Clostridium – Sciblogs Sciblogs Tue, 29 Oct 2013 10:06:53 +0000 en-US hourly 1 Clostridium – Sciblogs 32 32 Report of independent inquiry into Fonterra botulism scare released Tue, 29 Oct 2013 10:06:53 +0000 Today saw the release of the report by the independent inquiry into the contamination of whey powder that lead to the Fonterra botulism scare in August this year. At 165 pages its not exactly light reading. The inquiry team, comprised of Jack Hodder QC (a lawyer), Jacob Heida (an expert in whey production processes and standards for infant food ingredients) and Gabrielle Trainor (a crisis manager), have identified what led to the scare and list 33 recommendations for Fonterra to implement as a result of the crisis. The inquiry team are also clear that “no heads should roll” as:

“the errors in judgement which might be attributed to individual employees are essentially the result of gaps in Fonterra’s procedures and training”

What went wrong?

Quite a few things, it turns out. But here are just a few of them:

1. Fonterra’s whey powder was not being manufactured to the specifications required by at least one of its customers. This relates to the lack of specific tests for sulphite-reducing clostridia (SRC) – the class of bacteria which includes Clostridium botulinum as well as other clostridial species which can cause food spoilage. SRC levels are considered a general indicator of quality of hygiene standards and one of Fonterra’s customers had specific acceptable levels for SRCs in place. This is why the final product was tested in Australia, and then rejected by the customer, when found to exceed their levels.

2. The contamination happened because a plastic torch lens broke in a batch of whey powder during an examination of part of the processing equipment. Not all of the plastic was recovered so the workers requested that the batch be downgraded to cattle feed. This request was denied and the workers told to “rework” and filter the product to remove the “foreign matter”. Alas, this was not standard operation at the plant and the workers improvised to get the job done, using a piece of stainless steel pipe that hadn’t been used for 2 years, and two flexible pieces of hose not normally used. They had all been cleaned, but not enough to remove the Clostridial biofilm living in them.

3. The commissioning, design and limits of the C. botulinum testing were inadequate.

More details on the testing are given in Appendix C, and the inquiry members hold no punches:

“The Inquiry’s view is that the AgResearch test was not capable of achieving the outcome that Fonterra wanted.”

What Fonterra wanted was: “a letter stating that these organisms are either C. sporogenes or C. botulinum and does/not have the ability to produce BoNT [toxin].” But the report states that AgResearch did not have the necessary typing methods or the toxin antidotes necessary for the mouse bioassay. In fact, they go on to say that it would appear that no laboratory in New Zealand is sufficiently qualified to carry out such testing. This will be why MPI sent the samples overseas.

What should Fonterra do about it?

You can read the full list of recommendations for yourself, but here are some of the ones I find most interesting:

1. Ensure that Fonterra’s specifications (and associated testing) for potential food quality and safety issues across nutritional product ingredients manufactured for Fonterra are of “best in class” standard: consistent with the most rigorous requirements of customers, and with international best practice. That is, don’t supply product that doesn’t match the specifications requested by your customers. (Recommendation 1 of 33)

2. Overhaul the procedures for non-routine microbiological testing within Fonterra to ensure senior management oversight of proposals for low probability/high risk internal work, and that such proposals are thoroughly assessed in advance for utility and the full range of possible consequences. That is, make sure the bosses know what non-routine tests are being commissioned and what the consequences of a positive result will be. (Recommendation 8 of 33)

3. Establish a revised protocol for engagement of external scientific and diagnostic resources to ensure that any work commissioned is by institutions or practitioners of international standing, is appropriately undertaken for, and funded by, Fonterra (i.e. asking whether the proposed work is not better undertaken on a pan-industry basis), and that the analyses sought will yield results that are robust. That is, can whoever you outsource the testing to, actually deliver and their data be reliable? (Recommendation 9 of 33).

In Appendix E, Jacob Heida states this very clearly:

“..within Fonterra, there appears to have been little knowledge of the testing procedures for C. botulinum and the botulinum toxin (BoNT). Fonterra should review its procedures to ensure that testing is outsourced to properly accredited laboratories and that results are peer reviewed.”

I’m really pleased to see peer review mentioned here. This is certainly what many of us were calling for during the crisis – show us the data and the methods! There is a little more info on AgResearch’s testing in this report – but still no real detail, other than that they did do some genetic tests (inconclusive) and that 14 mice were used.

4. Establish and sustain relationships with a pool of scientific experts in food quality and safety who can speak to the media and the public in the event of an incident. (Recommendation 16 of 33)

This is elaborated on in Appendix F:

“Scientific advice on health and safety issues should be part of the IMT’s [Incident Management Team] resources. Appropriately media trained scientific experts should be part of and available to the IMT to demystify complex data and scientific terms. Fonterra should have a pool of experts across various specialisations readily available.”

5. The Board should encourage investment by “NZ Inc” (including the NZ Government, Fonterra itself and other food products firms) in ensuring that New Zealand has pre-eminent scientific and diagnostic resources for food quality and safety purposes. That is, NZ should have accredited labs! (Recommendation 32 of 33)

Only time will tell how many of the recommendations Fonterra will implement, but its a good sign that the company chose to publish the Inquiry’s report in full instead of keeping it hidden behind closed doors. But in case they do implement recommendation 16, or other companies reading this report decide to do something similar, I urge all you scientists out there to get some media training (like the SAVVY course run by the Science Media Centre..)

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Monday Micro – A bacterial link to Multiple Sclerosis? Sun, 20 Oct 2013 17:00:29 +0000 OAlogo


There were a number of possible stories to write about for this week’s Monday Micro, but in keeping with the spirit of Open Access week I have chosen one from a paper published in PLOS One, an Open Access journal. The paper describes, for the first time, the isolation of a strain of the bacterium Clostridium perfringens type B from a human – a woman presenting with symptoms of Multiple Sclerosis (MS) (1).

According to Wikipedia, MS is an inflammatory disease in which damage to the insulating covers of nerve cells in the brain and spinal cord disrupts the ability of parts of the nervous system to communicate. This disruption results in a wide range of symptoms, which may be physical, mental, or psychiatric, including visual dysfunction, incoordination and spastic paralysis. The cause of MS is not clear and there is no cure, with treatment aimed at improving function after an attack and preventing new attacks.

Clostridium perfringens is a Gram-positive, spore-forming bacterium related to C. botulinum and C. difficile. It is divided into a number of types which differ with regard to the toxins they carry. Type A carries the alpha toxin and is a common cause of food poisoning in humans, whereas types B and D carry the epsilon toxin (ETX), and are often carried in the gastrointestinal (GI) tracts of ruminant animals. The ETX is a protoxin converted to its active form in the GI tract by trypsin and chymotrypsinor or by a protease carried by C. perfringens. The active toxin is ~1,000 time more potent than the protoxin. In ruminants, once produced ETX is absorbed via the intestine, enters the blood stream and permeabilises the blood-brain barrier causing MS-like symptoms.

In their paper, Rumah and colleagues describe isolating C. perfringens type B from a woman with symptoms of MS (1). Furthermore, they investigated the GI carriage of C. perfringens in a population of MS patients and healthy controls, finding the type A strain present in about half of the healthy controls but less than a quarter of the MS patients. Next they analysed tissue bank blood and cerebrospinal fluid samples, and found that samples from MS patients were 10 times more likely to react to ETX than samples from healthy controls, suggesting that the immune systems of the MS patients had encountered ETX before (1).

So could this bacterial toxin be a cause of MS? Given its ability to cross the blood-brain barrier, and to cause MS-like symptoms in animals, this looks like a distinct possibility.


1. Rumah KR, Linden J, Fischetti VA, Vartanian T (2013) Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease. PLoS ONE 8(10): e76359. doi:10.1371/journal.pone.0076359

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Details of new botulinum toxin censored to stop ‘misuse’ Tue, 15 Oct 2013 09:36:11 +0000 Clostridium botulinum,* the bacterium that turned out not to be contaminating a heap of New Zealand made infant formula a few months ago, is at the centre of a new scare. This time it’s the discovery of a strain of the bacterium for which there is no treatment.

C. botulinum produces neurotoxins that prevent acetylcholine from being released from the motor nerve endings causing flaccid paralysis and symptoms of blurred vision, drooping eyelids, nausea, vomiting, diarrhea and/or constipation and cramps. In severe cases it leads to paralysis of the breathing muscles and causes respiratory failure. Until recently, seven different neurotoxins had been identified to date – known by the letters A-G. Toxin types A, B, E, and F cause disease in humans while types C and D cause disease in cows, birds, and other animals but not in humans. Treatment for patients with botulism is the delivery of the corresponding antitoxin to neutralise the particular toxin type causing their disease.

In two papers just published in the Journal of Infectious Diseases (alas, they are behind a paywall), Jason Barash, Stephen Arnon and colleagues from the California Department of Public Health describe a strain of C. botulinum (IBCA10-7060) isolated from an infant with botulism** (1, 2). IBCA10-7060 was found to produce two toxins, toxin-type B and a novel toxin which the authors have called type H. What first led them to suspect they had something new was when the antitoxins used to treat mice injected with either faecal extract or culture filtrate failed to save any of the animals.

The researchers have since sequenced the 2 botulinum toxin gene clusters of strain IBCA10-7060 and found that the sequence of the type H toxin differs substantially from the gene sequences for the 7 known toxin types A–G. But we are going to have to take their word for it, as the authors have taken the unusual step of censoring any genetic information about the novel toxin that could allow it to be misused as a biological weapon.

This from the accompanying editorial by David Hooper and Martin Hirsch:

It is general JID policy that gene nucleotide sequences must be submitted to the International Nucleotide Sequence Databases and that the accession numbers must appear in the final revision and published version of the manuscript. However, in this case, we made an exception. Because no antitoxins as yet have been developed to counteract the novel C. botulinum toxin, the authors had detailed consultations with representatives from numerous appropriate US government agencies (see Notes), as well as the JID editors, and received approval to publish both manuscripts, while withholding the key gene sequences until appropriate countermeasures were developed.

The worry is that someone could easily use the genetic sequence to synthesise the toxin gene DNA which could then be used to make a toxin for which there is currently no known antitoxin. So the gene sequence of the toxin is going to be withheld until an antitoxin has been produced. It’s worth at this point noting that, according to Wikipedia, botulinum toxin A is one of the most toxic substances known to man – 1 kilogram of it would be enough to kill the entire human population.

As an aside, its interesting to see that in this case the manuscripts were reviewed by a committee representing various branches of the US government***. Missing from the committee was anyone from the US National Science Advisory Board for Biosecurity, the very committee charged with providing advise and guidance on the potential of research to be misused to pose a biological threat to public health or national security. Curious.

So if the research is so dangerous, why publish in the first place? This is the first new botulinum toxin type to be identified in more than 40 years, so its discovery serves as a reminder that there are an awful lot of dangerous microbes that we don’t even know exist yet. But it also serves to tell the scientific and medical community that there exist novel and potentially untreatable strains of C. botulinum in the environment. It would be interesting to now find out how prevalent this new toxin type is, but to carry out studies like that will require the genetic information.

I understand why the genetic sequences are being withheld in this particular case, but all the same it makes me uneasy. Who gets to decide which other researchers around the world can have the censored information to help progress the discovery of an antitoxin, or look into the toxin’s prevalence? The country currently being held hostage by it’s own elected representatives?! That’s scary.

1. Barash JR, Arnon SS (2013). A Novel Strain of Clostridium botulinum That Produces Type B and Type H Botulinum Toxins. J Infect Dis.
2. Dover N, Barash JR, Hill KK, Xie G, Arnon SS (2013). Molecular Characterization of a Novel Botulinum Neurotoxin Type H Gene. J Infect Dis.

*As a quick reminder, C. botulinum is a spore-forming bacterium that doesn’t like oxygen. It can cause a rare but sometimes fatal illness called botulism, a result of the microbe’s ability to produce neurotoxins which are produced when C. botulinum spores germinate and the bacterium starts to actively grow, which it can only do in an environment with no oxygen. There are three main forms of illness caused by C. botulinum:
1. Foodborne botulism, caused by consuming food or drink contaminated with botulinum toxins,
2. Infant botulism, where the gastrointestinal tracts of babies becomes colonised by C. botulinum spores before their protective gut microbes establish are established (This is why parents are advised not to give honey to children under the age of 1),
3. Wound botulism, when wounds become infected with C. botulinum spores present in the environment.

**It’s not disclosed if the infant lived or died.

***1. Drug Development Section, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH);
2. Enteric and Hepatic Diseases Branch, NIAID, NIH;
3. Bacteriology and Mycology Branch, NIAID, NIH;
4. Office of Biotechnology Activities, Office of Science Policy, NIH;
5. National Counterproliferation Center, Office of the Director of National Intelligence;
6. Office of the Assistant Secretary for Preparedness and Response, Department of Health and Human Services (DHHS);
7. Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, DHHS;
8. Threat Characterization and Attribution Branch, Chemical and Biological Defense Division, Science and Technology Directorate, Department of Homeland Security;
9. US Army Medical Research Institute of Infectious Diseases;
10. Division of Select Agents and Toxins, Office of Public Health Preparedness and Response, Centers for Disease
Control and Prevention, DHHS;
11. Biological Countermeasures Unit, Federal Bureau of Investigation Weapons of Mass Destruction Directorate,
Department of Justice.

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Botulism scare: how is Clostridium botulinum and its toxin detected? Mon, 05 Aug 2013 10:31:44 +0000 With news that all Nutricia Karicare Infant Formula Stage 1 and Stage 2 Follow-on formula should be avoided, the Fonterra botulism scare continues to rumble on. Fonterra Managing Director Gary Romano appeared on Campbell Live tonight where he said that it was the toxin rather than the bacterium Clostridium botulinum that had been identified in the whey powder. This is in contrast to everything that has been said so far and must be a mistake; it would make the scare a much bigger one than it is at the moment.

If it was the bacterium that was identified as the contaminant, it was most likely to be spores, produced by the bacterium as a way of surviving adverse conditions. The spores can hang around in the environment for years, until conditions are right for them to germinate and produce actively growing C. botulinum which can then produce the toxins that cause botulism*. As the conditions for germination and growth are very specific, the likelihood of spores germinating are actually relatively low, hence why botulism is extremely rare in developed countries. If the powder was contaminated with one of the actual toxins, a kilogram of which could kill the entire human population, then the chances of contracting botulism become much more real. Given that no cases have been reported to date, this scenario seems unlikely.

Fonterra have said that they have traced the contamination back to three batches of whey powder produced in May 2012 and questions are now being asked about why it took the company until the 31st July 2013 to inform the government and its customers of the contamination. Speaking on Campbell Live tonight, Gary Romano said that one particular standard test came back with a higher than usual result so they investigated, despite the result not actually being outside of the allowable limits. So what are the standard tests for C. botulinum? The US Food and Drug Administration (FDA) information on testing for C. botulinum toxin dates back to 2001, and states how to test for the bacterium itself and the toxin.

Testing for viable C. botulinum

C. botulinum comes in four ‘varieties’ known as groups I, II, III and IV. Most outbreaks of human botulism are caused by group I or II, while group III organisms mainly cause disease in animals. Group I and II strains can be differentiated on the basis of their ability to break down proteins and sugars. Group I organisms can break down proteins, while Group II organisms break down sugars instead. To complicate matters further, seven different types of toxin (A to G) have been identified, not all of which are harmful to humans.

In order to see if the bacteria are present, samples are used to inoculate an enrichment media which will contain all the nutrients C. botulinum needs to grown and which has had all the dissolved oxygen removed. The FDA suggests using cooked meat medium which is then incubated at 35°C. It’s worth noting here that the four different groups of C. botulinum have different optimum temperatures for growth**. After 5 days of incubation, the culture should be examined for turbidity, gas production, and digestion of meat particles. If there is any bacterial growth, some of the culture should then be Gram-stained and examined microscopically to look at the shape and size of the bacterial cells, and whether there is any evidence of spore formation. Apparently, a typical clostridial cell resembles a tennis racket! The FDA guidelines suggest testing the culture for the presence of toxin after it has been growing for 5-15 days.

It is worth remembering that as C. botulinum is so rarely found in dairy products, samples wouldn’t have been grown in this enrichment media as a first port of call. It’s likely they would have started more generically like for the Clostridium family, other species of which include the hospital superbug C. difficile, C. perfringens which can cause food poisoning and gas gangrene, and C. tetani which causes tetanus.

So to recap:
Step 1: Does the sample contain a species of Clostridia?
Step 2: Is it C. botulinum?
Step 3: Which group does it belong to?
Step 4: Does it produce a toxin, and if so, which type?

Whilst definitive, you can begin to see how it can take a little time to get a final confirmation that a sample contains C. botulinum, and the smaller the level of contamination, the harder it will be. No wonder then that the food industry is keen for more rapid, and sensitive diagnostic tests. A paper published in the Journal of Medical Microbiology in 2010 developed a real-time PCR assay that could simultaneously detect any of the A, B, E and F toxin genes in a given sample at a sensitivity of about 130–840 fg genomic DNA, which equates to about 50-330 genomes (1).

Testing for toxin

The gold standard for toxin testing seems to be a mouse bioassay. What this means is that suspected toxin samples are injected into the abdominal cavity of mice at different dilutions, and the animals monitored for signs of illness over a 48 hour period. If the toxin is present, mice injected with the sample solution will show signs of botulism. The time it takes for them to become unwell will depend on the concentration of toxin present in the sample injected. To find out which toxin is present, groups of mice are also injected with antitoxins to each of toxins A, B, E and F. If the correct antitoxin is injected alongside the sample, the toxin will be neutralised and those animals will not show any signs of botulism. Again this assay will take some time to perform.

While searching for diagnostic assays, I came across a paper in the journal Veterinary Record (2) in which the authors state that the numbers of cases of botulism in cows is increasing. They report finding toxin and C. botulinum from milk and udder samples. The article is not open-access so I’ve only been able to read the abstract and I’m not sure what types of C. botulinum they found but it may just be the types that are not harmful to humans.


1. Satterfield et al (2010). A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F. Journal of Medical Microbiology. 59: 55-64.

2. H. Böhnel, and F. Gessler (2013).Presence of Clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism. Veterinary Record, 172:397 doi:10.1136/vr.100418


**Group I = 35-40 degrees, Group II = 18-25 degrees, Group III = 40 degrees.

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Fonterra scare: the other side of Botox Sun, 04 Aug 2013 10:48:24 +0000 Fonterra is currently in the midst of its third scare in as many years, with news that over 30 tonnes of whey powder concentrate manufactured at the Hautapu plant in Waikato has been contaminated with the soil bacterium Clostridium botulinum. The contamination happened back in May 2012, caused by a faulty pipe, and there are reports that the three contaminated batches could have already been exported to Australia, China, Malaysia, Saudi Arabia, Thailand and Vietnam. China has stopped the importation of all New Zealand milk powders, while Russia has suspended the import and circulation of Fonterra products. Here in New Zealand, Nutricia has recalled two Karicare baby milks, their Stage 1 Infant Formula for babies aged 0-6 months and their Stage 2 Gold+ Follow on Formula for babies 6-12 months*. Fonterra animal feed subsidiary NZAgbiz has also recalled a small amount of calf milk replacer sold in the North Island.

C. botulinum is a Gram-positive bacterium, which cannot tolerate any more than trace amounts of oxygen**. The bacterium produces spores which are very hard to kill and thus help the bacterium survive adverse conditions. C. botulinum is a worry because it can cause botulism, a rare but sometimes fatal illness. Botulism is caused by the ability of C. botulinum to produce several neurotoxins (types A-G) which prevent acetylcholine from being released from the motor nerve endings causing flaccid paralysis and symptoms of blurred vision, drooping eyelids, nausea, vomiting, diarrhea and/or constipation and cramps. In severe cases it leads to paralysis of the breathing muscles and causes respiratory failure. Toxin types A, B, E, and F cause disease in humans while types C and D cause disease in cows, birds, and other animals but not in humans. The toxins are produced when C. botulinum spores germinate and the bacterium starts to actively grow, which it can only do in an environment with no oxygen.

2. Infant botulism, where the gastrointestinal tracts of babies becomes colonised by C. botulinum spores before their protective gut microbes establish are established. This is why parents are advised not to give honey to children under the age of 1,
3. Wound botulism, when wounds become infected with C. botulinum spores.
No wonder then that the Karicare infant milk powders have been recalled.

Botulinum toxin A is one of the most toxic substances known to man. According to Wikipedia, 1 kilogram of it would be enough to kill the entire human population! Incredible then that this deadly toxin is voluntarily injected into the faces of millions in the pursuit of youth. They know it by its trade name – Botox. But it’s not just used for treating wrinkles, botulinum toxin A is also used to treat spasms, migraines, squints, excessive blinking and excessive sweating.

Ironic then that a marvel of modern medicine is likely to cost our economy millions, a reminder that New Zealand needs to look beyond milk powder and as Shaun Hendy says “Get off the grass“.

*Recalled products:
Karicare Infant Formula Stage 1 (0-6 months) with batch numbers 3169 and 3170 (use by 17 06 2016 and 18 06 2016).
Karicare Gold+ Follow On Formula Stage 2 (6–12 months) with batch number D3183 (use by 31 12 2014).

**called an obligate anaerobe

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