Identification of salmonella

Details Identification of salmonella Identification of salmonella

1999-07-08

2002-04-09

Gitomer, Ralph (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S034000

Reexamination Certificate

active

06368817

ABSTRACT:

The present invention relates to processes for identifying the presence of Salmonella species in a sample, as well as culture media suitable for such identification processes.
Members of the genus Salmonella constitute the most important causes of food poisoning in the UK. At present, the only effective means of diagnosis involves cultural isolation of the causative organism from faeces. This however is not straightforward as specialised media and reagents are required to isolate relatively small numbers of Salmonellae from a massive amount of commensal flora in the guts. Selective media have been developed for this purpose which rely on the visualisation of simple biochemical features such as production of hydrogen sulphide or non-fermentation of lactose.
A useful review of five plating media for isolation of Salmonella species and a comparison against Hektoen enteric agar, a standard medium, is described by Dusch et al in J. Clin. Microbial. (1995) 33(4), 802 to 804. All but one of the media are solid (standard agar concentration) whilst one is a semi solid reduced agar concentration medium. For the solid media, the compounds which are produced in the presence of microbial growth are selected so as to be visible to the naked eye. In order that the visualised compounds are associated with microbial colonies, those compounds must be non-diffusible in the culture medium. These media typically test for two different biochemical characteristics of bacterial colonies and the results are such that positive and negative results of each of the two tests can be observed with positive or negative results of the other test. Some of the biochemical tests observe the activity of specific enzymes by the use of chromogenic substrates which are uncoloured or non-fluorescent but which generate enzymic reaction products which are coloured or fluorescent and can hence be observed in the presence of the substrates. Sometimes the enzymic reaction product may react with a further component of the culture medium to generate the visible product, for instance metal ions or pH indicators, where the reaction product is an acid or base.
It is known to include in the culture medium substrates for two different enzymes which have different enzymic reaction products, each of which can be observed in the presence of the other (and of each of the substrates themselves).
One enzyme substrate which is commonly used in the identification of Salmonella is a substrate for &bgr;-galactosidase. Salmonella is generally negative for this enzyme activity, but most other members of the Enterobacteriaceae are positive. One &bgr;-galactosidase substrate whose enzymic reaction product is non-diffusible is 5-bromo-4-chloro-3-indolyl-&bgr;-D-galactopyranoside (X Gal). Other indoxyl and halogenated indoxyl compounds are useful as substrates and have reaction products which are visible and non-diffusible in agar culture media.
X-Gal is used as a substrate in Rambach medium, described inter alia in U.S. Pat. No. 5,194,374. It is used in combination with an alkanediol, which is metabolised by Salmonella to form an acid reaction product which is visualised by the incorporation of a pH indicator such as neutral red.
In EP-A-0516817, a culture medium for detecting Salmonella comprises a chromogenic &bgr;-galactosidase substrate and, in addition, glucuronate and a pH indicator. This mixed medium is alleged to be more selective than Rambach medium since almost all Salmonella species tested, but few other bacterial species ferment glucuronic acid resulting in a lowering of the pH.
In WO-A-94/0 1952, a 5-bromo-4-chloro-3-indolyl compound which is a substrate for an esterase enzyme is used to identify Salmonellae, which are positive for such enzymes. The substrate is an ester of a C
7-10
-fatty acid. It is suggested that the medium may be supplemented to eliminate non-Salmonella bacteria, such as using properties relating to cleavage or metabolism of &bgr;-galactosides and &bgr;-glucosides (for both of which Salmonella is negative).
Rambach medium and the X-gal glucuronic acid combination were found by Dusch et al to have less than optimal sensitivities. A further medium comprising xylose, lysine and Tergitol 4 has very good sensitivity and specificity. The culture medium includes the surfactant Tergitol 4 to inhibit Proteus, and determining hydrogen sulphide formation from sodium thiosulphate in the medium which is visualised by the incorporation of ferric ions.
It is known that Salmonella species produce &agr;-galactosidase, but it is likely that that enzyme would be considered a poor marker for Salmonella since it is produced by many related genera, such as Escherichia, Citrobacter, Klebsiella, Enterobacter and Shigella.
In Acta Microbiol Hung. (1988) 35(4), 389-395 Ketyi, I. discusses the &agr;-galactosidase activity of various species of entero-bacteria including Salmonella, Shigella and
E
-
coli
. He indicated that enzymic activity is a general feature of Enterobacteriaceae. He used melibiose, as an indicator of &agr;-galactosidase positive strains. Melibiose is not a chromogenic compound.
In WO-A-9630543 a chromogenic &bgr;-galactosidase substrate is used in combination with a mixture of sugars including mannitol, with xylose and melibiose for identifying Salmonella. The sugars are cleaved to form acids and the growth medium contains a pH indicator. However the acids which change the pH are products of a series of enzymic reactions on the product of sugar metabolism.
James et, al in App. Env. Microbiol. (1996), 62(10) 3868-170 and in J. App. Microbiol.(1997),82, 532-536, describe a new &bgr;-galactosidase substrate for use in place of X-Gal. The substrate is a derivative of cyclohexenoesculetin, of which the aglycone released by hydrolysis by &bgr;-galactosidase forms a black-brown complex with ferric ions in the medium. The new substrate, CHE-Gal, gave good correlation with X-Gal, that is high specificity and high sensitivity for detecting &bgr;-galactosidase activity.
These cyclohexenoesculetin substrates and other esculetin derivatives are described further and claimed in WO-A-9741138 (not published at the priority date of the present invention).
One aspect of the present invention is based on the need for culture media which are very sensitive to Salmonella whilst being highly specific, thereby minimising subsequent confirmatory tests. These types of test often need to be carried out with the inadequately specific media of the prior art. A second aspect of the invention is based on the provision of a medium comprising two chromogenic substrates which gives readily observable results. A visual determination can be easily made of the presence and absence of enzymic reaction products of each substrate regardless of the presence or absence of the enzymic reaction product of the other substrate.
According to a first aspect of the present invention there is provided a new process in which the following steps are carried out:
1. a sample suspected of containing Salmonella bacteria is cultured in the presence of a nutrient,
2. the bacterial culture is contacted with each of two enzyme substrates,
3. the presence of the enzymic reaction products of each of the substrates is accessed after step 2 to determine whether or not growth of Salmonella species has taken place, in which the first substrate is a substrate for an enzyme for which Salmonella is negative, the process being characterised in that the second substrate is a substrate for &agr;-galactosidase and in that both substrates are chromogenic.
The present inventors believe that it is the first time that &agr;-galactosidase has been used as a marker for Salmonella using an &agr;-galactosidase specific chromogenic substrate, that is a substrate for which the enzymic product of the reaction in the presence of &agr;-galactosidase is chromogenic without being subjected to further enzymic reactions. Thus, the inventors have recognised the utility of combining &bgr;-galactosidase and &agr;-galactosidase as markers for detecting Salmonella species. The method is useful for carrying

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