Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This work investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4 and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45 % reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium adhered bacteria

A Lepape

Alberta Faustino

Alexandra Moura

Ana Nicolau

AR Costa

B Weber-Dabrowska

C Loc Carrillo

CP Champagne

Dulce Oliveira

E Domann

H Ceri

H Nishikawa

JA Wagenaar

JD Schilling

Joana Azeredo

M Pezzlo

MA Mulvey

Mariana Henriques

MC Healey

N Cerca

PS Stewart

RJ Atterbury

Sanna Sillankorva

VN Krylov

WE Huff

Current Microbiology

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria

Sillankorva, S

Oliveira, D

Moura, A

Henriques, M

Faustino, A

Nicolau, A

Azeredo, J

Institutional repository of Hospital de Braga

Efficacy of a Broad Host Range Lytic BacteriophageAgainst E. coli Adhered to UrotheliumSanna Sillankorva • Dulce Oliveira •Alexandra Moura • Mariana Henriques •Alberta Faustino • Ana Nicolau • Joana AzeredoReceived: 24 September 2010 / Accepted: 22 November 2010 / Published online: 8 December 2010 Springer Science+Business Media, LLC 2010Abstract Persistent urinary tract infections (UTI) areoften caused by E. coli adhered to urothelium. This type ofcells is generally recognized as very tolerant to antibioticswhich renders difficult the treatment of chronic UTI. Thisstudy investigates the use of lytic bacteriophages as alter-native antimicrobial agents, particularly the interaction ofphages with E. coli adhered to urothelium and specificallydetermines their efficiency against this type of cells. Thebacterial adhesion to urothelium was performed varyingthe bacterial cell concentrations and the period and con-ditions (static, shaken) of adhesion. Three collection bac-teriophages (T1, T4, and phiX174 like phages) were testedagainst clinical E. coli isolates and only one was selectedfor further infection experiments. Based on the lyticspectrum against clinical isolates and its ability to infectthe highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage causednearly a 45% reduction of the bacterial population after2 h of treatment. This study provides evidence thatbacteriophages are effective in controlling suspended andadhered cells and therefore can be a viable alternative toantibiotics to control urothelium- adhered bacteria.IntroductionIn the recent years, there has been a spread of multidrugresistant bacteria, including bacteria resistant to the lastresort antibiotics [1]. Therefore, prevention and control ofbacterial resistance requires new antimicrobial agents, theprudent use of existing ones, new vaccines and enhancedpublic health efforts to reduce transmission of bacterialresistance. Also, the use of bacteriophages as an alterna-tive to antibiotherapy is again redrawing attention. Aconsiderable number of publications demonstrate thatphage preparations for veterinary purposes can efficientlycontrol the bacterial loads in infected animals [2–5] andstrongly supports the concept of phage therapy as analternative or complementary strategy to antibiotherapy.Furthermore, orally administered phages have beenrecovered from the blood and physical fluids, includingurine [6, 7] and phages administered by intraperitonealinjection have been detected in different organs, such as:liver, kidneys, spleen, lungs, muscles, brain, and blood [8].Despite these attractive characteristics, there is limitedinformation on phage infection of bacteria adhered toepithelial cells. The study described herein focuses theefficacy of phage to kill its target host when the last isfound adhered to urothelium. For this, the efficacy of threeDSMZ collection phages (T1, T4 and phiX174) against apool of 69 E. coli strains isolated from urinary tractinfections was studied and only the broadest host rangephage was used to perform the infection experiments withsuspended or urothelium adhered E. coli.S. Sillankorva (&)  D. Oliveira  A. Moura  M. Henriques A. Nicolau  J. AzeredoDepartment of Biological Engineering, University of Minho,Braga, Portugale-mail: s.sillankorva@deb.uminho.ptA. MouraInstituto Piaget, Escola Superior de Saúde Jean, Piaget/VilaNova de Gaia, PortugalA. FaustinoSão Marcos Hospital, Braga, Portugal123Curr Microbiol (2011) 62:1128–1132DOI 10.1007/s00284-010-9834-8Materials and MethodsBacteria, Bacteriophage, and MediaThe bacteriophages and respective hosts were purchasedfrom the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Germany. Both bacteriophages T1(DSM 5801) and T4 (DSM 4505) have as host Escherichiacoli DSM 613, while phage phiX174 (DSM 4497) hasashost E. coli DSM 13127. The media used for E. coligrowth were either LB media or artificial urine (0.65 gl-1of Magnesium chloride hexahydrate, 0.65 g l-1 of Calciumchloride monohydrated, 4.6 g l-1 of sodium chloride,2.3 g l-1 of sodium sulfate, 0.65 g l-1 of Sodium Citratedihydrated, 0.20 g l-1 of Disodium oxalate, 2.8 g l-1 ofpotassium dihydrogen phosphate, 1.6 g l-1 of potassiumchloride, 2.0 g l-1 of NH4Cl, 12 g l-1 of Urea, 1.1 g l-1of Creatinin, 0.1 g l-1 of Tryptic Soy Broth (TSB)). Pha-ges were propagated in MGC media (5.0 g/l of peptone,3.0 g/l of meet extract and 0.8 g/l of NaCl). The clinicalisolates of E. coli used in this study, were isolated frompatients with urinary tract infections.Lytic Spectra of the Phages and Selectionof the Broadest Host Range PhageThe three phages (T1, T4, and phiX174) were testedagainst the 69 clinical E. coli isolates using the phagespot test procedure [9]. In brief, 3 ml of MGC top-agar(MGC media with 0.6% agar) were added to 100 llovernight cultures of each clinical isolate and poured ontoa dish containing MGC agar (MGC media with 1.2%agar). The agar was allowed to solidify after which 10 lleach phage was spotted on the bacterial lawn of the dif-ferent isolates. The drop was allowed to dry and theplates were incubated overnight at 37C. Phage sensitiveE. coli showed clear or turbid areas where the phagesolutions were spotted.Antibiotic Sensitivity TestThe susceptibility of the 69 E. coli clinical isolates to eightantimicrobial agents was determined by the disc diffusionmethod on modified Mueller–Hinton II agar according tothe European Committee on Antimicrobial SusceptibilityTesting (EUCAST).Stability of Phage in Artificial Urine100 ll of T1 phage were added to 400 ll of artificial urineand incubated at 37C during 24 h. Samples were takenafter 0, 3, 6, 9, and 24 h and the phage titre was performedaccording to Adams [10].Epithelial Cells and Growth ConditionsEpithelial cell line from human bladder (TCC-SUP fromDSMZ) was grown on D-MEM, supplemented with 15% offetal bovine serum (FBS, Gibco-Invitrogen) and 1%Streptomycin/Penicillin (Gibco-Invitrogen). Cells weregrown in 24 well microplates at 37C and 5% of CO2, untilthey reached an 80% of confluence. A number of 1 9 105cells per well, determined by tripan blue exclusion staining,was achieved.E. coli Adhesion to Epithelial CellsThe TCC-SUP epithelial cell monolayers were washedwith phosphate buffered saline (PBS 0.1 M, pH 7.4). After,500 ll of E. coli 613 diluted suspensions (104 up to 109CFUml-1) or 500 ll of PBS (control), were added to eachmonolayer. Adhesion assays were performed during 30, 60,120, and 180 min, at 37C and under agitation (120 rpm).At each time point, the wells were washed three times withPBS, to remove unattached bacteria. Bacteria were enu-merated by CFU determination, after tripsinization ofepithelial cells. All experiments were carried out intriplicate.Phage Infection of Urothelium Adhered and SuspendedBacteriaUrothelium was grown at 37C in 24 well ELISA platesuntil a concentration of 105 cells/well, after which E. coliwere allowed to adhere for 2 h. Afterwards, the adheredbacteria were phageinfected during 2 h varying the MOIused (0.1 and 1) and varying also the phage infectionconditions (static and dynamic). The plates were incubatedat 37C and the efficacy of phage infection was assessedthrough determination of the concentration of bacteria.Phage infection of exponential E. coli cultures were alsoperformed in order to compare the lytic efficacy of adheredagainst suspended E. coli. All experiments were carried outin triplicate.ResultsAntibiotic and Bacteriophage Susceptibilityof the 69 Clinical Isolates of E. ColiThe susceptibility of the 69 clinical E. coli isolates ispresented in Fig. 1. The resistance profiles of the E. coliclinical isolates revealed that more than 60 percent of thestrains were resistant to ampicillin (AMP). Furthermore,the strains were most susceptible to nitrofurantoin (FD)(Fig. 1). Based on these results, the 69 isolates wereS. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage 1129123divided in two groups: the first containing the resistantstrains (Fig. 2a) and the second group containing the iso-lates susceptible to the antibiotics tested (Fig. 2b) andtested for susceptibility to three DSMZ culture collectionphages: T1, T4, and phiX174, respectively. Phage T1showed aptitude to infecting the highest percentage ofantibiotic resistant E. coli strains (Fig. 2a) while phage T4lysed a larger number of antibiotic susceptible isolates(Fig. 2b). Overall the T1 and T4 phages were the mostefficient phages showing activity against 43.5 and 41.1% ofthe clinical isolates tested whereas phage phiX174 exhib-ited a narrower range of activity, being able to infect only9.4% of the isolates tested. According to these results,phage T1 was selected for further experiments since it wascapable of lysing the broadest range of hosts.Growth of E. coli and Stability of Phage T1 in UrineThe growth characteristics of E. coli DSMZ 613, phageT1’s host, in artificial urine were determined. This bacte-rium has a doubling time of approximately 53 min and aspecific growth rate of 0.78 h-1. Stability tests performedwith phage T1 in artificial urine showed that the phageremained stable for 24 h and maintained its infectionability, as confirmed by plaque forming unit tests per-formed after different periods of exposure to artificial urine(data not shown).Phage Infection of Bacteria Suspended or Adheredto UrotheliumIn vitro phage infection experiments of bacteria adhered tothe urothelium were performed under static and dynamic(under the influence of shaking) conditions. Prior to thephage infection experiments on E. coli cells, the phagemultiplicity of infection (MOI) was optimized. E. coli wereinfected, under dynamic conditions (120 rpm), with fivedifferent MOIs ranging from 0.01 up to 10 and cell lysiswas followed by optical density measurements (data notshown). Two multiplicities of infection were selected forall further experiments: 0.1 and 1, respectively and theinfection period selected was 2 h. Furthermore, two dif-ferent E. coli concentrations which resulted in approxi-mately 105 CFUml-1 of adhered cells after 60 min werealso selected for the adhesion and phage infection studies,106 and 107 CFUml-1, respectively (data not shown). It isimportant to have this concentration of adhered cells sincetypically a urinary tract infection is characterized by apositive uroculture of more than 105 CFUml-1[11, 12].The reduction percentages of bacterial cells adhered tourothelium (A columns in Figs. 3, 4) following phageinfection were compared with the reduction valuesobtained with suspended cultures (S columns in Figs. 3, 4).Under static conditions, cell reductions of both adhered andsuspended bacteria following phage infection were ingeneral very low. The use of different MOIs did notinfluence greatly the average reduction percentagesobtained. However, it was clear that phages were slightlymore efficient in lysing suspended E. coli than those foundadhered to urothelium, independently on the inoculumconcentration used. The higher inoculum concentration(107 CFUml-1) resulted in a higher percentage of cellreduction clearly due to the presence of slightly more cellsFig. 1 Susceptibility of the 69 clinical E. coli isolates to 8 antibiotics(AMP ampicillin, NXM norfloxacin, AUG amoxicillin/clavulanate,FD nitrofurantoin, SXT TMP-SMX, CP cephalothin, CIP ciproflox-acin, TET tetracycline)Fig. 2 Susceptibility of the 69 clinical E. coli isolates to bacteriophage T1, T4, and phiX174. Susceptibility of the: a antibiotic resistant andb antibiotic susceptible strains to phages1130 S. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage123adhered to the urothelium than in the experiments where 106CFUml-1 was used as inoculum. Although the reduction ofadhered cells by phages under static conditions was low, thiswork evidences that phages can kill to some extent cellsadhered to urothelium. Comparatively to the phage experi-ments carried out under static, under dynamic conditions thephage was significantly more efficient in infecting E. coliadhered to urothelium (Fig. 4). Under static infection con-ditions, the average cell reduction percentages obtained,using inoculum concentrations of 106 and 107 CFUml-1,were approximately 10 and 20%, while under dynamicconditions those percentages were about 30 and 35%(compare Figs. 3, 4). Furthermore, like in the experimentsperformed under static conditions, the differences obtainedusing a MOI of 0.1 and 1.0 were insignificant.DiscussionUrinary tract infections (UTI) are among the most commonbacterial infections affecting a large proportion of theworldwide population. These infections are mainly causedby E. coli that have the capacity to colonize and invadeepithelial cells [13]. Presumably, this tactic of invasionallows the development of a cellular niche where micro-organism can persist in a protected pathway [14] especiallyagainst the action of the antibiotics, as already demon-strated both in vitro and in vivo [15, 16]. In fact, thephysiological alterations of bacteria triggered by the sessilemode of live can confer protection against antibiotics[17–19]. Specifically it has already been shown that bac-teria adhered to epithelium can be up to 100 times moretolerant to antibiotics [19, 20]. In this study the authorsdescribe the use of bacteriophages to reduce the loads ofE. coli adhered to urothelium and evaluate their efficacyafter infection of cells using two different phage-host ratiosand under two distinct conditions, static, and shaken,respectively. The results after the 2 h of phage treatmentwere similar using an initial MOI of 0.1 or 1 since bothphage concentrations led to approximately the same per-centage of cell reduction. Furthermore, phage T1 wassignificantly more efficient in infecting E. coli adhered tourothelium under shaken conditions most likely to anenhancement of the contact between the phage and therespective host. Contrarily to the infection of adheredE. coli, where the efficacy of phage T1 was affected by theapplication condition used (static or dynamic), the infec-tion of planktonic cells with phage T1 under shaken andnon-shaken conditions was comparable. Although in thepresent study, under static conditions, the adhered bacteriashowed to be less sensitive to the phages overall the effi-cacy of phage T1 against adhered bacteria was similar tothe infection of planktonic cells. Thus, contrarily to anti-biotics, which hardly remove bacteria from existing bio-films [21], this study shows that phages are able to causecell lysis of biofilm bacteria.Besides the ability of phage T1 to infect adhered andplanktonic E. coli cultures, this work also shows thatphages deposited in culture collection have the ability tokill E. coli from different origins than the deposited hostsince it was able, together with T4 and phiX174, to infect avariety of antibiotic resistant clinical isolates.References1. Lepape A, Monnet DL (2009) Experience of European intensivecare physicians with infections due to antibiotic-resistant bacte-ria. Euro Surveill 14:1–32. Atterbury RJ, Van Bergen MA, Ortiz F, Lovell MA, Harris JA,De BA, Wagenaar JA, Allen VM, Barrow PA (2007) Bacterio-phage therapy to reduce salmonella colonization of broilerchickens. Appl Environ Microbiol 73:4543–45493. Huff WE, Huff GR, Rath NC, Donoghue AM (2006) Evaluationof the influence of bacteriophage titer on the treatment of coli-bacillosis in broiler chickens. Poult Sci 85:1373–1377Fig. 4 Percentage of cell reduction after phage infection underdynamic conditions of E. coli suspensions and cells adhered tourothelium using MOIs of 0.1 and 1. Phage infection was performedon bacterial suspensions (S) with two different concentrations (106and 107 cellsml-1) and on cells adhered (A) to urothelium for 2 husing inoculum concentrations of 106 and 107 cellsml-1Fig. 3 Percentage of cell reduction after phage infection under staticconditions of E. coli suspensions and cells adhered to urotheliumusing MOIs of 0.1 and 1. Phage infection was performed on bacterialsuspensions (S) with two different concentrations (106 and 107cellsml-1) and on cells adhered (A) to urothelium for 2 h usinginoculum concentrations of 106 and 107 cells ml-1S. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage 11311234. Loc Carrillo C, Atterbury RJ, el-Shibiny A, Connerton PL, DillonE, Scott A, Connerton IF (2005) Bacteriophage therapy to reduceCampylobacter jejuni colonization of broiler chickens. ApplEnviron Microbiol 71:6554–65635. Wagenaar JA, Van Bergen MA, Mueller MA, Wassenaar TM,Carlton RM (2005) Phage therapy reduces Campylobacter jejunicolonization in broilers. Vet Microbiol 109:275–2836. Krylov VN (2001) Phage therapy in terms of bacteriophagegenetics: hopes prospects safety limitations. Russian J Genet37:715–7307. Weber-Dabrowska B, Dabrowski M, Slopek S (1987) Studies onbacteriophage penetration in patients subjected to phage therapy.Arch Immunol Ther Exp 35:563–5688. Nishikawa H, Yasuda M, Uchiyama J, Rashel M, Maeda Y,Takemura I, Sugihara S, Ujihara T, Shimizu Y, Shuin T,Matsuzaki S (2008) T-even-related bacteriophages as candidatesfor treatment of Escherichia coli urinary tract infections. ArchVirol 153:507–5159. Champagne CP, Gardner N (1995) The spot test method for thein-plant enumeration of bacteriophages with paired cultures ofLactobacillus delbrueckii subsp bulgaricus and Streptococcussalivarius subsp. thermophilus. Int Dairy J 5:417–42510. Adams MH (1959) Bacteriophages. Interscience Publishers, NewYork11. Domann E, Hong G, Imirzalioglu C, Turschner S, Kuhle J,Watzel C, Hain T, Hossain H, Chakraborty T (2003) Culture-independent identification of pathogenic bacteria and polymi-crobial infections in the genitourinary tract of renal transplantrecipients. J Clin Microbiol 41:5500–551012. Pezzlo M (1988) Detection of urinary tract infections by rapidmethods. Clin microbiol Rev 1:268–28013. Healey MC, Hwang HH, Elsner YY, Johnston AV (1991) Amodel for demonstrating the adhesion of actinobacillus-seministo epithelial-cells. Canadian J Veter Res 55:121–12714. Finlay BB, Falkow S (1997) Common themes in microbialpathogenicity revisited. Microbiol Mol Biol Rev 61:136–13915. Mulvey MA, Schilling JD, Hultgren SJ (2001) Establishment of apersistent Escherichia coli reservoir during the acute phase of abladder infection. Infect Immun 69:4572–457916. Schilling JD, Lorenz RG, Hultgren SJ (2002) Effect of tri-methoprim-sulfamethoxazole on recurrent bacteriuria and bacte-rial persistence in mice infected with uropathogenic Escherichiacoli. Infect Immun 70:7042–704917. Cerca N, Martins S, Cerca F, Jefferson KK, Pier GB, Oliveira R,Azeredo J (2005) Comparative assessment of antibiotic suscep-tibility of coagulase-negative staphylococci in biofilm versusplanktonic culture as assessed by bacterial enumeration or rapidXTT colorimetry. J Antimicrob Chemother 56:331–33618. Cerca N, Martins S, Sillankorva S, Jefferson KK, Pier GB, Oli-veira R, Azeredo J (2005) Effects of growth in the presence ofsubinhibitory concentrations of dicloxacillin on Staphylococcusepidermidis and Staphylococcus haemolyticus biofilms. ApplEnviron Microbiol 71:8677–868219. Costa AR, Henriques M, Oliveira R, Azeredo J (2009) The role ofpolysaccharide intercellular adhesin (PIA) in Staphylococcusepidermidis adhesion to host tissues and subsequent antibiotictolerance. Eur J Clin Microbiol Infect Dis 28:623–62920. Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A(1999) The calgary biofilm device: new technology for rapiddetermination of antibiotic susceptibilities of bacterial biofilms.J Clin Microbiol 37:1771–177621. Stewart PS, Costerton JW (2001) Antibiotic resistance of bacteriain biofilms. Lancet 358:135–1381132 S. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage123

English

Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium

Sillankorva, Sanna

Oliveira, D.

Moura, A.

Henriques, Mariana

Faustino, A.

Nicolau, Ana

Azeredo, Joana

Universidade do Minho: RepositoriUM

Efficacy of a Broad Host Range Lytic BacteriophageAgainst E. coli Adhered to UrotheliumSanna Sillankorva • Dulce Oliveira •Alexandra Moura • Mariana Henriques •Alberta Faustino • Ana Nicolau • Joana AzeredoReceived: 24 September 2010 / Accepted: 22 November 2010 / Published online: 8 December 2010 Springer Science+Business Media, LLC 2010Abstract Persistent urinary tract infections (UTI) areoften caused by E. coli adhered to urothelium. This type ofcells is generally recognized as very tolerant to antibioticswhich renders difficult the treatment of chronic UTI. Thisstudy investigates the use of lytic bacteriophages as alter-native antimicrobial agents, particularly the interaction ofphages with E. coli adhered to urothelium and specificallydetermines their efficiency against this type of cells. Thebacterial adhesion to urothelium was performed varyingthe bacterial cell concentrations and the period and con-ditions (static, shaken) of adhesion. Three collection bac-teriophages (T1, T4, and phiX174 like phages) were testedagainst clinical E. coli isolates and only one was selectedfor further infection experiments. Based on the lyticspectrum against clinical isolates and its ability to infectthe highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage causednearly a 45% reduction of the bacterial population after2 h of treatment. This study provides evidence thatbacteriophages are effective in controlling suspended andadhered cells and therefore can be a viable alternative toantibiotics to control urothelium- adhered bacteria.IntroductionIn the recent years, there has been a spread of multidrugresistant bacteria, including bacteria resistant to the lastresort antibiotics [1]. Therefore, prevention and control ofbacterial resistance requires new antimicrobial agents, theprudent use of existing ones, new vaccines and enhancedpublic health efforts to reduce transmission of bacterialresistance. Also, the use of bacteriophages as an alterna-tive to antibiotherapy is again redrawing attention. Aconsiderable number of publications demonstrate thatphage preparations for veterinary purposes can efficientlycontrol the bacterial loads in infected animals [2–5] andstrongly supports the concept of phage therapy as analternative or complementary strategy to antibiotherapy.Furthermore, orally administered phages have beenrecovered from the blood and physical fluids, includingurine [6, 7] and phages administered by intraperitonealinjection have been detected in different organs, such as:liver, kidneys, spleen, lungs, muscles, brain, and blood [8].Despite these attractive characteristics, there is limitedinformation on phage infection of bacteria adhered toepithelial cells. The study described herein focuses theefficacy of phage to kill its target host when the last isfound adhered to urothelium. For this, the efficacy of threeDSMZ collection phages (T1, T4 and phiX174) against apool of 69 E. coli strains isolated from urinary tractinfections was studied and only the broadest host rangephage was used to perform the infection experiments withsuspended or urothelium adhered E. coli.S. Sillankorva (&)  D. Oliveira  A. Moura  M. Henriques A. Nicolau  J. AzeredoDepartment of Biological Engineering, University of Minho,Braga, Portugale-mail: s.sillankorva@deb.uminho.ptA. MouraInstituto Piaget, Escola Superior de Sau´de Jean, Piaget/VilaNova de Gaia, PortugalA. FaustinoSa˜o Marcos Hospital, Braga, Portugal123Curr Microbiol (2011) 62:1128–1132DOI 10.1007/s00284-010-9834-8Materials and MethodsBacteria, Bacteriophage, and MediaThe bacteriophages and respective hosts were purchasedfrom the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Germany. Both bacteriophages T1(DSM 5801) and T4 (DSM 4505) have as host Escherichiacoli DSM 613, while phage phiX174 (DSM 4497) hasashost E. coli DSM 13127. The media used for E. coligrowth were either LB media or artificial urine (0.65 gl-1of Magnesium chloride hexahydrate, 0.65 g l-1 of Calciumchloride monohydrated, 4.6 g l-1 of sodium chloride,2.3 g l-1 of sodium sulfate, 0.65 g l-1 of Sodium Citratedihydrated, 0.20 g l-1 of Disodium oxalate, 2.8 g l-1 ofpotassium dihydrogen phosphate, 1.6 g l-1 of potassiumchloride, 2.0 g l-1 of NH4Cl, 12 g l-1 of Urea, 1.1 g l-1of Creatinin, 0.1 g l-1 of Tryptic Soy Broth (TSB)). Pha-ges were propagated in MGC media (5.0 g/l of peptone,3.0 g/l of meet extract and 0.8 g/l of NaCl). The clinicalisolates of E. coli used in this study, were isolated frompatients with urinary tract infections.Lytic Spectra of the Phages and Selectionof the Broadest Host Range PhageThe three phages (T1, T4, and phiX174) were testedagainst the 69 clinical E. coli isolates using the phagespot test procedure [9]. In brief, 3 ml of MGC top-agar(MGC media with 0.6% agar) were added to 100 llovernight cultures of each clinical isolate and poured ontoa dish containing MGC agar (MGC media with 1.2%agar). The agar was allowed to solidify after which 10 lleach phage was spotted on the bacterial lawn of the dif-ferent isolates. The drop was allowed to dry and theplates were incubated overnight at 37C. Phage sensitiveE. coli showed clear or turbid areas where the phagesolutions were spotted.Antibiotic Sensitivity TestThe susceptibility of the 69 E. coli clinical isolates to eightantimicrobial agents was determined by the disc diffusionmethod on modified Mueller–Hinton II agar according tothe European Committee on Antimicrobial SusceptibilityTesting (EUCAST).Stability of Phage in Artificial Urine100 ll of T1 phage were added to 400 ll of artificial urineand incubated at 37C during 24 h. Samples were takenafter 0, 3, 6, 9, and 24 h and the phage titre was performedaccording to Adams [10].Epithelial Cells and Growth ConditionsEpithelial cell line from human bladder (TCC-SUP fromDSMZ) was grown on D-MEM, supplemented with 15% offetal bovine serum (FBS, Gibco-Invitrogen) and 1%Streptomycin/Penicillin (Gibco-Invitrogen). Cells weregrown in 24 well microplates at 37C and 5% of CO2, untilthey reached an 80% of confluence. A number of 1 9 105cells per well, determined by tripan blue exclusion staining,was achieved.E. coli Adhesion to Epithelial CellsThe TCC-SUP epithelial cell monolayers were washedwith phosphate buffered saline (PBS 0.1 M, pH 7.4). After,500 ll of E. coli 613 diluted suspensions (104 up to 109CFUml-1) or 500 ll of PBS (control), were added to eachmonolayer. Adhesion assays were performed during 30, 60,120, and 180 min, at 37C and under agitation (120 rpm).At each time point, the wells were washed three times withPBS, to remove unattached bacteria. Bacteria were enu-merated by CFU determination, after tripsinization ofepithelial cells. All experiments were carried out intriplicate.Phage Infection of Urothelium Adhered and SuspendedBacteriaUrothelium was grown at 37C in 24 well ELISA platesuntil a concentration of 105 cells/well, after which E. coliwere allowed to adhere for 2 h. Afterwards, the adheredbacteria were phageinfected during 2 h varying the MOIused (0.1 and 1) and varying also the phage infectionconditions (static and dynamic). The plates were incubatedat 37C and the efficacy of phage infection was assessedthrough determination of the concentration of bacteria.Phage infection of exponential E. coli cultures were alsoperformed in order to compare the lytic efficacy of adheredagainst suspended E. coli. All experiments were carried outin triplicate.ResultsAntibiotic and Bacteriophage Susceptibilityof the 69 Clinical Isolates of E. ColiThe susceptibility of the 69 clinical E. coli isolates ispresented in Fig. 1. The resistance profiles of the E. coliclinical isolates revealed that more than 60 percent of thestrains were resistant to ampicillin (AMP). Furthermore,the strains were most susceptible to nitrofurantoin (FD)(Fig. 1). Based on these results, the 69 isolates wereS. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage 1129123divided in two groups: the first containing the resistantstrains (Fig. 2a) and the second group containing the iso-lates susceptible to the antibiotics tested (Fig. 2b) andtested for susceptibility to three DSMZ culture collectionphages: T1, T4, and phiX174, respectively. Phage T1showed aptitude to infecting the highest percentage ofantibiotic resistant E. coli strains (Fig. 2a) while phage T4lysed a larger number of antibiotic susceptible isolates(Fig. 2b). Overall the T1 and T4 phages were the mostefficient phages showing activity against 43.5 and 41.1% ofthe clinical isolates tested whereas phage phiX174 exhib-ited a narrower range of activity, being able to infect only9.4% of the isolates tested. According to these results,phage T1 was selected for further experiments since it wascapable of lysing the broadest range of hosts.Growth of E. coli and Stability of Phage T1 in UrineThe growth characteristics of E. coli DSMZ 613, phageT1’s host, in artificial urine were determined. This bacte-rium has a doubling time of approximately 53 min and aspecific growth rate of 0.78 h-1. Stability tests performedwith phage T1 in artificial urine showed that the phageremained stable for 24 h and maintained its infectionability, as confirmed by plaque forming unit tests per-formed after different periods of exposure to artificial urine(data not shown).Phage Infection of Bacteria Suspended or Adheredto UrotheliumIn vitro phage infection experiments of bacteria adhered tothe urothelium were performed under static and dynamic(under the influence of shaking) conditions. Prior to thephage infection experiments on E. coli cells, the phagemultiplicity of infection (MOI) was optimized. E. coli wereinfected, under dynamic conditions (120 rpm), with fivedifferent MOIs ranging from 0.01 up to 10 and cell lysiswas followed by optical density measurements (data notshown). Two multiplicities of infection were selected forall further experiments: 0.1 and 1, respectively and theinfection period selected was 2 h. Furthermore, two dif-ferent E. coli concentrations which resulted in approxi-mately 105 CFUml-1 of adhered cells after 60 min werealso selected for the adhesion and phage infection studies,106 and 107 CFUml-1, respectively (data not shown). It isimportant to have this concentration of adhered cells sincetypically a urinary tract infection is characterized by apositive uroculture of more than 105 CFUml-1[11, 12].The reduction percentages of bacterial cells adhered tourothelium (A columns in Figs. 3, 4) following phageinfection were compared with the reduction valuesobtained with suspended cultures (S columns in Figs. 3, 4).Under static conditions, cell reductions of both adhered andsuspended bacteria following phage infection were ingeneral very low. The use of different MOIs did notinfluence greatly the average reduction percentagesobtained. However, it was clear that phages were slightlymore efficient in lysing suspended E. coli than those foundadhered to urothelium, independently on the inoculumconcentration used. The higher inoculum concentration(107 CFUml-1) resulted in a higher percentage of cellreduction clearly due to the presence of slightly more cellsFig. 1 Susceptibility of the 69 clinical E. coli isolates to 8 antibiotics(AMP ampicillin, NXM norfloxacin, AUG amoxicillin/clavulanate,FD nitrofurantoin, SXT TMP-SMX, CP cephalothin, CIP ciproflox-acin, TET tetracycline)Fig. 2 Susceptibility of the 69 clinical E. coli isolates to bacteriophage T1, T4, and phiX174. Susceptibility of the: a antibiotic resistant andb antibiotic susceptible strains to phages1130 S. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage123adhered to the urothelium than in the experiments where 106CFUml-1 was used as inoculum. Although the reduction ofadhered cells by phages under static conditions was low, thiswork evidences that phages can kill to some extent cellsadhered to urothelium. Comparatively to the phage experi-ments carried out under static, under dynamic conditions thephage was significantly more efficient in infecting E. coliadhered to urothelium (Fig. 4). Under static infection con-ditions, the average cell reduction percentages obtained,using inoculum concentrations of 106 and 107 CFUml-1,were approximately 10 and 20%, while under dynamicconditions those percentages were about 30 and 35%(compare Figs. 3, 4). Furthermore, like in the experimentsperformed under static conditions, the differences obtainedusing a MOI of 0.1 and 1.0 were insignificant.DiscussionUrinary tract infections (UTI) are among the most commonbacterial infections affecting a large proportion of theworldwide population. These infections are mainly causedby E. coli that have the capacity to colonize and invadeepithelial cells [13]. Presumably, this tactic of invasionallows the development of a cellular niche where micro-organism can persist in a protected pathway [14] especiallyagainst the action of the antibiotics, as already demon-strated both in vitro and in vivo [15, 16]. In fact, thephysiological alterations of bacteria triggered by the sessilemode of live can confer protection against antibiotics[17–19]. Specifically it has already been shown that bac-teria adhered to epithelium can be up to 100 times moretolerant to antibiotics [19, 20]. In this study the authorsdescribe the use of bacteriophages to reduce the loads ofE. coli adhered to urothelium and evaluate their efficacyafter infection of cells using two different phage-host ratiosand under two distinct conditions, static, and shaken,respectively. The results after the 2 h of phage treatmentwere similar using an initial MOI of 0.1 or 1 since bothphage concentrations led to approximately the same per-centage of cell reduction. Furthermore, phage T1 wassignificantly more efficient in infecting E. coli adhered tourothelium under shaken conditions most likely to anenhancement of the contact between the phage and therespective host. Contrarily to the infection of adheredE. coli, where the efficacy of phage T1 was affected by theapplication condition used (static or dynamic), the infec-tion of planktonic cells with phage T1 under shaken andnon-shaken conditions was comparable. Although in thepresent study, under static conditions, the adhered bacteriashowed to be less sensitive to the phages overall the effi-cacy of phage T1 against adhered bacteria was similar tothe infection of planktonic cells. Thus, contrarily to anti-biotics, which hardly remove bacteria from existing bio-films [21], this study shows that phages are able to causecell lysis of biofilm bacteria.Besides the ability of phage T1 to infect adhered andplanktonic E. coli cultures, this work also shows thatphages deposited in culture collection have the ability tokill E. coli from different origins than the deposited hostsince it was able, together with T4 and phiX174, to infect avariety of antibiotic resistant clinical isolates.References1. Lepape A, Monnet DL (2009) Experience of European intensivecare physicians with infections due to antibiotic-resistant bacte-ria. Euro Surveill 14:1–32. Atterbury RJ, Van Bergen MA, Ortiz F, Lovell MA, Harris JA,De BA, Wagenaar JA, Allen VM, Barrow PA (2007) Bacterio-phage therapy to reduce salmonella colonization of broilerchickens. Appl Environ Microbiol 73:4543–45493. Huff WE, Huff GR, Rath NC, Donoghue AM (2006) Evaluationof the influence of bacteriophage titer on the treatment of coli-bacillosis in broiler chickens. Poult Sci 85:1373–1377Fig. 4 Percentage of cell reduction after phage infection underdynamic conditions of E. coli suspensions and cells adhered tourothelium using MOIs of 0.1 and 1. Phage infection was performedon bacterial suspensions (S) with two different concentrations (106and 107 cellsml-1) and on cells adhered (A) to urothelium for 2 husing inoculum concentrations of 106 and 107 cellsml-1Fig. 3 Percentage of cell reduction after phage infection under staticconditions of E. coli suspensions and cells adhered to urotheliumusing MOIs of 0.1 and 1. Phage infection was performed on bacterialsuspensions (S) with two different concentrations (106 and 107cellsml-1) and on cells adhered (A) to urothelium for 2 h usinginoculum concentrations of 106 and 107 cells ml-1S. 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Microbiol Mol Biol Rev 61:136–13915. Mulvey MA, Schilling JD, Hultgren SJ (2001) Establishment of apersistent Escherichia coli reservoir during the acute phase of abladder infection. Infect Immun 69:4572–457916. Schilling JD, Lorenz RG, Hultgren SJ (2002) Effect of tri-methoprim-sulfamethoxazole on recurrent bacteriuria and bacte-rial persistence in mice infected with uropathogenic Escherichiacoli. Infect Immun 70:7042–704917. Cerca N, Martins S, Cerca F, Jefferson KK, Pier GB, Oliveira R,Azeredo J (2005) Comparative assessment of antibiotic suscep-tibility of coagulase-negative staphylococci in biofilm versusplanktonic culture as assessed by bacterial enumeration or rapidXTT colorimetry. J Antimicrob Chemother 56:331–33618. Cerca N, Martins S, Sillankorva S, Jefferson KK, Pier GB, Oli-veira R, Azeredo J (2005) Effects of growth in the presence ofsubinhibitory concentrations of dicloxacillin on Staphylococcusepidermidis and Staphylococcus haemolyticus biofilms. ApplEnviron Microbiol 71:8677–868219. Costa AR, Henriques M, Oliveira R, Azeredo J (2009) The role ofpolysaccharide intercellular adhesin (PIA) in Staphylococcusepidermidis adhesion to host tissues and subsequent antibiotictolerance. Eur J Clin Microbiol Infect Dis 28:623–62920. Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A(1999) The calgary biofilm device: new technology for rapiddetermination of antibiotic susceptibilities of bacterial biofilms.J Clin Microbiol 37:1771–177621. Stewart PS, Costerton JW (2001) Antibiotic resistance of bacteriain biofilms. Lancet 358:135–1381132 S. Sillankorva: Efficacy of a Broad Host Range Lytic Bacteriophage123

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Efficacy of a Broad Host Range Lytic Bacteriophage Against E. coli Adhered to Urothelium

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Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium

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