WO1999020286A1

WO1999020286A1 - A composition for the treatment of legionella pneumophila and a method for such treatment

Info

Publication number: WO1999020286A1
Application number: PCT/US1998/022371
Authority: WO
Inventors: Gilbert Theo Hinze
Original assignee: Radical Waters Ip (Pty) Ltd; Davis, Joanne, T.
Priority date: 1997-10-23
Filing date: 1998-10-23
Publication date: 1999-04-29
Also published as: AU1114499A

Abstract

This invention relates to a composition and method for treating Legionella Pneumophila comprising an electro-chemically activated anion-contained aqueous solution.

Description

lit : A COMPOSITION FOR THE TREATMENT OF LEGIONELLA PNEUMOPHILA AND A METHOD FOR SUCH TREATMENT

FifflH nf the Invention .

This mvenπon relates to a composition for the treatment of Legionella

pneumophila, a method for treating Legionella pneumophila and the use of

such composition m the preparation of a medicament for treating Legionella

pneumophila.

Background to the Invention

So-called Legionnaire' s disease became known 1976 after an outoreak of a

serious respira_tory disease, diagnosed as having been caused by Legionella

pneumophila. Current treatment m humans includes Erythromycm with the

addition of Rifampicm m non-responding cases.

Legionella bacteria have a wide natural distribution m water and their growtn

is promoted by o_ther micro-organisms, including Pseudomonas species, which

provide nutrients and protect them from adverse conditions, including the effect

of biocidal treatment of water.

The Legionella bacteria can infect humans by means of an aerosol, moving into

the breathing zone of persons and deposition of the aerosol into the lungs.

Other sources of infection include recreational waters, residential and industrial

waters, air-conditionmg systems, humidifiers, respiratory therapy apparatus,

dental wa_ter supply lines and resuscitation systems. Current control measures against infection include super heating, hyper-

chloπnation and chlorine gasification. However, no operating, maintenance,

cleaning and decontamination procedures presently exist that are generally

regarded as safe work practices.

Obiect of the Invention :

It is accordingly the object of this invention to provide a composition for

treating Legionella as well as an associated method for treating same.

Brief Summary of the Invention :

According to a first aspect of the invention there is provided a composition for

treating Legionella Pneumophila compπsing an electro-chemically activated

anion-containing aqueous solution.

The anion-containing solution, or so-called anolyte, may be obtained from the

electrolysis of an aqueous solution of a salt. The salt may be sodium chloride.

In particular it may be non-iodated sodium chloride or potassium chloride.

The anion-containing solution and the associated cation-containing solution

may be produced by an electro-chemical reactor or so-called electrolysis

device. The electro-chemical reactor may include a through flow, electro-chemical cell

having two co-axial cylindrical electrodes with a co-axial diaphragm between

them so as to separate an annular inter electrode space into a catalytic and an

analytic chamber.

The anolyte may have a redox potential of above + 600 mV and preferably

about +750 mV and may have a pH of about 6,5 - 7,5. The anolyte may

include any one of more or radical anion species from the group consisting of

CIO ; CIO^" ; HCIO; OH^" ; H0₂- ; H,0₂ ; 0₃ ; S₂0₈ ²" and Cl,O₆P

According to a second aspect of the invention there is provided a method for

treating Legionella Pneumophila comprising the steps of atomising a suitable

dosage of an electro-chemically activated, anion-containing aqueous solution;

and

dispensing the atomised dosage of aqueous anion-containing solution into an

atmosphere to be treated, the aqueous solution being substantially as herein

defined.

According to a third aspect of the invention there is provided the use of an

electro-chemically activated anion-containing aqueous solution in the

preparation of a medicament for use in the treatment of Legionella

Pneumophila in humans. Detailed Description of Preferred Embodiment :

A preferred embodiment of the invention will now be described by means of

three non-limiting examples only.

An electro-chemical reactor, including a through flow, electro-chemical cell

having two co-axial cylindrical electrodes with a co-axial diaphragm between

them so as to separate an annular inter-electrode space into a catalytic and an

analytic chamber, was used to produce anolyte and catholyte solutions.

Example 1

Anolyte solution with varying characteristics was used as shown in the

respective examples.

A series of trials have been conducted whereby various dilutions of aqueous

anion-containing solutions have been seeded with Legionella pneumophila

(Serotype 1) organisms and the microcidal effects of treatment with anolyte

have been observed after incubation for a period of 4 days (96 hours) at a

temperature of 37° C . The efficacy of the treatment with anolyte at the various dilutions and times of

exposure was established by the presence or absence of Legionella cultures on

the infected BCYE culture medium.

Three replicates of each of the dilutions and of the control groups were seeded

with a pure culture of Legionella Pneumophila (Serotype 1), resulting in

counts of above 7 million parts per millilitre (TNTC).

Samples were collected at pre-determined time intervals and transferred onto

the growth medium before being incubated for 4 days (96 hours) at ±37°C.

As can be seen from the Table for Example 1, anolyte was microcidal at

levels between +998 mV and +407 mV (i.o.w. at a dilution rate of more than

1 - 10).

Example 2

Further tests were then conducted to narrow down the ranges of efficacy using

a reducing-oxidation potential (ORP) as the monitoring (measuring) and on a

similar basis as set out in Example 1. As is illustrated in the Table for Example 2, it is deduced that :

1. A contact time of about 5 minutes at about 750 mV and a contact time

of about 30 minutes at about +607mV is completely microcidal against

Legionella Pneumophila (Serotype 1); and

2. The microcidal effect of anolyte is directly proportional to the ORP of

the dilution.

Example 3

P.aeruginosa and S.aureus (Methicillin resistant) strains were cultured overnight

on blood agar plates. Both of these strains were obtained from clinical

specimens obtained during routine laboratory investigations at the General

Hospital in Johannesburg, South Africa.

The L. pneumophila strain was cultured for 3 days on BCYE agar as it is a

slow-growing organism. This isolate was obtained from the ATCC (American

type culture collection) reference stock cultures, designated ATCC 33155.

These plate cultures were used for preparation of the liquid suspensions in

Ringer's solution. A suitable inoculum of each of the 3 test strains was removed from the agar

plates with a nichrome loop and emulsified in 1/40 strength Ringer's buffer.

These were then homogenised in a vortex mixer (the 1/40 Ringer's buffer is

suitable for diluting the fastidious Legionella as well as the S.aureus and

P.aeruginosa). Using a 0.5 McFarland's standard opacity tube, which is the

equivalent to 150 million organisms/ml, the capacity of the three cultures in

suspension was adjusted to an opacity to give a final count (i.e. after adding

to the Ringer's solution or Ringer' s-anolyte solution) of approximately 1

million colony forming units per ml (1 x 10⁶ cfus per ml - called the "high

count challenge). A second set was prepared with a 1/10 dilution (1 x 10⁵ cfus

- called the "low count challenge").

Anolyte solutions were prepared as follows :

1 : 1 - 1 part Ringer's 1/40 + 1 part anolyte (2.0 mi + 2.0 ml)

1 :50 - 49 parts Ringer's 1/40 + 1 part anolyte. (4.9 + 2.0)

1 : 100 - 1 part 1 :5 anolyte + 1 part Ringer's (2.0 ml + 2.0 ml)

1 :150 - 1 part anolyte + 2 parts Ringer's (1.0 ml + 2.0 ml).

These dilutions were distributed in 100 x. 1 quantities in 5 ml disposable

plastic test tubes in triplicate for each set of organisms.

A thiosulphate neutraliser was made up by adding 2 crystals per 10 ml (which is also the amount used in the British Public Health Service Laboratories or

PHLS including that of John Lee's Legionella Unit) and distributed in lOμl

quantities in plastic disposable test tubes.

All cultures were pre-tested microbiologically to determine whether any effect

such as a decrease in the number of viable organisms recovered would occur,

using any of the reagents such as thiosulphate neutraliser or a 30 minute

exposure to Ringer's buffer.

This test was done in triplicate as follows :

Test tube dilutions O O O O O

Ringer's 1 : 1 1 :5 1 : 100 1 : 150

lOOμl only anolyte anolyte anolyte anolyte

To each set of test tubes containing either the anolyte dilutions or plain

Ringer's (i.e. the control), 1 drop (lOμl) of culture was added. As the same

conditions were being applied to both the test and the control samples, no

special calculation was required for volume adjustment from lOμl to HOμl

when the culture was added. From the Ringer's only control tube, a further 1/100 dilution in Ringer's was

made at the appropriate time interval (see below) to facilitate counting, should

the original plate count be too high to observe individual cfus.

At the appropriate time in intervals, (5 mins and 30 mins post-exposure) lOμ

1 of organism in anolyte dilution / Ringer's only (control) was removed and

mixed with the 10 μl of thiosulphate neutraliser. This "mix" was seeded onto

a petri dish (blood agar for the S.aureus and P.aeruginosa and BCYE for the

Legionella). the plates were spread over the entire surface with a sterile

nichrome spreader.

The blood agar plates were incubated for 48 hours at 37°C aerobically and the

BCYE plates at the same temperature for 5 days aerobically in a sealed jar with

a very moist atmosphere.

Colonies were counted using a colony counter with a magnifying lens and a

grid.

No significant difference in the number of cfus/ml of the untreated (control)

organisms were obtained after (a) being left in Ringer's solution for a 30

minute period and (b) treatment with sodium thiosulphate when compared with

counts taken immediately after preparation of the suspensions. Thus any drop in cfus was purely due to the effect of the anolyte.

The "high count challenge" dose gave the following numbers of cfus/ml

S.aureus 3.4 10⁶ cfus/ml

P.aeruginosa 1.2 x 10⁶ cfus/ml

L. pneumophila 2.7 x 10⁶ cfus/ml

The "low count challenge" dose gave the following numbers of cfus/ml :

S. aureus 2.9 x 10⁵ cfus/ml

P. aeruginosa 2.2 x 10⁵ cfu\ml

L. pneumophila 4.9 x 10⁵ cfu/ml

All cultures with a concentration of 10⁵ cfus/ml showed no growth (became

non- viable) after being exposed to any of the dilutions of anolyte (1: 1, 1 :50,

1 :100, 1 :150) for both the 5 and 30 minute periods.

The results of these cultures containing 10⁶ cfus/ml treated in the same manner

with anolyte dilutions were as set in the Table for Example 3.

It is envisaged that the following methods of treatment could be used : It is envisaged that the following methods of treatment could be used :

1. By dosing anolyte onto elements such as a condenser used in air-

conditioning systems;

2. By fogging anolyte into air-conditioning ducts or into the atmosphere

eg. in an operation theatre, etc.; and

3. By patients inhaling fogged anolyte, thereby exposing the Legionella

organism to the anolyte in the alveoli of the lungs.

It will be appreciated that many variations in detail are possible without

departing from the scope and/or spirit of the invention as claimed in the claims

hereinafter.

Claims

CLAIMS :

1. A composition for treating Legionella Pneumophila comprising an

electro-chemically activated anion-contained aqueous solution.

2. A composition as claimed in claim 1 , wherein the anion-containing

solution is obtained from the electrolysis of an aqueous solution of a

salt.

3. A composition as claimed in claim 1 , wherein the anion-containing

solution is produced by an electro-chemical reactor, the electro┬¼

chemical reactor including a through flow, electro-chemical cell

having two co-axial cylindrical electrodes with a co-axial diaphragm

between them so as to separate an annular inter electrode space into

a catalytic and an analytic chamber.

4. A composition as claimed in claim 1 , wherein the solution has a

redox potential of above + 600 mV and a pH of about 6,5 - 7,5.

5. A method for treating Legionella Pneumophila comprising the steps

of atomising a suitable dosage of an electro-chemically activated,

anion-containing aqueous solution; and dispensing the atomised dosage of aqueous anion-containing solution into an atmosphere to be

treated.

6. The use of an electro-chemically activated anion-containing aqueous

solution in the preparation of a medicament for use in the treatment

of Legionella Pneumophila in humans.