14-1 Introduction

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The term enteric bacteria (or enterics) is generally used in reference to organisms of the Family Enterobacteriaceae, many members of which occur in the enteric tract of humans and animals in health and disease. Other residents of the intestinal tract (e.g., Clostridium, Bacteroides and various lactobacilli and streptococci) also might be called enterics, but convention reserves the term for the Family Enterobacteriaceae. All members of this family possess these characteristics in common:

  • gram-negative
  • rod-shaped
  • facultatively anaerobic
  • positive for true catalase and cytochromes
  • ferment glucose by one of two major pathways to a variety of end products
  • oxidase-negative
  • possess the enterobacterial common antigen in the cell wall

Enterics can be found in a variety of natural habitats, not just in the intestinal tract. Selective isolation of these organisms is assisted by the use of media containing agents which inhibit gram-positive organisms. MacConkey Agar is a popular selective medium, although this medium can also support the growth of many gram-negative organisms other than the enterics. (Neisseria is one of a number of gram-negative bacteria which cannot grow on MacConkey Agar.) There is no one medium on which all enterics will grow with the exclusion of all other organisms.

Most enterics are motile by peritrichous flagella; two major exceptions are Klebsiella and Shigella. Most will reduce nitrate to nitrite but never to nitrogen gas. Depending on the specific organism, various sugars may be fermented and/or respired (yielding energy - see Exp. 5.1), and one or more amino acids (such as lysine, ornithine, arginine, glutamic acid and histidine) may be decarboxylated. Fermentation and decarboxylation are anaerobic processes and will result in acid and alkaline reactions, respectively. Another anaerobic process - production of hydrogen sulfide from thiosulfate - is possessed by some of the enterics.

For any organism in the enteric family, glucose (and other carbohydrates, depending on the organism) is fermented to pyruvic acid and beyond by either the mixed-acid or butanediol pathways. End-products of the mixed-acid type of fermentation include lactic, acetic, succinic and formic acids and ethanol. A very low pH is rapidly achieved and maintained. After incubation in a standardized medium (MR-VP Broth), the addition of methyl red (a pH indicator) results in a red color (a positive reaction), an indication of pH 4.4 or below. Organisms possessing the butanediol type of fermentation produce the same end products and low pH by one day of incubation but neutral products (acetoin and/or 2,3-butanediol) and carbon dioxide are then formed at the expense of one or more of the acids. After an additional day of incubation, the pH is at 6.2 or above where the addition of methyl red results in a yellow color. The Voges-Proskauer (VP) test indicates the presence of neutral products directly. Therefore, with rare exceptions, an MR-positive organism will be VP-negative, and an MR-negative organism will be VP-positive.

Many enterics produce gas during carbohydrate fermentation which collects in a Durham tube. This gas is the result of the enzyme formic hydrogenlyase which splits formic acid into hydrogen and carbon dioxide.

As expected for most chemoheterotrophic bacteria, ammonia is produced from aerobic deamination of one or more amino acids such as those found in peptones and yeast extract. The combination of this alkaline product with acids produced from carbohydrate fermentation produce a net reaction which is important in the interpretation of some differential media such as Kligler Iron Agar. As for many carbohydrates, amino acids may be utilized as sources of carbon and energy (the latter via respiration).

Thus it may be seen that an unknown isolate may be identified to the enteric family and ultimately to genus and species by the application of the appropriate tests, many of which are used in this exercise. Certain other gram-negative rods may resemble the enterics superficially. For example, Pseudomonas shares many habitats with enterics and is often found on the plating media utilized in enteric isolation, but it can be easily recognized by its strictly aerobic nature, its failure to ferment any sugar and its (usually) oxidase-positive reaction. Vibrio, Aeromonas and the luminescent (light-producing) genus Photobacterium, all of which are facultatively anaerobic, appear even more closely related to the enterics, but they are distinguished easily by the oxidase test.

Selected Genera of the Family Enterobacteriaceae

Escherichia, Enterobacter and Klebsiella: These organisms are often considered together, as most strains of these genera are distinguished easily by their ability to ferment lactose rapidly to acid and gas. These strains are the true coliforms, useful as indicators of water quality (see Exercise 15). Escherichia coli is the predominant facultatively anaerobic organism in the large intestine, and its presence in water, food or anywhere indicates fecal contamination and the possibility of associated intestinal pathogens. E. coli may cause diseases of the urinary tract, and certain strains cause severe intestinal disease. The various species of Enterobacter and Klebsiella are widely distributed on plants and in soil and are often found in the gastrointestinal tract. The finding of these organisms in drinking water indicates surface soil contamination. K. pneumoniae is an occasional cause of pneumonia in humans.

Genetically, Shigella and E. coli are virtually identical. Were they to be discovered and named today, they would be grouped into one species. However, Shigella has been set apart traditionally for its consistent non-motility, its failure to ferment lactose and its cause of bacillary dysentery, characterized by severe abdominal pain and bloody diarrhea. Only a few ingested cells are needed to result in disease.

Citrobacter is a commonly-found non-pathogenic enteric. In food and medical laboratories, colonies of typical strains (those which are lactose-negative and H2S-positive) appear identical to those of Salmonella on most isolation media. Subsequent biochemical testing will differentiate these genera easily. Certain other strains of Citrobacter which can ferment lactose rapidly to acid and gas are thus considered coliforms and are occasionally isolated in Experiment 15.

Salmonella is a genus of pathogenic organisms infecting humans and many mammals, birds and reptiles. Organisms in this genus are so genetically similar that they are now considered as belonging to one or two species. Subdivision of the genus has been made to at least seven genetically-distinct subgroups (subspecies) and further to well over 2000 serovars (formerly called serotypes). Each serovar is distinguished by a unique combination of cell wall (O) and flagellar (H) antigens (see Exp. 14.2). Serovar recognition is important in epidemiology. Often an outbreak or epidemic caused by strains of one serovar can be traced to a common source. Decades ago, it was the practice to consider each serovar a species. Now, most serovars are designated for convenience with names which are written like species names. The three serovars Salmonella typhimurium, S. enteritidis and S. heidelberg are responsible for about half of the cases of human Salmonella infection in the United States.

Most serovars of Salmonella cause gastroenteritis of varying degrees of severity, with or without bacteremia. The source of gastroenteritis is usually contaminated food containing over 106 cells/g (or ml). Symptoms generally appear in 8 to 30 hours after ingestion and include nausea, fever, diarrhea, and abdominal pain, usually subsiding in one to two days. Certain host-adapted, biochemically-distinct serovars (which may be termed bioserovars) cause life-threatening illnesses such as typhoid fever by S. typhi, hog cholera by S. choleraesuis (also pathogenic to humans) and fowl typhoid by S. gallinarum.

Organisms of the genus Edwardsiella are known to cause disease in humans and a variety of warm and cold-blooded vertebrates. E. tarda is an occasional opportunistic pathogen for humans, causing wound infections and occasional gastroenteritis. E. tarda and E. ictaluri have caused massive infections of commercially-raised catfish with considerable economic loss.

Yersinia includes the species Y. pestis, the causative agent of bubonic plague. Other diseases produced by Yersinia include a severe gastroenteritis in humans and red-mouth disease in trout and salmon.

The Proteus group of enterics (Proteus, Providencia and Morganella) is easily distinguished by the ability to deaminate the amino acid phenylalanine. These organisms are relatively non-pathogenic but are capable of causing urinary tract infections and occasional gastroenteritis. Fecal matter, sewage and soil, especially where animal protein is decomposing, are common sources for these organisms. Scombroid food poisoning, caused by the decarboxylation by Morganella morganii of histidine to histamine (which causes severe allergy-like symptoms), is associated with certain fish of high histidine content such as tuna and mackerel.

Erwinia is a genus of plant pathogens which produce a variety of diseases including wilts and soft rots. Erwinia has been implicated in cases of septicemia caused by contaminated intravenous fluids.

Serratia is a widespread organism found in soil, water and clinical material. Some strains produce a bright, red pigment (prodigiosin) as was seen in Experiment 6. Serratia tends to hydrolyze proteins, nucleic acids and chitin rapidly. Some insect diseases and cases of human pneumonia are caused by Serratia.

Many additional genera are also included in the family Enterobacteriaceae which now contains about thirty genera and well over a hundred species! The luminescent organism Photorhabdus is found frequently in association with nematodes. Hafnia and Obesumbacterium are occasional brewery contaminants. Others are mainly of obscure clinical or environmental interest and are rarely isolated. In the mid 1990's, an unusual group of strains (the G30 Biogroup) isolated from six Wisconsin lakes was discovered at the U.W. Department of Bacteriology and was shown by the Centers for Disease Control to be a distinct enteric genus based on genotypic and phenotypic studies. It is presently established as CDC Enteric Group 121. (You may often see as yet unnamed enteric groups referred to when you read about clinical organisms.) Upon formal publication the designation will be Aquamonas haywardensis (water unit from Hayward).

Overview of isolation and identification (with reference to our procedure).

The principles of enrichment and isolation continue to apply to the enterics. For example, in clinical and food microbiology laboratories, where enteric pathogens such as Salmonella and Shigella are implicated in a disease or a contaminated food, the procedure may be outlined as follows:

  1. Obtain specimen. If the specimen is expected to contain a large, active population of the suspected organism (such as a diarrhea specimen from an individual suffering from an intestinal infection), one may go directly to step 3.
  2. Inoculate specimen into selective enrichment broth medium (or media) such that the organisms to be looked for are given every opportunity to grow with minimum interference from the growth of other organisms. The selective agents included (also in the following plate media) are primarily intended to inhibit gram-positive organisms. Depending on the amounts and types of selective agents included, some highly-selective media may inhibit certain gram-negative organisms (including some enterics) as well.
  3. Streak onto selective-differential plate media for isolated colonies. Once these are obtained, careful selection is made of colonies to be picked for further analysis.
  4. One must pick only from the very top of the colonies. Contaminating organisms on the surface of the medium may be present. Though inhibited, they are still viable! If in doubt about purity, one can streak onto a non-selective medium.
  5. Typical strains of Salmonella and Shigella do not ferment lactose or sucrose. Therefore, if either of these organisms is suspected, one may simply pick non-acid-producing colonies off media which contain these sugars. However, many other gram-negative rods do not ferment lactose or sucrose including Pseudomonas which does not ferment anything. Also, some exceptional strains of Salmonella are lactose-positive. Anticipating this probability, a supplementary medium is used which does not differentiate according to fermentation reactions (Bismuth Sulfite Agar).
  6. Inoculate preliminary biochemical screening media from the chosen colonies. One has no way of identifying specific organisms on the plates. Also, one would not wish to waste time and money by inoculating all of the media necessary for identification from each chosen colony! Therefore, one or more media such as Triple Sugar Iron Agar (or Kligler Iron Agar), Lysine Iron Agar and (sometimes) Motility Indole Ornithine Medium are inoculated from each colony. Each of these media yields several useful pieces of information about the colony picked. With any of these media, non-fermenting organisms such as Pseudomonas are recognized and discarded, thus saving time and money in the coming procedures.
  7. Perform serological tests. As explained in the introduction, serovars of Salmonella are distinguished by the identification of their O and H antigens, and a suspected culture of Salmonella may be tested with antibodies, singly and in combinations, for identification to whatever level is desired: genus, serological group or serovar. Serological identification of Shigella and certain other enterics may also be attempted.
  8. Perform confirmatory biochemical tests. Various differential media can be inoculated from one of the preliminary screening media.

In Experiment 14.1, an abbreviated version of the above will be performed on a mixed unknown consisting of three organisms. Two of the three organisms are enterics to be identified. The third is a strain of Pseudomonas which will be recognized sooner or later as a non-enteric and then discarded during or before the third period. We will forego any selective enrichment broth media and streak the mixture directly onto the plates for isolated colonies. In Experiment 14.2 we will perform a serological procedure to identify a strain of Salmonella to a serological group, but we will not be doing this for any of the unknowns. There are no enteric pathogens among the unknowns. Make sure nothing is identified ultimately as Salmonella or Shigella. Refer to the appendices at the end of Experiment 14 concerning the various media and organisms.

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