#streptococcus agalactiae

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more bacteria people doodles 🦠

currently dwelling in the streptococci family

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pyogenes' background

biblically accurate design

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Isolating Group B Streptococcus from a food (fish) sample

Recently (Straits Times, 27 Nov 2015), food stalls in Singapore were stopped from selling Chinese raw fish dishes unless certain conditions were met.  This was because a Group B Streptococcus (GBS) stain responsible for causing invasive human infection (please see a previous post) had been found in some fish samples along the food supply chain.

Shortly afterwards (Straits Times, 5 Dec 2015), a ban was placed on the use of freshwater fish in raw fish dishes sold ready-to-eat. 

We had previously tried to culture GBS from fish without success (though we had positive PCR results to suggest that small numbers of GBS were present). We took this as a bit of a challenge and decided to have another go. So here is a sample of raw fish purchased from a stall before the ban was implemented. The method we use is a modification of that described by van der Mee-Marquet et al, 2009. I should stress this experiment was done out of interest and not part of an official investigation.

Remember 1. this is not a sterile sample, there may be bacteria other than GBS in the sample which we are not interested in. We need to select out the GBS from the other bacteria.

2. because the fish is not actually infected, the number of GBS (if they are present at all) may be very low, we need to enrich their numbers.

The first step is to weigh out 25 g of the sample.

We then put the sample in a stomacher bag (basically a robust plastic bag) and add 225 ml of Todd Hewitt broth which also contains 8 µg/ml of polymyxin B and  32 µg/ml nalidixic acid (the original method uses gentamicin 8 µg/ml and colistin 5 µg/ml). Todd Hewitt broth is a liquid medium for cultivating Streptococci. The polymixin B and nalidixic acid are antibiotics to suppress the growth of Gram-negative bacteria (GBS being a Gram-positive coccus).

The sample is macerated with the broth within the stomacher bag using a device called a stomacher (see above). The resulting mixture (the primary enrichment) is then placed in an incubator.

After incubating for 24 hours at 37°C…the incubator will become very smelly! One ml of the primary enrichment is removed and transferred to 5 ml of brain heart infusion broth containing 8 µg/ml of polymyxin B and 32 µg/ml nalidixic acid (the secondary enrichment) and incubated for a further 24 hours at 37°C (below). 

You might think having 2 enrichment steps a bit excessive. I certainly did, and wrote to the authors of the original paper to find out if it was really necessary. They replied that the second step is required because in their experience the numbers of GBS may be so low that primary enrichment is not enough.

After incubation, a loopful of the secondary enrichment is removed and streaked onto a selective agar. In our case we used chromID® Strepto B by bioMérieux. The plate is incubated for a further 24-48 hours at 37°C (I find sometimes the color of the colonies is better visualized after 48 hrs incubation). This plate contains antibiotics to suppress other bacteria but also three chromogenic substances so that GBS colonies should appear pale pink or red.

Can you spot the GBS colonies?

Well here they are (white arrows). 

The first thing you notice is that in this particular example, they are not really red. More like magenta or purple with a reddish tinge.  I’m not sure if the fact that they are surrounding by bacteria with blue colonies is altering the color somewhat.

The second thing is that there only a few colonies of GBS surrounded by many colonies of other bacteria (the blue ones). So when the counts are low you have to look really hard!

Anyway after we re-isolate the GBS colonies onto a fresh chromID® Strepto B plate without the other competing bacteria, we get ‘proper’ red colonies. These were confirmed to be GBS by MALDI-TOF.

This all goes to show it is not easy to isolate GBS from a food sample unless there is very gross contamination. One has to be very painstaking at looking for suspect colonies and be prepared to test many colonies that turn out to be some other bacteria (usually Lactococci).

Strictly speaking in this particular example we have only shown that the GBS can be found in the food sample. From this experiment alone, we cannot exclude that it came from the food handler, the environment of the stall or the vegetables that were served together with the fish. To demonstrate that GBS actually came from fish would require further investigation to check for carriage of GBS in food handlers, and sampling of fish further up the food supply chain. 

This has in fact already been done. Quoting a joint statement released by the National Environment Agency (NEA), the Ministry of Health (MOH) and the Agri-Food & Veterinary Authority of Singapore (AVA), the local press reported that  “food handlers are unlikely to be the source of the bacteria which caused the spike in GBS infections. Stool samples of 82 food handlers and fishmongers were tested and none were found to carry the ST283 strain”. 

Furthermore, “Between August and October this year, nearly 400 fish samples across the supply chain - from fishery ports to wholesalers, wet markets, supermarkets and food outlets - were tested by the authorities.GBS was detected in 20.1 per cent of these samples, while 4.1 per cent of total samples tested positive for the ST283 strain.The fish that tested positive for the ST283 strain of the bacteria are Song fish (Asian bighead carp), Toman fish (snakehead), and tilapia, which are all freshwater species” (Straits Times, 27 Nov 2015).

Straits Times. GBS infection: Food stalls told to stop selling Chinese-style raw fish dishes unless fish is from safe suppliers. 27 Nov 2015.

Straits Times. NEA bans use of all freshwater fish in ready-to-eat raw fish dishes from Dec 5. 5 Dec 2015.  

van der Mee-Marquet N, Domelier AS, Salloum M, Violette J, Arnault L, Gaillard N, Bind JL, Lartigue MF, Quentin R; Bloodstream Infection Study Group of the Reseau des Hygienistes de la Region Centre. Molecular characterization of temporally and geographically matched Streptococcus agalactiae strains isolated from food products and bloodstream infections. Foodborne Pathog Dis. 2009 Dec;6(10):1177-83. (no free access)