
 staphylococcus aureus is one of the most significant bacterial pathogens and can cause diseases ranging from minor and surgical site infections   to potentially life - threatening endocarditis [ 24 ] and bacteraemia [ 58 ] .
it is a particular problem in hospitals as a consequence of the emergence and dissemination of multidrug - resistant forms such as methicillin - resistant s. aureus ( mrsa ) , vancomycin intermediate susceptibility s. aureus ( visa ) , and heterogenous visa ( hvisa ) .
the prevalence of these antibiotic resistant forms means that the discovery of novel chemotherapeutic agents to combat these pathogens is of key importance [ 9 , 10 ] .
the lantibiotics ( lanthionine - containing antibiotics ) are a group of posttranslationally modified antimicrobial peptides of which nisin and lacticin 3147 are among the most extensively investigated .
a number of lantibiotics have been noted to exhibit potent antimicrobial activity against staphylococci of clinical relevance . in agar
diffusion assays , the type i lantibiotics epidermin , pep5 , epicidin k7 , and epilancin 280 display impressive levels of activity against coagulase negative staphylococci ( cns ) , and it has been suggested that their potential could be exploited to prevent the colonization of medical devices .
nisin has also been shown on several occasions to possess significant anti - staphylococcus activity . when tested against 20 mrsa strains
, one study revealed that the minimum inhibitory concentration ( mic ) of nisin a ranged between 1.5 and 16  mg / l , while a more recent investigation revealed mics of 0.54.1 
the in vitro activity of other forms of nisin ( nisin f , q , and z ) against mrsa has also recently been highlighted .
the in vivo activity of a number of lantibiotics against staphylococci has also been investigated .
the effectiveness of the epidermin - like mutacin b - ny266 was tested on mice infected by intraperitoneal ( ip ) injection with 3.1  10  cfu of s. aureus smith / mouse . immediately after injection ,
mutacin b - ny266 was administered , also via the ip route , at concentrations of 110  mg / kg of mouse and was found to be protective .
more recently , it has been established that microbisporicin , in addition to having potent in vitro activity ( mic  0.13  g / ml ) , effectively controls murine septicemia caused by s. aureus in female cd-1 mice ( 2325  g ) .
the mice were infected via the ip route with 1  10  cfu of s. aureus smith 819 atcc 19636 in 0.5 mls gastric hog mucin .
microbisporicin was then administered intravenously or subcutaneously ( sc ) 1015  mins after infection at final concentrations of 1015 
the effective dose 50 ( ed50 ) of microbisporicin was found to be 2.1  mg / kg regardless of whether it was administered via iv or sc .
ed50 values were determined on the bases of survival of the mice to the seventh day .
higher doses of microbisporicin ( 200  mg / kg ) led to the survival of all animals treated and were nontoxic .
nisin f effectively controlled the mrsa strain , s. aureus k , in immunocompromised wistar rats following the introduction of 4  10  s. aureus cells into the nostrils of the rats for 4 consecutive days before treating with 8192 arbitrary units ( au ) of nisin f intranasally for the subsequent 4 days .
in contrast , however , when 1 x10  s. aureus xen 36 cells were injected intraperitoneally , the administration of a lower concentration of nisin f ( 640 au ) after 4 hours succeeded in inhibiting the growth of the pathogen for only 15 minutes after which time the pathogen reemerged . finally , short- and long - term in vivo studies with mersacidin established that this lantibiotic quite effectively inhibited mrsa introduced intranasally into immunocompromised ( hydrocortisone - treated ) balb / c mice . for the short term trial ,
the mice were infected on days 5 , 7 , and 9 with 3  1010  cfu of the s. aureus strain .
the mice were then treated intranasally with mersacidin ( 1.66  mg / kg per treatment ) twice a day on days 10 , 11 , and 12 . for the longer trial ,
the mice were challenged with s. aureus on days 5 , 7 , 9 , 30 , 32 , and 34 and subsequently treated with mersacidin on days 35 , 36 , and 37 . in both cases the mersacidin treatment successfully inhibited mrsa - induced rhinitis .
notably , a comparison of the in vitro and in vivo activity of mersacidin against a number of mrsa strains indicates that mersacidin more effectively inhibits s. aureus   in vivo .
these peptides are active as a consequence of the synergistic activity of two lanthionine - containing peptides [ 22 , 23 ] .
lacticin 3147 has been found to exhibit potent in vitro activity against a range of pathogenic bacteria including clostridium difficile , vancomycin - resistant enterococci , propionibacterium acne , penicillin - resistant pneumococcus , and streptococcus mutans [ 14 , 2426 ] as well as pathogenic mycobacteria such as mycobacterium   avium subsp paratuberculosis and mycobacterium tuberculosis h37ra .
of greatest relevance to this study is the fact that lacticin 3147 possesses anti - staphylococcus activity .
the lantibiotic itself , when incorporated into a teat seal , protects against s. aureus - associated bovine mastitis [ 28 , 29 ] , while use of a lacticin 3147-producing lactococcus lactis dpc 3251 within a teat dip inhibits s. aureus both in   vitro and also in vivo .
the in vitro activity of lacticin 3147 against clinical mrsa isolates has also been established with mics ranging from 1.9 to 15.4  mg / l . despite lacticin 3147 being one of the most extensively studied lantibiotics , its ability to control a systemic infection caused by s. aureus , or indeed any other pathogen , has not been investigated . here
we address this issue using balb / c mice infected via the ip route with s. aureus xen 29 , a strain of methicillin sensitive s. aureus ( mssa ) that has been genetically modified to express the photorhabdus luminescens lux genes to facilitate in vivo imaging .
the ability of subcutaneously administered lacticin 3147 to control infection was assessed by in vivo imaging and microbiological analysis of the organs of sacrificed animals .
the in vitro activity of lacticin 3147 and vancomycin ( employed as a positive control ) against s. aureus xen 29 was assessed through mic determination assays carried out in triplicate as described previously   with purified lacticin 3147 , prepared via hplc , again as described previously .
s. aureus xen 29 ( derived from the parental pleural isolate s. aureus 12600 ; xenogen corporation , almeda , ca ) possesses a copy of the modified luxabcde operon of p. luminescens integrated at a single site on the chromosome .
s. aureus xen 29 was cultured overnight in brain heart infusion ( bhi ) broth aerobically at 37c from an isolated colony growing on bhi agar containing 200  g / ml kanamycin . on the day of the trial ,
the overnight culture was subcultured ( 1  :  100 dilution ) into fresh bhi and grown to log phase ( od600 nm of 0.5 ) .
this culture was diluted to facilitate the ultimate administration of the culture in the form of a 1  10  cfu/100  l dose in 0.5% hog gastric mucin ( sigma aldrich ) .
mice were fed a standard rodent diet ad libitum and all animal studies were approved by the animal experimentation ethics committee .
13 balb / c female mice ( 7 weeks old , 15  g   2  g in weight ) were divided into 3 groups ( a , b , c ; n = 3 , 5 , and 5 , resp . ) . at t0 mice in groups
a  c received the 1  10 cfu dose ( 100  l volume ) via the ip route in 0.5% gastic hog mucin ( sigma aldrich ) . at t1.5 
mg / kg of ltn and 43.8  mg / kg of ltn , corresponding to 30.76  mm lacticin 3147/kg ) in a single dose and a second dose at t3hrs ( 25.425  mg / kg ltn and 21.90  mg / kg ltn ; 15.382  mm lacticin 3147/kg ) .
vancomycin ( 50  mg / kg ; 33.6  mm / kg ) was administered at t1.5  hrs and at t3hrs to the mice in group b while the mice in group a received pbs ( once ) as a control .
both antimicrobials and pbs were adminisitered subcutaneously in 100  l doses . in vivo imaging
was carried out at two time points that is , 3 hours and 5 hours postinfection .
mice were anaesthetized for bioluminescent imaging via the inhalation of aerosolized isoflurane mixed with oxygen .
the mice were then transferred to the ivis chamber ventral side up , and luminescence was measured over a 3-to-5  mins exposure time .
the imaging system measures the number of photons reaching each detector of the charged - couple device camera , and the ivis software translates these data into false color images that display regions of intense luminescence with red , moderate luminescence in yellow and green and mild luminescence in blue .
the images contained herein are photographic images with an overlay of bioluminescence that uses this computer - generated color scale .
these organs were mechanically disrupted and serial dilutions made which were subsequently plated in 100  l volumes on tsa  kan plates in order to enumerate the staphylococci present in each organ .
luminescent images were quantified with ivis imaging software . the total flux ( number of photons / s / cm )
was calculated by a user defined area ( region of interest ) covering the infection site .
the flux was averaged across all mice from each respective group . the reduction in luminescence
was quantified and represents a comparison with the luminescence from mice administered phosphate - buffered saline control at the same time point .
the mean and standard error of the mean ( sem ) of the luminescence at the final time point and bacterial counts for the mice were calculated for all groups .
differences in the bioluminescence and bacterial counts analyzed through a one - way analysis of variance , followed by the holm - sidak posttest ( sigma stat , version 3.5 ) .
the ability of subcutaneously injected lacticin 3147 to control a systemic s. aureus infection following the introduction of the pathogen into the murine peritoneal cavity was investigated .
this involved in vivo imaging to detect levels of light emitted by the pathogen within mice and through the postmortem microbiological analysis of organs .
negative and positive controls were employed in the form of mice treated with pbs and the glycopeptide antibiotic vancomycin , respectively .
the target strain s. aureus xen 29 is a methicillin sensitive isolate which has been employed previously to facilitate an investigation of acute in vivo infections [ 3236 ] .
prior to commencement of the study , the in vitro sensitivity of xen 29 to lacticin 3147 was assessed .
l for vancomycin and lacticin 3147 , respectively ( table 1 ) . for in vivo studies , mice received a dose of 1  10 cfu and , 1.5  hrs postinfection , were administered lacticin 3147 ( 50.85 
mg / kg of ltn and 43.8  mg / kg of ltn ) , vancomycin ( 50  mg / kg ) , or pbs . at t3hrs ,
the mice were subject to ivis imaging , and second doses of lacticin 3147 ( 25.425  mg / kg and 21.90  mg / kg ) and vancomycin ( 50  mg / kg ) were administered to the relevant mice .
ivis analysis of the progression of the s. aureus xen 29 infection showed that the pathogen spreads systemically and eventually also occupies the thoracic cavity in mice injected with pbs 5  hrs ( t5hrs ) after injection of the pathogen .
a significant ( p = 0.000116 ) reduction in the rlu measurements corresponding to the thoracic region of the lacticin 3147 treated group was evident when compared to that of the pbs ( negative ) control group ( figure 1 ) at this time point highlighting the ability of the lantibiotic to prevent systemic spread of the s. aureus xen 29 infection .
in contrast , lacticin 3147 does not significantly reduce rlu values corresponding to the peritoneal cavity relative to the control .
it may be that lacticin 3147 is deficient in penetrating the peritoneal cavity ( figure 1 ) . to further ascertain lacticin 3147 efficacy , culture - based analysis of staphylococcal levels in the organs
lacticin 3147 treatment resulted in a significant reduction ( p < 0.05 in all cases ; figure 1 ) in pathogen numbers in the liver , spleen , and kidneys of the mice treated relative to the pbs - treated controls ( figure 1 ) . as expected , vancomycin brought about a significant reduction in s. aureus levels relative to the pbs - treated controls as determined by both bioimaging and culture - based analysis .
notably , numbers of s. aureus in the spleens of lacticin 3147- and vancomycin - treated mice were statistically indifferent . however , vancomycin treatment more successfully lowered s. aureus numbers in the liver and kidneys . while both lacticin 3147 and vancomycin bind lipid ii , [ 10 , 37 , 38 ] differences exist with respect to their mechanism of action .
vancomycin binds to the c - terminal d - ala - d - ala motif of the pentapeptide of lipid ii   whereas , on the basis of similarities between ltn and mersacidin , it is proposed that lacticin 3147 binds to the sugar phosphate head group of lipid ii . furthermore , lacticin 3147 is also capable of forming pores in the membranes of target cells [ 37 , 38 ] . it should be noted that while similar mg / kg doses of lacticin 3147 and vancomycin were employed in this study , our in vitro investigations established that vancomycin is 19 times more potent than lacticin 3147 against xen29 ( mic values ; 1.013  mg / l and 19.1  mg /
thus the dose of vancomycin administered in vivo corresponded to 100-fold that of the in vitro mic whereas lacticin 3147 was administered at a level 8-fold greater than its in vitro mic .
this may explain the enhanced ability of vancomycin with respect to clearance of xen 29 from the peritoneal cavity .
this is the first occasion upon which the impact of lacticin 3147 against a systemic infection has been assessed and thus it is also the first instance of its administration subcutaneously .
it may be that lacticin 3147 can not travel to the peritoneum to eradicate the infection but can prevent the spread of infection throughout the blood stream . as stated previously , mersacidin has successfully been shown to inhibit a systemic mrsa infection in mice when administered via the subcutaneous route . however , mersacidin is a one - component lantibiotic and is also globular which may provide facile delivery through the skin .
it , like vancomycin , is quite a small peptide with a molecular weight of 1 , 825  da .
lacticin 3147 consists of 2 peptides with molecular weights of 3305  da ( ltn ) and 2847  da ( ltn ) , and it may be that the larger size of the individual peptides or a specific difficulty relating the transport of one of the components to the peritoneal cavity may be an issue .
mutacin b - ny266 has also been shown to protect against s. aureus in the peritoneum , but this lantibiotic was administered intraperitoneally , and thus transfer to the site of infection was not an issue . in conclusion
, here we have provided evidence that lacticin 3147 could be employed to treat systemic infections .
both culture- and bioluminescence - based analyses reveal that the lantibiotic significantly reduces numbers of the s. aureus xen29 relative to the negative control by preventing the dissemination of the pathogen .
although these results are more promising than those described when nisin f was employed in a similar manner ( 19 ) , differences with respect to the strains of s. aureus employed , concentrations of lantibiotic , and other factors mean that a direct comparison of outcomes is not possible .
while further investigations are required , over longer periods of time , to more extensively assess the clinical potential of lacticin 3147 , it is worth noting that lacticin 3147 possesses many physicochemical properties that favour its in vivo application .
these include excellent activity over a broad ph range , especially at physiological ph ( ph 7 ) , the absence of cytotoxicity towards eukaryotic cells , its broad spectrum of activity at nanomolar concentrations , its alternative mode of action , and the presence of ( methyl)lanthionine bridges that confer structural rigidity to lantibiotics and reduce proteolytic attack .
these properties , accompanied by its ability to inhibit a systemic s. aureus infection , make lacticin 3147 a promising candidate for potential applications in human medicine .
 the objective of this study was to investigate the in vivo activity of the lantibiotic lacticin 3147 against the luminescent staphylococcus aureus strain xen 29 using a murine model . 
 female balb / c mice ( 7 weeks old , 17  g ) were divided into groups ( n = 5 ) and infected with the xen 29 strain via the intraperitoneal route at a dose of 1  106  cfu / animal .   
 after 1.5  hr , the animals were treated subcutaneously with doses of phosphate - buffered saline ( pbs ; negative control ) or lacticin 3147 . 
 luminescent imaging was carried 3 and 5 hours postinfection . 
 mice were then sacrificed , and the levels of s. aureus xen 29 in the liver , spleen , and kidneys were quantified . 
 notably , photoluminescence and culture - based analysis both revealed that lacticin 3147 successfully controlled the systemic spread of s. aureus in mice thus indicating that lacticin 3147 has potential as a chemotherapeutic agent for in vivo applications . 
1. Introduction
2. Materials and Methods
3. Results/Discussion