Germ of a new food microbiology

 Think of a food microbiologist you know.  I’m guessing that you now have in mind someone wearing a white laboratory coat, surrounded by food samples, Petri dishes and agar slopes, using an inoculating loop and making smears on microscope slides.  This might be accurate in most cases.

In the 80s, I read an article entitled “Germ of a New Food Microbiology”.  The author’s argument was that the so-called “Standard Plate Count” gives us less information on food safety than just about any other analysis.  There is no indication of how the microbial population will perform in the hands of the consumer;  the SPC is anything but total - many of the bacteria in the sample may not be able to grow on the count medium or at the incubation conditions; single cells and clumps will both be counted as one cell; if we use selective media to count specific types of bacteria, perhaps pathogens, they may not grow if they have been stressed during processing or storage; sample preparation, incubation and transfer to selective media may involve several days, meaning that the count may be obtained only after a significant proportion of the shelf life is over.  We also get no information on how the consumer might react after eating the food

The situation hasn’t changed very much.  Any modern analytical technique still has to be able to correlate with the plate count because of the way food safety is measured..  

The development of rapid microbiological methods now has a long history.
In many cases, the development has involved reducing the number of steps in the process, automating manipulative procedures and reducing the scale of operations to reduce costs.

Many so-called ‘rapid methods’ will give a result within hours of being set up.  However, they may involve significant technician time.

Modern methods often involve molecular techniques, particularly the polymerase chain reaction (PCR) and sequencing.  PCR relies on the ability of DNA polymerase to replicate a portion of a DNA molecule, using specific primers that bind to complementary strands of the target DNA.  The laboratory process usually involves a thermal cycler.  The replication of regions between the primer binding sites results in an exponential amplification of the target, so that within a few hours, millions of copies are produced.  

Recent developments have enabled real-time detection of the products by means of fluorescent reporter molecules that bind to the amplified products.  The progress of the amplification can be followed by monitoring the increase in fluorescence as the number of cycles increases.  The larger the number of target molecules in the sample, the fewer numbers of cycles are required to reach a detection threshold, so the number of cycles required indicates the level of contamination of the sample.

Though the PCR technique can detect a single molecule of DNA, there are some hurdles to overcome.  If we are looking to detect numbers of bacteria in the range <3 cfu/g to 102 cfu/g  either large samples or enrichment of the samples is required.  However, if we want to retain the relationship between the initial numbers in the food sample and enumeration by PCR, enrichment cannot be used.  In addition, the food matrix itself may interfere with the replication process.   

Taking this a step further, we can now analyse the population in particular parts of the processing equipment, using small portable real-time sequencers that allow microbiome-based monitoring of surfaces within the plant.  In turn, this may enable the identification of sources of the contamination and allow timely intervention by suitable control measures.

In order to get away from assessment of food safety based on plate counts, we need to formulate microbiological reference criteria based on these new methods and introduce them to our microbiological food standards.

The material presented here represents a great simplification of the techniques involved.  If the article has inspired you to seek further information, you will find hundreds of explanatory articles on the Internet - just search for RT-qPCR, and Microbiome-based environmental monitoring etc.

More on antibiotic resistant bacteria in natural waters and wild-harvested foods.

I wrote an article on 05/12/2012 about our isolation of antibiotic-resistant genes in bacteria in water and in stream and river muds, comparing pristine waters with polluted ones along a stretch of a river in the Waikato region of New Zealand.

I recently read another article by Jack Heinemann and Sophie Joy van Hamelsveld from University of Canterbury in Stuff:  https://www.stuff.co.nz/pou-tiaki/300930926/antibioticresistant-bacteria-in-wild-cockles-and-watercress-putting-people-at-risk-of-serious-illness.  It's worth a read.

The article raises yet another concern about antibiotic resistance.  The testing of water for recreational use does not guarantee that mahinga kai, wild-harvested foods, such as shellfish, are safe to eat.  Shellfish can concentrate bacteria from the water to high levels, even when the tested water appears to be safe.

Is the food still safe to eat?

 For the past few days, New Zealand has suffered from high winds and extensive flooding as a result of Cyclone Gabrielle, which in some coastal areas has coincided with high tides.  Many areas have been without power for extended periods and thousands of people have had to leave their homes, in some cases being rescued from the roofs of their houses.

I was interviewed by a radio journalist who wanted to ask questions about the safety of food and water. Any food that has been contaminated by the flood waters is, of course, not fit to eat.  But what of foods that were in refrigerators and freezers?

This is a difficult question to answer.  If the power has been off for a few hours and the refrigerator has not been opened, the food will probably be safe to eat, however, some areas are not likely to have power restored for several days.  Some foods, such as yoghurt are naturally preserved by their acid content and will be shelf-stable even at room temperature for a few days.  Hard cheeses will also be safe.  Similarly, milk in sealed bottles will be OK for a couple of days.  Vegetables are also shelf-stable for several days at room temperature, provided that they have not been in the water.  Eggs have their shells to protect the contents, but it would be worth washing the shells to prevent contamination of the contents when they are cracked open.  Perhaps a good guide is to think of supermarket displays - if the food such as fruit, potatoes, tomatoes and cabbages is sold from open counters, it can reasonably be expected to last for several days.

The big concern will be fish and meat.  If the frozen food has thawed out completely, it may still be safe to eat if it is thoroughly cooked.  if the food still has ice crystals in it, refreezing is possible.  Beef and pork steaks are essentially sterile inside, so it is only necessary to cook the outside, remembering to cook the edges too.  Fish is normally sold in fillets, so again, frying or barbecuing will render it safe, however, the food cannot be stored for another few days before consumption.  I would be more concerned about chicken, which is always difficult to cook throughout because of the uneven thickness of drumsticks etc.  In all cases, the critical thing is to get the temperature of the food above about 75C throughout.  This will kill all vegetative (non-sporulated) bacterial cells.  If a thermometer is used, it must be clean and inserted into the thickest part of the food.  More care is required with minced meat.  During the mincing operation, the outside of the meat is mixed in with the interior portions, so thorough cooking is required - medium-rare hamburgers are out! 

Another, though not infallible guide is to look at the food and smell it - if it smells off, then don't eat it.  Things like bacon sometimes become a bit slimy as a result of bacterial growth on the surface.  If it is fried properly, it will be safe to eat, but may have off-flavours.

Of course, canned foods are safe, provided that there are no holes in the can. Similarly, sealed packages of snack foods, such as 'chippies' will be OK to eat and may make the kids feel a bit happier.  It is a good idea always to have a supply of canned foods in the cupboard so that they are available in emergency situations like ours.

Another major concern is water.  Some water treatment plants have been out of action for a couple of days as a result of power outages and some watersheds may have been swamped by flood waters.  A number of water supply authorities have said that the tap water is safe to drink.  However, if there is any doubt about its safety, it should be boiled.  I was asked about water for making up baby formula.  In that situation, I would always recommend using boiled water.  Rural households often collect water from the house roof and store it in large tanks.  In my opinion, this water will be safe, provided that the roof and tanks have not been inundated, but once again, if there is any doubt and the power is on, it should be boiled.

All of these questions will become more relevant when people are allowed to return to their dwellings. There may be a temptation to try to salvage food from refrigerators and cupboards.  If there is any doubt, the safest thing to do is to dump it, as food poisoning will put even more stress on health services at this time.

Our next concern will be resupply.  Many roads are impassable, leaving some towns completely cut off.  Supermarkets are very short on stock, even if they have power to operate the tills.  Foods may be in short supply for a while, but in addition, orchards and vegetable farms have been severely damaged, so shortages will continue for months.  There may be more reliance on imports.

Odd Spot:  if the cyclone were not enough to challenge the population, we also had a magnitude 6.3 earthquake yesterday.  It was centred about 50km NE of Paraparaumu in the North Island, so it was under the sea.  There have been no reports of injuries or damage, but it's something we could all have done without!

Will in vitro meat be the new SCP?

 In the 1960s, several companies began development of Single Cell Protein (SCP) as a protein source for human and animal feed.  SCP refers to protein produced by microorganisms, such as bacteria, yeasts and unicellular algae.  This was not a new concept and can be traced back at least as far as 1936.

My first job was as a fermentation experimental officer at ICI in the UK, helping to develop the ICI Single Cell Protein Process.  Several other companies were developing SCP for human and animal consumption.  ICI intended to produce 1m te/yr by the 1990s; Shell was working on producing SCP as a by-product of gas oil dewaxing.  Several other processes were based on mycoprotein from Fusarium sp.- ‘Quorn’, and algae.  These projects involved very significant effort and investment; it has been estimated that by 1983, the main players had invested in R&D a staggering $US 2.9 x 10⁸ in today’s terms.

Image shows the ICI production fermenter for SCP.  60m tall with a capacity of 3,000m³.   I do not own the copyright of this image.

The new processes were beset with problems.  Bacterial cells contain relatively large amounts of RNA, which can result in gout and kidney stones in consumers, as purines increase plasma uric acid, so RNA reduction was required.  Some products had undesirable taste, and considerable downstream processing was required to produce acceptable texture and mouthfeel. Consumer acceptance of food made from bacteria was also difficult to achieve.  However, one of the greatest hurdles was the scale of operation required and the resulting cost of the product.  Most of these processes, while being technological marvels, were not commercially successful.  In fact,  as far as I am aware, the only product now on the market is Quorn.

Against this background, we see in vitro meat (IVM) or cultured meat, being developed as a sustainable food.   It is also referred to as ‘Lab-grown meat’, illustrated by lumps of tissue apparently grown in Petri dishes, though this is perhaps somewhat misleading, as the scale of operation to produce sufficient product to be economically viable would require a facility similar in size to a modern dairy plant. To produce IVM, stem cells are collected from living animals and cultured in a reactor, using suitable culture media, which must be made up of food-grade components.  Typically, vitamins, antibiotics, growth factors, such as calf serum, horse serum and chicken embryo extract are included, (though I have seen a recent report stating that calf serum is not necessary). The stem cells will differentiate into muscle cells under the influence of certain hormones.  Obviously, strict sterility must be maintained, or contaminating bacteria will grow in the highly nutritious medium.  The developing cells will require a supply of oxygen, carbon dioxide will be produced in the metabolic processes, and heat will have to be removed.

Merely getting the muscle cells to grow is only part of the process - Since consumers want something that resembles  meat as closely as possible, some form of three-dimensional scaffolding is usually required for the cells to fuse together to form organised tissues.

For many people, the decision to purchase IVM will come down to taste, texture, appearance and cost.  Estimates suggest that IVM will be twice the price of farmed meat.   I saw recently a report that a burger pattie could cost around $11.35  I will be happy to eat IVM, but not if the product costs a lot more than my favourite grass-fed beef fillet steak.

Got the munchies?

 Hands up anyone who, at any time during their lifetime, has scraped the cake mixing bowl and eaten the residues, or eaten raw cookie dough.  Yes, I see a lot of you with your hands up!  In a survey by Ardent Mills, Minnesota, 73% of respondents admitted to eating raw homemade cookie dough and 57% allowed children to lick the bowl and spoon after mixing cakes, while 65% admitted to eating store-bought cookie dough without cooking.  In a 2009 outbreak of Escherichia coli O157 food poisoning in 30 U.S. states, 77 patients, mostly young females contracted the illness and 35 were hospitalised.  A common factor revealed in the investigation was consumption of commercial raw cookie dough produced in a single plant.  As a result, 3.6 million packages of ready-to-bake cookie dough were recalled.

I used to beg my Mum not to use a spatula to scrape all the mixture from the bowl.  Mum didn’t know much about food safety; she had no formal training and over the years had learned how to bake.  Many younger people are probably in the same boat.  Most of us know that you can possibly pick up Salmonella food poisoning from eating undercooked eggs or chicken, and that the foods likely to give you food poisoning include leafy greens and sprouts, raw shellfish and raw milk, though the latter usually ignites a flame war if you publish comments.

So if you are asked why you should not eat raw cookie dough, what will you say is the risk?  Salmonella in the uncooked eggs, right?  What about the flour?   In 2016, ten million pounds of raw flour were recalled owing to contamination with Escherichia coli.  That year, the U.S. FDA and Centres for Disease Control and Prevention investigated infections by E.coli O121 occurring across the country.  Some serotypes of this bacterium can produce Shiga toxins.  A great deal is now known about Shiga toxins, but for our purposes here, we can say that ingestion of the toxin results in abdominal pain and watery diarrhoea, but may also cause haemorrhagic colitis, which is far more serious.  

The April 23 edition of Morbidity and Mortality Weekly Report discussed multi-state outbreaks of E. coli O26:H11 infections linked to raw flour in 2019. The investigators initially thought that ground beef was the source of the infection - patients interviewed reported eating ground beef and leafy greens.  However, by the use of modern microbiological analytical techniques - Pulsed Field Gel Electrophoresis and Core Genome Multilocus Sequence Typing - they were able to show  that the STEC O26:H11 isolates were different from those strains that had caused the ground beef illnesses in 2018.

How does flour, which is dry and doesn’t support bacterial growth, come to be involved in E. coli food poisoning transmitted in raw cookie dough?  Wheat flour is grown outdoors and may be contaminated from soil or birds and animals.  Cattle and deer have no cell receptors for Shiga toxin, so may carry the toxigenic bacteria without ill effect.  The milling process generally doesn’t include a lethal process step, so E. coli and Salmonella can survive into the finished product.  These vegetative cells are killed during cooking, but may be present in raw dough and cake batter.

Heat treatment of flour has been used in the past to destroy pathogens, but is expensive and has a negative impact on the properties of gluten, potentially making the flour unsuitable for baking.  Various other treatments have been proposed, including cold plasma, electron beam and gamma irradiation, but these methods may meet with consumer resistance.

A company based in Ontario, Canada has recently developed an organic, non-thermal treatment for flour that is a liquid added at the tempering stage of milling.  However, as far as I am aware, this treatment is not yet used in commercial flour production.

What about making home-made raw cookie dough safe?  It is extraordinarily difficult to find information on how to pasteurise raw flour at home to make raw cookie dough treats.  One blogger has described home pasteurisation of flour, using a 1200 watt microwave oven to heat the flour to 71C with a 55 second treatment.  She based this treatment on advice from FDA and I estimate that the flour receives an F0 of around 4.  

FDA is now pushing the message “Don’t eat any raw cookie dough, cake mix, batter, or any other raw dough or batter product that is supposed to be cooked or baked” and “Follow package directions for cooking products containing flour at proper temperatures and for specified times”.
Indeed, some packages of flour in US now have a label warning consumers to “Cook before sneaking a taste”.

Still want to risk it?

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Hello Fresh fish causes food poisoning in New Zealand

 At least three people have been treated in hospital for scombroid poisoning after eating Trevally from a meal kit supplied by Hello Fresh.

Histidine in the fish tissue can be converted to histamine by histidine decarboxylase, found in Escherichia coli, Morganella morganii, Proteus, Pseudomonas and Klebsiella species, which may occur naturally in the gills, skin and gut.  If the fish is not properly handled after being caught and during transport and distribution, allowing the temperature to rise for an extended period, these bacteria can grow and produce sufficient histamine to cause an allergic reaction in the consumer.  

Scombroid poisoning was originally named because Scombridae fish naturally contain higher levels of histidine.  The range of symptoms varies, but includes nausea, headaches, vomiting and diarrhoea and possibly itching and a burning sensation in the lips.

Unfortunately, the fish may not appear to be spoiled and the enzyme remains active even after the bacteria have been killed,  continuing to produce histamine under cold storage.  Activity can  resume when frozen fish is thawed.  Histamine cannot be destroyed by cooking, so control of storage temperature is essential throughout the food chain.