By John Lindquist. Based on material in the Laboratory Manual for the Food Microbiology Laboratory (1998 edition, edited by John L. and published at the University) which was used in Bacteriology/Food Science 324 at the University of Wisconsin – Madison.
The family Enterobacteriaceae comprises a very large group of morphologically and physiologically similar bacteria. They are of great importance to the food microbiologist, as some of these organisms are involved in food spoilage, some are food-borne pathogens and some are indicators of fecal contamination of food products. Because of their high degree of importance, it is necessary that the food microbiologist be able to isolate and identify these organisms quickly and efficiently. The similarity in physiology of the many species requires the analyst to be knowledgeable of, and to be able to employ, the wide variety of differential (including serological) tests used in the identification process.
These organisms are simply referred to as the "enterics," but one must realize that the enteric tract must not be assumed to be the habitat for all members of the group. Aside from the intestinal canal, enterics are found throughout nature including plants, soil and water. Each species has its preferred habitats. One species which is commonly associated only with the intestinal tract is Escherichia coli, the indicator of fecal contamination. Certain strains of this species cause severe food-transmitted diseases.
All enterics are gram-negative rods. Occasionally they may be mistaken for cocci, but the observation of a relatively "young" culture generally yields definitively rod-shaped cells. These organisms are true "facultative anaerobes." Therefore they are expected to be benzidine-positive, catalase-positive and able to ferment at least glucose. As indicated in the previous exercise, they are oxidase-negative, and motile enterics are so by means of peritrichous flagellation. (Non-motile enterics include the genera Shigella and Klebsiella). Most can respire anaerobically, using nitrate as the terminal electron acceptor; nitrate is then reduced to nitrite and no further. Enterics are unique in possessing the "enterobacterial common antigen" in the cell wall.
The family Enterobacteriaceae can be defined on a genetic basis and, indeed, the definition of each newly discovered species is primarily based on its genetic distinctiveness. The validity of most of the older, well-known species has held up under the new genetic characterization. Once a species is so defined, careful choice must be made of easily-testable phenotypic characteristics to associate with the species. Studies of the genetic characterizations of the enterics serve as models for the definition of organisms in other bacterial groups.
Regarding the catabolism of glucose and other sugars, two major types of fermentation are found among the enterics. Organisms which possess the mixed-acid type of fermentation convert pyruvate (obtained from the glycolytic pathway) to a variety of acids (lactic, acetic, succinic, formic) and ethanol. Those which possess the butanediol type of fermentation convert pyruvate to these products plus "neutral products" (acetoin and/or 2,3-butanediol) and carbon dioxide. The methyl red test is used to test for the relative pH attained from glucose fermentation in a standard medium (MR-VP Broth); a positive reaction (a very low pH due to high concentration of acids) indicates the mixed acid type of fermentation. It is essential that the medium be incubated for at least two days to allow fully for the differentiation between the two fermentation types. The Voges-Proskauer test indicates the neutral products formed by the butanediol type.
Whether or not a certain sugar is fermented by a particular organism can be determined by the use of a "fermentation broth" which is a complex medium to which one particular sugar is added as well as a suitable pH indicator. Acid from fermentation is detectable by a change in the pH indicator to its "acid color." Peptones and/or extracts are generally employed as nutritive ingredients (providing mainly nitrogenous compounds), and enterics may grow in the medium without additional sugar, provided they can utilize the amino acids as sources of energy (by respiration) and carbon. Enterics of either fermentative group (mixed acid or butanediol) may have the enzyme formic hydrogenlyase which converts formic acid to gaseous hydrogen and carbon dioxide; organisms possessing this enzyme are detectable by the gas bubble seen in the Durham tubes placed in the fermentation broths of catabolized sugars.
Alkaline products formed by these organisms may be detected in various media, and the net result of concurrent acid and alkaline reactions is responsible for the results seen in certain of these media. All enterics will perform aerobic deamination of one or more of the amino acids found in peptones, yeast extract, beef extract and similar organic nitrogenous materials. Ammonium is then produced with a consequent alkaline reaction which, if not over-neutralized by an acidic reaction from fermentation, will be detected by an alkaline shift in the color of the pH indicator. A similar alkaline reaction may be seen under anaerobic conditions if decarboxylation of amino acids takes place. To test whether or not a particular organism decarboxylates a certain amino acid, the amino acid in question (lysine and ornithine are commonly tested) is included in relatively high concentration in a medium containing a pH indicator. Formation of a highly alkaline diamine may be detected easily. Decarboxylation of lysine, ornithine and histidine will result in the production of cadaverine, putrescine and histamine, respectively. Scombroid food poisoning is a result of bacterial decarboxylation of histidine found in certain fish (tuna, mackerel, and related species) containing a high concentration of this amino acid; the enteric Morganella morganii is the primary organism implicated, and it will readily decarboxylate the histidine during improper storage of the fish. The resulting histamine causes the severe allergy-like symptoms associated with this syndrome.
The differential isolation and identification media incorporate ingredients relating to specific physiological processes relevant to identification of specific groups of organisms. As an example, XLD Agar is particularly suited for the isolation of Salmonella in that certain characteristics of the organism are exploited to result in a colonial appearance which is not matched by too many other organisms. Our differential media page has explanations of some of the various media.
Certain physiological groups of organisms may be recognized within the family Enterobacteriaceae. The true coliforms are those enterics which ferment lactose vigorously to acid and gas at 35-37°C within one or two days. Most strains found in the genera Escherichia, Enterobacter and Klebsiella fit the description of coliforms and are used as pollution indicators in water and food analysis. (Many strains of Citrobacter are also considered coliforms.) Certain other enterics may ferment lactose, but minimal gas production and/or a lower temperature optimum for most of these organisms preclude them from being termed "coliforms."
Salmonella and Shigella stand out as the major pathogens of the family. Both are causative agents of serious forms of gastroenteritis. Salmonella is particularly important to the consumer as the organism is found very commonly in poultry and other meats. Any raw poultry product must be assumed to contain salmonellae. Prevention of cross-contamination between raw and cooked poultry (and other foods) is essential in avoiding salmonellosis as are proper storage and cooling. Most isolates of Salmonella and Shigella do not ferment lactose or sucrose, therefore inclusion of these sugars in plating media assists in their isolation; non-fermenting colonies will then be favored for further testing.
Another physiological group comprises the genus Proteus and its relatives, Morganella (noted above) and Providencia. These organisms often appear on plating media for the isolation of Salmonella and Shigella and may appear non-fermenting. A distinguishing characteristic of these organisms is their possession of the enzyme phenylalanine deaminase for which a test may be made easily. Many organisms in this group also hydrolyze urea rapidly.
Other important genera include Yersinia, a food-borne pathogen responsible for many intestinal infections and Erwinia which includes species which cause disease in or spoilage of edible plants. The genera Hafnia and Obesumbacterium have been found as major spoilage organisms in the brewing industry. Edwardsiella has been found to cause some serious extraintestinal infections and also has been implicated as a possible cause of gastrointestinal illness. Massive infections of catfish and other freshwater fish destined for market have been caused by Edwardsiella.
Further information can be obtained from sources such as Bergey's Manual, The Prokaryotes and the Manual of Clinical Microbiology.
An outline which has gone along with food microbiology lab lectures on the isolation of enterics in general (and of coliforms and Salmonella in particular) starts here.
|Selected Groups of Bacteria|
Bacteriology 102 Website
Additional notes on "enterics" & "lactics"
|Page last modified March, 2020.|
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison