- Email: [email protected]
In Vitro Analysis of the Bactericidal Activity of Escherichia Coli Nissle 1917 Against Pediatric Uropathogens
In Vitro Analysis of the Bactericidal Activity of Escherichia Coli Nissle 1917 Against Pediatric Uropathogens Douglas W. Storm,* Stephen A. Koff, Dennis J. Horvath, Jr., Birong Li and Sheryl S. Justice From the Department of Urology, Naval Medical Center (DWS), San Diego, California, and Division of Urology (SAK) and Center for Microbial Pathogenesis, The Research Institute (DJH, BL, SSJ), Nationwide Children’s Hospital and Ohio State University School of Medicine (SAK, SSJ), Columbus, Ohio
Abbreviations and Acronyms QIR ⫽ quiescent intracellular reservoir UTI ⫽ urinary tract infection Study received institutional review board approval. Supported by an intramural grant through the Research Institute, Nationwide Children’s Hospital. * Correspondence: Department of Urology, Naval Medical Center San Diego, 34800 Bob Wilson Dr., San Diego, California 92134-5000 (telephone: 619-532-7231; FAX: 619-532-7234. e-mail: [email protected]).
Purpose: The usefulness of prophylactic antibiotics to prevent recurrent urinary tract infections in children was recently questioned. Some groups have attempted to use probiotics, most commonly lactobacillus, to prevent recurrent infections by altering the intestinal bacterial reservoir with variable results. Mutaflor® is a possible alternative probiotic in which the active agent is Nissle 1917. Nissle 1917 is a commensal Escherichia coli strain that eradicates pathogenic bacteria from the gastrointestinal tract. Due to its ability to alter the intestinal biome we hypothesized that Mutaflor may have the potential to prevent recurrent urinary tract infections. Thus, we used an in vitro assay to analyze the effectiveness of Nissle 1917 for eradicating pediatric uropathogens. Materials and Methods: We established a collection of 43 bacterial pediatric uropathogens. With each isolate a microcin-type assay was performed to determine the effectiveness of Nissle 1917 on bacterial growth inhibition and competitive overgrowth. Results: Nissle 1917 adversely affected the growth of 34 of the 43 isolates (79%) isolates. It inhibited the growth of 21 isolates and overgrew 13. The percent of species adversely affected by Nissle 1917 was 40% for Pseudomonas, 50% for E. coli, Enterococcus and Staphylococcus, 100% for Klebsiella and Enterobacter, and 0% for Citrobacter and Serratia. Conclusions: Nissle 1917, the active agent in Mutaflor, inhibited or out competed most bacterial isolates. These mechanisms could be used in vivo to eradicate uropathogens from the gastrointestinal tract. Further study is needed to determine whether Mutaflor can prevent recurrent urinary tract infections in children. Key Words: urinary tract infections, probiotics, Escherichia coli, gastrointestinal tract, microcin
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URINARY tract infection remains a common problem in children with recurrence in 30% to 50% and renal scars in up to 56%.1,2 The goal of preventing recurrent infections and renal damage has not been fully realized in this population despite the use of prophylactic antibiotics and treatment for bladder dysfunction and/or
constipation.3 Moreover, the literature has recently questioned the efficacy and safety of prophylactic antibiotics.4 –7 Given these findings, other therapeutic measures such as probiotics have been considered. Replacing intestinal uropathogens with a new colonic flora consisting of nonuropathogenic probiotic containing bac-
0022-5347/11/1864-1678/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 1678-1683, October 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.04.021
AND
RESEARCH, INC.
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
teria could prevent further UTIs.8 –10 To date oral formulations of lactobacillus have been the most commonly prescribed probiotic but have shown only limited success in preventing recurrent UTIs.11–13 Mutaflor is a probiotic containing Nissle 1917, a commensal, nonpathogenic Escherichia coli strain, as the active component. Mutaflor contains 2.5 to 25 ⫻ 109 cfu Escherichia coli Nissle 1917 in an enteric coated gelatin capsule. Mutaflor has efficaciously and safely treated children of all ages with inflammatory bowel disease and diarrhea.14 –17 Previous in vitro and in vivo studies have demonstrated successful Nissle 1917 colonization in the bowel and displacement of pathological intestinal bacteria.18 –21 Also, Nissle 1917 boosts innate immune responses.22,23 With this background we wondered whether Mutaflor could also be useful in preventing recurrent UTIs in children. This prompted our in vitro study to investigate its antibacterial activity against other bacteria.
1679
Figure 1. Microcin assay steps. A, target background pediatric uropathogen strain is spread on Luria broth plate. B, toothpick previously dipped in Luria broth containing Nissle 1917 strain is stabbed into background strain and plate is incubated overnight.
examined the next day. A positive result was defined as an area of clearing (growth inhibition) around the Nissle 1917 strain or overgrowth of the Nissle 1917 strain over the background (fig. 2). For comparison the microcin assay was repeated separately using the Nissle 1917 strain and the laboratory adapted E. coli strain MG1655 as the target backgrounds, and each uropathogen as the antagonist. Assays were completed in quadruplicate.
Bacterial Overgrowth Competition Assay
MATERIALS AND METHODS Acquisition of Nissle 1917 Isolate A sample strain of Nissle 1917 E. coli was sent to our laboratory and grown statically in Luria broth (Fisher Scientific™) at 37C upon arrival. Bacterial medium was placed in individual 1 ml aliquots and frozen at ⫺80C for future analysis.
Bacterial Isolate Collection Pediatric bacterial specimens were collected and used to determine the in vitro bactericidal characteristics of Nissle 1917. As children presented to our hospital with signs and symptoms of UTI, isolates were gathered in a prospective manner. Urine specimens were sent to the hospital clinical microbiology laboratory, where the causative microorganism was identified and a bacterial burden of at least 105 cfu/ml urine was established. Bacterial sensitivity and resistance patterns were also determined. Plated specimens were brought to our research laboratory, where individual isolates were grown statically in Luria broth overnight at 37C and stored at ⫺80C. Samples were collected with institutional review board approval.
Microcin Assay A way in which bacteria inhibit other microbial growth is by producing proteinaceous toxins called microcins, which function as antimicrobial agents to eliminate other bacteria from their environment. We used this assay to determine to what degree microcin production by Nissle 1917 would affect the growth of the pediatric uropathogens. The microcin assay was previously validated by Pugsley.24 The individual pediatric uropathogen strains and the Nissle 1917 strain were grown separately in Luria broth statically at 37C overnight. Each target uropathogen was then spread on a Luria broth agar plate (fig. 1, A). The antagonist, Nissle 1917, was stabbed into the background (fig. 1, B). The plate was incubated at 37C overnight and
In cases of Nissle 1917 overgrowth the assay was repeated to verify the growth advantage of Nissle 1917. Since this assay required a method to differentiate the Nissle 1917 E. coli strain from the pediatric uropathogens, we introduced a kanamycin resistance marker into the Nissle 1917 strain. A kanamycin resistant gene was inserted into the Nissle 1917 chromosome by standard P1 phage transduction using methods previously described by Justice et al.25 The P1 phage was inserted into a site previously reported to have no effect on bacterial growth.25 This was verified by growth curve calculations showing no difference in the growth rate before or after P1 phage insertion. We verified that the Nissle strain was resistant to kanamycin by observing overnight growth when the strain was streaked on a Luria broth plate containing kanamycin. Only isolates overgrown by Nissle 1917 on microcin assay were subjected to this competition assay. These uropathogen isolates were known to be susceptible to kanamycin since they were reported to be kanamycin sensitive at the time of the UTI in the child. The uropathogen isolates and the kanamycin resistant Nissle 1917 strain were individually grown overnight at 37C in Luria broth. The next morning the bacterial cultures were diluted in phosphate buffered saline (MP Biomedicals, Solon, Ohio) to a concentration of 2 ⫻ 108 cfu/ml (optical density 0.072 nm). The diluted Nissle 1917 strain and a diluted uropathogen (5 l each) were added to 5 ml (1 ⫻ 102 cfu/ml per strain) Luria broth and incubated at 37C overnight. At 14 hours this bacterial co-culture was serially diluted and plated on plain Luria broth agar plates, which were incubated overnight. Since the plates contained no antibiotics, Nissle 1917 and pediatric uropathogen colonies grew, representing the combined total bacterial colony count. Since the colonies were similar in size and morphology, we could not differentiate Nissle 1917 colonies from uropathogen colonies. To enumerate the number of Nissle
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
Figure 2. Microcin assay using different uropathogen strain as background with Nissle 1917 strain stabbed into background. A, Nissle 1917 cleared surrounding uropathogen. B, Nissle 1917 strain overgrew uropathogen.
1917 colonies we performed patch plating, as described by Snyder and Champness.26 All colonies from the plain Luria broth agar plate were transferred to a Luria broth agar plate containing kanamycin. Since the Nissle 1917 colonies were kanamycin resistant, they were expected to grow while the uropathogen colonies, which were kanamycin sensitive, would not survive. After patch plating the plates were incubated at 37C overnight and the next morning the surviving colonies were counted. This number, representing the Nissle 1917 colonies, was compared to the total number of patch plated colonies. In this fashion we calculated a percent showing the growth characteristics of the 2 strains.
RESULTS Pediatric Uropathogen Library We collected 43 bacterial isolates known to have caused pediatric UTIs in patients with a mean age of 86 months (range 1 to 204). Of the patients 59% presented with clinical evidence of cystitis while 41% presented with pyelonephritic symptoms, including nausea, vomiting, flank pain, leukocytosis and/or fever greater than 38C. Figure 3 shows the bacterial subtypes in this library. The distribution and types of bacterial uropathogens represent a wide range of common bacteria known to cause UTIs in children. To further classify the pediatric uropathogens we used methods previously described by Johnson and Stell27 to identify the proportion of several virulence genes in our bacterial library. The genes analyzed were pAi, FimH, pap, ibeA, FyuA, sfa/foc, hlyA and kpsmTII. We focused on these genes since they have an important role in bladder and kidney adherence, and provide various mechanisms to subvert the host immune response. The percents of these virulence genes were 61% for pAi, 93% for FimH, 27% for pap, 39% for ibeA, 29% for sfa/foc, 10% for hlyA and 56% for kpsmTII. These results identified a wide range of virulence genes in the uropathogens. Assay Results Microcin. We performed a microcin assay for each of the 43 bacterial strains and observed a zone of clear-
ance surrounding the Nissle 1917 strain in 21 (49%) (see table and fig. 2, A). The maximum zone of inhibited growth was 34 mm in diameter, indicating strong killing action on the part of Nissle 1917. Indeed, Nissle 1917 showed a broad spectrum of activity against related and unrelated bacterial species (see table). The Nissle 1917 strain also showed overgrowth of an additional 13 uropathogens (30%) (see table and fig. 2, B), suggesting that the Nissle 1917 strain also outcompeted certain uropathogens by its faster growth rate. Nissle 1917 adversely affected the growth of 79% of the pediatric bacterial isolates. Overgrowth. To eliminate possible metabolic requirements across the various uropathogen species we focused only on the overgrown E. coli uropathogen strains. Also, since the colony morphology of the Nissle 1917 and the uropathogenic E. coli strains was similar in size and shape, this would ensure that we were blinded to the colonies that had been patch plated. We tested 7 E. coli uropathogens. Nissle 1917 outcompeted 6 of these E. coli isolates and represented 60% to 84% of the total colony population. Nissle 1917 Susceptibility to Uropathogen Microcins To ensure that the uropathogens were unable to kill the Nissle 1917 strain by their microcin production 30
Number of Bacterial Isolates
1680
20
E Coli Pseudomonas Enterococcus Klebsiella Staphylococcus Serratia Citrobacter Enterobacter
10
0
Figure 3. Bacterial strains in pediatric uropathogen library
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
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Results of microcin assay using Nissle 1917 as antagonist with bacterial uropathogen strain as background, and uropathogen strain as antagonist with Nissle 1917 and MG1655 as background No. Uropathogen Antagonist, Nissle ⫹ MG1655 Background (%)
No. Nissle Antagonist, Uropathogen Background (%) No. Strains*
Inhibited by Nissle
Overgrown by Nissle
Totals
Inhibited Nissle Growth
Inhibited MG1655 Growth
26 4 5 2 2 1 2
13 (50) 2 (50) 2 (40) 2 (100) 0 1 (100) 1 (50)
7 (27) 2 (50) 2 (40) 0 1 (50) 0 1 (50)
20 (77) 4 (100) 4 (80) 2 (100) 1 (50) 1 (100) 2 (100)
3 (12) 0 0 0 0 0 0
4 (15) 0 1 (20) 0 2 (100) 0 0
E. coli Enterococcus Pseudomonas Klebsiella Serratia Enterobacter Staphylococcus
* The 1 Citrobacter strain assayed was not affected by Nissle 1917 and did not affect Nissle 1917 or MG1655.
we performed the microcin assay using Nissle 1917 as the target background strain and the uropathogens as the antagonist. For comparison the assay was repeated using the laboratory adapted E. coli strain MG1655 as the background. Only 7% of the pediatric isolates showed an area of Nissle 1917 growth inhibition with a maximum zone of inhibition of only 2 to 4 mm (see table). Notably no nonE. coli uropathogens affected the growth of Nissle 1917. In contrast, 16% of the pediatric isolates showed growth inhibition when MG1655 was the target organism (see table). This suggests that Nissle 1917 is highly resistant to microcins produced by other bacterial strains.
DISCUSSION In this study Nissle 1917 interfered with the growth of 79% of the uropathogens collected from children with UTIs in the form of growth inhibition in 49% and overgrowth in 30%. The bactericidal effect of Nissle 1917 was observed for almost all of the bacterial species (see table). The only species not affected was Citrobacter. However, we only had 1 Citrobacter isolate and it is unclear whether the apparent immunity is species specific or simply due to an underrepresentation of Citrobacter isolates. Microcin production is a common bacterial defense, although to date fewer than 10 specific microcins have been identified in the E. coli species.28 It is likely that the uropathogens that were not inhibited produce the same microcins as Nissle 1917, resulting in their resistance. Given the limited number of microcins and the frequency that bacteria produce microcins, the 49% growth inhibition by Nissle 1917 is impressive. In addition to growth inhibition, we identified the overgrowth of several pediatric isolates. The competitive advantage of Nissle 1917 that enabled it to overgrow 30% of the uropathogens occurred when Nissle 1917 was placed in a microbial environment
where the background uropathogen was previously well established, and also when Nissle 1917 and the uropathogens were simultaneously co-cultured. In addition, when Nissle 1917 served as the background organism in the microcin assay, few uropathogens could inhibit its growth. This reveals the ability of Nissle 1917 to persist despite competition from other bacteria. Furthermore, Nissle 1917 showed a bactericidal effect against strains with various virulence genes, and uropathogens causing cystitis and pyelonephritis. Nissle 1917 inhibited or overgrew 80% of the strains causing cystitis and interfered with 86% of the pyelonephritic isolates. These results reveal that through entirely different bactericidal mechanisms of action Nissle 1917 successfully eradicated most known pediatric uropathogens. In reality our study may have underestimated the protective effect of the Nissle strain against uropathogens. Previous in vitro and in vivo studies suggest that Nissle 1917 may be protective by other means. Altenhoefer18 and Oelschlaeger19 et al noted in cell culture that Nissle 1917 decreased the ability of several bacterial pathogens to invade an intestinal epithelial cell layer. Nissle 1917 also improves the innate defense system of the colon by stimulating desmosome and tight junction proteins, and inducing the production of colonic antimicrobial peptides.22,23 Through these mechanisms Nissle 1917 makes the bowel less permeable to bacterial invasion and boosts natural defenses against pathogenic organisms. These additional mechanisms of action might further potentiate the protective effect of Nissle 1917 against uropathogens. Most commonly, lactobacillus based probiotics have been previously studied to prevent recurrent UTIs. The aim in using these probiotics is to recolonize the bowel and vagina with lactobacillus. Background studies showed that in adults and children with UTIs colonic lactobacillus colony counts are decreased before probiotic use.11,12
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BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
However, compared to adults the vaginal lactobacillus microflora do not appear to be altered in children who are susceptible to UTIs.11 Thus, lactobacillus based probiotics may not be as effective in young females. Also, while some studies in adults showed that these probiotics may reestablish lactobacillus in the colonic and vaginal microflora, it remains unclear whether this is actually efficacious in inhibiting UTIs.12 Upon review minimal data have established the lactobacillus mechanism of action against uropathogens or its clinical usefulness in preventing recurrent UTIs.12 In contrast, from our analysis and previous research it appears that Nissle 1917 may be efficacious in preventing and eliminating the colonization of pathogenic bacteria in the bowel.18,19,22,23 Such a decrease in the fecal load of uropathogens could then decrease the incidence of recurrent UTIs. While Mutaflor appears to be a promising prophylactic agent against recurrent UTIs derived from intestinal bacteria, it may not be efficacious in cases of recurrent infection resulting from dormant bacteria already in the bladder. After an acute UTI E. coli may establish a latent infection in bladder transitional cells in the form of QIRs.9,10 These QIRs may persist for months, resist antibiotic treatment and serve as a source of UTI recurrence.9,29 Since QIRs reside in bladder transitional cells rather than in the gut, this reservoir would not be susceptible to the effect of Nissle 1917. This could potentially allow
recurrent infection despite an excellent Mutaflor effect on colonic bacterial flora. Another potential concern is that oral administration of Mutaflor may result in Nissle 1917 E. coli actually causing UTIs. This appears unlikely since genomic analysis of Nissle 1917 verified the absence of any pathogenicity factors typically found in uropathogenic E. coli.29,30 Also, it has been studied in children of all ages, including neonates, without any apparent overt side effects, including Nissle 1917 induced UTIs.20
CONCLUSIONS In our controlled in vitro study Nissle 1917 E. coli, the active agent in the probiotic Mutaflor, inhibited or outcompeted most bacterial isolates in our library. These findings along with those previously identified in the treatment of inflammatory and infectious bowel disease suggest that Mutaflor may be a useful agent in the prevention of recurrent UTIs in children. While additional studies are needed to validate and further define the clinical effectiveness of Mutaflor, based on these preliminary in vitro findings we believe that future trials evaluating its therapeutic application are warranted.
ACKNOWLEDGMENTS Ulrich Sonnenborn, Ardeypharm, Herdecke, Germany, provided the Nissle 1917 E. coli sample and permission to study the strain.
REFERENCES 1. Coulthard MG, Lambert HJ and Keir MJ: Occurrence of renal scars in children after their first referral for urinary tract infection. BMJ 1997; 315: 918.
6. Conway PH, Cnaan A, Zaoutis T et al: Recurrent urinary tract infections in children: risk factors and association with prophylactic antimicrobials. JAMA 2007; 298: 179.
2. Rushton HG: The evaluation of acute pyelonephritis and renal scarring with technetium 99m-dimercaptosuccinic acid renal scintigraphy: evolving concepts and future directions. Pediatr Nephrol 1997; 11: 108.
7. Montini G, Rigon L, Zucchetta P et al: Prophylaxis after first febrile urinary tract infection in children? A multicenter, randomized, controlled, noninferiority trial. Pediatrics 2008; 122: 1064.
3. Tamminen-Mobius T, Brunier E, Ebel KD et al: Cessation of vesicoureteral reflux for 5 years in infants and children allocated to medical treatment. The International Reflux Study in Children. J Urol 1992; 148: 1662. 4. Garin EH, Olavarria F, Garcia Nieto V et al: Clinical significance of primary vesicoureteral reflux and urinary antibiotic prophylaxis after acute pyelonephritis: a multicenter, randomized, controlled study. Pediatrics 2006; 117: 626. 5. Pennesi M, Travan L, Peratoner L et al: Is antibiotic prophylaxis in children with vesicoureteral reflux effective in preventing pyelonephritis and renal scars? A randomized, controlled trial. Pediatrics 2008; 121: e1489.
8. Justice SS, Hunstad DA, Cegelski L et al: Morphological plasticity as a bacterial survival strategy. Nat Rev Microbiol 2008; 6: 162. 9. Hunstad DA and Justice SS: Intracellular lifestyles and immune evasion strategies of uropathogenic Escherichia coli. Ann Rev Microbiol 2010; 64: 203. 10. Mysorekar IU and Hultgren SJ: Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proc Natl Acad Sci USA 2006; 103: 14170. 11. Lee JW, Shim YH and Lee SJ: Lactobacillus colonization status in infants with urinary tract infection. Pediatr Nephrol 2009; 24: 135.
12. Reid G and Bruce AW: Probiotics to prevent urinary tract infections: the rationale and evidence. World J Urol 2006; 24: 28. 13. Abad CL and Safdar N: The role of lactobacillus probiotics in the treatment or prevention of urogenital infections—a systematic review. J Chemother 2009; 21: 243. 14. Henker J, Muller S, Laass MW et al: Probiotic Escherichia coli Nissle 1917 (EcN) for successful remission maintenance of ulcerative colitis in children and adolescents: an open-label pilot study. Z Gastroenterol 2008; 46: 874. 15. Henker J, Laass MW, Blokhin BM et al: Probiotic Escherichia coli Nissle 1917 versus placebo for treating diarrhea of greater than 4 days duration in infants and toddlers. Pediatr Infect Dis J 2008; 27: 494. 16. Kruis W, Schutz E, Fric P et al: Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 1997; 11: 853.
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
17. Henker J, Laass M, Blokhin BM et al: The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers. Eur J Pediatr 2007; 166: 311. 18. Altenhoefer A, Oswald S, Sonnenborn U et al: The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol 2004; 40: 223. 19. Oelschlaeger TA, Altenhoefer A and Hacker J: Inhibition of Salmonella typhimurium invasion into intestinal cells by probiotic E. coli strain Nissle 1917. Gastroenterology 2001; 120: A326. 20. Lodinova-Zadnikova R and Sonnenborn U: Effect of preventive administration of a nonpathogenic Escherichia coli strain on the colonization of the intestine with microbial pathogens in newborn infants. Biol Neonate 1997; 71: 224. 21. Schulze J, Lorenz A and Mandel L: Colonisation of Escherichia coli in different gnotobiotic animal models. Microbial Ecol Health Dis 1992; 5: iv.
22. Wehkamp J, Harder J, Wehkamp K et al: NFkappaB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium. Infect Immun 2004; 72: 5750. 23. Cichon A, Enders C, Sonnenborn U et al: DNAmicroarray-based comparison of cellular responses in poarized T84 epithelial cells triggered by probiotics: E. coli Nissle 1917 (EcN) and Lactobacillus acidophilus PZ 1041. Gastroenterology 2004; 126: A-578. 24. Pugsley AP: Escherichia coli K12 strains for use in the identification and characterization of colicins. J Gen Microbiol 1985; 131: 369. 25. Justice SS, Hunstad DA, Seed PC et al: Filamentation by Escherichia coli subverts innate defenses during urinary tract infection. Proc Natl Acad Sci USA 2006; 103: 19884. 26. Snyder L and Champness W: Conjugation. In: Molecular Genetics of Bacteria, 3rd ed. Wash-
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ington, D.C.: ASM Press 2007; chap 5, pp 243-275. 27. Johnson JR and Stell AL: Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis 2000; 181: 261. 28. Gordon DM and O’Brien CL: Bacteriocin diversity and the frequency of multiple bacteriocin production in Escherichia coli. Microbiology 2006; 152: 3239. 29. Schilling JD, Lorenz RG and Hultgren SJ: Effect of trimethoprim-sulfamethoxazole on recurrent bacteriuria and bacterial persistence in mice infected with uropathogenic Escherichia coli. Infect Immun 2002; 70: 7042. 30. Grozdanov L, Raasch C, Schulze J et al: Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol 2004; 186: 5432.
Abbreviations and Acronyms QIR ⫽ quiescent intracellular reservoir UTI ⫽ urinary tract infection Study received institutional review board approval. Supported by an intramural grant through the Research Institute, Nationwide Children’s Hospital. * Correspondence: Department of Urology, Naval Medical Center San Diego, 34800 Bob Wilson Dr., San Diego, California 92134-5000 (telephone: 619-532-7231; FAX: 619-532-7234. e-mail: [email protected]).
Purpose: The usefulness of prophylactic antibiotics to prevent recurrent urinary tract infections in children was recently questioned. Some groups have attempted to use probiotics, most commonly lactobacillus, to prevent recurrent infections by altering the intestinal bacterial reservoir with variable results. Mutaflor® is a possible alternative probiotic in which the active agent is Nissle 1917. Nissle 1917 is a commensal Escherichia coli strain that eradicates pathogenic bacteria from the gastrointestinal tract. Due to its ability to alter the intestinal biome we hypothesized that Mutaflor may have the potential to prevent recurrent urinary tract infections. Thus, we used an in vitro assay to analyze the effectiveness of Nissle 1917 for eradicating pediatric uropathogens. Materials and Methods: We established a collection of 43 bacterial pediatric uropathogens. With each isolate a microcin-type assay was performed to determine the effectiveness of Nissle 1917 on bacterial growth inhibition and competitive overgrowth. Results: Nissle 1917 adversely affected the growth of 34 of the 43 isolates (79%) isolates. It inhibited the growth of 21 isolates and overgrew 13. The percent of species adversely affected by Nissle 1917 was 40% for Pseudomonas, 50% for E. coli, Enterococcus and Staphylococcus, 100% for Klebsiella and Enterobacter, and 0% for Citrobacter and Serratia. Conclusions: Nissle 1917, the active agent in Mutaflor, inhibited or out competed most bacterial isolates. These mechanisms could be used in vivo to eradicate uropathogens from the gastrointestinal tract. Further study is needed to determine whether Mutaflor can prevent recurrent urinary tract infections in children. Key Words: urinary tract infections, probiotics, Escherichia coli, gastrointestinal tract, microcin
1678
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URINARY tract infection remains a common problem in children with recurrence in 30% to 50% and renal scars in up to 56%.1,2 The goal of preventing recurrent infections and renal damage has not been fully realized in this population despite the use of prophylactic antibiotics and treatment for bladder dysfunction and/or
constipation.3 Moreover, the literature has recently questioned the efficacy and safety of prophylactic antibiotics.4 –7 Given these findings, other therapeutic measures such as probiotics have been considered. Replacing intestinal uropathogens with a new colonic flora consisting of nonuropathogenic probiotic containing bac-
0022-5347/11/1864-1678/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 1678-1683, October 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.04.021
AND
RESEARCH, INC.
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
teria could prevent further UTIs.8 –10 To date oral formulations of lactobacillus have been the most commonly prescribed probiotic but have shown only limited success in preventing recurrent UTIs.11–13 Mutaflor is a probiotic containing Nissle 1917, a commensal, nonpathogenic Escherichia coli strain, as the active component. Mutaflor contains 2.5 to 25 ⫻ 109 cfu Escherichia coli Nissle 1917 in an enteric coated gelatin capsule. Mutaflor has efficaciously and safely treated children of all ages with inflammatory bowel disease and diarrhea.14 –17 Previous in vitro and in vivo studies have demonstrated successful Nissle 1917 colonization in the bowel and displacement of pathological intestinal bacteria.18 –21 Also, Nissle 1917 boosts innate immune responses.22,23 With this background we wondered whether Mutaflor could also be useful in preventing recurrent UTIs in children. This prompted our in vitro study to investigate its antibacterial activity against other bacteria.
1679
Figure 1. Microcin assay steps. A, target background pediatric uropathogen strain is spread on Luria broth plate. B, toothpick previously dipped in Luria broth containing Nissle 1917 strain is stabbed into background strain and plate is incubated overnight.
examined the next day. A positive result was defined as an area of clearing (growth inhibition) around the Nissle 1917 strain or overgrowth of the Nissle 1917 strain over the background (fig. 2). For comparison the microcin assay was repeated separately using the Nissle 1917 strain and the laboratory adapted E. coli strain MG1655 as the target backgrounds, and each uropathogen as the antagonist. Assays were completed in quadruplicate.
Bacterial Overgrowth Competition Assay
MATERIALS AND METHODS Acquisition of Nissle 1917 Isolate A sample strain of Nissle 1917 E. coli was sent to our laboratory and grown statically in Luria broth (Fisher Scientific™) at 37C upon arrival. Bacterial medium was placed in individual 1 ml aliquots and frozen at ⫺80C for future analysis.
Bacterial Isolate Collection Pediatric bacterial specimens were collected and used to determine the in vitro bactericidal characteristics of Nissle 1917. As children presented to our hospital with signs and symptoms of UTI, isolates were gathered in a prospective manner. Urine specimens were sent to the hospital clinical microbiology laboratory, where the causative microorganism was identified and a bacterial burden of at least 105 cfu/ml urine was established. Bacterial sensitivity and resistance patterns were also determined. Plated specimens were brought to our research laboratory, where individual isolates were grown statically in Luria broth overnight at 37C and stored at ⫺80C. Samples were collected with institutional review board approval.
Microcin Assay A way in which bacteria inhibit other microbial growth is by producing proteinaceous toxins called microcins, which function as antimicrobial agents to eliminate other bacteria from their environment. We used this assay to determine to what degree microcin production by Nissle 1917 would affect the growth of the pediatric uropathogens. The microcin assay was previously validated by Pugsley.24 The individual pediatric uropathogen strains and the Nissle 1917 strain were grown separately in Luria broth statically at 37C overnight. Each target uropathogen was then spread on a Luria broth agar plate (fig. 1, A). The antagonist, Nissle 1917, was stabbed into the background (fig. 1, B). The plate was incubated at 37C overnight and
In cases of Nissle 1917 overgrowth the assay was repeated to verify the growth advantage of Nissle 1917. Since this assay required a method to differentiate the Nissle 1917 E. coli strain from the pediatric uropathogens, we introduced a kanamycin resistance marker into the Nissle 1917 strain. A kanamycin resistant gene was inserted into the Nissle 1917 chromosome by standard P1 phage transduction using methods previously described by Justice et al.25 The P1 phage was inserted into a site previously reported to have no effect on bacterial growth.25 This was verified by growth curve calculations showing no difference in the growth rate before or after P1 phage insertion. We verified that the Nissle strain was resistant to kanamycin by observing overnight growth when the strain was streaked on a Luria broth plate containing kanamycin. Only isolates overgrown by Nissle 1917 on microcin assay were subjected to this competition assay. These uropathogen isolates were known to be susceptible to kanamycin since they were reported to be kanamycin sensitive at the time of the UTI in the child. The uropathogen isolates and the kanamycin resistant Nissle 1917 strain were individually grown overnight at 37C in Luria broth. The next morning the bacterial cultures were diluted in phosphate buffered saline (MP Biomedicals, Solon, Ohio) to a concentration of 2 ⫻ 108 cfu/ml (optical density 0.072 nm). The diluted Nissle 1917 strain and a diluted uropathogen (5 l each) were added to 5 ml (1 ⫻ 102 cfu/ml per strain) Luria broth and incubated at 37C overnight. At 14 hours this bacterial co-culture was serially diluted and plated on plain Luria broth agar plates, which were incubated overnight. Since the plates contained no antibiotics, Nissle 1917 and pediatric uropathogen colonies grew, representing the combined total bacterial colony count. Since the colonies were similar in size and morphology, we could not differentiate Nissle 1917 colonies from uropathogen colonies. To enumerate the number of Nissle
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
Figure 2. Microcin assay using different uropathogen strain as background with Nissle 1917 strain stabbed into background. A, Nissle 1917 cleared surrounding uropathogen. B, Nissle 1917 strain overgrew uropathogen.
1917 colonies we performed patch plating, as described by Snyder and Champness.26 All colonies from the plain Luria broth agar plate were transferred to a Luria broth agar plate containing kanamycin. Since the Nissle 1917 colonies were kanamycin resistant, they were expected to grow while the uropathogen colonies, which were kanamycin sensitive, would not survive. After patch plating the plates were incubated at 37C overnight and the next morning the surviving colonies were counted. This number, representing the Nissle 1917 colonies, was compared to the total number of patch plated colonies. In this fashion we calculated a percent showing the growth characteristics of the 2 strains.
RESULTS Pediatric Uropathogen Library We collected 43 bacterial isolates known to have caused pediatric UTIs in patients with a mean age of 86 months (range 1 to 204). Of the patients 59% presented with clinical evidence of cystitis while 41% presented with pyelonephritic symptoms, including nausea, vomiting, flank pain, leukocytosis and/or fever greater than 38C. Figure 3 shows the bacterial subtypes in this library. The distribution and types of bacterial uropathogens represent a wide range of common bacteria known to cause UTIs in children. To further classify the pediatric uropathogens we used methods previously described by Johnson and Stell27 to identify the proportion of several virulence genes in our bacterial library. The genes analyzed were pAi, FimH, pap, ibeA, FyuA, sfa/foc, hlyA and kpsmTII. We focused on these genes since they have an important role in bladder and kidney adherence, and provide various mechanisms to subvert the host immune response. The percents of these virulence genes were 61% for pAi, 93% for FimH, 27% for pap, 39% for ibeA, 29% for sfa/foc, 10% for hlyA and 56% for kpsmTII. These results identified a wide range of virulence genes in the uropathogens. Assay Results Microcin. We performed a microcin assay for each of the 43 bacterial strains and observed a zone of clear-
ance surrounding the Nissle 1917 strain in 21 (49%) (see table and fig. 2, A). The maximum zone of inhibited growth was 34 mm in diameter, indicating strong killing action on the part of Nissle 1917. Indeed, Nissle 1917 showed a broad spectrum of activity against related and unrelated bacterial species (see table). The Nissle 1917 strain also showed overgrowth of an additional 13 uropathogens (30%) (see table and fig. 2, B), suggesting that the Nissle 1917 strain also outcompeted certain uropathogens by its faster growth rate. Nissle 1917 adversely affected the growth of 79% of the pediatric bacterial isolates. Overgrowth. To eliminate possible metabolic requirements across the various uropathogen species we focused only on the overgrown E. coli uropathogen strains. Also, since the colony morphology of the Nissle 1917 and the uropathogenic E. coli strains was similar in size and shape, this would ensure that we were blinded to the colonies that had been patch plated. We tested 7 E. coli uropathogens. Nissle 1917 outcompeted 6 of these E. coli isolates and represented 60% to 84% of the total colony population. Nissle 1917 Susceptibility to Uropathogen Microcins To ensure that the uropathogens were unable to kill the Nissle 1917 strain by their microcin production 30
Number of Bacterial Isolates
1680
20
E Coli Pseudomonas Enterococcus Klebsiella Staphylococcus Serratia Citrobacter Enterobacter
10
0
Figure 3. Bacterial strains in pediatric uropathogen library
BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
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Results of microcin assay using Nissle 1917 as antagonist with bacterial uropathogen strain as background, and uropathogen strain as antagonist with Nissle 1917 and MG1655 as background No. Uropathogen Antagonist, Nissle ⫹ MG1655 Background (%)
No. Nissle Antagonist, Uropathogen Background (%) No. Strains*
Inhibited by Nissle
Overgrown by Nissle
Totals
Inhibited Nissle Growth
Inhibited MG1655 Growth
26 4 5 2 2 1 2
13 (50) 2 (50) 2 (40) 2 (100) 0 1 (100) 1 (50)
7 (27) 2 (50) 2 (40) 0 1 (50) 0 1 (50)
20 (77) 4 (100) 4 (80) 2 (100) 1 (50) 1 (100) 2 (100)
3 (12) 0 0 0 0 0 0
4 (15) 0 1 (20) 0 2 (100) 0 0
E. coli Enterococcus Pseudomonas Klebsiella Serratia Enterobacter Staphylococcus
* The 1 Citrobacter strain assayed was not affected by Nissle 1917 and did not affect Nissle 1917 or MG1655.
we performed the microcin assay using Nissle 1917 as the target background strain and the uropathogens as the antagonist. For comparison the assay was repeated using the laboratory adapted E. coli strain MG1655 as the background. Only 7% of the pediatric isolates showed an area of Nissle 1917 growth inhibition with a maximum zone of inhibition of only 2 to 4 mm (see table). Notably no nonE. coli uropathogens affected the growth of Nissle 1917. In contrast, 16% of the pediatric isolates showed growth inhibition when MG1655 was the target organism (see table). This suggests that Nissle 1917 is highly resistant to microcins produced by other bacterial strains.
DISCUSSION In this study Nissle 1917 interfered with the growth of 79% of the uropathogens collected from children with UTIs in the form of growth inhibition in 49% and overgrowth in 30%. The bactericidal effect of Nissle 1917 was observed for almost all of the bacterial species (see table). The only species not affected was Citrobacter. However, we only had 1 Citrobacter isolate and it is unclear whether the apparent immunity is species specific or simply due to an underrepresentation of Citrobacter isolates. Microcin production is a common bacterial defense, although to date fewer than 10 specific microcins have been identified in the E. coli species.28 It is likely that the uropathogens that were not inhibited produce the same microcins as Nissle 1917, resulting in their resistance. Given the limited number of microcins and the frequency that bacteria produce microcins, the 49% growth inhibition by Nissle 1917 is impressive. In addition to growth inhibition, we identified the overgrowth of several pediatric isolates. The competitive advantage of Nissle 1917 that enabled it to overgrow 30% of the uropathogens occurred when Nissle 1917 was placed in a microbial environment
where the background uropathogen was previously well established, and also when Nissle 1917 and the uropathogens were simultaneously co-cultured. In addition, when Nissle 1917 served as the background organism in the microcin assay, few uropathogens could inhibit its growth. This reveals the ability of Nissle 1917 to persist despite competition from other bacteria. Furthermore, Nissle 1917 showed a bactericidal effect against strains with various virulence genes, and uropathogens causing cystitis and pyelonephritis. Nissle 1917 inhibited or overgrew 80% of the strains causing cystitis and interfered with 86% of the pyelonephritic isolates. These results reveal that through entirely different bactericidal mechanisms of action Nissle 1917 successfully eradicated most known pediatric uropathogens. In reality our study may have underestimated the protective effect of the Nissle strain against uropathogens. Previous in vitro and in vivo studies suggest that Nissle 1917 may be protective by other means. Altenhoefer18 and Oelschlaeger19 et al noted in cell culture that Nissle 1917 decreased the ability of several bacterial pathogens to invade an intestinal epithelial cell layer. Nissle 1917 also improves the innate defense system of the colon by stimulating desmosome and tight junction proteins, and inducing the production of colonic antimicrobial peptides.22,23 Through these mechanisms Nissle 1917 makes the bowel less permeable to bacterial invasion and boosts natural defenses against pathogenic organisms. These additional mechanisms of action might further potentiate the protective effect of Nissle 1917 against uropathogens. Most commonly, lactobacillus based probiotics have been previously studied to prevent recurrent UTIs. The aim in using these probiotics is to recolonize the bowel and vagina with lactobacillus. Background studies showed that in adults and children with UTIs colonic lactobacillus colony counts are decreased before probiotic use.11,12
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BACTERICIDAL ACTIVITY OF ESCHERICHIA COLI NISSLE 1917
However, compared to adults the vaginal lactobacillus microflora do not appear to be altered in children who are susceptible to UTIs.11 Thus, lactobacillus based probiotics may not be as effective in young females. Also, while some studies in adults showed that these probiotics may reestablish lactobacillus in the colonic and vaginal microflora, it remains unclear whether this is actually efficacious in inhibiting UTIs.12 Upon review minimal data have established the lactobacillus mechanism of action against uropathogens or its clinical usefulness in preventing recurrent UTIs.12 In contrast, from our analysis and previous research it appears that Nissle 1917 may be efficacious in preventing and eliminating the colonization of pathogenic bacteria in the bowel.18,19,22,23 Such a decrease in the fecal load of uropathogens could then decrease the incidence of recurrent UTIs. While Mutaflor appears to be a promising prophylactic agent against recurrent UTIs derived from intestinal bacteria, it may not be efficacious in cases of recurrent infection resulting from dormant bacteria already in the bladder. After an acute UTI E. coli may establish a latent infection in bladder transitional cells in the form of QIRs.9,10 These QIRs may persist for months, resist antibiotic treatment and serve as a source of UTI recurrence.9,29 Since QIRs reside in bladder transitional cells rather than in the gut, this reservoir would not be susceptible to the effect of Nissle 1917. This could potentially allow
recurrent infection despite an excellent Mutaflor effect on colonic bacterial flora. Another potential concern is that oral administration of Mutaflor may result in Nissle 1917 E. coli actually causing UTIs. This appears unlikely since genomic analysis of Nissle 1917 verified the absence of any pathogenicity factors typically found in uropathogenic E. coli.29,30 Also, it has been studied in children of all ages, including neonates, without any apparent overt side effects, including Nissle 1917 induced UTIs.20
CONCLUSIONS In our controlled in vitro study Nissle 1917 E. coli, the active agent in the probiotic Mutaflor, inhibited or outcompeted most bacterial isolates in our library. These findings along with those previously identified in the treatment of inflammatory and infectious bowel disease suggest that Mutaflor may be a useful agent in the prevention of recurrent UTIs in children. While additional studies are needed to validate and further define the clinical effectiveness of Mutaflor, based on these preliminary in vitro findings we believe that future trials evaluating its therapeutic application are warranted.
ACKNOWLEDGMENTS Ulrich Sonnenborn, Ardeypharm, Herdecke, Germany, provided the Nissle 1917 E. coli sample and permission to study the strain.
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