CA2330814A1 - Bacteriophage assay - Google Patents

Abstract

There is provided an assay suitable for the typing of bacterial strains. In the assay a predetermined amount of phage is combined with a bacterial isolate of unknown strain, the mixture being located in a suitable container. The mixture of phage and bacteria is conveniently held in a liquid or semi-liquid medium facilitating interaction of the two species. The extent of bacterial growth in the presence of the phage is measured by conventional means, preferably by means of an OD reading. Desirably the phage is retained in the selected container, which is conveniently a micro-titre plate, through use of a fixant such as 5 % gelatin.

Description

3 The present invention is concerned with the 4 identification of bacteria, both by species and by sub-s type, and to a new method of bacterial identification 6 which relies upon bacteriophage specificity.

8 The control and epidemiology of bacterial outbreaks is 9 becoming increasingly important and much effort is currently expended in identification of bacteria by 11 species and sub-type. With the apparent continued rise 12 in antibiotic resistant bacterial strains the need for 13 careful and accurate identification of bacteria is 14 becoming ever more critical.
16 Strain typing has been defined as "A pre-requisite to 17 studying the epidemiology of bacterial pathogens and, 18 ultimately, the development of control strategies" (gee 19 Smith et al, (1995) AEM 61:4263).
21 Typically identification of bacterial species and sub-22 types involves methods such as classifying bacteria 23 according to their ability to grow using selected 24 carbon sources, the specificity of bacteriophages for particular bacteria, or involves genetic analysis of 1 the bacterial genome and comparison thereof to known 2 genomic sequences (for example using techniques such as 3 RFLP, RAPD, ERIC, PCR-RFLP or the like). For example 4 strain differentiation in Erwinia carotovora subspecies atroseptica (hereinafter referred to as "Eca") is 6 usually performed by serology, phage typing, carbon 7 source utilization, genetic analysis or a combination 8 of such techniques. All of the currently used methods 9 are relatively time-consuming, causing delay in the positive identification of a sample. Minimisation of 11 any delay may be vital for successfully controlling the 12 spread of bacterial infections in the population 13 generally or in selecting a suitable treatment regime 14 for a patient.
16 For example, strain typing of bacteria may be carried 17 out using bacteriophages (hereinafter referred to 18 simply as "phages"). A phage is any virus whose host 19 is a bacterium. Most bacteria can be infected by phages, which are a highly diverse group of viruses. A
21 given phage can only infect one or a few strains or 22 species of bacteria and this limitation of phage 23 infectivity forms the basis of strain typing using 24 phages. The outcome of phage infection depends upon the phage and its host cell, but can be classified into 26 two main groups as follows:

28 Virulent phages . induce lysis of the host cell.

Temperate phages: can establish a stable non-lytic 31 relationship with the host cell.

33 Conventionally, strain typing of an unknown strain of 34 bacteria via phage infection involves plating out a single colony of bacteria obtained from a test sample 36 onto an agar dish and, once a bacterial lawn is 1 established, introducing inocula of a specific phage at 2 discrete points. The inoculated plate is then 3 incubated again before being examined by eye and the 4 extent of degradation or lysis of the bacteria at the points where phage has been introduced is graded by the 6 technician. The effect of various phages on the test 7 bacteria are analysed.

9 The grading used to establish the extent of phage action on a bacterial colony will vary from complete 11 lysis (and thus death) of the bacteria (due to 12 successful replication of the phage) through to no 13 effect noticeable to the eye (when the phage is unable 14 to interact with the bacteria of the sample). Various grades between these two extremes also exist and to a 16 large extent the accuracy of the test results depends 17 upon the skill, experience and perception of the person 18 reading the results and performing the grading 19 procedure. Unfortunately, the subjective nature of the grading system means that ultimately the phage typing 21 system lacks accuracy.

23 A typical analysis of E.coli 0157: H7 by conventional 24 phage typing methods is reported by Khakhira et al., in Epidermiol. Infect. (1990) 105:511-520, see especially 26 Table 1 of this reference. In this analysis there was 27 visual assessment of 62 phage types and an attempt to 28 assign a positive value to each result. The complexity 29 of conventional bacterial strain typing by phage interaction is clear from the typical analysis results 31 depicted in Table 1.

33 We have now found that the highly specific interaction 34 between phages and bacteria can be used in a much more effective assay in which the results of the 36 phage/bacteria interaction is determined through 1 measurement of bacterial growth, rather than bacterial 2 death. This novel approach to phage typing enables 3 conventional techniques for observing bacterial 4 populations, such as determining the optical density of a sample, to be successfully employed. Consequently 6 the results do not rely on a subjective analysis but on 7 a direct and reproducible reading of a characteristic 8 of the test sample.

In the assay of the invention a predetermined amount of 11 phage is combined with an isolate of bacteria, the 12 mixture being located in a suitable container. The 13 mixture of phage and bacteria is conveniently held in a 14 liquid or semi-liquid medium facilitating interaction of the two species. Conveniently the phage is located 16 in the container and the bacteria added to the phage.
17 However the assay is not limited to this approach and 18 also encompasses, for example, the bacteria being 19 located in the container and the phage added thereto.
21 In one aspect the present invention provides an assay 22 to identify bacteria present in a sample, said assay 23 comprising the following steps:

(a) isolating a single colony of said bacteria;

27 (b) combining said isolated bacteria with a 28 selected bacteriophage in a container, the 29 combination of bacteria and phage being incubated in a medium containing the nutrients required for 31 bacterial growth and which enables phage/bacteria 32 interaction; and 34 (c) determining the extent of bacterial growth.
36 Where the phage interacts with the bacteria of the test 1 sample, the phage will infect the bacteria and, 2 depending upon the virulence of the phage, will either 3 cause death of the bacteria or will slow bacterial 4 reproduction. If the phage is unable to infect the 5 bacteria, bacterial growth will be unaffected. Thus, 6 determination of the extent of bacterial growth 7 following incubation with the phage is a direct 8 correlation of the interaction of the phage and 9 bacteria. Since the phage will interact only with specific bacteria, the extent of bacterial growth in 11 the assay provides information on the species or sub-12 type of the bacteria.

14 Thus the assay of the present invention will have utility as an in vitro method of diagnosis for 16 bacterially based infections or diseases in plants, 17 animals and humans. Additionally the assay has utility 18 as a means of monitoring food or medicines etc. for 19 bacterial contamination.
21 Suitable phages are commercially available from culture 22 collections and conventional phage typing systems. In 23 addition phages specific for particular bacteria can be 24 engineered using routine techniques in the laboratory due to the phages' ability to rapidly mutate producing 26 host range mutants. Alternatively suitable phages can 27 be isolated from the natural habitat of the bacteria in 28 question and again standard techniques and 29 methodologies are well known and within the ability of the skilled technician.

32 One important feature of the present assay is to retain 33 the phage of interest in a non-replicative state to 34 avoid mutation of the phage prior to the assay.
Similar requirements are imposed on the conventional 36 method of phage typing and have not been found to be 1 unduly onerous since if no bacteria are present, the 2 phage will be unable to replicate or mutate.

4 Suitable media for incubation of the phage/bacteria combination include those conventionally used for 6 growing bacterial cultures, for example nutrient broth 7 (NB) or Luria Bertani (LB) broth. Conveniently the 8 medium is liquid at the temperature of incubation since 9 this assists the mixture of phage with the bacteria.
It is essential that the medium chosen is compatible 11 with the method used to determine the extent of 12 bacterial growth. Thus if the optical density of a 13 sample is used as a measure of bacterial growth, the 14 medium chosen must enable the penetration of light through the sample. Liquid media are generally 16 suitable for optical density measurements.

18 Conveniently, determination of the extent of bacterial 19 growth may be obtained by measuring the optical density (OD) of the sample in accordance with conventional 21 practice. The growth of the bacterial colony may be 22 determined by obtaining the difference between an 23 initial reading taken as soon as the bacteria and phage 24 have been combined together and a second reading taken after the period of incubation. Alternatively a 26 control may be used to standardise a single reading of 27 the sample taken following the period of incubation.
28 Suitable control standards include the growth medium 29 alone or the growth medium together with phage. A
positive control consisting of bacteria and growth 31 medium only may also be useful to obtain percentage 32 growth readings.' (Percentage growth is the median 33 absorbance phage (n) well minus the base value (ie the 34 ratio obtained when complete lysis occurs) divided by the median absorbance control well minus the base 36 value). An alternative expression is infection ratios 1 (see Table 1). (The Infection Ratio is the (mean) 2 Absorbance control wells divided by the (mean) 3 Absorbance phage (n) well.) To improve accuracy of the 4 readings obtained it may be advantageous to run parallel tests in duplicate or triplicate and then 6 average the results for each sample/phage combination.

8 Suitable optical density readings would normally be 9 taken at wave lengths of 590-630 nm, usually at 595-600 nm. Equipment for reading the optical density of a 11 sample is commercially available. Suitable apparatus 12 includes microtitre plate readers.

14 In one preferred embodiment the phage is pre-located in the selected container and retained therein, for 16 example by means of a fixant, by physical entrapment or 17 by chemical interaction (eg ionic attraction) with the 18 surface of the container. For example, we have 19 successfully located the phage in microtitre plates using 5% gelatin as a fixant. Other polymers which may 21 be suitable fixants include PVP, PVPP, alginate, 22 albumins, starches, PVA, guar gum and the like.
23 Conveniently the fixant used will physically retain the 24 phage within the container during storage but on addition of the bacteria in a liquid growth medium the 26 fixant will dissolve or otherwise release the phage for 27 interaction with the bacteria. Other means of 28 retaining the phage includes electrostatic attraction 29 of the charged phage particle with oppositely charged groups on the surface of the container or retention of 31 the phage on a porous surface, for example on a porous 32 membrane. Alternatively, the phage may be freeze dried 33 onto the container and the freeze dried phage may then 34 optionally be coated. Again freedom of the phage to interact with the bacteria is important.

1 Such techniques of retaining the phage within the 2 selected container has the advantage of enabling the 3 container to be stored for relatively long periods of 4 time without any concern that cross-contamination of particular phage into an adjacent container may occur 6 or that the phages will lose their viability. As a 7 result it is possible to prepare multiple containers, 8 for example microtitre plates, in which the containers 9 have been pre-loaded with phages of different specificities. In this way a profile of the bacterial 11 colony under test may be obtained. Microtitre plates 12 have the additional advantage of co-operating with 13 standard laboratory equipment such as microtitre OD
14 readers.
16 In the assay of the present invention the ratio of 17 bacteria:phage is important and it may be necessary to 18 perform routine test dilution of the bacterial colony 19 to obtain a suitable ratio. This ratio is necessary to a) allow bacteria to grow and become infected, while 21 preventing external cell lysis by phage enzymes, b) to 22 prevent overgrowth of the bacteria in the presence of 23 too few phages.

Table 1 shows an exemplary micro-titre plate layout of 26 phages and bacterial strains indicating optical density 27 values. Column 0 contains no phage. Columns 1-11 28 contain different phages. Row A contains the positive 29 control strain, sensitive to all the phages; row B
contains growth medium only, rows C to E contain 3 31 replicates of strain 1; rows F to H contain 3 32 replicates of strain 13. Well AO (control strain in 33 the absence of phage) is used to obtain infection 34 ratios.
36 According to a further aspect the present invention 1 provides a container having a specific phage subtype 2 located therein, said phage being retained in said 3 container by a fixant, physical entrapment or chemical 4 interaction with the surface of the container. In a preferred embodiment the present invention provides a 6 microtitre plate, wherein wells of said plate are said 7 containers. Preferably different specific phage 8 subtypes are located in different wells on the same 9 plate.
11 Such pre-prepared plates may be designed to identify 12 particular sub-types of bacteria (eg E.coli 0157) from 13 other related sub-types of the same species.
14 Alternatively the plates may be designed to identify bacterial species. The plates may be stored until 16 required.

18 The present invention may have utility in the following 19 situations:
21 a) typing bacterial plant pathogens for 22 epidemiological analysis. Examples include 23 Pseudomonas spp, Xanthomonas spp, Erwinia spp, 24 (including Erwinia carotovora subspecies atroseptica) and the like.

27 b) typing human and animal bacterial pathogens for 28 epidemiological analysis. Examples include 29 Salmonella, Campylobacter, Staphylococcus, Streptococcus, Escherichia, Pseudomonas, Listeria, 31 Shigella, Vibrio, Serratia, Bacillus, Klebsiella, 32 Mycobacterium and the like.

34 c) typing bacteria which cause water and/or food contamination or which are present in hospitals 36 and/or food processing areas, or monitoring for 1 such bacteria. A specific example concerns 2 Lactococcus spp. which play an important role in 3 the milk fermentation process during the 4 production of cheese and cultured dairy products.
5 These processes are susceptible to phage 6 contamination and so typing can be used to monitor 7 the phage susceptibility of the cultures used.

9 d) rapid screening of Mycobacterium tuberculosis 10 isolates using microtitre plates containing 11 specific phages for phage therapy.

13 e) typing biological pesticides such as Bacillus 14 thuringi ensi s .
16 Phages may be located in a container along with other 17 tests in different wells so that on a single, eg 18 microtitre, plate toxin types, plasmid types, specific 19 identifying sequences of DNA/RNA, chemical susceptibility and resistance, and antibody 21 specificities may be tested.

23 A suitable protocol for the use of such plates is set 24 out below.
26 Protocol (assuming isolated bacterial colonies) (see 27 Figs. 1, 2 and 3);

29 1) Grow bacterial isolates overnight in a suitable growth medium.
31 2) Dilute the cells to pre-determined cell density in 32 same medium (for example Luria Bertani broth).
33 3) Add the cells (150~C1 per well) to a pre-prepared 34 microtitre plate containing phages in wells thereof using a multi-channel pipette or other 36 means.

1 4) Incubate the plate overnight.
2 5) Read the plate in a micro-titre plate reader or 3 stack the plate on an automatic feeder.
4 6) Once the plate has been read, identify the bacterial isolates of interest from the output 6 information (conveniently a printout or computer 7 display) which indicates bacterial growth in each 8 well and compares the interaction of the sample 9 with the phage sub-types used relative to known I0 standards (for example, pre-selected profiles).

12 Thus, in one embodiment of the invention phages 13 specific to the bacterium of interest are added to 14 wells in a micro-titre plate, fixed in a matrix which allows the phages to survive but prevents them from 16 cross-contaminating other wells in the plate. Suitable 17 fixation has been achieved using 5°s gelatin, although 18 other matrices or methods of fixing the phages could be 19 employed, eg polyvinylpyrrolidone (PVP) or freeze drying. Once the phages have been fixed into the wells 21 the plates can be stored until needed. The storage 22 time depends on the viability of the phages but would 23 typically be at least 6 months and may be several 24 years.
26 To type a bacterial isolate it is grown overnight in a 27 suitable growth medium and diluted in that medium to a 28 pre-determined concentration. The optical density is 29 taken before dilution to estimate the number of cells present and the dilution factor needed, since after 3I dilution too few cells are present to obtain an 32 accurate reading. A suitable concentration would be in 33 the region of 103-105 cells/ml to prevent overgrowth of 34 the bacteria in the wells. An appropriate volume of the diluted cells (typically 150~c1) is then added to 36 each individual well using a multi-channel pipette.

1 Each well (other than the non-phage containing 2 controls) will also contain one phage type of a total 3 of up to approximately 45 different phage types 4 (conveniently one phage type of a total of 19 or 11 different phage types, depending upon the need for 6 replication of each assay (see Fig. 4)) with each phage 7 type being located in a separate well. Controls 8 include (1) cells in the absence of phage (to ensure 9 cells are able to grow in the absence of phage), and (2) phage plus growth medium in the absence of cells 11 (to monitor plate contamination). All samples and 12 controls may be performed in triplicate to increase 13 reproducibility. After over-night incubation at a 14 temperature suitable for phage adsorption and replication, the micro-titre plate is read on a plate 16 reader at 595 nm and an OD reading obtained for each 17 well (see Table 1). The temperature used for 18 incubation will depend upon the bacteria under study.
19 For example an incubation temperature of approximately 25°C would be suitable for Erwinia and an incubation 21 temperature of approximately 37°C would be suitable for 22 E.coli. In the absence of phage action, cells grow and 23 produce OD values of approximately 0.5 (although the 24 extent of cell growth and thus the OD reading may vary between bacterial strains). In the presence of phage 26 action, cells are either killed or their growth is 27 reduced, thereby lowering OD values. In the event of 28 total cell death OD values can be as low as those of-29 the control (2) where no bacteria were added.
31 A computer program can be set up for analysis of the 32 incoming data and it is envisaged that a database of 33 the results for various phage/bacteria readings could 34 be established. The median of OD values may be taken (assuming at least three replicates) and the 36 "percentage growth" calculated by comparing the median 1 OD value with the OD of bacterial growth in the absence 2 of phage (positive control) (Table 2).
3 Percentage growth = T - C1 4 Cz ' C1 6 Where:
7 T - median of phage/bacteria OD readings.
8 C1 = lowest OD value obtained after complete cell lysis.
9 Cz = median of OD readings for bacteria in the absence of phage.

12 Percentage growth values will be used to produce a 13 "growth profile" for a particular strain (Figs. 5-10).
14 The assay of the invention therefore offers much greater discriminatory power than traditional phage 16 typing which in most cases gives a positive or negative 17 result only.

19 If a particular strains) is being sought/tracked, this strains infection profile could be pre-selected and the 21 program asked to identify all plates/rows containing 22 similar profiles. For example, this would be very 23 useful for epidemiological analysis of a particularly 24 pathogenic strain during a hospital infection. In addition, a database containing all previously tested 26 isolates, together with location and date of isolation 27 etc., could be searched and patterns of dissemination 28 identified. Although some variation in percentage 29 growth occurs (Figs. 5-7 and 10), such variation could be taken into account by the computer program when 31 assessing profile similarities.

33 Extensive testing of the system has already been 34 undertaken for use with the plant pathogen Erwinia carotovora subspecies atroseptica (Eca) and the human 36 pathogen E.coli 0157:H7, and has been found to be 1 simple, rapid and reproducible (Figs. 5-7 and 10). Eca 2 is widespread in temperate regions and its control is 3 of commercial importance to the potato seed industry as 4 Eca is responsible for soft rot of potato tubers in the field and in storage, as well as blackleg of potato 6 plants.

8 E.coli 0157: H7 causes potentially fatal food poisoning 9 and is a problem worldwide. Much attention is currently being given to the epidemiology of this 11 pathogen on farms, in the food industry and in 12 hospitals.

14 Recently there has been some interest in the use of phages as a means of controlling infections of 16 antibiotic resistant strains of bacteria. The assay of 17 the present invention can be used as a~ means of 18 identifying a phage which is efficient at causing cell 19 death in any particular bacterial species or sub-type.
21 In a further aspect the present invention also provides 22 an assay to identify a bacteriophage able to combat 23 replication of a specific bacterial species, said assay 24 comprising:
26 (a) isolating a single colony of said bacteria;

28 (b) combining said isolated bacteria with a 29 selected bacteriophage, said combination being held in a liquid medium containing the nutrients 31 required for bacterial growth and incubating said 32 combination;

34 (c) determining the extent of bacterial growth;
and 1 (d) selecting any bacteriophage which has 2 significantly depressed the extent of bacterial 3 growth.

5 Optionally, the assay may be combined with other tests 6 on the same plate for rapid analysis of bacterial 7 isolates.

9 The present invention also provides a method of 10 treating a bacterial infection in a plant, animal or 11 human, said method comprising selecting a bacteriophage 12 specific to the bacterial strain causing the infection 13 by means of the assay as decribed above and 14 administering a suitable dose of the bacteriophage so 15 selected.

17 The present invention will now be further described 18 with reference to the following (non-limiting) Example 19 and Figures in which:

24 Fig. 1 Procedure for novel phage typing system.
26 Fig. 2 Schematic representation of novel phage typing 27 system. Phages are fixed in microtitre-plate wells to 28 allow storage and convenience of use. Bacterial 29 colonies are diluted to a predetermined density and a fixed volume added to each well. After over-night 31 incubation, appropriate to the bacteria being typed, 32 plates are read in a microtitre plate reader followed 33 by data storage in a database for later retrieval.

Fig. 3 Schematic representation of novel phage typing 36 system. If phages fail to infect bacterial cells, 1 these cells grow in media increasing its optical 2 density. If phages infect cells, Iysogeny of lysis may 3 occur reducing or preventing cell growth and altering 4 optical density accordingly.
6 Fig. 4 Micro-titre plate layout of phages and bacterial 7 stains. Column 0 contains no phage. Columns 1-11 8 contain different phages. Row 1 contains the positive 9 control strain, sensitive to all the phages; row 2 contains growth medium only, rows 3 to 6 contain 3 11 replicates of strain 1; rows 7 to 10 contain 3 12 replicates of strain 2. Well AO (control strain in the 13 absence of phage) is used to obtain infection ratios.

Fig. 5 Analysis of five E.coli 0157:H7 strains, A) 16 1291, 3895 and 3939, B) 3602, and C) 3946, all of which 17 belong to phage type 28 when analysed by conventional 18 phage typing but show three types when analysed by new 19 method. In A, bars show standard deviations of greater than 5%.

22 Fig. 6 Analysis of E.coli 0157: H7 strain 3694 (phage 23 type 54 by conventional phage typing) together with six 24 RDNC strains, 643, 644, 645, 646, 647 and 648 all of which appear to fall into the same phage type as 3694.
26 Bars show standard deviations greater than 5%.

28 Fig. 7 A) Analysis of E.coli 0157: H7 strain 3964 (phage 29 type 2 by conventional phage typing) repeated five times with three replicates in each case. B) Analysis 31 of seven strains, five belonging to phage type 2 (322, 32 1563, 3487, 3932 and 3964) and two classified as RDNC
33 by conventional phage typing. Bars show standard 34 deviations of greater than 5%.
36 Fig. 8 Growth profiles of E.coli 0157: H7 strains from 1 A) RDNC strain 921, B) RDNC strain 936, C) RDNC strain 2 3690, D) strain 3890 (phage type 4 by conventional 3 phage typing), E) Non-0157 E.coli strain control 530 4 OI11. Using conventional phage typing strains A-D do not fit into any of the conventional phage types and 6 are classified accordingly as RDNC (react but do not 7 conform). Each strain was analysed in triplicate on a 8 single microtitre plate.

Fig. 9 Analysis of Erwinia carotovora subsp.

11 atroseptica strains A) SCRI 1 and SCRI 98, B) SCRI 1050 12 and SCRI 48, C) SCR I 13 and SCRI 87. In each pair A, B

13 and C, both .strains give identical phage types by 14 conventional phage typing. Each strain was analysed in triplicate.

17 Fig. 10 Analysis of Erwinia carotovora subsp.

18 atroseptica strains A) SCRI 1039 representing 14 19 replicates from 12 different microtitre plates and B) SCRI 1043 representing 16 replicates all from different 21 microtitre plates. Bars represent standard deviations 22 of greater than 5%.

26 Methods 28 Computer-assisted phage typing: Phages at routine test 29 dilution, determined for both sets of phages, were added to 5 % sterile gelatin, previously, and 5 ~.1 31 added to selected wells in a 96 well sterile microtitre 32 plate. In the case of Eca phages only, microtitre 33 plates were stored at 4'C for up to one month until 34 required. Based on optical density values at 595 nm and 630 nm respectively, Eca and E. coli 0157 cells were 36 diluted to ca. 10' cells ml-1 and 150 ~Cl added to 1 microtitre plate wells before overnight incubation at 2 25'C and 37'C respectively. The following day the 3 optical density of each well was read on a 4 spectrophotometry (Dynatec) at 595 nm or 630 nm, for Eca and E. coli respectively. Each strain was tested in 6 triplicate. Since 11 phages and 26 phages were used for 7 Eca and E.coli respectively, plate layouts were 8 designed differently (Fig. 4).

Analysis of data: The.median of three optical density 11 values for each strain was taken and the median value 12 in the presence of a particular phage minus the base 13 value (the value obtained in the case of complete cell 14 death in the presence of a phage) was divided by the 25 median value of the control wells containing no phages 16 minus the base value. This value was then multiplied by 17 100 and termed "percentage growth".

19 i.e. percentage growth -median optical density phage [n] well - base value x 100 21 median optical density control well - base value 23 Results E.coli: The percentage growth was obtained for each E.
26 coli 0157 isolate and results compared to conventional 27 phage typing (Table 3). In general, isolates belonging 28 to a particular phage type gave related patterns using 29 the novel method. However, the advantage of the novel system over conventional phage typing was seen when: a) 31 isolates with a conventional phage type showed 32 different growth patterns using the novel method, e.g.
33 3946, 3602 (phage type 28) showed different patterns to 34 other isolates within this phage type, indicating that either these isolates had been mis-classified by the 36 conventional method or that the novel method was able 1 to differentiate between isolates within this phage 2 type (Fig. 5). Similar findings were seen within phages 3 type 2 and 49; b) all RDNC (React but Does Not Conform) 4 isolates were typeable using the novel system. These isolates, so called because of their failure to give 6 consistent phage typing patterns and therefore their 7 failure to fall into a conventional phage type, were 8 differentiable using the novel method. Five RDNC
9 isolates, 644, 645, 646, 647 and 648, isolated from a single source/outbreak, gave growth patterns consistent 11 with phage type 54 (Fig. 6). Similarly RDNC isolates 12 714 and 785, again isolated from a single 13 source/outbreak, gave growth patterns consistent with 14 phage type 2 (Fig. 7). Four other RDNC isolates gave unique growth patterns using the novel system which did 16 not match any of the phage types tested (Fig. 8).
17 However, of these four isolates, one had a unique 18 pattern compared to other RDNC isolates when typed by 19 conventional phage typing, and another RDNC isolate was the only isolate tested to possess verotoxin 1 but not 21 verotoxin 2 {Table 3). The novel method was found to 22 be highly reproducible when percentage growth values 23 were compared between isolates falling into a given 24 phage type {Figs. 5, 6, 7).
26 Eca: Phage typing using the novel method produced a 27 large variation in phage types for Eca. Interestingly, 28 a number of isolates which gave identical phage typing 29 patterns using the conventional method, were clearly distinguishable using the novel method (Fig. 9), thus 31 adding to the discriminating power of the method. As in 32 the case of E. coli, the novel method proved to be 33 highly reproducibility. For example, percentage growth 34 values, when tested on 14 and 16 replicates of Eca SCRI
1039 and SCRI 1043 respectively, showed little 36 variation in standard deviation between replicates. In 1 the case of SCRI 1043, this deviation was below 5 % for 2 all phages (Fig. 10).

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Claims (21)

1. An assay to identify bacteria present in a sample, said assay comprising the following steps:

(a) isolating a single colony of said bacteria;

(b) combining said isolated bacteria with a selected bacteriophage in a container, the combination of bacteria and phage being incubated in a medium containing the nutrients required for bacterial growth and which enables phage/bacteria interaction; and (c) determining the extent of bacterial growth.

2. An assay as calimed in Claim 1 wherein the incubation medium is nutrient broth or Luria Bertani broth.

3. An assay as claimed in either one of Claims 1 and 2 wherein the extent of baterial growth is determined by measuring the optical density of the sample.

4. An assay as claimed in Claim 3 wherein the optival density is read using light of wavelength 590 nm to 630 nm.

5. An assay as claimed in any one of Claims 1 to 4 wherein the bacteria and phage are combined together in a well of a micro-titre plate.

6. An assay as claimed in any one of Claims 1 to 5 wherein the phage is pre-located in said container and retained therein by means of a fixant, by physical entrapment or by chemical interaction with the surface of the container.

7. An assay as claimed in Claim 6 wherein said bacteria is retained by using 5% gelatin as fixant.

8. An assay to identify a bacteriophage able to combat replication of a specific bacterial species, said assay comprising:
(a) isolating a single colony of said bacteria;
(b) combining said isolated bacteria with a selected bacteriophage, said combination being held in a liquid medium containing the nutrients required for bacterial growth and incubating said combination;
(c) determining the extent of bacterial growth;
and (d) selecting any bacteriophage which has significantly depressed the extent of bacterial growth.

9. An assay as claimed in Claim 8 wherein the incubation medium is nutrient broth or Luria Bertani broth.

10. An assay as claimed in either one of Claims 8 and 9 wherein the extent of bacterial growth is determined by measuring the optical density of the sample.

11. An assay as claimed in Claim 10 wherein the optival density is read using light of wavelength 590 nm to 630 nm.

12. An assay as claimed in any one of Claims 8 to 11 wherein the bacteria and phage are combined together in a well of a micro-titre plate.

13. An assay as claimed in any one of Claims 8 to 12 wherein the phage is pre-located in said container and retained therein by means of a fixant, by physical entrapment or by chemical interaction with the surface of the container.

14. An assay as claimed in Claim 13 wherein said bacteria is retained by using 5% gelatin as f ixant .

15. A method of treating a bacterial infection in a plant, animal or human, said method comprising selecting a bacteriophage specific to the bacterial strain causing the infection by means of the assay of any one of Claims 8 to 14 and administering a suitable dose of the bacteriophage so selected.

16. A container having a specific phage subtype located therein, said phage subtype being retained in said container by a fixant, physical entrapment or chemical interaction with the surface of the container.

17. A container as claimed in Claim 16 wherein said phage subtype is retained in said container by using 5% gelatin as fixant.

18. A container as claimed in either one of Claims 16 and 17 which is a microtitre plate and wherein 9 different phage subtypes are located in wells of said plate, each well containing a maximum of a single phage subtype.

19. A container as claimed in either one of Claims 16 and 17 which is a microtitre plate and wherein 11 different phage subtypes are located in wells of said plate, each well containing a maximum of a single phage subtype.

20. A container as claimed in either one of Claims 16 and 17 which is a microtitre plate and wherein up to 45 different phage subtypes are located in wells of said plate, each well containing a maximum of a single phage subtype.

21. A container as claimed in any one of Claims 16 to 20 for use in the assay defined in any one of Claims 1 to 14.