By John Lindquist. Based primarily 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 genus Salmonella is of great concern to the food industry. Processed foods are not permitted to contain any Salmonella cells. The reason for this "zero tolerance" is that salmonellae are responsible for severe, acute gastroenteritis. Salmonella is responsible for a major portion of the cases of gastroenteritis each year in the United States.
Salmonellae are widespread in nature. They are commonly found in the intestinal tract of mammals, birds and reptiles. Pork and beef are often found to harbor salmonellae. Poultry continues to be one of the primary reservoirs of the organism. Any raw poultry product should be assumed to contain salmonellae and therefore must be handled appropriately. Eggs are contaminated with salmonellae when laid. Pasteurization of the egg is designed to inactivate the organism, though one should not casually assume eggs are then necessarily free of it. In recent years, there have been numerous outbreaks of gastroenteritis credited to Salmonella-contaminated eggs.
Taxonomically, the species concept has been applied with difficulty to members of this genus. Through much of the 1970's and 80's, the three-species concept was advocated by many with the recognition of S. typhi, S. cholerae-suis and S. enteritidis. The last-named species, whose name was derived from a very early-established serovar (serotype), was basically a dumping ground for the vast majority of strains isolated, generally from cases of gastroenteritis. Many now agree there should only be one or two recognized species in the genus Salmonella, as the various strains are so closely-related genetically.
The name "Arizona" appears to have been applied in one form or another to a group of Salmonella-like organisms since the late 1930's according to the 7th edition of Bergey's Manual (1957) where it was called Paracolobactrum arizonae. Serological similarity with Salmonella was usually a noted feature along with the atypical (for Salmonella) tendencies to ferment lactose and liquefy gelatin. Later names included Arizona arizonae, Arizona hinshawii and Salmonella arizonae before being totally absorbed into the modern Salmonella scheme as two of the several subspecies. Its DNA has been characterized as being so similar to other salmonellae that it can be lumped into the same Salmonella species.
Seven genetically and biochemically-defined subspecies of Salmonellaare recognized. However, the "handle" for any isolated strain of Salmonella is a further subdivision, the serovar – also known as serotype – a designation which reflects the testable antigenic makeup of the organism, involving the identification of the cell wall ("O") and flagellar ("H") antigens. Each identified strain belongs to one of over 2000 recognized serovars. All serovars are designated with an antigenic formula, a series of numbers and letters which refer to the recognized antigens. Most serovars are also given names which are often written for convenience as if they were (but they really are not!) species names – for example, Salmonella typhimurium. The convention now is to capitalize the serovar designation and indicate it (without italicizing it) after the genus, species or subspecies name; using the previous example it would be written thusly: ser. Typhimurium. See the table below.
Although the term serovar is used predominantly in this discussion, this fairly-recent admonition by some Salmonella and nomenclatural authorities to use "var"-suffixed terms has been resisted by many bacteriologists – notably those at the CDC – who prefer to stick with the more descriptive, familiar and euphonious term serotype.
In the usual listing in the literature of the various subspecies of Salmonella, the first embraces the vast majority of isolated serovars. Most of the serovars of this subgenus are responsible for salmonellosis (gastroenteritis) in humans, a prime concern to the food microbiologist. These organisms are virtually identical genetically, and they tend to possess the same phenotypic characteristics. Exceptions to this rule occur with "host-adapted" serovars – i.e., those which have become adapted in their evolution to certain animals with (often) a consequent shifting of certain physiological properties. Following are some host-adapted serovars with their names written out like species names (for convenience, as discussed above). Major phenotypic variations are highlighted:
The following review explains host-adaptation as it relates to Salmonella: Andreas J. Baumler, Renee M. Tsolis, Thomas A. Ficht, and L. Garry Adams. 1998. Evolution of Host Adaptation in Salmonella enterica. Infect. Immun. 66: 4579-4587.
Salmonellosis results from the ingestion of viable Salmonella cells. Generally, about 108 cells must be ingested, although there are reports of as few as 100 cells being able to cause gastroenteritis. The disease is referred to as a toxico-infection, as one must ingest viable cells and have these cells colonize the small intestine. During their growth in the intestine, they release an enterotoxin, resulting in severe vomiting and diarrhea. Onset of symptoms occurs about 12 hours after ingestion of the contaminated food with symptoms lasting 2 to 3 days. The disease is generally self-limiting.
The isolation and identification of Salmonella can be a very time-consuming and expensive process and includes the use of a number of selective and differential media. Foods should not be released by the manufacturer until declared free of salmonellae. This requires holding the product, at consumer expense, for a protracted period of time. Research continues to develop rapid, reliable methods to detect salmonellae.
The method traditionally used by the Food and Drug Administration begins with a nonselective enrichment in lactose broth. A selective broth enrichment follows on the next day. On the third day, the selective enrichment is streaked onto several different selective-differential plating media to initiate the isolation of any salmonellae present. (The sample can be plated directly if a large, active population of salmonellae is suspected.) Should likely colonies be found, they are subcultured into Triple Sugar Iron (TSI) Agar, Lysine Iron Agar (LIA) and Brain Heart Infusion (BHI) Broth. If the results of the TSI Agar and LIA indicate the probability of Salmonella, the BHI Broth culture is employed in the serological confirmation of the presence of the organism with the use of polyvalent flagellar antisera. Tests for identity of cell wall antigens may also be done, using cells from the TSI Agar. However, certain other genera contain strains which share the same cell wall antigens as many salmonellae, so this test is not definitive in identifying Salmonella. Further confirmation involves tests with various differential media; this is especially useful when a non-flagellated strain is encountered. The entire process usually takes about six days to complete.
The antigenic formula summarizes the unique combination of testable antigens associated with a serovar. As an example, the antigenic formula for the serovar Salmonella typhimurium – 1, 4, 5, 12 : i : 1, 2 – is constructed from the following sets of detected cell wall (O) and flagellar (H) antigens:
|O antigens detected: 1, 4, 5 and 12|
H antigens detected – phase 1: i
H antigens detected – phase 2: 1 and 2
By "phase 1" and "phase 2" we mean two sub-populations of cells where the cells in each possess a certain antigen (or set of antigens) associated with their flagella. In testing for the various H antigens possible in a culture of a Salmonella isolate, the antigens of the majority sub-population are initially detected by the use of individual, specific antibodies. By spot-inoculating the culture on a solid medium containing these antibodies, cells of the majority sub-population can be immobilized while cells of the minority sub-population (if present) can swarm out onto the medium (as their flagella are not hindered by the antibodies in the medium) and proliferate, such that they can be isolated and tested for their H antigens. This has been termed the "phase-reversal technique."
The example above shows a "diphasic" serovar. Examples of "triphasic" and "quadriphasic" serovars are included in the table below.
In the following procedure, the test for cell wall ("O") antigens can be done on an isolate likely to be Salmonella. With a wax pencil, circular areas are marked off on the surface of a glass slide. These marks should be drawn heavily in order for the circular areas to contain cell suspensions which must not be allowed to run into each other or off the slide. After a drop of cells suspended in saline is placed in each circle, a drop of antiserum is subsequently added to each. (Alternatively, the antiserum drops can be placed on the slides first, and the cells can be suspended directly into the antiserum.) Where there is a reaction between antibodies in the antiserum and their homologous antigens on the cell wall of the bacteria, the cells will clump together ("agglutinate"), and the drop will appear to contain many small particles. The reaction is best observed from underneath the slide, and this can be accomplished with a mirror or by placing the slide in a petri dish and then holding the dish above eye level. In this photo, one drop is seen to contain agglutinated cells, and the other retains its original milky appearance.
The test for flagellar ("H") antigens is done in tubes, and we have no photo of this test as yet to show here. Strains of some other genera (e.g., Citrobacter) may possess one or more of the same O antigens of Salmonella; the H test is much more specific for Salmonella identification.
This table is based on the system wherein one species of Salmonella is recognized and is called Salmonella cholerae-suis. A competing system designates the same species as Salmonella enterica and the first subspecies as Salmonella enterica subsp. enterica. Subspecies are defined and differentiated biochemically and genetically. Also termed "subgroups," the subspecies names are preceded by the numerical subgroup designation.
Several host-adapted, biochemically aberrant serovars which do not have unique antigenic formulae (and must then be distinguished by their biochemical reactions) are shown in this table – i.e., S. pullorum, S. gallinarum, S. paratyphi-C, S. cholerae-suis and S. typhi-suis.
Irrespective of subspecies designation, the serovars are grouped according to their O antigens: Those which share O antigen 2 are in Group A, those which share O antigen 4 are in Group B, those which share O antigens 6 and 7 are in Group C1, etc.
A more comprehensive list of serovars can be found in Bergey's Manual of Systematic Bacteriology, Volume 1 (1984) upon which this table is based. The newer ninth edition of Bergey's Manual of Determinative Bacteriology (1994) sets Salmonella bongori off as a separate (second) species. Recognizing the two species, the 1998 edition of the Difco Manual has an updated serotype list. For the ultimate list according to a controlling authority, the WHO (World Health Organization) Collaborating Centre for Reference and Research on Salmonella periodically publishes the Antigenic Formulas of the Salmonella Serovars.
|subspecies||antigenic formula||serovar name|
|1. subsp. cholerae-suis||1,2,12:a:1,5||ser. Paratyphi-A|
|6,7:y:e,n,z15:z47:z50||ser. Mikawasima 1|
|13,23:d:1,7||ser. Grumpensis 2|
|30:i:e,n,z15||ser. Mjordan 3|
|47a,47b:z45:z4,z23:z6||ser. Bere 4|
|2. subsp. salamae||1,9,12:l,w:e,n,x||ser. Daressalaam|
|3a. subsp. arizonae 5||51:z4,z23:-||(none given)|
|3b. subsp. diarizonae 5||40:k:e,n,x,z15||(none given)|
|4. subsp. houtenae||11:z4,z32:-||(none given)|
|43:z4, z23:-||ser. Houten|
|5. subsp. bongori||48:z35:-||ser. Bongor|
|6. subsp. indica||1,6,14,25:a:e,n,x||ser. Ferlac|
1 an example of a quadriphasic serovar.
|Selected Groups of Bacteria|
Bacteriology 102 Website
|Page last modified on 9/11/01 at 8:30 AM, CDT.|
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison