage

lowastlowast

(b)










Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article evaluation of the efficacy of a...

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