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WO1997032034A1 - Control of microorganism growth - Google Patents

Control of microorganism growth Download PDF

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Publication number
WO1997032034A1
WO1997032034A1 PCT/GB1997/000409 GB9700409W WO9732034A1 WO 1997032034 A1 WO1997032034 A1 WO 1997032034A1 GB 9700409 W GB9700409 W GB 9700409W WO 9732034 A1 WO9732034 A1 WO 9732034A1
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WO
WIPO (PCT)
Prior art keywords
sample
organism
micro
inhibitor
sphingosine
Prior art date
Application number
PCT/GB1997/000409
Other languages
French (fr)
Inventor
Peter Jeremy Stephens
Original Assignee
Oxoid Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oxoid Limited filed Critical Oxoid Limited
Priority to AU20055/97A priority Critical patent/AU2005597A/en
Publication of WO1997032034A1 publication Critical patent/WO1997032034A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to methods for controlling micro-organism growth, and especially to methods for enriching the microorganism population of a sample prior to analysis thereof to detect the possible presence of a target organism.
  • non-target organisms can mask the presence of a target organism.
  • Previous stressful conditions such as processing of the sample material, for example pasteurisation or chemical treatment, may have injured the cells of the target organism to an extent which inhibits their ability to proliferate quickly, although they retain the capability of proliferating eventually to dangerous levels under appropriate conditions.
  • the non-target organisms can proliferate readily and, for example by competing for nutrients, so inhibit the proliferation of the injured target organism cells that it remains impossible to detect the presence of the target organism.
  • the cell division cycle inhibitor(s) should be present in a total amount which arrests or slows down cell division of at least most, preferably all, species of organisms likely to be encountered in the sample material.
  • the cell division cycle inhibitor should, of course, not be present in an amount which is toxic to the target organism, even when injured. The practical result is that cell division of potentially dominent microorganisms in the sample is delayed for a period sufficient to permit injured target microorganism cells to repair.
  • microorganism growth in the sample can be permitted, if appropriate in the presence of selective inhibitors to discourage the growth of non-target organisms,
  • the repaired target cells, if present, can compete effectively and hence proliferate satisfactorily to provide an analysis sample containing detectable levels of the target organism.
  • the invention provides a method of enriching the microorganism population of a sample, such as a foodstuff or beverage, prior to analysis thereof to detect the possible presence of a target microorganism therein, which method comprises the steps of prolonging the lag phase of the microorganism cell-division cycle of at least the majority of species present in the sample for a time sufficient to enable injured cells of the target microorganism to repair themselves, and thereafter permitting growth of microorganisms to occur in the sample.
  • the invention provides a method of enriching the microorganism population of a sample prior to analysis thereof to detect the possible presence of a target microorganism therein, in which method an inhibitor of the microorganism cell-division cycle is provided in the sample in a sub-toxic amount sufficient to prolong the lag phase of the general microorganism cell-division cycle for a time sufficient to enable cells of the target microorganism which may have been injured, for example by previous processing of the sample material, to repair themselves, and following said sufficient time growth of microorganisms present in the sample is permitted.
  • growth of microorganisms present in the sample is permitted by providing an activator of the general microorganism cell-division cycle in an amount at least sufficient to counteract any residual effect of the inhibitor.
  • one or more agents which inhibit the growth of non-target microorganisms that may be present in the sample material are added to the sample.
  • cell-division is delayed by incorporating an inhibitor of protein kinase activity in the pre-enrichment sample.
  • An inhibitor of protein kinase C activity is preferred.
  • Ideal inhibitors are sphingosines, such as D-sphingosine, analogues of sphingosines, hexadecylphosphocholine, l-0-hexadecyl-2-acetyl-rac- glycerol, l-0-hexadecyl-2-methyl-rac-glycerol and l-O-hexadecyl-2-methyl-sn-glycerol.
  • the amount of inhibitor required will depend on the nature of the sample material, the organisms likely to be present, and the effectiveness of the chosen inhibitor. In general, however, the inhibitor should be present in the culture medium, containing the sample material, in a final concentration of at least about 0.1 ⁇ M, preferably at least about 1 ⁇ M, more preferably at least about 5 ⁇ M.
  • Suitable reagents are activators of protein kinase C (or as mentioned previously, protein kinases ingeneral) include: diacylglycerols such as l-oleoyl-2- acetyl-sn-glycerol,l-oleoyl-2-acetyl-rac-glycerol,l-stearoyl-2-arachidonyl-sn-glycerol (SAG), 1,2-dihexanoyl-sn-glycerol, 1,2-dioctanoyl-rac-glycerol, 1,2-dioctanoyl-sn- glycerol, 1 ,2-didecanoyl-rac-glycerol, 1 ,2-didecanoyl-sn-glyceroland 1 ,2-dioleoyl-rac- glycerol and phorbol esters such as 12-0-tetradecanoyl-phorbol- 13 -acetate
  • a preferred reagent is l-stearoyl-2-arachidonyl-sn-glycerol (SAG).
  • SAG l-stearoyl-2-arachidonyl-sn-glycerol
  • the enrichment culture medium contains both a protein kinase inhibitor and a counteracting agent, for example a sphingosine and SAG.
  • a protein kinase inhibitor for example a sphingosine and SAG.
  • a combination of inhibitor and counteracting agent such as this enables the effect of the inhibitor in prolonging the lag phase to be modulated, thus controlling the extent of prelongation, which can be selected to best suit the nature of the sample material and the target/non- target organisms likely to occur in it.
  • the duration of the delay in the lag phase is at least about 6 hours. Generally a delay of from about 6 to about 12 hours will enable a sufficient number of injured target cells to repair.
  • the delaying effect of the cell-cycle inhibitor can wear off after an elapse of time.
  • delay can be arranged to terminate after a number of hours found by experiment to be appropriate for the target organism and typical sample material in question. This is easily built into a routine analysis procedure, for example in manufacturing control procedures, where the nature of the sample material is constant.
  • termination of the delay can be brought about by a positive action, for example dilution (e.g. by addition of sterile medium devoid of the inhibitor) and/or the addition of one or more nutrients or reagents that encourage growth, thus displacing the balance is favour of growth.
  • the invention also provides a composition for addition to sample material, or to a conventional enrichment culture medium, said composition comprising a general microorganism cell-division cycle inhibitor which, when said composition is added to sample material, causes the lag phase to be prolonged for a time sufficient to enable cells of a target microorganism which may have been injured, for example by previous processing of the sample material to repair themselves.
  • This composition can, for example, be in the form of a supplement for addition to a culture medium. This supplement is usually in the form of a small quantity of dry sterile solids, often freeze-dried.
  • a typical commercial product is a glass ampoule containing the solids, which in use is broken open and the solids are dissolved in a small quantity of sterile water, perhaps containing ethanol or the like to assist dissolution of the solids.
  • the resulting solution is added to the bulk culture medium, for example a commercially-available buffered peptone water.
  • An important embodiment of the invention is therefore an ampoule containing a cell-cycle inhibitor, preferably a sphingosine, in sterile re-hydratable form.
  • the ampoule also contains SAG.
  • a filler, such as PVP, may also be present to provide sufficient bulk for reagent handling purposes.
  • the composition additionally comprises one or more traditional agents which inhibit the growth of non-target microorganisms that may be present in the sample material.
  • Traditional selective agents that can be added include: novobiocin, sodium desoxycholate, malachite green, pH reducing agents, a w (water activity) reducing agents, brilliant green, bile salts, selenite, crystal violet and Tergitol 4.
  • the invention also provides a complete micro-organism culture medium (i.e. a medium containing nutrients) containing a cell-division cycle inhibitor in an amount sufficient to prolong the lag phase for a time sufficient to enable injured cells of a target micro-organism to repair themselves.
  • the inhibitor is preferably an inhibitor of protein kinase activity, more preferably of protein kinase C activity.
  • the inhibitor is a sphingosine, preferably in an amount of at least about O.l ⁇ M.
  • the medium additionally contains SAG, preferably in an amount of at least about O. l ⁇ M.
  • An important embodiment of the invention is a method of detecting the presence of Salmonella typ imurium in a sample likely to contain Escherichia coli, wherein during an enrichment procedure intended to encourage the establishment of a detectable Salmonella population a protein kinase activity inhibitor, especially a sphingosine, is used to prolong the lag phase of the cell-division cycle for a time sufficient to enable injured Salmonella to repair themselves and thereby to compete effectively with E.coli during the sample enrichment.
  • a protein kinase activity inhibitor especially a sphingosine
  • the invention can be applied to advantage in the analysis of a very wide range of sample materials in which the presence of micro-organisms can be of significance, for example foodstuffs, beverages, ingredients for use in such products, consumer goods such as cosmetics and personal products (e.g. toothpastes, soaps, skin cleansing/treatment compositions, shampoos), clinical samples (e.g. faeces, body fluids such as blood, serum and urine), and environmental samples (e.g. water, effluents or samples from factories or other industrial premises).
  • foodstuffs beverages
  • ingredients for use in such products for example foodstuffs, beverages, ingredients for use in such products, consumer goods such as cosmetics and personal products (e.g. toothpastes, soaps, skin cleansing/treatment compositions, shampoos), clinical samples (e.g. faeces, body fluids such as blood, serum and urine), and environmental samples (e.g. water, effluents or samples from factories or other industrial premises).
  • consumer goods such as cosmetics and personal products (e.g.
  • the invention is not limited to the detection of particular micro-organism species, but can be applied generally.
  • target organisms include Liste ⁇ a, Salmonella, Escherichia coli (for example, species producing Vero-cytotoxin), Campy lobacter, Yersinia and Vibrio species.
  • FIGS. 1 to 4 of the accompanying drawings show the results of experiments (EXAMPLE A) in which the lag phase of microorganism (Listeria monocytogenes) growth was controlled by the presence of sphingosine or combinations of sphingosine and SAG:
  • Figure 1 shows the effect on growth of various concentrations of sphingosine alone.
  • Figure 2 shows the effect on growth of combinations of 5 ⁇ M sphingosine (S) and various concentrations of SAG.
  • Figure 3 is an enlarged portion of Figure 2, covering the first 30 hours of growth only.
  • Figure 4 shows the effect on growth of combinations of 10 ⁇ M sphingosine (S) and various concentrations of SAG.
  • Figures 5 and 6 show results of experiments (EXAMPLE B) in which the lag phase of growth of heat-injured Salmonella was compared with that of fully viable £. coli.
  • Figure 5 shows the effect of various concentrations of sphingosine on the growth of heat-injured Salmonella typhimurium (10 0 3 organisms/ ml).
  • TPB tryptic phosphate broth
  • D-sphingosine (Sigma) was added to each well, from a stock solution in ethanol, to give the desired progressively varying concentration of sphingosine and an ethanol concentration that was 1 % v/v. Growth was measured using the Bioscreen, which measures increasing bacterial numbers as increasing absorbance at a light wavelength of 600nm. Growth was measured at 30°C for up to 90h. Control experiments (no sphingosine) were performed in TPB with an ethanol concentration of 1 % v/v. Experiments with both the inhibitor and the activator (SAG) were performed by adding both reagents prior to analysis in the Bioscreen thus giving an ethanol concentration of 2% v/v. The corresponding control experiment also had an ethanol concentration of 2% v/v.
  • a commonly-encountered strain of Salmonella typhimurium was cultured until the population, at about 1-5 xlO 8 organisms per ml, had reached the mid-exponential phase of growth. Approximately 10ml of this culture was heat-injured using a heating apparatus composed of a narrow bore stainless steel coil submerged in a thermostatically controlled waterbath. The coil was flushed several times with sterile distilled water and pre-heated to 53.5°C. The 10ml sample of the Salmonella culture was loaded into the coil using a disposable syringe. This pre-heating, and rapidity of loading, ensured that the total equilibration time was less than one second.
  • the culture was heated within the coil for 15 minutes.
  • the heat- injured cells were then expelled from the coil into 24 times volume of commercially-available Buffered Peptone Water (Oxoid) (hereinafter "BPW") using a second syringe. Every expulsion displaced 200 ⁇ l of culture from the coil.
  • BPW Buffered Peptone Water
  • Every expulsion displaced 200 ⁇ l of culture from the coil.
  • the first 4 or 5 ejections were discarded because these cells had been in part of the coil that projected from the waterbath and had therefore not been exposed to the correct temperature.
  • D-sphingosine (Sigma) was dissolved in absolute alcohol to give a stock solution of concentration 0.5mM. This was diluted 1 in 100 in BPW as a serial dilution to give a sphingosine concentration of 5 ⁇ M and a final ethanol concentration of 1 % .
  • a Bioscreen was used to investigate the effect of this concentration of inhibitor on the length of lag in heat- injured Salmonella typhimurium and a typical competitor organism commonly found in contaminated food samples: Escherichia coli.
  • the E.coli culture was prepared in the same manner as the Salmonella culture, but not subjected to the heat treatment.
  • sample culture was 10-fold serially diluted into either BPW and or into BPW containing the inhibitor.
  • a 100-well Bioscreen plate was set up for each sample (i.e. control and inhibited) at organism populations of about 10 2 -10 3 cells/ml.
  • the Bioscreen was programmed to measure the O.D. of every well at 600nm every 15 min for 48h.
  • the accompanying graphical drawings are based on mean O.D. readings.

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Abstract

An improved sample enrichment procedure which increases the chances of detecting pathogenic organisms, such as Salmonella, involves the addition of a cell-cycle inhibitor, especially a protein Kinase C inhibitor such as sphingosine, to the culture medium in an amount that prolongs the lag phase of microorganism growth for a time (several hours) sufficient to permit repair of target organism cells that may have been injured by previous stressful conditions. The repaired cells can thereafter compete effectively with dominant non-target species that may also be present in the sample.

Description

CONTROL OF MICRO-ORGANISM GROWTH
FIELD OF THE INVENTION
This invention relates to methods for controlling micro-organism growth, and especially to methods for enriching the microorganism population of a sample prior to analysis thereof to detect the possible presence of a target organism.
BACKGROUND TO THE INVENTION
For public safety and quality control purposes the analysis of samples of consumer products, such as foodstuffs and beverages, for the presence of pathogenic microorganisms is conducted on an extremely wide scale. Because pathogenic microorganisms such as Listeria and Salmonella are capable of proliferating very rapidly under the right conditions, the presence of even a single viable cell of such organisms in a foodstuff may give rise to serious infection after the foodstuff has been stored and ingested by a human. It is therefore necessary for such organisms to be detectable even when present at extremely low levels. In trying to achieve this sensitivity of detection, it is common practice for samples to be subjected to "pre- enrichment" prior to analysis. During enrichment the sample is incubated under conditions which encourage microorganism growth so that any organisms present can proliferate in the sample and attain population levels which are more readily detectable.
A difficulty with this procedure is that the sample may contain a variety of comparatively innocuous microorganism species. The presence of these irrelevant organisms (herein referred to as "non-target" organisms) can mask the presence of a target organism. Previous stressful conditions, such as processing of the sample material, for example pasteurisation or chemical treatment, may have injured the cells of the target organism to an extent which inhibits their ability to proliferate quickly, although they retain the capability of proliferating eventually to dangerous levels under appropriate conditions. When the sample is subjected to conventional enrichment the non-target organisms can proliferate readily and, for example by competing for nutrients, so inhibit the proliferation of the injured target organism cells that it remains impossible to detect the presence of the target organism. A false deduction may therefore be made that the sample material is free of the target organism. The so-called "Jameson Effect", reported and studied since the 1960's is an example of this: when proliferating microorganisms enter the early stationary phase of their growth, the proliferation of other microorganisms, whose growth has not yet reached the stationary phase, is inhibited. See, for example, Jameson, J.Hve.Cambs. (1962) Vol. 60 p 193-207. Thus, injured cells which need time to repair and hence commence proliferation much later than healthy cells, may be prevented from proliferating sufficiently to attain a detectable population level. Hitherto, no satisfactory technical solution to the "Jameson Effect" in enrichment cultures has been found.
It is already common practice to add selective inhibitors, especially various antibiotics, to sample materials in order to inhibit the proliferation of commonly encountered non- target organisms. By inhibiting such competitors, growth of the target organism is encouraged. However, if previous treatment of the sample material has resulted in the target organism cells being injured, the selective agents intended to inhibit the proliferation of the non-target organisms may also have a counter-productive effect on the target organism, because even "selective" antibiotics tend to have some degree of inhibitory effect on all microorganisms.
GENERAL DESCRIPTION OF THE INVENTION
By the invention we provide a sample enrichment procedure in which repair of injured target organism cells is encouraged whilst, at the same time, proliferation of non-target organisms is discouraged. We achieve this by the presence of one or more cell division cycle inhibitors during enrichment. The cell division cycle inhibitor(s) should be present in a total amount which arrests or slows down cell division of at least most, preferably all, species of organisms likely to be encountered in the sample material. The cell division cycle inhibitor should, of course, not be present in an amount which is toxic to the target organism, even when injured. The practical result is that cell division of potentially dominent microorganisms in the sample is delayed for a period sufficient to permit injured target microorganism cells to repair. Thereafter microorganism growth in the sample can be permitted, if appropriate in the presence of selective inhibitors to discourage the growth of non-target organisms, The repaired target cells, if present, can compete effectively and hence proliferate satisfactorily to provide an analysis sample containing detectable levels of the target organism.
By the invention we provide a method of enriching the microorganism population of a sample, such as a foodstuff or beverage, prior to analysis thereof to detect the possible presence of a target microorganism therein, which method comprises the steps of prolonging the lag phase of the microorganism cell-division cycle of at least the majority of species present in the sample for a time sufficient to enable injured cells of the target microorganism to repair themselves, and thereafter permitting growth of microorganisms to occur in the sample.
In a preferred embodiment, the invention provides a method of enriching the microorganism population of a sample prior to analysis thereof to detect the possible presence of a target microorganism therein, in which method an inhibitor of the microorganism cell-division cycle is provided in the sample in a sub-toxic amount sufficient to prolong the lag phase of the general microorganism cell-division cycle for a time sufficient to enable cells of the target microorganism which may have been injured, for example by previous processing of the sample material, to repair themselves, and following said sufficient time growth of microorganisms present in the sample is permitted.
Optionally, growth of microorganisms present in the sample is permitted by providing an activator of the general microorganism cell-division cycle in an amount at least sufficient to counteract any residual effect of the inhibitor. Preferably one or more agents which inhibit the growth of non-target microorganisms that may be present in the sample material, are added to the sample.
In a preferred embodiment, cell-division is delayed by incorporating an inhibitor of protein kinase activity in the pre-enrichment sample. An inhibitor of protein kinase C activity is preferred. Ideal inhibitors are sphingosines, such as D-sphingosine, analogues of sphingosines, hexadecylphosphocholine, l-0-hexadecyl-2-acetyl-rac- glycerol, l-0-hexadecyl-2-methyl-rac-glycerol and l-O-hexadecyl-2-methyl-sn-glycerol. The amount of inhibitor required will depend on the nature of the sample material, the organisms likely to be present, and the effectiveness of the chosen inhibitor. In general, however, the inhibitor should be present in the culture medium, containing the sample material, in a final concentration of at least about 0.1 μM, preferably at least about 1 μM, more preferably at least about 5μM.
After the lag phase has been prolonged for a time sufficient to allow substantial repair of any injured target cells in the sample, normal microorganism growth can be permitted. If desired, this may be encouraged by addition to the sample at this stage of one or more reagents that counteract the effect of the protein kinase C activity inhibitor. Suitable reagents are activators of protein kinase C (or as mentioned previously, protein kinases ingeneral) include: diacylglycerols such as l-oleoyl-2- acetyl-sn-glycerol,l-oleoyl-2-acetyl-rac-glycerol,l-stearoyl-2-arachidonyl-sn-glycerol (SAG), 1,2-dihexanoyl-sn-glycerol, 1,2-dioctanoyl-rac-glycerol, 1,2-dioctanoyl-sn- glycerol, 1 ,2-didecanoyl-rac-glycerol, 1 ,2-didecanoyl-sn-glyceroland 1 ,2-dioleoyl-rac- glycerol and phorbol esters such as 12-0-tetradecanoyl-phorbol- 13 -acetate. A preferred reagent is l-stearoyl-2-arachidonyl-sn-glycerol (SAG). Preferably this is added to the culture medium in a concentration of at least about 0.1 μM, preferably at least about 1 μM, more preferably at least about 10 μM.
As a more preferred option, the enrichment culture medium contains both a protein kinase inhibitor and a counteracting agent, for example a sphingosine and SAG. A combination of inhibitor and counteracting agent such as this enables the effect of the inhibitor in prolonging the lag phase to be modulated, thus controlling the extent of prelongation, which can be selected to best suit the nature of the sample material and the target/non- target organisms likely to occur in it.
Preferably the duration of the delay in the lag phase is at least about 6 hours. Generally a delay of from about 6 to about 12 hours will enable a sufficient number of injured target cells to repair.
Although this would appear to extend the whole culturing procedure, in pratice this initial delay is more than compensated for by benefits, in terms of enhanced probability of detection of the target organism, and enhanced speed of detection in subsequent stages of the conventional detection procedure.
We have noted that the delaying effect of the cell-cycle inhibitor can wear off after an elapse of time. Thus, by controlling the choice and amount of inhibitor, delay can be arranged to terminate after a number of hours found by experiment to be appropriate for the target organism and typical sample material in question. This is easily built into a routine analysis procedure, for example in manufacturing control procedures, where the nature of the sample material is constant. Alternatively, termination of the delay can be brought about by a positive action, for example dilution (e.g. by addition of sterile medium devoid of the inhibitor) and/or the addition of one or more nutrients or reagents that encourage growth, thus displacing the balance is favour of growth.
After resumption of normal micro-organism growth, conventional procedures leading to detection and identification of the target organism can be followed.
The invention also provides a composition for addition to sample material, or to a conventional enrichment culture medium, said composition comprising a general microorganism cell-division cycle inhibitor which, when said composition is added to sample material, causes the lag phase to be prolonged for a time sufficient to enable cells of a target microorganism which may have been injured, for example by previous processing of the sample material to repair themselves. This composition can, for example, be in the form of a supplement for addition to a culture medium. This supplement is usually in the form of a small quantity of dry sterile solids, often freeze-dried. A typical commercial product is a glass ampoule containing the solids, which in use is broken open and the solids are dissolved in a small quantity of sterile water, perhaps containing ethanol or the like to assist dissolution of the solids. The resulting solution is added to the bulk culture medium, for example a commercially-available buffered peptone water. An important embodiment of the invention is therefore an ampoule containing a cell-cycle inhibitor, preferably a sphingosine, in sterile re-hydratable form. Optionally, the ampoule also contains SAG. A filler, such as PVP, may also be present to provide sufficient bulk for reagent handling purposes.
Optionally, the composition additionally comprises one or more traditional agents which inhibit the growth of non-target microorganisms that may be present in the sample material. Traditional selective agents that can be added include: novobiocin, sodium desoxycholate, malachite green, pH reducing agents, aw (water activity) reducing agents, brilliant green, bile salts, selenite, crystal violet and Tergitol 4.
The invention also provides a complete micro-organism culture medium (i.e. a medium containing nutrients) containing a cell-division cycle inhibitor in an amount sufficient to prolong the lag phase for a time sufficient to enable injured cells of a target micro-organism to repair themselves. The inhibitor is preferably an inhibitor of protein kinase activity, more preferably of protein kinase C activity. Ideally the inhibitor is a sphingosine, preferably in an amount of at least about O.lμM. Beneficially, the medium additionally contains SAG, preferably in an amount of at least about O. lμM.
An important embodiment of the invention is a method of detecting the presence of Salmonella typ imurium in a sample likely to contain Escherichia coli, wherein during an enrichment procedure intended to encourage the establishment of a detectable Salmonella population a protein kinase activity inhibitor, especially a sphingosine, is used to prolong the lag phase of the cell-division cycle for a time sufficient to enable injured Salmonella to repair themselves and thereby to compete effectively with E.coli during the sample enrichment.
The invention can be applied to advantage in the analysis of a very wide range of sample materials in which the presence of micro-organisms can be of significance, for example foodstuffs, beverages, ingredients for use in such products, consumer goods such as cosmetics and personal products (e.g. toothpastes, soaps, skin cleansing/treatment compositions, shampoos), clinical samples (e.g. faeces, body fluids such as blood, serum and urine), and environmental samples (e.g. water, effluents or samples from factories or other industrial premises).
Similarly, the invention is not limited to the detection of particular micro-organism species, but can be applied generally. Examples of target organisms include Listeήa, Salmonella, Escherichia coli (for example, species producing Vero-cytotoxin), Campy lobacter, Yersinia and Vibrio species.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 to 4 of the accompanying drawings show the results of experiments (EXAMPLE A) in which the lag phase of microorganism (Listeria monocytogenes) growth was controlled by the presence of sphingosine or combinations of sphingosine and SAG:
Figure 1 shows the effect on growth of various concentrations of sphingosine alone.
Figure 2 shows the effect on growth of combinations of 5 μM sphingosine (S) and various concentrations of SAG.
Figure 3 is an enlarged portion of Figure 2, covering the first 30 hours of growth only.
Figure 4 shows the effect on growth of combinations of 10 μM sphingosine (S) and various concentrations of SAG.
Figures 5 and 6 show results of experiments (EXAMPLE B) in which the lag phase of growth of heat-injured Salmonella was compared with that of fully viable £. coli.
Figure 5 shows the effect of various concentrations of sphingosine on the growth of heat-injured Salmonella typhimurium (10 03 organisms/ ml).
Figure 6 shows comparable results for viable E.coli.
EXAMPLE A
Listeria monocytogenes Scott A was grown to late exponential phase in tryptic phosphate broth (herein after "TPB") at 30°C. TPB is described in Conner et al, Applied and Environmental Microbiology (1986) Vol. 52 p 59-63. The culture was then diluted in fresh TPB to a concentration of approximately 5 x 106 cells/ml. At this concentration a signal is detectable in a Bioscreen automated turbidimeter and a direct measurement of lag is possible. A small portion (400μl) of this sample was added to each well of a Bioscreen multi-well plate. D-sphingosine (Sigma) was added to each well, from a stock solution in ethanol, to give the desired progressively varying concentration of sphingosine and an ethanol concentration that was 1 % v/v. Growth was measured using the Bioscreen, which measures increasing bacterial numbers as increasing absorbance at a light wavelength of 600nm. Growth was measured at 30°C for up to 90h. Control experiments (no sphingosine) were performed in TPB with an ethanol concentration of 1 % v/v. Experiments with both the inhibitor and the activator (SAG) were performed by adding both reagents prior to analysis in the Bioscreen thus giving an ethanol concentration of 2% v/v. The corresponding control experiment also had an ethanol concentration of 2% v/v.
EXAMPLE B HEAT INJURY
A commonly-encountered strain of Salmonella typhimurium was cultured until the population, at about 1-5 xlO8 organisms per ml, had reached the mid-exponential phase of growth. Approximately 10ml of this culture was heat-injured using a heating apparatus composed of a narrow bore stainless steel coil submerged in a thermostatically controlled waterbath. The coil was flushed several times with sterile distilled water and pre-heated to 53.5°C. The 10ml sample of the Salmonella culture was loaded into the coil using a disposable syringe. This pre-heating, and rapidity of loading, ensured that the total equilibration time was less than one second.
The culture was heated within the coil for 15 minutes. The heat- injured cells were then expelled from the coil into 24 times volume of commercially-available Buffered Peptone Water (Oxoid) (hereinafter "BPW") using a second syringe. Every expulsion displaced 200μl of culture from the coil. The first 4 or 5 ejections were discarded because these cells had been in part of the coil that projected from the waterbath and had therefore not been exposed to the correct temperature.
D-sphingosine (Sigma) was dissolved in absolute alcohol to give a stock solution of concentration 0.5mM. This was diluted 1 in 100 in BPW as a serial dilution to give a sphingosine concentration of 5μM and a final ethanol concentration of 1 % .
AUTOMATED GROWTH CURVE ANALYSIS
A Bioscreen was used to investigate the effect of this concentration of inhibitor on the length of lag in heat- injured Salmonella typhimurium and a typical competitor organism commonly found in contaminated food samples: Escherichia coli. The E.coli culture was prepared in the same manner as the Salmonella culture, but not subjected to the heat treatment.
In every experiment, the sample culture was 10-fold serially diluted into either BPW and or into BPW containing the inhibitor. A 100-well Bioscreen plate was set up for each sample (i.e. control and inhibited) at organism populations of about 102-103 cells/ml. The Bioscreen was programmed to measure the O.D. of every well at 600nm every 15 min for 48h. The accompanying graphical drawings are based on mean O.D. readings.
Five μM sphingosine in 1 % ethanol showed no evidence of toxicity to heat-injured Salmonella typhimurium, nor did it affect the length of lag or growth rate on entry into exponential phase. The presence of 5μM sphingosine was, however, seen to have a substantial inhibitory effect on Escherichia coli, increasing the detection by approximately 18 hours.

Claims

1. A method of enriching the micro-organism population of a sample, such as a foodstuff or beverage, prior to analysis thereof to detect the possible presence of a target micro-organism therein, which method comprises the steps of prolonging the lag phase of the micro-organism cell-division cycle of at least the majority of species present in the sample for a time sufficient to enable injured cells of the target micro¬ organism to repair themselves, and thereafter permitting growth of micro-organisms to occur in the sample.
2. A method of enriching the micro-organism population of a sample, such as a foodstuff or beverage prior to analysis thereof to detect the possible presence of a target micro-organism therein, in which method an inhibitor of the micro-organism cell-division cycle is provided in the sample in a sub-toxic amount sufficient to prolong the lag phase of the general micro-organism cell-division cycle for a time sufficient to enable injured cells of the target micro-organism to repair themselves, and following said sufficient time growth of micro-organisms present in the sample is permitted.
3. A method according to claim 2, wherein the inhibitor is an inhibitor of protein kinase activity, preferably of protein kinase C activity.
4. A method according to any one of the preceding claims, wherein the target micro-organism is a pathogenic bacterium, such as Salmonella or Listeria.
5. A method according to any one of the preceding claims, wherein the lag phase is prolonged by the use of one or more sphingosines.
6. A method according to claim 5, wherein prolongation of the lag phase by the sphingosine(s) is modulated by the presence of l-stearoyl-2-arachidonyl-sn-glycerol
(SAG).
7. A method according to any one of the preceding claims, wherein one or more agents which inhibit the growth of non- target micro-organisms that may be present in the sample material, are added to the sample.
8. A micro-organism culture medium containing a cell-division cycle inhibitor in an amount sufficient to prolong the lag phase for a time sufficient to enable injured cells of a target micro-organism to repair themselves.
9. A culture medium according to claim 8, wherein the inhibitor is an inhibitor of protein kinase activity, preferably protein kinase C activity.
10. A culture medium according to claim 9, wherein the inhibitor is a sphingosine, preferably in an amount of at least about O. lμM.
11. A culture medium according to any one of claims 8 to 10, additionally containing SAG, preferably in an amount of at least about O. lμM.
12. A supplemental composition for use in micro-organism culture, especially sample enrichment, comprising an ampoule containing a cell-cycle inhibitor, especially a sphingosine, in sterile re-hydratable form.
13. A supplemental composition according to claim 12, containing a sphingosine and SAG.
14. A method of detecting the presence of Salmonella typhimurium in a sample likely to contain Escherichia coli, wherein during an enrichment procedure intended to encourage the establishment of a detectable Salmonella population a protein kinase activity inhibitor, especially a sphingosine, is used to prolong the lag phase of the cell-division cycle for a time sufficient to enable injured Salmonella to repair themselves and thereby to compete effectively with E.coli during the sample enrichment.
PCT/GB1997/000409 1996-02-28 1997-02-17 Control of microorganism growth WO1997032034A1 (en)

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WO2016210436A1 (en) * 2015-06-26 2016-12-29 Saber Biotics, Llc Selective media and uses thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0877092A1 (en) * 1997-04-08 1998-11-11 Becton, Dickinson and Company Rapid microorganism detection method
WO2016210436A1 (en) * 2015-06-26 2016-12-29 Saber Biotics, Llc Selective media and uses thereof
US10752933B2 (en) 2015-06-26 2020-08-25 Saber Biotics, Llc Methods for the identification, characterization, and use of inhibitors of the fructose-asparagine utilization pathway

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