( previously known as enterobacter sakazakii ) is a gram - negative opportunistic pathogen belonging to the enterobacteriaceae family .
it is known to cause serious infections including bacteremia , enterocolitis , meningitis and even sepsis , especially in premature newborns .
recently , a case study reporting cystitis caused by c. sakazakii in the elderly has been published .
the lethality of cronobacter infections is very high ( 4080% ) and the incidence is relatively low ( 8.7 per 100 000 low birth weight neonates ) ; however , it is assumed that the number of infections caused by cronobacter is underreported .
antibiotic therapy is effective if applied soon after infection , but resistance against some therapeutics has already been reported .
it can be expected that alternative approaches will be needed soon , since the antimicrobial resistance is rising .
the resistance of cronobacter strains to higher temperature and osmotic stress during desiccation enables their survival in dried food .
in addition , cronobacter strains have adhesive properties and are able to invade human cells .
the current threat of antibiotic resistance has led to re - evaluation of bacteriophages as antibacterial agents potentially usable for treatment and/or prevention of bacterial infections .
advantages of this approach are the specificity of phages bypassing the collateral damage induced by antibiotics on natural microflora , and the virtually single - administration efficacy of lytic phages .
phage therapy of staphylococcal and enterococcal infections has already been tested in clinical trials , with few reported unwanted side effects independent of the route of application .
studies testing phage cocktails for the treatment of chronic venous leg ulcers and chronic ear infection are currently in progress .
additionally , promising results of animal experiments show that phages might play a role in the biocontrol of pathogens in clinical medicine .
it has been demonstrated in vitro that contamination of powdered milk formulas with cronobacter can be effectively eliminated with selected cronobacter - specific phages . to our knowledge , the potential of phage therapy to control cronobacter infection in animal models of human diseases has not yet been tested ; therefore the aim of our study was to prove the effects of phage therapy on cronobacter - induced urinary tract infection ( uti ) in a murine model .
the bacterial strain used in this study was isolated from a food matrix ( obtained from food research institute , bratislava ) in luria - bertani ( lb ) medium overnight at 37c .
the strain c. turicensis 290708/07 was identified using the biochemical api 20e identification system ( biomerieux , marcy - letoile , france ) and characterized by molecular methods as described in detail by turcovsky et al . .
strain for phage propagation , isolation and examination throughout the study . for in vivo experiments , bacteria were centrifuged ( 6000 g/10 min/4c ) after overnight cultivation and resuspended in phosphate buffer saline ( pbs ) .
the number of colony - forming units was determined by standard plate counts of serial dilutions on lb agar or macconkey agar plates .
phages used in the study were isolated from sewage collected from 2 wastewater treatment plants in petralka and devnska nov ves , bratislava , slovakia ( further in text as p2 , d2 , respectively ) .
pure phage cultures were obtained by repeated isolation from single plaques grown on indicator strain in double agar overlay plates .
a mixture of the 2 phages with the highest lytic activity toward the indicator strain was used in the in vivo experiment .
incubated indicator bacterial lawns were dropped with 10 l ( 10 pfu / ml ) of phage preparation , following incubation at 37c overnight . on the next day , bacterial lawns were examined for lysis zones .
the bacterial strains used in this test contained  10 e. coli strains , 24 enterobacter cloacae strains , 2 citrobacter strains and 7 different salmonella enterica strains .
forty - seven c57bl/6ncrl 1011-week - old female mice were used for the experiment ( anlab ltd . , prague , czech republic ) .
animals were housed in a room with a 12/12 h light dark cycle and had ad libitum access to water and food pellets .
animals were divided into 4 groups as follows : crono ( n=13 ) with c. turicensis transurethral application , crono - phage ( n=14 ) with c. turicensis transurethral application and intraperitoneal phage administration , phage ( n=10 ) with intraperitoneal phage administration and a ctrl group ( n=10 ) as a mock - infected control group .
all experiments were approved by the ethics committee of the institute of molecular biomedicine , comenius university in bratislava , slovakia .
the mice were briefly anaesthetized with isoflurane and instilled via 28 g plastic transurethral catheters ( outer diameter 0.35 mm , inner diameter 0.28 mm ) with a volume of 100 l containing either 110 colony - forming units / ml of c. turicensis or sterile lb medium in the experimental group .
simultaneously , 100 l of either cronobacter  mixture of phages ( 110 plaque - forming units / ml ) , or sterile pbs were administered intraperitoneally .
tissues were homogenized in 300 l of sterile pbs using tissuelyser ii ( qiagen , hilden , germany ) , and aliquots were used for cultivation and biochemical analyses .
all chemicals were from sigma - aldrich ( st louis , usa ) , if not otherwise stated .
advanced glycation end - products ( ages ) as markers of carbonyl stress were determined spectrofluorometrically , using the specific fluorescence of schiff base and amadori products after addition of phosphate buffer saline ( pbs ) at ex=370 nm and em=430 nm directly in homogenate tissues .
thiobarbituric acid reacting substances ( tbars ) as a marker of oxidative damage of lipids were determined as described by ohkawa et al . .
briefly , 20 l of homogenate , 30 l distilled h2o , 20 l of 0.67% thiobarbituric acid ( sigma - aldrich , germany ) and 20 l of glacial acetic acid were mixed in 96-well microtitration plate .
after 45 minutes of incubation at 94c ( mastercycle pro s , eppendorf , hamburg , germany ) , 100 l of n - butanol ( merck , usa ) was added and the absorbance was measured after centrifugation ( 2000 g/10 min/4c ) in the upper phase at 532 nm .
ferric reducing activity of the tissue ( frat ) as a marker of antioxidant status was measured as described by erel et al . .
twenty l of sample was added into 200 l of freshly prepared frat reagent ( 3 mol / l acetate buffer , ph 3.6 ; 10 mmol / l of tripyridyl - s - triazine dissolved in 40 mmo / l hcl ; 20 mmol / l fecl3 ; and 6h2o and h2o in ratio 1:1:1:9 , respectively ) warmed to 37c and measured after 4 min of incubation at 593 nm .
proteins were quantified using the bradford method . as a standard , bovine serum albumin ( fermentas , vilnius , lithuania ) was used and 5 l of tissue homogenates were mixed with 250 l of bradford reagent ( fermentas , vilnius , lithuania ) .
all measurements were done on a sapphire ii instrument ( tecan , grdig , austria ) .
total rna was isolated from tissues using the trireagent ( mrc , cincinnati , usa ) .
concentration and purity of isolated rna was evaluated spectrophotometrically by nanodrop ( thermo fisher scientific , usa ) .
one - step rt real - time pcr with specific primers was used for the relative quantification of gene expression ( quantitect sybr green rt - pcr kit , qiagen , hilden , germany ) .
gtccctttcactcactggcc ; primer r  gagtgcctcttctgccagttc ) and monocyte chemoattractant protein-1 ( mcp-1 ; primer f 
peptidylprolyl isomerase a ( ppia ; primer f  ttcgagctctgagcactgg ; primer r  ccagtgccattatggcgt ) was used as a housekeeping gene .
differences between groups were evaluated with anova and post - hoc student t - test with bonferroni correction of multiple comparison bias .
the bacterial strain used in this study was isolated from a food matrix ( obtained from food research institute , bratislava ) in luria - bertani ( lb ) medium overnight at 37c .
the strain c. turicensis 290708/07 was identified using the biochemical api 20e identification system ( biomerieux , marcy - letoile , france ) and characterized by molecular methods as described in detail by turcovsky et al . .
strain for phage propagation , isolation and examination throughout the study . for in vivo experiments , bacteria were centrifuged ( 6000 g/10 min/4c ) after overnight cultivation and resuspended in phosphate buffer saline ( pbs ) .
the number of colony - forming units was determined by standard plate counts of serial dilutions on lb agar or macconkey agar plates .
phages used in the study were isolated from sewage collected from 2 wastewater treatment plants in petralka and devnska nov ves , bratislava , slovakia ( further in text as p2 , d2 , respectively ) .
pure phage cultures were obtained by repeated isolation from single plaques grown on indicator strain in double agar overlay plates .
a mixture of the 2 phages with the highest lytic activity toward the indicator strain was used in the in vivo experiment .
incubated indicator bacterial lawns were dropped with 10 l ( 10 pfu / ml ) of phage preparation , following incubation at 37c overnight . on the next day , bacterial lawns were examined for lysis zones .
the bacterial strains used in this test contained  10 e. coli strains , 24 enterobacter cloacae strains , 2 citrobacter strains and 7 different salmonella enterica strains .
forty - seven c57bl/6ncrl 1011-week - old female mice were used for the experiment ( anlab ltd . , prague , czech republic ) .
animals were housed in a room with a 12/12 h light dark cycle and had ad libitum access to water and food pellets .
animals were divided into 4 groups as follows : crono ( n=13 ) with c. turicensis transurethral application , crono - phage ( n=14 ) with c. turicensis transurethral application and intraperitoneal phage administration , phage ( n=10 ) with intraperitoneal phage administration and a ctrl group ( n=10 ) as a mock - infected control group .
all experiments were approved by the ethics committee of the institute of molecular biomedicine , comenius university in bratislava , slovakia .
the mice were briefly anaesthetized with isoflurane and instilled via 28 g plastic transurethral catheters ( outer diameter 0.35 mm , inner diameter 0.28 mm ) with a volume of 100 l containing either 110 colony - forming units / ml of c. turicensis or sterile lb medium in the experimental group . simultaneously
, 100 l of either cronobacter  mixture of phages ( 110 plaque - forming units / ml ) , or sterile pbs were administered intraperitoneally .
tissues were homogenized in 300 l of sterile pbs using tissuelyser ii ( qiagen , hilden , germany ) , and aliquots were used for cultivation and biochemical analyses .
all chemicals were from sigma - aldrich ( st louis , usa ) , if not otherwise stated .
advanced glycation end - products ( ages ) as markers of carbonyl stress were determined spectrofluorometrically , using the specific fluorescence of schiff base and amadori products after addition of phosphate buffer saline ( pbs ) at ex=370 nm and em=430 nm directly in homogenate tissues .
thiobarbituric acid reacting substances ( tbars ) as a marker of oxidative damage of lipids were determined as described by ohkawa et al . .
briefly , 20 l of homogenate , 30 l distilled h2o , 20 l of 0.67% thiobarbituric acid ( sigma - aldrich , germany ) and 20 l of glacial acetic acid were mixed in 96-well microtitration plate . after 45 minutes of incubation at 94c ( mastercycle pro s , eppendorf , hamburg , germany ) , 100 l of n - butanol ( merck , usa ) was added and the absorbance was measured after centrifugation ( 2000 g/10 min/4c ) in the upper phase at 532 nm .
ferric reducing activity of the tissue ( frat ) as a marker of antioxidant status was measured as described by erel et al . .
twenty l of sample was added into 200 l of freshly prepared frat reagent ( 3 mol / l acetate buffer , ph 3.6 ; 10 mmol / l of tripyridyl - s - triazine dissolved in 40 mmo / l hcl ; 20 mmol / l fecl3 ; and 6h2o and h2o in ratio 1:1:1:9 , respectively ) warmed to 37c and measured after 4 min of incubation at 593 nm .
bovine serum albumin ( fermentas , vilnius , lithuania ) was used and 5 l of tissue homogenates were mixed with 250 l of bradford reagent ( fermentas , vilnius , lithuania ) .
all measurements were done on a sapphire ii instrument ( tecan , grdig , austria ) .
total rna was isolated from tissues using the trireagent ( mrc , cincinnati , usa ) .
concentration and purity of isolated rna was evaluated spectrophotometrically by nanodrop ( thermo fisher scientific , usa ) .
one - step rt real - time pcr with specific primers was used for the relative quantification of gene expression ( quantitect sybr green rt - pcr kit , qiagen , hilden , germany ) . genes of interest were tumor necrosis factor - alpha ( tnf- ; primer f 
gtccctttcactcactggcc ; primer r  gagtgcctcttctgccagttc ) and monocyte chemoattractant protein-1 ( mcp-1 ; primer f 
peptidylprolyl isomerase a ( ppia ; primer f  ttcgagctctgagcactgg ; primer r  ccagtgccattatggcgt ) was used as a housekeeping gene .
differences between groups were evaluated with anova and post - hoc student t - test with bonferroni correction of multiple comparison bias .
electron microscopy of used bacteriophages p2 and d2 showed that both are members of the podoviridae family , order caudovirales , with 18020 nm head diameter and 6010 nm long non - contractile tail .
host specificity tests were performed on 41 different strains , including salmonella , e. coli , citrobacter and enterobacter cloacae .
neither of these phages was able to infect citrobacter , salmonella or e. coli strains .
phages showed different ability to lyse enterobacter cloacae strains ( p2  25% , d2  38% ) .
cultivation analysis revealed that bacterial colonies were found only in bladders and kidneys of mice that were injected with cronobacter cells .
mean bacterial count in the kidney was 734 cfu and phage therapy decreased the count by 70% ( z=3.06 ; p=0.002 , figure 1a ) . in the bladder the mean bacterial load was cca 900 cfu in both crono and crono + phage groups ( z=0.55 ; p=0.58 ;
no significant differences were found between the phage and crono + phage groups in the number of phage plaques in the kidney ( z=0.32 ; p=0.75 ; figure 1c ) , but were higher in the crono + phage group , although not significant due to high interindividual variability ( z=1.53 ; p=0.13 ; figure 1d ) .
ages in renal cortex as a marker of carbonyl stress were not altered by infection with cronobacter , but were significantly reduced by phage therapy in comparison to the infected mice ( by 57% ; t=2.46 ; p=0.02 ; figure 2a ) .
malondialdehyde as a marker of lipoperoxidation was doubled in the kidneys of infected mice ( t=4.15 ; p=0.0009 ; figure 2b ) and lowered with phage therapy by 39% ( t=2.94 ; p=0.008 ; figure 2b ) .
groups did not differ in frat as a marker of antioxidant status in the renal tissue ( figure 2c ) .
no significant differences were found in any of the analyzed markers of oxidative stress in the bladder ( figure 2d  f ) .
analysis of expression of tnf alpha and mcp-1 revealed that both cytokines were upregulated in the bladder of infected mice ( tnf alpha by 72% ; t=3.02 ; p=0.007 ; mcp-1 by 52% ; t=3.03 ; p=0.007 ) .
these differences were reduced by phage therapy ( tnf alpha by 35% ; t=3.58 ; p=0.002 ; figure 3c ; mcp-1 by 22% ; t=2.53 ; p=0.02 ; figure 3d ) . in the renal cortex only the infection - induced increase in expression of mcp-1 was significant ( by 48% ; t=2.57 ; p=0.02 ; figure 3b ) . the decrease due to phage therapy was significant for tnf alpha ( by 36% ; t=2.78 ; p=0.02 ; figure 3a ) .
although seldom life - threatening , chronic uti is associated with high morbidity and thus represents a frequent problem in primary care .
it even represents a common cause of sepsis in critically ill or otherwise immuno - compromised patients and may be a cause of nonspecific complaints with negative initial urinalysis in the elderly . in most cases , appropriate choice and dosing of antibiotics or chemotherapeutics is sufficient for successful treatment ; however , with increasing incidence of uti caused by resistant strains , the need for alternative options becomes urgent .
host specificity of phages is high and can be narrowed by genetic engineering or by in vitro evolution and selection .
the safety profile seems to be far better in comparison to antibiotics , at least according to already published data .
phage particles do not invade or actively affect eukaryotic cells , but may induce a humoral immune response .
long - term studies dealing with safety of phage administration are , nevertheless , needed .
however , uti in mice is a suitable model for testing the ability of phage therapy to conquer an infection in vivo , mainly due to physiologically sterile environment and a reproducible read - out parameter in the form of renal colonization .
ascending nephritis was partially prevented in our experiment , indicating that the applied phages could be usable in other infection models .
this assumption has to be verified in separate studies , as the conditions in the urinary tract differ from those in other organs or tissues .
the observed 70% reduction of cronobacter colonies in the kidneys 24 hours after a single phage dose is consistent with other similar results observing phage efficacy in the treatment of wound infections .
the ability of phages to survive in the urinary tract 24 hours after administration has already been shown .
there was no difference in cronobacter colonization of the bladder between treated and infected groups .
this might indicate that the kinetics of phages after intraperitoneal administration could be improved by choosing another application route . on the other hand ,
similar to other studies using the transurethral uti model , the variance , especially in the cultivation analysis , is high .
in addition to simple reproduction of the results , a similar confirmatory experiment is planned on larger experimental animals , including rats , dogs and other vertebrates . according to our knowledge
this is the first study showing that phage therapy decreases oxidative stress in urinary tract infection .
it has been shown that phage therapy can attenuate the effects of oxidative burst caused by activated phagocytes . whether the observed reduction in oxidative stress in our experiment is direct or due to reduced inflammation
the clinical significance of this antioxidative effect is questionable , but might gain importance in chronic infections where oxidative damage of tissues plays an important role in the pathogenesis .
data from the presented study show that the phage therapy was effective in the prevention of renal infection in a model of cronobacter - induced urinary tract infection in mice . inflammation and oxidative stress were reduced in the kidney and partially reduced in the bladder .
future studies should focus on long - term effects and safety , as well as on the potential use of phage therapy of cronobacter infections in models of enterocolitis , meningitis or sepsis .
 summarybackgroundcronobacter spp . is an opportunistic pathogen causing rare but dangerous cases of meningitis , sepsis and urinary tract infection . 
 phage therapy overcomes antibiotic resistance and represents an alternative approach to standard antimicrobial treatment . 
 there are no published studies on the use of phages against cronobacter spp . in vivo . 
 the aim of our study was to prove the effects of isolated cronobacter - specific phages on renal colonization in a model of urinary tract infection in mice.material/methodsurinary tract infection was induced by transurethral application of cronobacter turicensis ( 1011 cfu / ml ) . 
 simultaneously , isolated cronobacter - specific phages were administered intraperitoneally ( 1011 pfu / ml ) . 
 after 24 hours , kidneys and bladder were collected and used for cultivation and analysis of gene expression and oxidative stress markers.resultsphage therapy reduced the number of cronobacter colonies in the kidney by 70% . 
 higher levels of malondialdehyde were reduced by phage therapy without affecting the antioxidant status . 
 the expression of pro - inflammatory cytokines tumor necrosis factor - alpha and monocyte chemoattractant protein-1 increased by the infection and was attenuated by phage therapy.conclusionsphage therapy proved effective in the prevention of ascending renal infection in a murine model of urinary tract infection . 
 long - term effects and safety of the treatment are currently unknown . 
 further studies should test phage therapy in other cronobacter infection models . 