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Salmonella diagnostics/analyses

SVA performs analyses of salmonella in faecal samples, environment and feed samples, mainly from individual animals, herds, slaughterhouses, and food.

Salmonella culture takes three days before the first ocular inspection of the culture plates can be made. Hence, if no suspected salmonella colonies are detected the analysis is completed at the earliest after three days. If there is a suspicion of a positive finding the analysis may take a few more days.

SVA is a National Reference Laboratory for salmonella (NRL). This means that other Swedish laboratories must send in suspected salmonella isolates, from animals, feed or food, for confirmation of the suspicion to the NRL where the isolates also will be serotyped.

The methods used for salmonella analyses depend on the customer’s request and type of sample.
The methods have a high sensitivity and even a low concentration of salmonella can be detected in most types of samples. The choice of method is also dependant on if the sample is taken in official control or not. The time it takes to conduct the analysis is also an important factor.
The methods are based either on culture of bacteria or detection of salmonella-specific gene sequences.

Culture methods for detection of salmonella

To be sure to detect salmonella, culture is performed in several steps or on various specific media. This is to increase the possibility of finding salmonella for example in faeces where there is an abundance of other bacteria.

Upon arrival in the laboratory 25 grams of the sample is put in a pre-enrichment broth. The broth is incubated at 37°C for one day. The next step is to take a sample from the broth and transfer it to a selective medium that inhibits the growth of most other bacteria but not salmonella. The sample is incubated for another day at 41.5°C. This higher temperature serves as a further selection for salmonella. Depending on the method, the details differ. 

If the method described by the Nordic Methodology Committee on Food Analysis (NMKL No 71 edition 5) is used, further culture for one day at 37°C on two new types of salmonella- specific media is done. These are called XLD and BG. At this stage the result (i.e. colony growth on the plates) is checked for the first time. If no bacterial colonies with typical appearance of salmonella are detected the sample is classified as “Salmonella not detected”. If there are, on the other hand, suspected colonies, confirmatory test are done. These consist of biochemical tests and so-called agglutination tests. These tests finally determine if salmonella has been detected in the sample.

If the method according to ISO 6579/A1:2007 is used, the sample is already on day two (after pre-enrichment) been dripped onto a semi liquid medium called MSRV.

Salmonella has so-called flagella. These are small threadlike structures enabling the bacteria to move.  If there is salmonella in the sample, expanding zones of bacterial growth are formed around the drops. The MSRV-plates are checked after one day. If suspected zones are detected, material from these is spread on to XLD- and BG-plates in the same manner as described above under “NMKL.” If no suspected colonies are detected the MSRV-plates are incubated for an additional day. If no typical zones are seen by then the analysis is completed and the sample is classified as “Salmonella not detected.”

If there are any suspected colonies on the XLD- and BG-plates after one day of culture, confirmatory biochemical tests and agglutination tests are performed. These finally determine if salmonella has been detected in the sample.

Consequently, it takes three days from the start of the analysis until the first preliminary result is available. If no suspected salmonella bacteria have been detected this result is final. If suspected salmonella colonies are detected the analysis may take several more days.

PCR-methodology for detection of salmonella

PCR techniques may also be used for detection of salmonella-specific DNA sequences. However, these methods do not provide information about serotype and antibiotic resistance. Such information is needed and thus confirmatory cultures must be performed on positive samples.

SVA performs two different types of PCR-methods

In the first method the PCR is carried out after the first day of incubation (pre-enrichment). Since the PCR-method can be carried out during one workday, the result is ready after two days if the sample is negative. If the PCR is positive, the sample must be cultured for typing and antibiotic susceptibility testing.

In the second method PCR is performed after two or three days of culture on selective media, as described in the text on culture methods.  

If salmonella is detected the bacterial isolate is always subjected to serotyping, to determine what serotype of salmonella it is. There are only two species of salmonella; Salmonella enterica and Salmonella bongori. These can then be divided into different so-called serovars. There are currently more than 2500 different named serovars of Salmonella enterica and about 20 of Salmonella bongori. New ones are constantly detected. Salmonella enterica is the only species detected in samples associated with animals or humans.

This typing is done via agglutination with specific sera specific for the different surface structures on the bacterium (so-called O-antigen) and flagellar structures (so-called H-antigen). By studying the patterns obtained, the salmonella strain can be typed and named. Some names are linked to disease, for example, Salmonella Abortusequi and Salmonella Typhimurium. Others are named after the place where they were first detected such as Salmonella Uppsala and Salmonella London.

There are several other ways to detect relatedness between different isolates of salmonella.

Another way to identify relatedness between different strains of salmonella is to examine the similarity at a genetic level. For this purpose there are several different molecular biological methods.

Gene sequencing means that the sequence of the different nucleotides are determined either in specific locations in the  genome or in the entire genome.

Another way is to compare how many times a specific nucleotide sequence occurs within a specified area in the bacterium’s genes. By determining this for five different nucleotide sequences within five different areas on the gene, a pattern for the isolate is created. Gene sequence A occurs a certain number of times within area one, gene sequence B occurs a certain number of times within area two and so on. This pattern, listed in numerical groups (for instance: 2-12-9-7-211), can then be compared to the patterns of other isolates. This analysis is called MLVA (multiple loci variable number tandem repeat analysis).

Pulsed field gel electrophoresis (PFGE) is another molecular biological method to determine genetic similarity between different bacterial isolates. This analysis involves cutting the bacterial genome into segments with different enzymes and then letting the segments migrate through a gel using pulsing electrical fields. You then get a specific pattern depending on how far different gene segments manage to migrate in the gel (depending on their sizes). Comparison of this pattern between different isolates is used to assess the isolates’ genetic similarity.
 
Typing of salmonella bacteria is especially important in epidemiological investigations and in tracings of disease. The similarities or differences, in terms of serotype/phage type/PFGE-pattern/MLVA-profile, are used to determine how closely related the isolates are. The results may then be used to strengthen or reject the suspicion of different routes of transmission.

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