CA1298556C - Lactobacillus compositions and method for treating and preventing urinary tract and ileal or colon infections - Google Patents

Abstract

A B S T R A C T

This invention relates to lactobacillus compositions and methods employing said compositions for treating or preventing urinary tract or ileal or colon conduit infections.
More particularly, this invention relates to the ability of certain strains of lactobacilli to adhere to epithelial cells and to produce an inhibitor against the growth of pathogenic bacteria as means of treating or preventing urinary tract, ileal or colon conduit infections.

Description

~91~5S~

Background of the Invention This invention relates to lactobacillus cornpositions and methods employing said compositions for treating or preventing urinary tract and ileal or colon conduit infections. More particularly, this invention relates to compositions and methods ernploying certain strains of lactobacilli which have the ability to adhere to epithelial cells and associated mucus and to produce an inhibitor against the growth of pathogenic bateria as a means of treating or preventing urinary tract infections, catheter-associated urinary tract infections, and ileal and colon conduit infections.
It is well known that indigenous, non-pathogenic bacterla predominate on epithelial cells and associated mucus in the healthy state, and that pathogenic organisms predominate in the stages leading to and during infections. The possibility that indigenous bacteria have a role in preventing infection has been postulated for many years, but few studies have been carried out to identify specific bacteria and their properties required for such an effect. Hata et al ~.S. Pate No. 4,314,995 investigated anaerobic, lactobacilli-like organisms as a means of treating a number of infectious diseases, but no consideration was given to the combined importance of bacterial adherence, competitive exclusion and inhibitor activity, and no discussion was included of urinary tract infections or ileal and colon conduit infections. As lactobacilli are only one of many microorganisms indigenous to the urinary tract, it could not be assumed that they would interfere with the adherence and growth of pathogenic bacteria ~k 9.~913~

on the epithelial surfaces of the urinary tract and ileal and colon conduits. Mutai et al. U.S ~atent No. 4,347,240 discloses a composition and method employing a strain of lactobacilli for inhibiting tumor growthO No suggestion is made for the use of lactobacillus to prevent or treat urinary tract or ileal or colon conduit infections.

_mmary of the Invention Specific strains of lactobacilli have been identified namely aerobic and microaerophilic isolates, which particularly after growth in urine and in brain heart infusion yeast extract medium, (a) adhere to epithelial cells, (b) competitively exclude pathogenic bacteria from adhering to epithelial cells when the lactobacilli are in the form of whole viable and non-viable cells and cell wall fragments, and (c) produce an inhibitor activity against the growth of pathogenic bacteria.
The present invention describes compositions and methods for using adherent and inhibitor-producing lactobacilli strains to treat or prevent infections of the urinary tract and of ileal and colon conduits.
In its broad aspect, the present invention includes method for the treatment or prevention of urinary tract or ileal or colon conduit infections of a human or lower animal which comprises administering a safe and effective amount of lactobacillus viable whole cells, non-viable whole cells, cell wall fragments, or inhibitory substances, said lactobacillus being one or more species which adheres to epithelial cells.
The lactobacillus preferably is aerobically or micraerophilically grown and the lactobacillus may be selected ~l~91~556 from the group consisting of L. casei, L. acidophilus, L.

plantarum, L. fermentum, L. brevis, L. jensenii and L~
~ _ crispatus.
More specifically, the lactobacillus may be aerobically grown and is selected Erom the group consisting of L casei var rhamnosus GR-l, L. casei var rhamnosus GR-2, L.
casei var rhamnosus GR-3, L. casei var rhamnosus GR-a~, L. case.i var rhamnosus RC-9, L. casei var rhamnosus RC-17, L. casei va alactosus ~C~-21, L. casei NRC 430 and L. casei ATCC 7469.
Alternatively, the lactobacillus may be microaero-philically grown and selected from the group consisting of L.
acidophilus RC-12, L. aci.dophilus RC-25, L. plantarum RC-l9, L.
~enii :~C-ll, L. ~ ATCC 4357 L. plantarum ATCC 8014 and L. fermentum A 60.
In another aspect of the invention, a pharmaceutical composition for treating or preventing urinary tract or ileal or colon conduit infections .in humans and lower animals comprises a safe and effective amount of one or more of the said aforementioned lactobacilli with a pharmaceutical carrier.
~ y "safe and effective amount" as used herein is meant an amount of lactobacillus high enough to significantly positively modify the condition to be treated but low enough to avoid serious side effects ~at a reasonable benefit/risk ratio,) within the scope of sound medical judgment. A safe effective amount of lactobacillus will vary with the particular condition being treated, the age and physical condition of patient being treated, the severity of the condition, the duration of treatmentr the nature of concurrent therapy, and the specific lactobacillus employed. We have found that at ~LZ9E~5iS6 least 1~ lactobacilli, and preferably 20 to 30, and more preferably more than ~0 said bacteria adhered to epithelial cells are desired. The effective amount of lactobacillus will thus be the minimum amount which will provide the desired attachrnent to epithelial cells. The presence of S x 10~
bacteria, as viable or non-viable whole cells, in 0.05 ml.
solution of phosphate buffered saline solution, or in 0.05 ml of suspension of agar, or the dry weight equivalent of cell wall fragments, has been found effective when adminlstered in quantities of ~rom about 0.05 ml. to about 20 ml.
By "pharmaceutical carrier" as used herein is meant one or more compatible solid or liquid filler diluents, or encapsulating substances. By "compatible" as used herein is meant that the components of the composition are capable of being commingled without interacting in a manner which would substantially decrease the pharmaceutical efficacy of the total composition under ordinary use situations.
Some examples of substances which can serve as pharmaceutical carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch;
cellulose and its derivatives such as sodium carboxymethy-cellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calclum sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oill olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol; agar; alginic acids; pyrogen - free water; isotonic saline, and phosphate buffer solution; as well as other non-toxic compatible ~9~3S~6 substances used in pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colouring agents, flavouring agents, lubricants, excipients, tabletting agents, stabili~ers, anti-oxidants and preservatives, can also be present.

Description of the Preferred Embodiments of the Invention A number of assays were devised to examine the ability of lactobacilli to adhere to epithelial cells, to competitively exclude pathogenic bacteria from adhering to epithelial cells, and to produce an inhibitor activity against the growth of pathogenic bacteria.
The invention will now be illustrated by means of the Eollowing non-limitative examples.

Example 1 Different growth media were tested to determine their efEect on adherence of lactobacilli to squamous and transitional epithelial cells. The organisms were grown aerobically overnight at 37C, then harvested and incubated with the epithelial cells, obtained from the urine of healthy women, and the adherence of the lactobacilli was assayed by a technique described in Reid et al., Journal of Infectious Diseases, September 1983.
Table 1 shows adherence to epithelial cells of L.
casei strain ~R-l grown in different culture media. This study established that aerobically grown lactobacilli attached in large numbers to epithelial cells, and that the organisms grew well in human urine, which is important for use of lactobacilli ~L298SS6 for treatment or prevention of urinary tract and ileal and colon conduit infections.

Growth media l,actobacilli per_epithelial cell _ _ _ . .. . _ . _ ..
BYE Broth 62.68 BYE Tween~ 48.83 r MRS Broth 48.40 B~I Broth 49.28 TS Broth 51.85 Urine 73 00 BYE Agar 63.78 BYE Tween 52.60 MRS Agar 48.13 BHI Agar 50.68 TS Aqar 60.73 Rogosa's Agar 38.08 BYE = brain heart infusion medium (BBL Microbiology Systems, Becton Dickinson & Co. Cockeysville, U.S.A.). with 0.5% yeast extract; Tween = 0.1% Tween 80, (BBL, USA); MRS = Lactobacillus MRS medium (Difco USA), BHI = brain heart infusion medium (BBL, USA); TS = trypticase soy (BBL, USA); Rogosa's = Rogosa SL, lactobacilli selective medium (Difco, USA).

Example 2.
Lactobacilli strains were isolated from the urethra, cervix and vagina of healthy women with no history of urogenital tract infections. The strains were identified as 6.

~Z~35~6 lactobacilli and speciated by methods described in the Virginia Polytechnic Institute (VPI) Laboratory Manual (Anaerobe Labo~ , 4th edition, 1977, published by VPI Anaerobe Laboratory, Blacksburg, Virginia). The adherence of aerobic and microaerophilic strains to epithelial cells was tested using an in vitro assay which employed uroepithelial cells from urine sediMent. It should be noted that the adherence values vary depending upon the receptivity of the host cells. The menstrual cycle affects this receptivity, and maximurn adherence occurs prior to ovulation and prior to menstruation. (Chan et al., Journal of Urology, March 1984). The uroepithelial cell used in the experiments of Table 2 were harvested from women on the tenth day of their menstrual cycle, to optimise subsequent attachment oE lactobacilli.
The results shown in Table 2 establish the high adherence capacity of aerobic and microaerophilic lactobacilli.

Strains Lactobacilli attached per epithelial cell .. . _ . _ _ .. . .. _ .
L. casei GR-l 44.50 L. casei GR-2 51.83 .~ _ L. casei GR-3 33.85 L. casei GR-4 37.93 L. casei RC-9 17.80 L. casei RC-17 118.70 L. casei XC-21 27.83 .

~IZ98S56 Strains Lactobacilli attached pcr epithelial cell L. acidolhilus ~C-12 17.43*

L. plantarum RC-l9 23 28*
.

L. acidophilus RC-2~ 14.88*
L. jensenil RC-ll 31.50*
L~ casei NR~ 43~ 30.35 L. case_ ATCC 746g 35.6~
L. acidophilus ATCC 4357 19.43*
L. plantarum ATCC 8014 18.23*
L. acidophilus T 13 14.00*
L. fermentum A 60 52.15 The lactobacllli were grown aerobically except where signified"*", which identifies organlsms grown microaerophilically in 10~ ~2' as this condition was found to improve the bacterial attachment of these particular lactobacilli strains. Comparative stud;es have shown that the aerobic strains tend to grow more rapidly and attach to f~ rc~aeroph,/~c epithelial cells in larger numbers than ~ o~ y~
strains. The ability of lactobacilli to grow rapidly in aerobic and microaerophilic conditions, particularly in urine medium, provides the organisms with an opportunity to attach to and colonize epithelial cells and associated mucus in the urinary tract and in ileal and colon conduits, enhancing their competition with pathogenic bacteria, which are known to grow rapidly under similar conditions.
Studies with batch cultures have shown that highly adherent L. casei GR-l and ~C-17 possess capsular material ~9~

surrounding their cells following growth in urine, urine supplemented with carbohydrates and brain heart infusion yeast extract agar. Chemostat cultures under carbon and nitrogen limitation also attached well to uroepithelial cells and produced capsules, and there was evidence of chaining. Lag, log and stationary phase cultures attached well to uroepithelial cells ln vitro, particularly stationary phase bacteria.
It is preferred that lactobacilli adhere to epithelial cells in at least greater than lU bacteria per cell, more preEerably greater than 25 and more preferably still greater than 40 bacteria per cell, using the adherence assay deseribed by Reid et al., (1983) and using epithelial cells harvested at peak times of receptivity, as defined by Chan et al., (1984), both referred to above.

Example 3.
The following studies were designed to test speeifie laeto~acillus isolates for their ability to competitively exelude eommon pathogenie baeteria from adhering to epithlelial eells. An in vitro assay was designed whieh involved ineubating laetobaeilli with uroepithelial eells for 30 minutes at 37C
prior to ineubation with the radiolabeled pathogens for 30 minutes at 37C, as deseribed by Chan et al., Infeetion and Immunity, January 198~. The pereentage in.~ibition cause by lactobacilli was measured from the numbers of radiolabeled organisms adhering to epithelial eells in test and eontrol samples, as shown in Table 3.
The results demonstrate the ability of laetobaeilli to eoi~petitively bloek pathogenie haeteria from adhering to ~98S5~i epithelial cells, which demonstrates that lactobacillus strains can be used to prevent urinary tract infections. Studies have shown that L. casei ~R-l can detach uropathogens from uroepithelial cells (up to 45% detachment) supporting the use of lactobacilli in treating infections.

Lactobacillus Pathogenic Bacteria % Inhibition of attachment of pathogen L. casei G~-l 94100 86 74 61 71 L. casei ~R-2 8481 lUO 58 59 79 -L. casei GR-3 5046 lOQ 83 37 77 L. casei RC-9 S728 L~T NT 69 NT
-L. casei RC-21 NT lOO NT NT 14 NT
... _ . . . .
L. casei ATCC 7469 42S8 53 30 66 75 L. plantarum ATCC 8014 5182 76 71 44 75 L. fermentum A ~0 5137 10~ 82 30 65 L. acidophilus T-13 420 21 74 0 63 L. fermentum B 54 390 53 50 0 64 l= E. coli strain C1212-77; 2= E. coli mannose sensitive .
strain; 3= Proteus mirabilis strain 28cii; 4= Klebsiella pneumoniae strain 3a; 5= Pseudomonas aeruginosa mucoid;
6= Pseudomonas aeruginosa non-mucoid; NT= not tested.
Lactobacilli were Eound to coaggregate with uropatho~ens, especiall~ mucoid Pseudomonas aeruginosa when incubated in urine. This is the first report of a coaggregation effect of urogenital bacteria, and varied for each urine sample, and for the combination of pathogens (E. coli, P.
rnlrabilis, Ps. aeruginosa, enterococci, K~ pneumoniae, P.

10 .

~Z98~

vu~garis, S. sapro~hyticus, S. aureus and S. epidermidis).
This eoaggregation effect may be factor in maintenance of a actor healthy urogenital tract, and may be~ important~ in preventing infection.

Example 4 -Data has been obtained demonstrating that laetobaeilli adherence to epithelial cells ls important in the exclusion of uropathogenic bacteria. Experiments determined that non-viable whole cells and cell wall fragments of lactobacilli attaehed to epithelial cells, and exeluded the attaehment of uropathogenie baeteria. These experiments have been carried out using a strain of lactobaeilli now identified as L. easei GR-l, grown aerobieally in urine. The laetobaeilli preparations were first ineubated with the uroepithelial eells from the urine sediment for 30 minutes at 37C prior to ineubation with the radiolabeled uropathogenie baeteria (E. eoli, K. pneumoniae. P.
aeruginosa) for 3~ minutes at 37C, as diselosed in Chan et al Infeetion_and Immunity, January 1985. The results demonstrated that formalin-killed whole cells gave 38 to 72% bloekage of uropathogenie baeterial adherence (Mean = 46%), acid-treated whole cells gave 5 to 31% blockage (Mean = 20%), and other eell wall fragments gave a range of from 4 to 75% bloekage (Mean =
34%~. The eell wall fragments ineluded lipoteiehoie acids, peptidoglycan, and fragments obtained by sonication, sodium dodeeyl sulfate (SDS) extraetion and S~S extraetion with aeid treatment. The laetobaeilli preparations prevented the pathogenie organisms from adhering to the epithelial eells by a steric hindranee effect and by competition and blockage of the 11 .

~Z9~35~6 receptor sites normally used the pathogenic bacteria. The results obtained using the lactobacilli whole cell and cell wall fragment preparations show that non-viable lactobacilli preparations attach to epithelial cells and can be used to exclude pathogenic bacteria from adhering to the cells ~xample ~
Studies have shown that uroepithelial cells from patients with a urinary tract infection are colonized with pathogenic bacteria (mean of 20 bacteria per cell in a study of 3/ patients). The administration of antibiotics often eradicates these organisms, unless they are encased in a microbial biofilm. ~owever, the bacteria-free uroepithelial cells are still receptive to bacterial adherence, as indicated ln Table 4, where E. coli strain C1212-77 was incubated wlth uroepithelial cells in vitro from women on antibiotic therapy, and assayed using the method of Reid et al., (1933), referred to above. The high attachment figures illustrate that the patients are still receptive to recolonization, and that other protective measures are required to exclude the uropathogens.

E. coli attached per epithelial cell Patient 1 76.90 Patient 2 77.68 Patient 3 ~2.30 Patient 4 71~60 Patient ~ 122.70 9i3SS~

TABLE 4 (Cont'd.) ~atient ~ 82.85 Patient 7 58.45 Patient 8 51.53 Patient 9 119.35 Patient 10 36.70 Patient 11 36.55 Similar high attachment figures were obtained for other uropathogens, including other E. coli strains, Proteus mirabilis, Klebsiella ~neumoniae and Pseudomonas aeruginosa.
Seven of these ll patients developed a recurrent UTI within the months following this study.
~ ased upon the data wh.ich show that lactobacilli whole cells and cell wall fragments cornpetitively excluded pathogenic ,p~e~o~o~J~zG~f~,7 bacter.ia from adhering to epithelial cells, ~ lon.izit-~en of the epithelial cells from patients (such as those ll shown in Table 4) with lactobacilli would have acted as a protective barrier against attachment with uropathogens.
Colonization of the epithelial cells and associated mucus with lactobacilli preparations can be used to prevent UTI
from recurring. In the case of the 11 patients in the study shown in Table 4, such treatment would be expected to have pxevented UTI from recurring in the 7 patients.

Example ~.
In addition to adhering to epithelial cells and preventing the colonization with pathogenic bacteria, lactobacilli possess other properties which prevent pathogens 13~

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from colonizing epithelial surfaces. Lactobacilli strains which adhered to epithelial cells were tested for an inhibitory activity, uslng an agar overlay technique similar to the technique disclosed in Performance Standards for Anti-microbial Discs Susceptibility Tests published by The National Committee for Clinical Laboratory Standards ~1979), modified in that the bottom layer contalned lactobacilli, the top layer was spread with E. coli, and antibiotic discs were not applied. One ml or 109 lactobacilli per ml. was inoculated into the bottom layer of agar; the top layer was added; the plates were incubated at 37C overnight, then the pathogenic bacteria (109 per ml.) were streaked onto the top layer, and the plates incubated at 37C overnight.
The growth of lactobacilli on the bottom layer of agar was found to inhibit growth of pathogenic bacteria on the top .
layer. The results presented in Table 5 illustrate that this inhibitory activity is an important factor in e~x~inatlo~ with bacterial adherence to enable lactobacilli to be used as an agent to treat or prevent infection.

L. casei GRl ++ ++ ++ -- ++ +-~ ++ +- ++ +-L. casei GR2 -~- +- ++ -- -- ++ -~+ -- ++ +-L. casei GR3 t- ++ +- -- ++ ++ ++ +- -- +-L. casei GR4 ++ ++ ++ +- ++ ++ ++ +- ++ +-~ . _ L casei RC9 t- ++ -~+ +- ++ ++ ++ +_ ++ +_ L. casei RC17 +- -~- ++ -- -- +- ++ ++ ++ +-L. casei RC21 +- -~- ++ -- -- ++ ++ +- ++ +-14.

~Z~1~5~

L. acidophilus RC12 +_ +_ +_ __ ++ ++ ++ + ++ +

L. plantarum RCl~ +- +- ~ - +- NT NT NT
L. acidophilus RC25 -~ +- -- -- +- +- NT NT ~T
L. jensenii RCl l +- +- -~- ++ -- ++ -- NT NT NT
L. casei NRC 430 r~ + ~ + ~ +
L. casei ATCC 7469 +- ++ ++ +- +++-~ ++ -- +- +-L. acidophilus ATCC 4357 ++ ++ ++ +- +-~++ ++ ++ ++ +-L. plantarum ATCC 8014 -1~ - -- +~+~ + ++ ++ +-L. fermentum A~0 -- -- -- __ __ __ __ __ ++ __ ++ = cornplete inhibition, +- = partial inhibition, -- = no inhibition; NT = not tested 1 = E coli C1212-77; 2 = E. col_ C1214-77; 3 = E. coli 734; 4 K. pneumoniae 3a; 5 = P. mirabilis 28 cii; 6 = Pseudomonas aeruginosa non-mucoid; 7 = Pseudomonas aeruginosa mucoid; 8 =
Staphylococcus saprophyticus YA; 9 = enterococci 1331; 10 =
Staphylococcus aureus 1750.

The inhibitory substance(s) produced by the lactobacilli are currently unidentified, but will be deEined as by-products of metabolism which prevent or inhibit the growth of pathogenic bacteria.
Further experiments with L. casei GR-l and L.
acidophilus 76 (ATCC 4357) have shown that the inhibitors are 1~9~3S~

heat stable to 1~0C and produced in media of various pH
values, especially pH 5.5. Brain heart infusion medium supplemented with yeast extract (0.5 to 2~) was found to cause maximum activity on agar, after aerobic, microaerophilic ~ C02) and anaerobic growth. The activity was present in media buffered for acid production with phosphate buffer (O.O~M, p~ ~.0) and Trizma buffers (0.05M, pH 7.5, 8.0, 8.5 and 8.7). In addition, the fatty acid levels of all the different lactobacillus strains tested showed various levels which did not coincide necessarily with the organism's ability to inhibit the growth of other bacteria. ~y making wells in the agar, it was possible to test the effect of enzymes, including trypsin, protease, papain, lipase, chymotrypsin, lysozyme and lactic dehydrogenase, and there was no blocking of the inhibition effect. tlydrogen peroxide was ruled out as inhibition resulted after anaerobic growth. Bacteriophage were ruled out as phage do not diffuse through agar, no plaques were seen on diminishing dilutions to extinction and a reverse-side agar technique excluded phage. The inhibitors had a bactericidal effect and also inhibited the growth of other lactobacillus strains. The strains also inhibited growth of yeasts and other vaginal bacteria from adults and children. Zones of inhibition were found by inoculating a single lactobacillus colony to the bottom agar layer and spreading the uropathogens onto the top layer. There was no evidence of resistant uropathogen colonies within the zones. Freeze dried cultures were able to produce inhibition upon growth. The E. coli Hu734 (inoculum of 104) did not grow in filter-sterilized supernatant from GR-l and 76 grown liquid cultures. The activity appears to be in the liquid culture as well as in agar.

16.

9 29~556 In patient studies in children and adult women, we have shown that lactobacilli can be found on the urogenital mUCQSa of women with urinary tract infections, but these strains are often unable to inhibit the growth of the infecting uropathogen. These observations suggest that inhibitor activity is important for prevention of infection.

Example 7.
The development of urinary tract infections is primarily caused by organisms colonizing the intestine, then sequentially colonizing the perineum, vaginal introitus, urethra and bladder. This is referred to as an ascending infection. In some patients, the infection ascends further into the ureters and kidneys to give rise to pyelonephritis.
There is no ideal animal model which can be used to study the ascending infections of humans. Therefore, the two animal systems described herein were used to examine the ability of lactobacilli to prevent pathogenic bacteria from colonizing the epithelium of the bladder and kidneys. However, tne bladder cells cornprise of transitional and squamous epithelial cells, the latter type being similar to those of the perineum, vaginal introitus and urethra. Therefore, the results from the animal experiments may also be used to demonstrate that the lactobacilli will competitively exclude uropathogens from colonizing epithelial cells of the type found in the perineum, vaginal introitus, urethra, bladder, ureters and kidneys. The results obtained from the animal experiments (Examples 7 and 8) support the use of lactobacilli preparations to prevent infections of the urinary tract.

17.

~2~855i6 Female Sprague ~awley rats were used as a model for chronic urinary tract infection (UTI) and transurethral instillation o~ uropathogenic bacteria, incorporated into an agar bead suspension, injected into the bladder. This resulted in lon~ term colonization of the uroepithelium of the bladder (up to log 7.3 vlable bacteria after 2 months) and kidneys up to log 8.9 bacteria after 2 months 5as discussed in Reid et al., Infection and Immunity, August 1985, incorporated herein by reference). ~owever, by instilling lactobacilli 3 weeks prior to challenge, the uropathogens were not found to attach and multiply, and ~TI was prevented in 84% of animals (21 protected out of 25 tested). Furthermore, although the lactobacilli attached to and colonized the uroepithelium, there was no adverse response detected ln the hosts against these non-pathogenic bacteria.

xample 8.
The second model was that of an acute UTI, using CBA
mice. A similar protocol was followed to that described for Example 7, except that lactobacilli incorporation of 5 x 109 bacteria in 0.05 ml. solution of phosphate buffered saline, and also the equivalent in agar beads, was tested. The lactobacilli were instilled two days prior to challenge, and the animals sacrificed after 24 hours. The results demonstrated that 19 of 30 mice did not develop infection after treatment with lactobacilli in a phosphate buffered saline suspension, whereas 28 of 30 control mice developed UTI. The animals given lactobacilli incorporated into agar beads also showed a degree of protection (67% protected). The strain of lactobacilli used in the mouse and rat model experiments was L.
casei var rhamnosus GR-l. Only 2% of mice treated with straln 1~ .

~Z9E~5~i6 GR-l and possessing 103 lactobacill:i ln the bladder and only 8% with 104 lactobacilli in the kidney were lnfected with E.
coli ~u 734. The fact that other strains of lactobacilli have been found to attach to epithelial cells and have also been found to produce active inhibitory activity against the growth of pathogenic bacteria demonstrates that adherent lactobacilli strains producing an inhibitor activity will be effective in competitively excluding pathogenic bacteria from the epithelial mucosa, and thus be effective for treating or preventing urinary tract infections.

Example 9 Patients requiring urinary diversion undergo an operation whereby a segment of ileum or colon is utilised to form a conduit which is connected to the ureters, and allows secretion of urine into a collecting bag. The majority of these patients develop infection in the conduit during and shortly after completion of antimicrobial therapy. As many as 14~ of patients develop pyelonephritis, due to ascension of the pathogens up the ureters into the kidney, Bruce et al., Journal of Urol_gy, 1984. Often, antimicrobial prophylaxis is unable to prevent this infection, and patients may suffer renal damage and progress to renal failure. The conduit infections are caused by a variety of pathogenic microorganisms. Examination of those patients who do not develop infection has shown that nonpathogenic bacteria colonize the conduit and mucus. In vitro experiments have shown that L. casei var rhamnosus GR-l attaches to ileal conduit tissue. Thus lactobacilli can be used to colonize the conduit epithelium and mucus, as a means of treating or preventing urinary conduit infections.

19.

~Z98~5~i Mucopolysaccharide substances are known to coat the surfaces of epithelial cells. These substances may contain receptors for different bacterial species. Often, mucopolysaccharides are free in the urine (for example~ Tamm Horsfall Glycoprotein) and in the ileal and colon conduit (for example, produced by goblet cells). The binding of adherent lactobacilli which produce inhibitory activity against the growth of pathogens, to those mucopolysaccharide substances will be expected to act as a barrier against colonization by pathogenic bacteria.

Example 10 The patients at most risk of acquiring an infection are those requiring long term and intermittant catheterization.
Catheter insertion causes trauma and acts as a focus for pathogenic bacteria to colonize the uroepitheliurn and the catheter itself in dense microcolonies which are resistant to antibiotic penetration. This leads to persistant infection.
An examination of the intrauterine devices talsen from 6 healthy patients with no history of pelvic or cervical infection showed that lactobacilli were colonizing the surfaces and no pathogens were isolated upon culture (enterococci were found in one case). These studies suggest that lactobacilli can coat prostheses (which are placed long term inside the body on a mucosal surface) without the patient developing infection. By utilising the adherence capacity of lactobacilli, it is possible to coat the uroepithelium and catheter surfaces thereby excluding pathogens from colonizing and causing an infection. The use of inhibitory substances produced by lactobacilli will also protect the host from colonization of both uroepithelium and catheter surfaces by the pathogenic organisrns.

Claims (36)

1. The use of lactobacillus viable whole cells, lactobacillus non-viable whole cells, lactobacillus cell wall fragments, or lactobacillus-derived inhibitory substances, or mixtures of one or more thereof, for the use of treatment or prevention of the urinary tract infection of a manual in need of such treatment or prevention.

2. The use according to Claim 1 wherein said lactobacillus is a species which is aerobically or microaerophilically grown.

3. The use according of Claim 2 wherein said lactobacillus is selected from the group consisting of L. casei L. acidophilus, L. plantarum, L. fermentum, L. brevis, L. jensenii, and L.
orispatus.

4. The use according to Claim 2 where said lactobacillus is a species which is aerobically grown.

5. The use according to Claim 4 wherein said lactobacillus is L. Casei.

6. The use according to Claim 4 wherein said lactobacillus is selected from the group consisting of L. casei var rhamnosus GR-1, L. casei var rhamnosus GR-2, L. casei var rhamnosus GR-3, L.

casai var rhamnosus GR-4, L. casei var rhamnosus RC-9, L. casei var rhamnosus RC-17, L casei var alactosus RC-21, L. casei NRC
430 and L. casei ATCC 7469.

7. The use according to Claim 2 wherein said lactobacillus is a species which is microaerophilioally grown.

8. The use according to Claim 7 wherein said lactobacillus is selected from the group consisting of L. acidophilus L.
plantarum, L. Jensenii and L. fermentum.

9. The use according to Claim 8 wherein said lactobacillus is selected from the group consisting of L. acidophilus RC-12, L.
acidophilus RC-25, L. plantarum RC-19, L. jensenii RC-11, L.
acidophilus ATCC 4357, L. plantarum ATCC 8014, L. fermentum A-60 and L. fermentum B-54.

10. The use according to one of Claims 1,2 or 3 wherein said infection is associated with the use of a urinary catheter.

11. The use according to Claim 1 wherein said lactobacillus is use in the form of viable whole cells.

12. The use according to Claim 1 wherein said lactobacillus is used in the form of non-viable whole cells.

13. The use according to Claim 1 wherein said lactobacillus is used in the form of cell wall fragments.

14. A prosthetic device useful in treatment or prevention of urinary tract infections of a mammal, said prosthetic device being biologically compatible and insertable into the urogenital tract of said mammal, and having at least a partial coating of a lactobacillus species, in the form of lactobacillus viable whole cells, lactobacillus non-viable whole cells, lactobacillus cell wall fragments or lactobacillus-derived inhibitory substances, which adheres to uroepithelial or vaginal epithelial cells.

15. The prosthetic device according to Claim 14 wherein said lactobacillus is a species which is aerobically or microaerophilically grown.

16. The prosthetic device according to Claim 15 wherein said lactobacillus is selected from the group consisting of L. casei, L. acidophilus, L. plantarum, L.fermentum, L. brevis, L. jensenii and L. crispatus.

17. The prosthetic device according to Claim 16 wherein said lactobacillus is L
casei.

18. The prosthetic device according to Claim 16 wherein said lactobacillus is selected from the group consisting of L. casei var rhamnosus GR-l, L. casei var rhamnosus GR-2, L. casei var rhamnosus GR-3, L. casei var rhamnosus GR-4, L.
casei var rhamnosus RC-9, L. casei var rhamnosus RC-17, L. casei var alactosus RC-21, L. casei NRC 430 and L. casei ATCC 7469.

19. The prosthetic device of Claim 15 wherein said lactobacillus is a species which is microaerophilically grown.

20. The prosthetic device of Claim 19 wherein said lactobacillus is selected from the group consisting of L. acidophilus RC-12, L. acidophilus RC-25, L. plantarumRC-l9, L. jensenii RC-ll, L. acidophilus ATCC 4357, L. plantarum ATCC 8014, L. fermentum A-60 and L. fermentum B-54.

21. The prosthetic device of Claim 14, Claim 15, Claim 16, Claim 17, Claim 18, Claim 19 or Claim 20 in the form of a catheter.

22. The prosthetic device of Claim 14, Claim 15, Claim 16, Claim 17, Claim 18, Claim 19 or Claim 20 in the form of an intraurethral device.

23. A composition suitable for treating or preventing urinary tract infections of a human or lower animal, which comprises one or more of lactobacillus viable whole cells, non-viable whole cells, cell wall fragments or inhibitory substances said lactobacillus being of one or more species of lactobacillus which adheres to uroepithelial or vaginal epithelial cells, and a pharmaceutically acceptable carrier.

24. The composition as claimed in Claim 23 wherein said lactobacillus is a species which is aerobically or microaerophilically grown.

25. The composition as claimed in Claim 24 wherein said lactobacillus is selected from the group consisting of L. casei, L acidophilus, L. plantarum, L. fermentum, L. brevis, L. jensenii and L. crispatus.

26. The composition as claimed in Claim 23 wherein said lactobacillus is aerobically grown.

27. The composition as claimed in Claim 26 wherein said lactobacillus is L. casei.

28. The composition as claimed in Claim 26 wherein said lactobacillus is selected from the group consisting of L. casei var rhamnosus GR-1, L. casei var rhamnosus GR-2, L. casei var rhamnosus GR-3, L. casei var rhamnosus GR-4, L. casei var rhamnosus RC-9, L. casei var rhamnosus RC-17, L. casei var alactosus RC-21, L.
casei NRC 430, L. casei ATCC 7469.

29. The composition as claimed in Claim 24 wherein said lactobacillus is microaerophilically grown.

30. The composition as claimed in Claim 29 wherein said lactobacillus is selected from the group consisting of L. acidophilus, L. plantarum, L. jensenii and L.
fermentum.

31. The composition as claimed in Claim 30 wherein said lactobacillus is selected from the group consisting of L. acidophilus RC-12, L. acidophilus RC-25, L.
plantarum RC-l9, L. jensenii RC-11, L. acidophilus ATCC
4357, L. plantarum ATCC 8014, L. fermentum A-60, and L.
fermentum B-54.

32. The composition as claimed in Claim 23 in the form of one of a cream or a suppository.

33. An inhibitory substance produced by one or more Strains of lactobacillus which adheres to uroeplthelial or vaginal epithelial cells, said substance being suitable for use in the treatment or prevention of urinary tract infections in mammals.

34. The substance according to Claim 33 wherein said lactobacillus is a species which 18 aerobically or microaerophilically grown.

35. The substance according to Claim 34 wherein said lactobacillus is selected from the group consisting of L. casei, L. acidophilus, L. plantarum, L. fermentum, L. brevis, L. jensenii and L. crispatus.

36. The substance according to one of Claims 33, 34 or 35 wherein the inhibitory substance has a molecular weight of at least between 12,000 - 14,000.