WO1997036603A1

WO1997036603A1 - Biocontrol agents for use in treatment of opportunistic infections

Info

Publication number: WO1997036603A1
Application number: PCT/AU1997/000211
Authority: WO
Inventors: James Harrison Aylward; Mary Anna Williamson
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 1996-04-02
Filing date: 1997-04-02
Publication date: 1997-10-09
Also published as: AUPN907296A0

Abstract

The invention relates to a new Lactobacillus casei strain which is effective in the treatment of fungal infection. It also relates to compositions comprising this strain and to methods of treating opportunistic infections particularly fungal infections.

Description

BIOCONTROL AGENTS FOR USE IN TREATMENT OF

OPPORTUNISTIC INFECTIONS

This invention relates to a biocontrol agent useful for treatment of opportunistic infections,

particularly anti-fungal infections. It also relates to methods of treating infections and compositions comprising the biocontrol agent of the invention.

Background of the Invention

Severe microbial infections are frequently seen in patients who are immunocompromised due to anti-cancer chemotherapy, immunosuppression in organ graft recipients, thermal injury, and in particular, Acquired Immune

Deficiency Syndrome (AIDS). The types of opportunistic infections encountered are persistent and varied, with a wide spectrum of disease manifestations. Fulminant infections, especially pneumonia, are common and are freqently the proximate cause of death. Typically, the organisms causing opportunistic infections are highly drug resistant. Examples of these infections are:- (i) bacteria: Gram-negative rods such as

Pseudomonas, Xanthomonas,

Achromobacter, Serratia, Klebsiella, and Proteus;

non-endospore forming anaerobic rods such as Bacteroides, Fusobacterium,

Staphylococcus (especially

methicillin-resistant S. aureus ("Golden Staph")) and cloxacillin- resistant S. epidermis; and non- tuberculous mycobacteria such as

Mycobacterium avium-intracellulare complex, M. scrofulaceum, M. bovis, M. kansasii, M. paratubercolis . (ii) fungi: Candida, Aspergillus, Cryptococcus, and the fungi in Table 1 (compiled by Libero Ajello, Ophthalmic Research, Emory University Eye Centre, N.E. Atlanta, GA 30322, USA, February

1994)., which are plant, insect, soil and animal/human pathogens

(iii) viruses: Herpesvirus, and Cytomegalovirus. (iv) protozoa: eg., Pneumocystis carinii and

Toxoplasma gondii..

Furthermore, plant and other non-human pathogens such as fungi which do not normally affect humans can themselves become opportunistic infectious agents in humans who are immuno- compromised.

The incidence of fungal infection is particularly high in AIDS sufferers, and can be life-threatening.

Lactic acid bacteria have been shown to act as effective biocontrol agents in relation to the treatment of opportunistic infections by stimulating the immune systems of immunocompromised hosts. These immunostimulatory effects are summarised in an article by Tomioka and Saito entitled "Lactic acid bacteria in the support of

immunocompromised hosts "In "The Lactic Acid Bacteria", BJB Wood Ed., Elsevier (1992). It was suggested in this article that Lactobacillus casei is the most potent of all Lactobacilli in terms of stimulating the immune system.

In Japanese specification 4-145026, L. casei suppressed the proliferation of both HIV infected and uninfected Molt-4 clone No. 8 T cells in culture. This cell line was derived from a premature stage of lymphocyte development. In contrast, a cell line derived from mature T-cells (MT-4) was stimulated to proliferate in vitro by the biocontrol agent, but at the same time suppressed HIV replication in infected cells. Thus, the authors

speculated that the biocontrol agent was especially

advantageous because in contrast to other immunity strengthening agents, it did not also accelerate HIV in vitro. It was thought that the biocontrol agent would reduce the number of patients suffering from AlDS-related complex (ARC) and AIDS complex (AC). In addition,

anecdotal evidence provided in this specification suggested that L. casei either inhibits the attachment of HIV to the host cell or inhibits proliferation of HIV by effecting stimulation of lymphocytes. However, there was no mention of treatment or prevention of fungal infection.

L. casei has also been shown to exhibit anti- tumour activity in animal models. Gann (75, 72-80) 1984 describes studies in which intravenous injection of L.

casei into mice suffering chemically-induced tumours resulted in total inhibition of tumour growth. In Cancer Immunol. Immunother (20, 18-22) 1985, studies in which intravenous injection of syngeneic mice and guinea pigs with L. casei resulted in the inhibition of implanted lung and liver tumours are described. Int. J. Immunopharmacol. (7, 108-109) 1985 reports studies in which intraperitoneal injection of syngeneic and allogeneic mice with L. casei prior to tumour inoculation resulted in the inhibition of tumour growth.

Reference also may be made to a double blind clinical trial described in European Urology (27, 104-109) 1995, in which L. casei was shown to be effective for preventing recurrence of superficial bladder cancer.

In Medicine and Biology (102, 309-314) 1981, reference is made to murine studies, in which live or heat- inactivated preparations of L. casei enhanced resistance to infection by Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae or Serratia marcescens. Host

resistance to these bacterial pathogens was enhanced at least ten fold by intraperitoneal injection of L. casei 7 days prior to infection.

In Medicine and Biology (104, 171-175) 1982, there is described studies in which L. casei was found to protect strongly against infection of mice by Listeria monocytogenes.

Reference also may be made to studies (reviewed by Tomioka and Saito, op . cit) in which L. casei protected mice against infection by Mycobacterium fortuitum and

Mycobacterium chelonae . In contrast, these studies did not show a protective effect against Mycobacterium avium.

An article by B. Perdigon and S. Alvarez (In

Probiotics R. Fuller, Ed. pp 145-180, 1992) refers to a murine model in which L . casei protected against infection by Salmonella typhimurium.

L . casei has also been shown to protect against pathogenic infection in immunodeficient mice.

Intraperitoneal injection of L . casei in mice,

immunocompromised by corticosteroid or a large tumour burden and challenged subsequently with a lethal dose of P. aeruginoβa resulted in a clearance of the infection

(Tomioka and Saito, op . cit).

Tomioka and Saito (op. cit) also describes the use of L. casei as a protective agent against bacterial infection in mice suffering severe burn wounds. Heat- inactivated L . casei, when added to burn cream, protected against a mixed infection of clinical isolates of P.

aeruginosa, E. coli, and S. aureus .

From the foregoing, it can also be demonstrated that live or heat-inactivated preparations of L . casei can act as adjuvants in respect of immune stimulation (see aforementioned references). The mechanism of this

stimulation is not fully understood but is considered to involve a complex pathway leading to the activation of T- cells, macrophages and natural killer cells.

In view of the increased incidence of microbial resistance to antibiotics and bactericidal agents and of fungi to anti-fungal agents, it is vital to have access to new effective agents, and particularly to compounds that can stimulate the immune system of the host. Active agents which can be consumed as part of the everyday diet would be highly desirable, since they enable ease of administration and provide improved compliance, as well as acting as preventative supplements.

Anti-fungal activity of the Lactobacillus species of bacteria has also not been demonstrated. We have surprisingly found that a specific strain of L. casei has broad spectrum anti-fungal activity.

Summary of the Invention

In one aspect, the invention provides a new and novel strain of L. casei, designated N94/49432. In

particular, it provides an anti-fungal or biocontrol agent comprising L . casei N94/49432.

The bacteria were isolated from Sour Dough

Baker's Starter (SDBS) mixture described in Australian Patent application no. 62776/94 incorporated herein by reference, and a sample has been deposited for the purposes of the Budapest Treaty at the Australian Government

Analytical Laboratories (AGAL) on 21 October 1994 and has been allocated Accession No. N94/49432.

The strain has an inhibitory effect against the growth of a number of fungal anthracnose isolates. Such isolates include Colletotrichum gloeosporioides, C.

acutatum, C. musae, Phoma, Geotrichum, Chaetomium,

Cunninghamella, Cladosporium, Alternaria, Dothiorella and Phomopsis . Other fungi which are also opportunistic pathogens are also inhibited by the bacteria of the

invention. These include, but are not limited to those shown in Table 1. The L. casei of the invention may be used in treatment of infection by human pathogens such as C. albicans, S. apiospermum, A . fumigatuε or the like.

Thus, in a second aspect, the invention provides a method of treatment of a microbial infection, comprising the step of administration of L. casei N94/49432 to a subject suffering from such an infection. Preferably, the infection is a fungal infection.

In a preferred embodiment, the amount of L. casei N94/49432 administered is preferably 10⁷ - 10¹⁰ cells per day per kg body weight. The appropriate dose can be easily determined by those skilled in the art. The administration may be via any route appropriate for the infection

concerned, including the oral, topical subcutaneous, intraperitoneal, intravenous or intramuscular routes.

The bacteria are desirably administered in the form of a composition comprising the novel bacteria and one or more pharmaceutically acceptable salts, carriers, excipients or auxiliaries. One or more other active agents may also be incorporated into this composition.

In a third aspect, the invention provides a composition comprising L. casei N94/49432, together with a pharmaceutically acceptable carrier.

The composition of the invention may be in the form of a liquid, tablet, liposome, powder, capsule, cream, ointment, nasal spray or any other form, provided that the active agent enables the subject to fight against the infection. Suitable types of formulations and methods for their preparation will be known to those skilled in the art for example by reference to "Remington: The Science and Practice of Pharmacy" 19th edition, A.R. Gennaro (ed). Mack Publishing Company, Easton, PA 18062, USA, 1995.

In a fourth aspect the invention provides a edible or drinkable composition in which the L . casei is incorporated into food products such as bread or other baked goods, cereals, dairy products such as milk, yoghurt, cheeses, drinks, or any type of product that can be

fermented. The L . casei of the invention can also be incorporated into any processed food such as processed meat or meat substitutes containing fermentable substances.

Other active agents which can be incorporated together with L . casei N94/49432 include, but are not limited to the wild yeast Hansenula anomala var anomala (NRRL Y-366), Saccharomyces cerevisiae, Lactobacillus parabuchneri, and Lactobacillus parabuchneri/brevis; other probiotic strains of bacteria eg Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus boulardii,

Lactobacillus GG, Streptococcus lactis, or Bifidobacterium; other so called "prebiotics" eg fructooligosaccharides or xylooligosaccharides; or other agents that stimulate the immune system eg coenzyme Q10, shark liver oil, tea

catechins, or Panax ginseng or acemannan from Aloe Vera leaf, Zinc salts, KLH (Keynole Limpet Hemocyanin), BCG (Bacillus Calmette-Guerin), mistletoe extract,

interleukins, or interferons or nitric oxide mediators; or other pharmaceutical anti fungal preparations eg the azoles, cryptolepine, terbinafine, ciclopiroxolamine, or amphoterin B. Agents which provide a synergistic, antimicrobial effect are especially desirable.

Other suitable agents or pharmaceuticals indicated in eg. the MIMS manual may also be used in conjunction with the composition of the invention.

The composition of the invention may be used in the treatment of infections by pathogens particularly fungi. It is particularly useful in treatment of

opportunistic infections in subjects who are immunocompromised due to surgery, radiation therapy, injury or the like. The invention is particularly useful in the treatment of AIDS patients who are prone to fungal

infection.

In a fifth aspect, the invention provides a coating or packaging in which L . casei is incorporated, for example, in items of hospital equipment that may come into contact with immuno-compromised patients such as drip lines, catheters, sutures, dressings and containers.

It will be clearly understood that the invention is also applicable to treatment of domestic, companion or zoo animals, including but not limited to cattle, horses, sheep, dogs, and cats, and to compounds derived from L . casei N94/49432 which exhibit anti-fungal activity as herein described.

Detailed Description of the Invention

The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the figures in which:-

Figure 1 shows the marked inhibition of the growth of C. gloeosporioides 9428A (plate Bb) and of C. musae 24168 (plate Ba) by L. casei N94/49432, compared with the growth of these fungal anthracnose isolates in the absence of L . casei 9313 (plates Ab and Aa). Conversely, growth of these fungal isolates was not inhibited by L. casei 9314 (plates Cb and Ca).

Figure 2 shows that L. casei 9313 inhibited the growth of C. gloeosporioides 23691 (plate Ab), C. acutatum 23459 (plate Bb), C. musae 24168 (plate Cb).

Figure 3 shows inhibition of Phoma species 92234

(plate Ab), unidentified fungal species 92225 (plate Bb) and Geotrichum species 92213 (plate Cb).

Figure 4 shows inhibition of an unidentified

fungal species 9203 (plate Ab),

Chaetomium species 92208B (plate Bb), C. acutatum 9209 (plate Cb).

Figure 5 shows inhibition of an unidentified

fungal species 92233 (plate A).

Figure 6 shows the inhibition of the

Cunninghamella species 92217 (plate B). Figure 7 shows inhibition of Cladosporium species

9204 (plate B).

Figure 8 shows inhibition of the AIternaria

species 9206B (plate B). Figure 9 shows inhibition of the Phoma species

92214 (plate A).

Figure 10 shows inhibition of a Dothiorella species

(plate A), and a Phomopsis species (plate

B).

Figure 11 shows inhibition of Collectorichum

gloeosporioides species 93-62 (plate B) and inhibition of Colletotrichum

gloeosporioides species SR-4 (plate D).

Figure 12 shows inhibition of Colletotrichum

gloeosporioides species UQ62 (plates B &

D). In contrast, plate C shows no inhibition of Colletotrichum

gloeosporioides species UQ62 by L .

sanfrancisco species 43332.

Figure 13 shows inhibition of Colletotrichum

gloeosporioides species 9429 (plate B) and inhibition of Colletotrichum

gloeosporioides species 23691 (plate D).

Figure 14 shows inhibition of Colletotrichum

gloeosporioides species 92210 (plate B) and inhibition of Colletotrichum musae species 24167 (plate D).

Figure 15 shows inhibition of the Colletotrichum aculatum species 92209 (plate B) and inhibition of an unidentified fungal species 92208A (plate D).

Figure 16 shows inhibition of the Phoma species

92232.

Example 1: Isolation of L. casei from Sour Dough

Baker's Starter (SDBS) Mixture.

L. casei strains 9313 and 9314 were isolated from the Sour Dough Baker's Starter (SDBS) mixture as described in Australian Patent application no. 62776/94. Briefly, MRS agar plates were prepared from MRS medium obtainable from Oxoid Australia. 52g of MRS medium was dissolved in 1000 ml of MilliQ water and 20g of agar was added. The mixture was autoclaved at 121°C for 15 minutes, and the plates were poured in a Class II cabinet, while the medium was still warm. After solidification, the SDBS mixture was streaked on to the MRS agar plates, using a sterile wire loop. After 48 hours incubation at 30°C morphologically distinct bacterial colonies were restreaked on to fresh plates. Distinct colonies were observed.

Example 2: Confirmation and Identification of L.

casei.

Identification of pure isolates designated strains 9313 and 9314 in Example 1 was confirmed by the Kluyver Laboratory of Biotechnology, University of

Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

The two strains of L. casei were subjected to a battery of standard identification tests (see Tables 2 and 3). They appeared identical by these tests, ie both were identified as L. casei subspecies casei.

This strain may be grown in clear medium to which yeast extractives have been added preferentially (such as pantothenic acid, pyridoxine, and nicotinic acid). In addition, there is evidence of a bacteria/yeast symbiosis and in this regard, the specific strain of L. casei

referred to above may be cultured in the presence of the wild yeast Hansenula anomala var anomala NRRL Y-366

(originally isolated from bakers yeast and used in the production of wine and sake) and/or Sacchromyces

cerivevisiae which is present in sour dough bread starter culture.

Example 3: Growth Inhibition Plate Assay: Effects of

L . casei on Plant Pathogens.

Inhibition assays with L. casei 9313 were performed as described in "Basic Plant Pathology Methods" O.D. Dhingra and J.B. Sinclair Eds, CRC Press Florida, 1985 pp 247-251, and in R.K.S. Wood, "The control of diseases of lettuce by use of antagonistic organisms. I. The control of Botrytis cinerea Pers., Annals of Applied Biology 38 203

(1951). Each of the fungal pathogens listed in Table 4 was spotted with the aid of a sterile wire loop in the centre of Yeast Extract - Malt Extract - Peptone - Glucose - Agar (YMPGA) - lactose medium plates or 5% MEA media (Oxoid) containing 2% agar, which are both suitable media for growth of both pathogen and antagonist. The composition of the YMPGA - lactose medium was as follows: 0.6% yeast extract (Difco), 0.3% malt extract (Oxoid), 1.0% peptone (Oxoid), 1% D-glucose (BDH), 0.6% tryptone (Difco), 2% lactose (United Milk, Tasmania), 0.000009% Tween 80

(Boehringer) 1.5% fresh yeast extractive (a 20% solution of fresh baker's yeast was autoclaved, allowed to settle and the clear upper portion was used) and 2% agar (Difco). L. casei strains either 9313 or 9314 were grown on MRS - agar plates for 48h. The colonies were removed from the surface of the plate with a sterile plastic loop and suspended in 20 ml of 50 mM sodium phosphate buffer, pH 7.0. Using a micro pipette and sterile tips, 5 microlitres of L. casei - phosphate buffer suspension was seeded at three equidistant points near the periphery of the culture plate. The plates were incubated at 30 degrees Celsius for 15 - 30 days whereafter the plates were examined for zones of inhibition which are shown in Figures 1 to 16.

Although both isolates were considered to be microbiologically identical using standard techniques shown in Tables 2 and 3, one (referred to herein as isolate 9313) performed spectacularly in growth inhibition assays against a suite of pathogenic plant fungi as shown in Figures 1 to 16. In contrast the other strain of L . casei (referred to herein as isolate 9314) was totally ineffective. The effective strain, L . casei 9313, was deposited at AGAL and granted Accession No. N94/49432.

Example 4: Growth Inhibition Plate Assay: Effects of

L. casei on Human Fungal Pathogen.

Three human opportunistic fungal pathogens were similarly tested: the pathogens tested were Candida

albicans, Scadosporium apiospermum or Aspergillus

fumigatus . The pathogens were grown on culture plates in a defined medium (B.P.A. Cammue, M.F.C. De Bolle, F.R.G.

Terras, P. Proost, J. VanDamme, S.B. Rees, J. Vanderleyden and W.F. Broekaert (1992) Isolation and characterisation of a novel class of plant antimicrobial peptides from

Mirabilis jalapa L. Seeds. Journal of Biological Chemistry 267 2228-2233).

Colonies of the pathogens were removed with a sterile wire loop and seeded in the centre of culture plates containing YMPGA - lactose medium or MEA - lactose medium. The composition of the MEA - lactose medium was as follows: 3% malt extract, 0.5% peptone, 0.6% tryptone, 2% lactose, 0.000009% Tween 80, 0.3% yeast extract, 1.5% fresh yeast extractives, and 2% agar. 5 microlitres of L . casei - phosphate buffer suspension was seeded at three

equidistant points near the periphery of the culture plate as described above.

Although the invention has been described in detail for the purposes of clarity and understanding, it will be understood by the person skilled in the art that various modifications and/or additions may be incorporated without departing from the scope of the invention

described.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An anti-fungal agent comprising L . casei .

2 . An anti-fungal agent comprising L . casei

N94/49432, or a biologically active derivative thereof.

3. An anti-fungal agent according to claim 2, wherein the L . casei N94/49432 is isolated from Sour Dough Baker's Starter mixture.

4. An anti-fungal agent according to any one of claims 1 to 3, further comprising a second anti-microbial agent.

5. An anti-fungal agent according to claim 4, wherein the second anti-microbial agent is selected from the group consisting of Sac char omyces cerevisiae,

Lactobacillus parabuchneri , Lactobacillus

parabuchneri/brevis, Lactobacillus acidophilus,

Lactobacillus bulgaricus, Lactobacillus boulardii,

Lactobacillus GG, Streptococcus lactis, Bifidobacterium, fructooligosaccharides, xylooligosaccharides, coenzyme Q10, shark liver oil, tea catachins, Panx ginseng, acemannan from Aloe Vera leaf, Zinc salts. Keyhole Limpet Hemocyanin, Bacillus Calmette-Guerin, mistletoe extract, interleukins, interferons or nitric oxide mediators, azoles,

cryptolepine, terbinafine, ciclopiroxolamine or amphoterin B.

6. A method of treating microbial infection,

comprising the step of administering an anti-fungal agent according to any one of claims 1 to 4 to a subject in need of such treatment.

7. A method of treating microbial infection,

comprising the step of administering an agent according to claim 5.

8. A method according to claim 6 or claim 7, wherein the infection is caused by one or more of the organisms of Table 1.

9. A method according to claim 6 or claim 7, wherein the infection is caused by one or more of the organisms of Table 4 .

10. A composition comprising an anti-fungal agent according to any one of claims 1 to 5, together with a pharmaceutically acceptable carrier.

11. A food product comprising an anti-fungal agent according to any one of claims 1 to 5.

12. A prophylactic device comprising an anti-fungal agent according to any one of claims 1 to 5.