Patent Application: US-201314421762-A

Abstract:
materials and methods for preventing and treating anastomotic leaks are disclosed . data establishes that pathogenic microbes interfere with establishing epithelial cell barriers in anastomoses and , more generally , with the reconnection of any two portions of like or different tissues comprising epithelia . suitable prophylactic and therapeutic composition comprise , e . g ., a phosphorylated high molecular weight polyethylene glycol compound .

Description:
the most feared complication following intestinal resection is anastomotic leakage . in high risk areas ( i . e ., esophagus / rectum ) where neoadjuvant chemoradiation is used , the incidence of anastomotic leaks remains unacceptably high ( about 10 %), even when performed by specialist surgeons in high - volume centers . the experiments disclosed herein tested the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become transformed in vivo to express a tissue - destroying phenotype . a model of anastomotic leak was developed in which rats were exposed to pre - operative radiation as in cancer surgery , underwent distal colon resection and then were intestinally inoculated with pseudomonas aeruginosa , a common colonizer of the radiated intestine . results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (& gt ; 60 %; p & lt ; 0 . 01 ) when colonized by p . aeruginosa , compared to radiated tissues alone ( 0 %). phenotype analysis comparing the original inoculating strain ( mpao 1 , termed p1 ) and the strain retrieved from leaking anastomotic tissues ( termed p2 ) demonstrated that p2 was altered in pyocyanin production and displayed enhanced collagenase activity , high swarming motility , and a destructive phenotype against cultured intestinal epithelial cells ( i . e ., apoptosis , barrier function , cytolysis ). comparative genotype analysis between p1 and p2 revealed a single nucleotide polymorphism ( snp ) mutation in the mext gene that led to a stop codon resulting in a non - functional truncated protein . replacement of the mutated mext gene in p2 with mext from the original parental strain p1 led to reversion of p2 to the p1 phenotype . no spontaneous transformation was detected during 20 passages in tsb media . also disclosed herein is a virulence - suppressing compound , peg / pi , which prevented p . aeruginosa transformation to the tissue - destructive phenotype and prevented anastomotic leak in rats . this work demonstrates that in vivo transformation of microbial pathogens to a tissue - destroying phenotype may have important implications in the pathogenesis of anastomotic leak . despite decades of refinements in technique , the development of ergonomic stapling devices , and the emergence of high - volume specialized surgeons working in high - volume centers , anastomotic leaks remain a real and present danger to patients . this is particularly evident in high risk areas of the gastrointestinal tract such as the esophagus and rectum , where the incidence of leak persists at 10 % and paradoxically appears to be increasing in incidence . results from the present study extend the observations by cohn nearly 60 years ago [ 4 ] and later confirmed by schardey in 1994 [ 5 ] and introduce a model which conforms to the molecular koch &# 39 ; s postulates that microbial phenotype , rather than microbial presence alone , plays a role in the tissue disruption that characterizes anastomotic leak . placing this finding in the context of the practice of high - risk gastrointestinal surgery is important as surgeons are operating on more complex and older patients who often have received adjunctive chemotherapy and radiation either before or after intestinal resection . the promiscuous use of antibiotics may also contribute to colonization by more pathogenic strains of bacteria at anastomotic sites , that when properly cued by host elements at the site of tissue injury , can become transformed to express a tissue destructive phenotype . such dynamic microbial virulence regulation that may be dependent on both spatial and regional context may explain , in part , the relative difficulty in predicting those at risk for anastomotic leak . the ability of p1 to transform to p2 and acquire high - swarming and enhanced ability to degrade collagen are undoubtedly important assets for p . aeruginosa to acquire as a mechanism to cause full thickness loss of anastomotic integrity . the data generated by the present study , however , are insufficient to determine the causative link between the p2 phenotype and anastomotic leak per se . although anastomotic tissues , with or without exposure to radiation , induced the p2 phenotype , p2 may only exert its full potential to cause leakage when it is further cued by radiated tissues , a hypothesis that currently cannot be tested in vitro . evidence for this is our recent observation that p . aeruginosa virulence is activated by soluble factors released from radiated intestinal epithelial cells . there are , however , major technical challenges to causally linking the p2 phenotype to anastomotic leak . p2 was introduced into the cecum of rats with anastomotic construction but without radiation and it did not cause anastomotic leak . in contrast to radiated rats , non - radiated rats maintain a normal microbiota and intact mucus layer , which may shield against the virulence effects of the p2 phenotype . additionally , once p2 is afforded the opportunity to adhere to anastomotic tissues in radiated mice , contact - dependent host tissue factors may induce transcriptional changes in p2 that then confer an even more invasive tissue - destroying phenotype . p . aeruginosa strain mpao1 was selected for the studies disclosed herein based on our work tracking dynamic virulence expression in p . aeruginosa in response to environmental stimuli and host tissue factors . at baseline , mpao1 is a low virulence expressing strain that in general requires exposure to local environmental cues and host tissue factors to cause severe injury such as lethality in worms ( c . elegans ) and gut - derived sepsis mice . as p . aeruginosa is one of the most common pathogens to colonize the gut following radiation , anastomotic leak was modeled with a microbial strain that is well - characterized , of low virulence potential at baseline , and with a publically available transposon library . the discovery of the involvement of mext in the p1 to p2 transition is intriguing . mext has been described as a mutational “ hot spot ” in p . aeruginosa [ 16 , 32 ]. mext belongs to a lysr - type transcriptional regulator whose expression determines the global transcription profile , including the mexef - oprn efflux pump , quorum - sensing system , and type iii secretion system . strains of p . aeruginosa with functional mext display nfxc - type antibiotic resistance that is characterized by increased resistance to chloramphenicol and fluoroquinolones , and the same profile was observed in the mpao1 strain ( p1 ). among other phenotypic characteristics of nfxc - type resistant p . aeruginosa strains is abolished swarming motility and attenuated pyocyanin , elastase , and rhamnolipids production . strain mpao1 ( p1 phenotype ) displayed characteristics similar to that described for nfxc - type strains , however its pyocyanin production , although indeed delayed in liquid media , was enhanced on agarized media . overall , nfxc - type strains are considered to display attenuated virulence . the known conversion between nfxc and non - nfxc strains is associated with the insertion of an 8 by sequence ( cggccagc ; seq id no : 5 ) in the mext gene . the sequence of the mpao1 genome harbors the insertion of the 8 by sequence in the mext gene that determines its p1 phenotype , similar to that described by kohler as the nfxc - type mutation [ 35 ]. the conversion of p1 to p2 , however , was not accompanied by the deletion of the 8 by insertion , but by the mutation c → a that reverted mext back to the non - functional state , indicating that several mechanisms of transformation exist . that the exact same mutation ( i . e ., c → a ) that reverted mext back to the non - functional state emerged both in vivo ( at the site of anastomosis ) and ex vivo ( co - incubation with anastomotic tissues ) appears to indicate the presence of an inducing factor ( s ) originating from the host , the microbe , or their interaction . paradoxically , the non - nfxc strain harboring the 8 by insertion is considered to be wild - type ( wt ) and the nfxc - type strain with a functional mext is considered to be mutant [ 35 ]. given that it would seem incongruous that a gene would emerge whose functionality is initially blocked , we considered that strains with the 8 by insertion to be the mutant , with the strain lacking this insertion identified as the wild - type strain . without wishing to be bound by theory , strong selection for the snp mutation at the site of the anastomosis as an adaptive response to local microenvironmental conditions present as a result of tissue injury and radiation , provides a mechanistic explanation of the switch from wt p1 to p2 . the possibility of spontaneous conversion of p1 to p2 was ruled out in the study described herein by subculturing both the p1 and p2 strains for 20 passages in rich nutrient tsb media . no p2 phenotype was detected in p1 populations and no p2 spontaneously reverted to p1 among 100 colonies selected for analysis at each point . therefore , the p1 to p2 conversion in the cecum ( 5 - 10 %) and its high rate of conversion at anastomotic sites (& gt ; 90 %) are both a function of the in vivo environment per se as well as the tissue injury , which obviously plays a more prominent role . it is important to keep in mind that the cecum of post - operative rats remains a stressed environment as the host has undergone general anesthesia and major intestinal surgery and metabolically is still in recovery phase . therefore , local cues within the cecum , albeit less when compared to the anastomotic site , contribute to the induction or selection for the p2 phenotype as a result of the effects of systemic host stress . data from the present study support an evolving principle in microbial pathogenesis that the gut represents a unique niche in which there is a spatialized ecologic feedback that leads to emergent traits among its colonizing microbes . in the ever - changing chaos of this complex ecosystem , it is easy to imagine that a patient being prepared for intestinal cancer surgery with purgatives , antibiotics , radiation , and chemotherapy , who then undergoes a traumatic tissue injury while exposed to healthcare - associated pathogens , will harbor microbes whose virulence might be triggered by unique host and physico - chemical cues . the discovery of the p2 snp confirms the importance of host factors as agents that play a key role in this response . therapies that seek to target microbial virulence expression may have an ecological advantage over antibiotics that indiscriminately eliminate all potential pathogens and the protective microbiota with the real risk of the emergence of resistance . host factors are expected to play a key role in microbial virulence expression leading to altered epithelial barriers and impaired anastomotic healing wherever anastomoses are found . therapies that seek to target microbial virulence expression are expected to have an ecological advantage over antibiotics that indiscriminately eliminate all potential pathogens , and the protective microbiota , with the real risk of the emergence of resistance . this disclosure provides data that facilitates unraveling the molecular details by which colonizing pathogens express enhanced virulence during surgical injury and contribute to anastomotic leak . more generally , the disclosure reveals mechanisms by which radiation and tissue injury increase susceptibility to infections . bacterial strains . pseudomonas aeruginosa strain mpao1 obtained from the transposon mutant library at the university of washington was used for initial inoculation in rats and is herein designated as the p1 strain . the transformed strain harvested from leaking rat anastomoses was designated a p2 strain as it is derived from the original mpao1 strain ( see results below ). p1 and p2 strains were used in the comparative in vitro experiments . for each experiment , strains were directly cultured from a 10 % glycerol stock stored at − 80 ° c . onto tryptic soy broth ( tsb ) agarized plates , and incubated at 37 ° c . ; cells growing overnight were used in all experiments according to the respective design . rat model of colorectal anastomotic leak . all experiments were approved by the institute for animal care and use committee at the university of chicago . all studies involving mice conformed to the animal welfare act and nih guidelines for the care and use of animals in biomedical research and with the university of chicago carlson veterinary guidelines . adult , male wistar rats 300 - 350 g ( charles river laboratory ) were used for all experiments . animals were allowed unrestricted access to rat chow and tap water throughout the experiments . in order to mimic the clinical practice of surgery for rectal cancer , rats were subjected to pre - operative fractionated pelvic radiation followed by a low colorectal resection and anastomosis . prior to irradiation rats were sedated ( 40 - 80 mg / kg ketamine , 5 - 10 mg / kg xylazine ; intraperitoneal injection , ip ), and then placed in the supine position beneath the radiation cone . a total of 25 gy of radiation , fractionated over 5 consecutive days ( 5 gy per day ; 1 . 47 gy per minute ), was delivered to the sigmoid colon and rectum using a phillips rt250 x - ray generator . all other abdominal organs were excluded from the radiation field using a lead shield . one week after the last day of irradiation , rats were subjected to a laparotomy using aseptic technique and a 0 . 5 cm segment of colon at the peritoneal reflection was resected and an end - to - end rectosigmoid anastomosis was performed using 13 interrupted 6 - 0 prolene sutures . after anastomosis formation , integrity was confirmed in all cases using a 5 ml saline rectal enema . in order to mimic nosocomial bowel contamination by pseudomonas aeruginosa , an overnight culture of strain mpao1 ( 200 μl of 10 7 cfu in 10 % glycerol ) was directly injected into the cecum with a 25 - gauge needle . the abdomen was closed in 2 layers using 4 - 0 vicryl . four groups of rats were studied : rats subjected to resection and anastomosis only ( group i ), rats subjected to resection and anastomosis + cecal p . aeruginosa ( group ii ), rats subjected to preoperative radiation + resection and anastomosis ( group iii ), and rats subjected to preoperative radiation + resection and anastomosis + cecal p . aeruginosa ( group iv ) ( fig1 a ). on postoperative day 6 , all animals were euthanized and the anastomotic site evaluated for gross leakage using a 5 ml rectal methylene blue enema followed by excision of the cecum and anastomotic segment for microbial and histologic examination . prevention of p . aeruginosa - mediated anastomotic leak with topical phosphate + peg ( peg / pi ). 5 % peg 15 - 20 dissolved in 25 mm potassium phosphate buffer , ph 6 . 0 , was administered via rectal enema ( 5 ml ) to the anastomotic suture site at the end of the surgery . histology . for histological evaluation , a 5 mm × 5 mm segment of tissue centered at the anastomotic suture line was removed and fixed in formalin overnight at 4 ° c . each tissue segment was then embedded in paraffin with the suture line mounted vertically within the block , cut into 5 μm sections , and stained with hematoxylin and eosin . light microscopy was performed using a zeiss axioskop and images where captured using a zeiss axiocam digital color camera ( 1 . 25 × magnification ). scanning electron microscopy ( sem ). to prepare for imaging with sem , tissues were dissected into ice cold pbs , transferred to 4 % paraformaldehyde solution ( usb 19943 ), and gradually dehydrated in 25 % ethanol - pbs ( i . e ., etoh - pbs ), 50 % etoh - pbs , 75 % etoh - pbs , 90 % etoh - pbs , and 100 % etoh for 40 minutes per each step . the samples were then transferred to 50 % etoh - hmds ( hexamethyldisilazane ted pella 18605 ) for 1 hr and then 100 % hmds for an additional hour . next , samples were transferred to freshly prepared 100 % hmds and maintained overnight in the hood to ensure evaporation . samples were then fixed to a carbon stubs ( ted pella 16111 - 9 , specimen mounts , aluminum , 9 mm high , ted pella carbon tape 9 mm , 16084 - 3 ), sputter coated with 80 % pt / 20 % pd to 12 nm with cressington sputter coater 208hr , and viewed in fei nova nano sem200 . wound healing assay . for wound healing experiments , iec - 18 cells ( atcc , cat . no . crl - 1589 ) were seeded onto collagen - coated plastic p35 dishes and grown to confluent monolayers . monolayers were scratched with a 10 μl pipette tip , incubated for 1 hour at 37 ° c ., and the initial wound width was then measured . 200 μl of p . aeruginosa strains ( mpao1 - p1 or mpao1 - p2 , od = 0 . 5 ) were added to culture dishes followed by incubation for 24 hours at 37 ° c ., and the wound width was then re - measured . for experiments involving peg / pi treatment , cell medium was removed from wounded cells after 1 hour of incubation and replaced with 2 ml of 5 % peg dissolved in dmem media supplemented with 25 mm phosphate buffer , ph 6 . 0 ) and incubated for 1 hour at 37 ° c ., after which media was replaced with antibiotic - free dmem media followed by bacterial inoculation as described . the values were expressed as the percentage of the initial wound healed . ldh release . to quantitatively measure cell lysis , the amount of lactate dehydrogenase ( ldh ) released from the cells was measured after 24 hours using cytotox 96 cytotoxicity assay ( promega , madison , wis .). caenorhabditis elegans killing assay . the c . elegans assay was performed as previously described [ 10 , 11 ] with slight modifications to include a shorter pre - fasting procedure that made c . elegans less susceptible to infection . briefly , synchronized l4 - young adult nematodes were transferred from e . coli op50 stock plates onto plain agarized plates , followed by a second transferring onto new plain agarized plates ( 60 mm diameter , falcon ). next , 1 ml of 100 μg / ml kanamycin was poured on the agar surface , and after 3 hrs worms were re - transferred to experimental p . aeruginosa lawns ( mpaop1 or mpao1 - p2 ) grown on ngm low phosphate agarized media ( agar 17 g / l ( fisher ), peptone 2 . 5 g / l ( sigma ), cholesterol 5 mg / l ( sigma ), nacl 3 g / l , mgso 4 1 mm , cacl 2 1 mm , ampicillin , 40 μg / ml ). plates with p . aeruginosa were incubated overnight at 37 ° c ., adjusted to room temperature for 1 hr , seeded with 5 pre - starved worms in 5 replicates per experiment performed , and incubated at 23 ° c . mortality of worms was then followed dynamically for 60 hr . random amplified polymorphic dna fingerprint analysis . to verify that the p . aeruginosa strain recovered from the tissue of our experimental animals was of a similar genetic background as the stock lab strain mpao1 , random amplified polymorphic dna ( rapd ) pcr fingerprinting was used as previously described [ 12 ]. dna was isolated from the stock pao1 strain and p . aeruginosa strain recovered from the anastomoses of experimental animals using an easy - dna kit ( invitrogen , carlsbad , calif .). primers 208 ( 5 ′- acggccgacc - 3 ′; seq id no : 6 ) and 272 ( 5 ′- agcgggccaa - 3 ′; seq id no : 7 ), nucleotides producing reproducible polymorphisms with p . aeruginosa , were used for pcr . collagenase assay . collagenase activity was assessed using an enzchek gelatinase / collagenase assay kit ( molecular probes , eugene , oreg .). p . aeruginosa strains were grown overnight in liquid tsb media and then diluted 1 : 100 . for the assay , 180 μl of diluted bacteria in liquid tsb was added to 20 μl of collagen substrate ( 100 μg / ml ; dq collagen , type i from bovine skin , fluorescein conjugate ; dq collagen , type iv from human placenta , fluorescein conjugate ). the negative control consisted of 180 μl tsb only added to 20 μl of collagen substrate . the reaction was measured every hour for 5 hours at an absorbance of 495 nm with a fluorescence microplate reader ( fl x800 , bio - tek instruments inc ), where the increase in fluorescence measured is proportional to proteolytic activity . values obtained for negative controls were subtracted from experimental samples to account for background fluorescence . all experiments were carried out in quintuplicate . at each time point , the od of each sample was measured ( at 600 nm ) in order to normalize to the amount of bacteria in each sample . apoptosis / necrosis assay . rat intestinal epithelial iec - 18 cell were grown to a full confluence on glass bottom culture dishes ( mattek ) in dulbecco modified essential medium ( dmem ) supplemented with 5 % fetal bovine serum , 1 % penicillin / streptomycin ( gibco ), and 0 . 01 u / ml insulin . then medium was replaced by antibiotic - free / fbs - free dmem medium , and iec - 18 monolayers were infected with either p1 or p2 to reach final concentration of 1 × 10 6 cfu / ml . cell were incubated for 3 hrs at 37c °, 5 % co 2 , followed by analysis for apoptosis and necrosis using apoptic & amp ; necrotic & amp ; healthy cells quantification kit ( biotium , inc .). images were obtained with axiovert 35 ( zeiss , germany ) fluorescent microscope . semi - quantitative analysis was performed by counting apoptotic and necrotic cells , and the counts were normalized to the number of nuclei stained by dapi . four fields of about 100 cells imaged from 4 independent dishes / group were included in the quantitative analysis . tight junction assay . iec - 18 monolayers were prepared and infected as described above . after 3 hrs of co - incubation with p1 or p2 , iec - 18 were washed twice with sterile pbs and fixed in 4 % paraformaldehyde for 20 min at room temperature followed by pbs washes 3 times . non - specific binding was blocked with blocking solution ( 1 % bsa , 0 , 1 % triton x - 100 in pbs ) for 30 min . tight junctions were labeled with rabbit anti - zo - 1 antibodies ( 1 : 250 ) ( invitrogen ) overnight at 4 ° c . cells were washed three times with pbs and incubated for 1 h at rt with secondary anti - rabbit antibodies conjugated to alexa fluor 488 ( 1 : 500 ) followed by pbs washes . cells were visualized using a leica dmire2 fluorescence microscope , sp2 laser scanning confocal ( leica microsystem , mannheim , germany ). swarming motility . medium for swarming motility assay consisted of 20 mm nh 4 cl , 12 mm na 2 po 4 , 22 mm kh 2 po 4 , 8 . 6 mm nacl , 1 mm mgso 4 , 1 mm cacl 2 , 11 mm dextrose , 0 . 5 % casamino acids , 0 . 5 % bacto - agar ( difco ) as previously described [ 13 ]. plates were allowed to dry overnight at room temperature and then bacterial strains were inoculated from overnight grown pia plates onto the swarming plates using a sterile toothpick followed by incubation at 30 ° c . pyocyanin production . in liquid media , pyocyanin was extracted into chloroform followed by re - extraction into 0 . 2n hcl , in which it was quantitated by the absorbance at 520 nm normalized to bacterial cell density , similar to previous descriptions [ 10 , 14 ]. in agarized media , equal square pieces of agarized media with lawns ( diameter 2 cm ) were extracted from plates , bacterial cells were removed in 1 ml of 0 . 9 % nacl , and pyocyanin was extracted from homogenized agar by chloroform , followed by re - extraction into 0 . 2n hcl and measuring at 520 nm . measurements were normalized to cell density measured by absorbance at 600 nm in 0 . 9 % nacl solutions . transformation of mpao1 to the mpao1 - p2 phenotype in ex vivo experiments . laparotomy and low colorectal anastomosis formation were performed in animals that were either exposed or not exposed to preoperative pelvic radiation as described above . on postoperative day 1 , all animals were sacrificed and the anastomotic segment and cecum ( 1 cm segment ) were removed . tissue segments ( cecum , colon anastomosis ) were homogenized in 1 ml sterile saline , incubated with strain mpao1 , and grown under static conditions , at 37 ° c . on day 3 of incubation , aliquots were plated onto pseudomonas isolation agar ( pia ) and 100 individual colonies were sub - cultured and evaluated for swarming motility . in selected experiments , tissue segments were homogenized in 1 ml of 5 % peg / pi . genome sequencing . genomic dna sequencing was performed on both mpa01 - p1 and mpa01 - p2 using the illumina gaiix sequencer to a total of 137 - and 115 - fold coverage , respectively . three libraries were created for each strain : lx36bp ( 370 / 340 million bp ), 2 × 100bp pe mate pairs with a 300 bp insert ( 320 / 180 mbp ) and 2 × 100bp pe mate pairs with a 3000 bp insert ( 380 / 420 mbp ). all libraries were quality - controlled and assembled de - novo using the mira assembler [ 15 ]. the mpa01 - p1 and mpa01 - p2 assemblies contained 6 . 26 mbp and 6 . 29 mbp , in 125 and 363 contigs respectively . these mpa01 - p1 and mpa01 - p2 assemblies had 99 . 99 and 99 . 95 % nucleotide identity to the pao1 / dsm - 1707 strain previously described [ 16 ]. the strain genomes were aligned to the reference strain nc — 002516 [ 17 ] using breseq 0 . 13 ( barrick , j . breseq , software ) and to each other to determine areas of difference . the sequences are deposited in sra as accession number sra049017 . these whole genome shotgun projects have been deposited at ddbj / embl / genbank under the accession numbers ahkm00000000 and ahkn00000000 . the version described in this report is the first version , i . e ., ahkm01000000 and ahkno 1000000 . replacement of mext in the p2 strain . the lambda red - based technique modified for p . aeruginosa [ 18 ] was used for replacement of mext in the p2 strain . the entire coding region of mext from mpa01 was amplified using primers forward 5 ′ cgg ata atg atc ggg ggt at 3 ′ ( seq id no : 8 ) and reversed 5 ′ ccg aat ttt tcc agc tcc tc 3 ′ ( seq id no : 9 ), and 10 μl of amplified mext - p1 was directly transformed in mapo1 - p2 / pucp18 - reds electrocompetent cells . transformants were selected by plating on pia containing 300 μg / ml of chloramphenicol . a cure of the plasmid was achieved on plates containing 10 % sucrose . transformants were verified for correct pcr insertion by mext sequencing using primers forward 5 ′ gcc tgt cag tga tcc tat gc 3 ′ ( seq id no : 10 ) and reversed 5 ′ gat cgc cga tga aca tgc 3 ′ ( seq id no : 11 ). statistical analysis . statistical analysis of anastomotic leak rate was performed using fisher &# 39 ; s exact test ( prism software ). significance for ex vivo studies was determined using 1 - way anova analysis . all other non - parametric data were analyzed using kruskal wallis and mann - whitney tests . kaplan - maier survival graph was analyzed using spss software . significance was determined as a p - value & lt ; 0 . 05 . intestinal exposure to radiation and p . aeruginosa causes spontaneous anastomotic leak in rats in order to define the role of p . aeruginosa on anastomosis healing , we first developed a novel anastomosis model in which rats were subjected to preoperative fractioned radiation similar to clinical practice . rats then underwent distal colon resection and anastomosis followed by intestinal inoculation with p . aeruginosa via cecal puncture at the end of the operation ( group iv ) ( fig1 a ). control groups included : rats subjected to resection and anastomosis only ( group i ), rats subjected to resection and anastomosis + cecal p . aeruginosa ( group ii ), rats subjected to preoperative radiation + resection and anastomosis ( group iii ). when the anastomoses of all rats were tested and directly examined on postoperative day ( pod ) six , rats in group iv demonstrated evidence of a significant incidence of spontaneous anastomotic leak with grossly visible disruption of the anastomotic suture line ( fig1 b ), dense adhesions to the anastomosis , immune cell infiltration ( fig1 c , fig8 ), and gross extravasation of injected intraluminal ( rectal ) contrast material ( methylene blue ) ( fig1 d ′). a major distinguishing characteristic in well - healing and intact anastomoses appeared to be the absence of dense adhesions to the external aspects of the anastomotic suture line , grossly intact and visible anastomotic healing both externally and internally , and lack of extravasation when intraluminal contrast was injected . gross histology confirmed these findings . when we opened the anastomoses and visualized the mucosa , anastomotic suture lines were intact in healed anastomosis but demonstrated focal disruptions in those that leaked . scanning electron microscopy ( sem ) of the mucosal of anastomotic suture lines in rats exposed to p . aeruginosa ( groups ii and iv ) ( fig2 ) demonstrated in group ii : an intact suture line at the anastomosis ( 35 ×), a smooth intact appearance of the epithelial surface ( 300 ×), and the presence of immune cells ( macrophages ) and few if any bacteria ( 3500 ×); in group iv : the suture line appears disrupted ( 35 ×), there is disintegration of the epithelial surface ( 300 ×), and there are abundant microbial cells at sites of disruption ( 3500 ×). taken together , these findings demonstrate that when p . aeruginosa is introduced to the proximal colon of rats subjected to preoperative radiation and a distal colon anastomosis , the microbe adheres to anastomotic sites and causes leakage . p . aeruginosa isolated from a disrupted anastomosis displays a tissue - destroying phenotype an experiment was conducted to determine whether strains of p . aeruginosa isolated from a leaking anastomosis would display an enhanced virulence phenotype . in particular , the phenotype of p . aeruginosa recovered from leaking anastomotic sites in rats subjected to radiation exposure and intestinal inoculation with p . aeruginosa ( group iv ) were compared to the inoculating mpao1 strain . on postoperative day 6 ( pod 6 ) leaking disrupted anastomoses were resected , homogenized and p . aeruginosa was recovered by plating tissues on pseudomonas isolation agar ( pia ). hundreds of single colonies were directly chosen from the plates and re - plated ( streaked ) on fresh pia plates . we found that majority of colonies displayed low pyocyanin production which we initially recognized as displaying attenuated virulence ( fig3 a , top panel ). to confirm this , the virulence of these colonies in c . elegans was tested and the surprising results showed enhanced killing of the yellow colonies compared to the initial strain ( fig3 b ). also observed were dendritic - like edges of lawns generated by the yellow colonies indicating that they possessed swarming motility . therefore , swarming assays were performed . high swarming motility was observed in these cells , in contrast to almost no swarming activity in the initial strain ( fig3 c ). the strains were then tested for their ability to disrupt cellular elements of anastomotic tissues ( epithelial cells , collagen ) and both an enhanced epithelial cell destruction ( fig3 d ) and a high level of collagenase ( fig3 e , f ) were observed among the yellow - appearing colonies . to verify that the strain recovered from leaking anastomotic tissues was genetically similar to the wilt - type strain used for intestinal inoculation ( mpao1 ), and not a pre - existing commensal in the rat gut , genetic fingerprint analysis ( rapd ) was performed , which confirmed their similarity ( fig3 g ). surprisingly , virtually all colonies isolated from disrupted anastomoses were attenuated in the production of the toxic metabolite pyocyanin grown on pia at high cell density . conversely , when grown in liquid culture , there was an increase in pyocyanin production compared to mpao1 ( fig3 a bottom panel , and fig9 ). spontaneous conversion of p1 and p2 in vitro was tested by subculture to 20 passages of each in rich nutrient tsb media . no p2 phenotype was detected in subcultures of p1 and conversely no p1 was detected in subcultures of p2 , and the pattern of pyocyanin production by p2 ( low on pia , high in liquid tsb ) was stably reproduced . we named the isolate from disrupted anastomotic tissues mpao1 - p2 ( herein termed p2 ) and the initial inoculating strain mpao1 - p1 ( herein termed p1 ). the ability of p2 to degrade collagens demonstrates its ability to cause a full thickness defect at the site of anastomotic injury . next the effect of p2 on apoptosis / necrosis and tight junctional integrity of cultured epithelial monolayers was assessed . rat intestinal epithelial eic - 18 cells were infected with 10 6 cfu p1 or p2 and incubated for 3 hrs followed by staining for apoptosis ( fitc - annexin v , green fluorescence ), necrosis ( etd - iii , red fluorescence ), nuclei ( hoechst 33342 , blue ), and tight junction ( zo - 1 immunostaining ). using confocal microscopy , we observed significant apoptotic cells in iec - 18 cells infected with p2 compared to p1 ( fig4 a , and fig1 demonstrating quantitative analysis of apoptotic cells ). while nuclei staining demonstrated the same amount of the cells in both groups ( fig4 b ). about 10 % of iec - 18 cells appear to be necrotic at 3 hrs when exposed to p2 infection confirming its cytotoxicity ( fig4 a ). p2 also caused a striking loss of tight junction integrity as judged by zo1 staining ( fig4 c ). taken together these results demonstrate that the p2 phenotype expresses a degree of virulence that is sufficient to disrupt healing anastomotic tissues from the most superficial elements ( epithelia ) to the submucosa and serosa ( collagen ). to determine the relative distribution of p1 versus p2 in the two treatment groups in which p . aeruginosa was injected into the cecum ( groups ii , iv ), we selectively cultured for p . aeruginosa from the cecum and anastomotic sites and then assessed retrieved strains for swarming . results demonstrate that cecal p . aeruginosa from both groups displayed a low incidence of the p2 phenotype (& lt ; 10 % of total recovered colonies ). in contrast , a high incidence (& gt ; 80 %) of the p2 phenotype was recovered ( by culture ) at anastomotic sites in both non - radiated ( group ii ) and radiated ( group iv ) rats indicating that surgical injury plays a role in the transformation to , or selection for , p2 . to determine if anastomotic tissues themselves can shift p1 to p2 , we performed experiments in which p1 was exposed to rat colon tissues ex vivo . tissues were obtained from reiterative studies in rats subjected to a colon resection and anastomosis with no exposure to p . aeruginosa and rats without anastomosis . cecum and colon segments were excised and homogenized in sterile saline . the p1 strain was added to the homogenate and the solution incubated at 37 ° c . after 72 hours , p . aeruginosa was recovered on pia , and then examined for pyocyanin production and swarming motility . 80 - 90 % of recovered colonies from anastomotic tissues were found to produce the p2 phenotype . no transformation was detected when p1 was incubated in saline alone and ˜ 10 % of colonies displayed the p2 phenotype when p1 was exposed to non - traumatized cecal tissues or non - anastomotic colon tissues . comparative analyses of p2 transformed in vivo and p2 transformed ex vivo demonstrated similar ability to destroy wounded epithelial cell monolayers ( fig5 ). these findings suggested that factors present within colonic tissues themselves may be responsible for the shift of p1 to p2 . to determine if the observed shift from p1 to p2 was secondary to a genotypic change , genome sequencing was performed . a single nucleotide mutation ( c → a position 2807731 in the nc002516 genome ) localized in the mext gene was identified ( fig6 a ). these results were further confirmed by direct sequencing of the amplified mext in p1 and p2 strains using primers forward 5 ′ gcctgtcagtgatcctatgc 3 ′ ( seq id no : 12 ) and reversed 5 ′ gatcgccgatgaacatgc 3 ′ ( seq id no : 13 ). sequencing of amplified mext from p2 strain isolated ex vivo demonstrated the same c → a snp . the mext gene is predicted to encode a full - length , 304 - residue mext protein , a regulator of the mexe - mexf - oprn multidrug efflux system of p . aeruginosa [ 19 ]. the genome sequence demonstrated the intact mext in p1 ( protein accession number pubseed : fig | 6666666 . 7915 . peg . 414 ) [ 20 ] while the snp ( c → a ) mutation in p2 was found to create an in - frame stop codon , limiting the possible products to a 44 - residue presumed non - functional truncated protein ( pubseed : fig | 6666666 . 7916 . peg . 276 ) , and a 242 - residue product resulting from re - initiation of translation at m63 ( protein accession number pubseed : fig | 6666666 . 7916 . peg . 277 ) ( genbank protein accession number for the intact mext protein from pao1 is aj007825 . 1 ). the functionality of mext can be assessed by the level of resistance to chloramphenicol and fluoroquinolones antibiotics that depends on mext - regulated expression of genes encoding the multi - drug efflux pump mexef - oprn [ 21 , 22 ]. we assessed antibiotic resistance of p1 and p2 and observed that the p2 strain showed a 10 - fold higher sensitivity to norfloxacin compared to the p1 strain ( mic 0 . 38 μg / ml for p1 and 3 . 8 for p2 ) and about 6 fold higher sensitivity to chloramphenicol ( fig9 ). to verify the snp mutation on the phenotype shift , we replaced mext - p2 by mext - p1 in strain p2 to create p2 / mext p1 . this replacement led to the reversion of p2 to p1 phenotype as determined by the pyocyanin production pattern ( high pyocyanin on agarized tsb , low pyocyanin in liquid tsb ), absence of swarming ( fig6 b - d ), high resistance to chloramphenicol ( fig6 e ), and attenuated collagenase activity ( fig6 f ). taken together , these data establish that the snp mutation in mext is responsible for transformation of the p1 to the p2 strain . prevention of the p1 → p2 transformation is associated with complete anastomotic healing high molecular weight polyethylene glycol , e . g ., peg 15 - 20 , attenuates virulence activation in p . aeruginosa in response to radiated epithelial cells and protects against post - radiated lethal sepsis . phosphate was shown to be important to the prevention of virulence activation and lethality in p . aeruginosa via its effect on phosphosensory and phosphoregulatory pathways that connect to quorum sensing [ 10 ], an effect that is maximized at a ph of 6 . 0 [ 11 ]. therefore a 5 % peg15 - 20 solution in 25 mm potassium phosphate buffer at ph 6 . 0 ( herein named peg / pi ) was tested in reiterative experiments . iec - 18 cells were pre - treated for 1 hour with 5 % peg / pi prior to p2 inoculation . a marked protective effect against p2 - induced cytotoxicity / disruption was observed . epithelial monolayers remained intact as judged by ldh released and demonstrated cellular migration across the wound to 50 % ( fig7 a , b ). 5 % peg / pi also prevented the transformation of p1 to p2 during exposure to anastomotic tissues ex vivo ( fig7 c ). finally , reiterative studies in rats exposed to pre - operative radiation followed by colon resection , anastomosis , and cecal injection with p . aeruginosa p1 ( group iv rats ) with 5 % peg / pi given as an enema demonstrated significantly attenuated anastomotic leak rates compared to rats given rectal saline ( fig7 d ). the causality between the transformed p2 phenotype in vivo and anastomotic leak in this model is indicated by the observation that no p2 strains were recovered from anastomotic tissues of rats treated with the peg / pi despite the compound having no microbicidal activity . this result was confirmed by the sem analysis of anastomotic tissues treated with 5 % peg / pi that demonstrated absence of bacteria on the epithelial surface and healed epithelial surfaces ( fig7 e ). taken together , these results indicate that in vivo transformation of p1 to p2 plays a key role in anastomotic leak in this model and is prevented by virulence - directed agents such as peg / pi . the promising results disclosed in example 7 led to experiments designed to assess the effect of the peg / pi therapeutic on anastomotic leak in vivo . rats were subjected to a distal colon resection and anastomosis . at time of surgery , the mesentery is ligated adjacent to the anastomosis to create ischemia ( devascularization ). three groups of animals were studied . conventional techniques well known in the art were employed unless otherwise indicated . group 1 underwent anastomosis only . group 2 underwent anastomosis and ischemia , and was administered peg / pi . group 3 underwent anastomosis and ischemia , and was not administered peg / pi . group 2 received 5 % peg in 25mm phosphate , which was administered via enema on postoperative day 0 . rats were sacrificed on postoperative day 6 to assess the amount of anastomotic healing or leakage . the results of this example ( depicted in fig1 ) demonstrate that peg / pi is effective at preventing the anastomotic leak in an organism that has experienced an ischemic event in combination with anastomosis . 1 . roos d , dijksman l m , oudemans - 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