Microbiome Research

For me the three kernels of research into the human microbiome are to characterise the microbes involved and their metabolites, understand microbe-microbe and host-microbiome interactions and devise interventions for the removal of harmful taxa and the reinstating of beneficial taxa. 

Figure 1. Circadian rhythm of the gut microbiome (left, from Reitmeier et al., 2020) and the germination of Clostridiodes difficile spores in response to bile salts (right, from Lawler et al., 2020).

Characterising the microbiome can be carried out using old-school microbiology (growth-dependent techniques such as plate counts), growth-independent methods (such as 16S rRNA gene sequencing [targeted or amplicon], shotgun sequencing, flow cytometry and imaging [confocal, laser-scanning]) and metabolomics (LC-MS/MS mass spectrometry, HPLC). The data generated from “omic” (not to mention “multiomic”) approaches is huge. Handling this big data is almost as problematic as understanding the microbiome itself – a system which is built on complex networks of interactions between thousands of actors. Powerful bioinformatics techniques are needed for rustling up and corralling this data. Tools such as artificial intelligence, machine learning, and network analysis are becoming indispensable in studying the microbiome.

Hot topics (in this author’s humble opinion) in microbiome research include:

• The identification of bacterial biomarkers associated with dysbiosis (preferably based on a cheap, rapid, simple, easy-interpretable test)
• Microbiome-based personalized medicine (e.g. understanding the difference between those patients who respond to drugs and those who don’t)
• The impact of drugs on gut microbiome
• The identification of new species with functions of interest
• Next-generation probiotics
• The effect of viruses on the microbiome
• The impact of pre-, pro-and post-biotics on the gut microbiome
• The prevention/amelioration of dysbiosis
• Personalised nutrition (for e.g. high-performance athletes, geriatric patients, those with cholesterol or diabetes)
• The development of new functional foods
• The identification of microbial gene products and metabolites that interact with human cells (e.g. anti-inflammatory or antiproliferative signal molecules).

I will present some recent ground-breaking research to do with the human microbiome.

Gopalakrishnan et al. (2018) in a study entitled “Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients” examined the oral and gut microbiome of melanoma patients undergoing anti–programmed cell death 1 protein (PD-1) immunotherapy and found that patients could be grouped into two categories based on their response to the treatment – responders and non-responders. Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus non-responders. Responders (who had a higher chance of recovering from their melanoma) displayed significantly higher diversity and relative abundance of bacteria of the Ruminococcaceae family compared to non-responders. Metagenomic studies (which looked at a large number of genes present in the gut microbiome) revealed functional differences in gut bacteria in responders, including the enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients mediated by their gut microbiome. In a nutshell: a healthy and high-functioning gut microbiome enabled the responders to react positively to the immunotherapy and aid in their immune system’s fight against their melanoma.

In a study which shows how specific each member of our microbiome is regarding the niche it occupies, Lawler et al. (2020) showed that spores of the pathogen, Clostridiodes difficile, will only germinate in the duodenum in the presence of primary bile salts. These primary bile salts are those which are directly secreted by the gall bladder. Bacteria other than C. difficile metabolise primary bile salts into secondary bile acids. If C. difficile senses the presence of secondary bile salts it won’t germinate: in other words when there is the possibility of other microbes present, C. difficile spores will remain dormant, fearing the competition represented by these other bugs. Location-wise, this means that C. difficile only has a short stretch of the duodenum in which to germinate. Now that we know its niche, we can target specific treatments to this area.

Finally, work by Reitmeier et al. (2020) has shown that your microbiome knows what time of the night and day it is and that its activity cycles in what is known as circadian rhythm. Not only did the authors find that specific gut microbes exhibit rhythmic oscillations in relative abundance, but that individuals with type-2 diabetes display perturbations in their gut microbiome’s cycling. This arrhythmic signature contributed to the classification and prediction of type-2 diabetes and the authors speculate that there may be functional links between circadian rhythmicity and the microbiome in metabolic diseases such as obesity and cancer.

The Microbiome and Disease

The more research that is done on the human microbiome the clearer it becomes that its malfunction can lead to disease. The range of diseases linked to an out-of-sorts microbiome (especially in terms of the gut) includes: autoimmune diseases (diabetes, rheumatoid arthritis, muscular dystrophy, multiple sclerosis, and fibromyalgia); metabolic diseases (obesity, chronic fatigue syndrome, cancer); infections (bacterial vaginosis, Clostridioides difficile infection); and gut-brain axis perturbations (anxiety, depression, autism). 

The increasing incidence of atopic diseases (eczema, asthma and food allergies) where the immune system goes into overdrive against harmless microbes or proteins from things such as pollen or food have been linked to an overabundance of hygiene in our modern, urban, industrialised societies. Lack of early-life exposure to microbial antigens in hygienic developed countries retards the microbial evolution of the infant gut, which in turn disrupts immune development. Species such as Bacteroides fragilis reportedly induce immunological tolerance through immune receptor signalling pathways and can be considered teachers of our immune system. When the right bugs such as B. fragilis are not present shortly after birth and during infancy, the immune system lacks these vital tutors to mould it and begins to behave oddly. The infant gut microbiota is affected in a positive sense by factors equated with a “dirtier” environment: being brought up with pets, residing in rural settings, and having many siblings have been shown to have protective effects against asthma and allergies. There is also a higher prevalence of atopic diseases in infants delivered by caesarean sections, formula fed infants* and those exposed to antibiotics. Cleanliness is not always next to godliness!

The technical term for a microbiome gone bad – a movement away from the “ideal” mix of microbes – is “dysbiosis!**”, and this has been linked to disease. Dysbiosis can be caused by a short, sharp shock such as the taking of a course of antibiotics or by gradual insults such as a high-fat diet, alcohol abuse or even stress. In general, high diversity of a microbiome can be equated to a healthy state, and low diversity to sickness. A rich and diverse gut microbiome is now recognised as being essential to our health and associated with resilience. Sometimes it is not the absolute numbers of certain bugs which equates to health but their ratio to other bugs. For example, a high fat diet (not to be recommended, obviously) reduces the proportion of phylum Bacteroidetes and increases the proportions of phylum Proteobacteria. A high-fibre diet***, increases the proportion of Firmicutes bacteria, and in many studies, a high level of these has been associated with a healthy gut.

The structure and composition of the gut microbiome is complex and, once matured at around three years of age, prevents the proliferation of potentially harmful bacteria from the environment. This is known as a “barrier function”, which also benefits from a rich and diverse microbiome. Bacteria are swallowed every day, but they are transitive in our gut ecosystem: pathogenic bacteria are eliminated, or chaperoned by the good bugs so that they cause no harm.

Finally, it is important to remember that the microbiome can be damaged by the disease process, leading to further disease symptoms.

*Entire books have been written on the importance of mother’s milk to the development of the gut microbiome. My advice to anyone having doubts about breastfeeding is to think of the bugs in the gut!

**Even bad breath seems to be caused by a perturbation of the mouth’s “ideal” mix of bugs!

***Any gut microbiome dude or dudes is practically Talibanic as regards believing that a high-fibre diet is essential, not only for a healthy gut, but for the health, both physical and mental, of the entire organism.

The Microbiome

If you’ve been keeping up with microbiology in any way over the last half dozen years you will surely have come across the term “microbiome”. Technically, this refers to the collection of genes of all the microbes that live on or in us but, in a loosening of the terminology, has come to mean the collection of those bugs themselves. There has been an explosion of research into the human microbiome since the advent of faster and cheaper methods of sequencing genes in the past decade (called next-generation sequencing) and what we are finding out about the bugs that live in or on is both astonishing and growing exponentially. Prior to the advent of next-generation sequencing any knowledge gained about these bugs was both hard-fought and extremely limited, as the vast majority of our symbionts are non-culturable – we cannot get them to grow on Petri dishes and study them using old-school microbiology.

We are a walking ecosystem, an ark of microbes. Here are some figures: we host as many bacteria, fungi and protozoa as we have cells of our own – about ten trillion (the number one followed by thirteen zeros); we carry two to three kilos of non-self around with us; there more than one thousand species present in each of our microbiome “zones” (e.g. the mouth, the skin); the size of the genome (the collection of genes) of the bugs on or in us is about two hundred times greater than our own genome; this microbiome is composed of more than three million protein-encoding genes; we have only been able to grow on artificial media about 10-20 percent of these bugs.

There are a number of different microbiomes on or in us: that of the nasal passages, the oral cavity, the skin, the gastrointestinal tract, and urogenital tract (including, as has been recently shown, the bladder). The microbiome is dynamic and changes in response to many internal and external agents, including environmental factors such as diet and use of antibiotics. The most dramatic changes in our microbiome composition occur in infancy and early childhood, when the microbiomes of our tissues are laid down. Important factors which have been shown to affect this seeding of the infant microbiome include: gestational age (full term or premature); mode of delivery (vaginal birth or caesarean section); type of feed (breast milk or formula feeds); maternal nutritional status (overweight or undernourished) and use of antibiotics during gestation. The complexity and plasticity of the infant microbiota has an impact on health later in life.

Let’s look at some of the things our microbiome does. In the gut the gazillions of bugs present help digest our food. For example, a guy called Bacteroides thetaiotaomicron breaks down the large, complex carbohydrates found in many plant foods –up to 250 different carbs. The bugs in our gut, mouth and on our skin and possibly other locations regulate our immune system. They are in constant communication with important regulator T-cells (Tregs) and are responsible for keeping excess inflammation in check as well as “training” the infant immune system. Just by their sheer physical presence, our microbiome’s bugs can protect us against other microbes that cause disease; this barrier function is particularly important in the case of the skin, vagina and nasal passages. In our gut many bugs produce vitamins – B vitamins (B12, thiamine and riboflavin) and vitamin K. They also produce short-chain fatty acids important in host metabolism and signalling to the immune system, analgesics, antioxidants and anti-inflammatory factors. Whatsmore, they play an important role in how the body metabolises drugs – a phenomenon called pharmacokinetics. 

There are two major groups (at the phylum level) of bacteria present in the human gut – Bacteroidetes and Firmicutes. Many studies focus on the relative numbers of members of these groups and draw conclusions on health status based on this. From my study of the field, whether one has a “healthy” microbiome is a far more complex matter than the ratio between groups of bugs. 

Two concepts are very important when thinking about the microbiome: specificity and redundancy. Specificity means that each species present has their own characteristic niche. For example, Clostridiodes difficile spores will only germinate in the duodenum in the presence of primary bile salts. Redundancy means that there are many bugs capable of carrying out any give role at any position in the microbiome. If, for example, one bug which munches the sugar, mannose, gets knocked out by that tequila you drank last night, another fella who is resistant to tequila will step into the breach and happily chew away on the mannose.

There is a great variety of environments in and on us humans. Think of the mouth: you have the wet and oxygenated tongue, the deep, dry recesses of the gums where oxygen is not so plentiful, the scummy, food-rich spaces between our teeth. Our gut is as varied in habitat as a continent. From the proximal to the distal part of intestine, the oxygen levels are dramatically decreased, resulting in the dominancy of anaerobic microbes in the colon. The aforementioned Bacteriodetes burn out oxygen, attenuating the detrimental actions of oxygen species against the obligate anaerobes including commensal Clostria. Acidity also varies from stomach (acid) to colon (basic) to caecum. Firmicutes (including segmented filamentous bacteria and lactic acid bacteria similar to the bugs found in cheese and yoghurt) and Proteobacteria (Enterobacteriaceae and Helicobacter spp.) can dominate in this proximal intestine.

Given the length of the human intestine is roughly five metres (fifteen feet) and the physico-chemical environment changes every couple of inches, it is not surprising that we are only beginning to understand the complexity of the microbiome present.

Domesticating Yeast

Humankind has domesticated dozens of species of animals and thousands of species of plants. Going all the way back to our Palaeolithic ancestors who took wolf cubs into their care, fed them scraps by the fireside and converted them into the faithful doggy friends we have today, and our Neolithic ancestors who, through a process of keeping the seeds of high-performing plants, bred what we now know as barley, wheat, rye, maize and oats, us humans have been selecting and shaping other species for millennia. We know all about the big species – from horses to hydrangeas. But what about the microbes?

Let’s talk about yeast. Without Saccharomyces cerevisiae to convert sugars into alcohol and carbon dioxide we would not have alcoholic beverages and our bread would be as flat as pancakes. The deliberate use of this yeast to transform foodstuffs is the first example of biotechnology, and the practice of pitching yeast into wort (malt juice) and dough has been carried out since at least the Neolithic, although the fact that it was a yeast that was responsible for beer, wine and fluffy bread was not discovered until the work of Louis Pasteur in the nineteenth century. There are many theories concerning when and in what sequence brewer’s and baker’s yeast, Saccharomyces cerevisiae, was domesticated. Did agriculture come first and did humankind learn to make alcoholic beverages from spare barley and wheat? Or have humans been brewing beer- and wine-like drinks even since our hunter-gatherer days? I have read a theory which states that agriculture owed its origins to brewers who wished for a more constant supply of high-quality grain from which to brew their beer.

It is beginning to look like, though, that the use of yeast for brewing preceded that of yeast for baking. Sometime after beer had become a staple in society, some smart bucko had the idea of adding some of the trub (yeasty sediment) from a brew to dough and – hey presto – leavened (fluffy) bread was invented. Traces of ancient yeast and the compounds they generate during their fermentations have been found in all sorts of ceramics from the Neolithic onwards. This proof of brewing from over eight thousand years ago is reinforced by the finding of Sumerian inscriptions from about four thousand BC which are essentially the tax returns for a batch of brews, as well as Egyptian hieroglyphic depictions of the dark art of making beer. But, just because our evidence for the existence of brewing only stretches back to the Neolithic, this does not mean there was no yeast-based biotechnology prior to this: the wooden bowls and vats and leather bladders in which beer would have been brewed prior to the invention of ceramics have not survived down through the millennia and writing and record keeping only begins with the dawn of agriculture in the Fertile Crescent. I can picture all sorts of weird and wonderful bladders of fermented honey (mead) and buckets of fermented extract of wheat, barley, rye, oats, pseudocereals, berries and whatever you were having yourself. 

A recent paper by Ana Pontes from Lisbon’s Universidade Nova and colleagues attempts to shed some light on the inter-relatedness of the many strains of yeast we humans now use and how these may have arisen. Their findings are highly detailed but intriguing. They found that S. cerevisiae strains can be placed into nineteen groups based on their genetic relatedness and that these groups very closely correspond to the uses to which we humans put the yeast. So that the S. cerevisiae we use to make wine are all related, those which we use to ferment olives are similarly a distinct group, the bread yeasts all slot in together, the same for sake yeasts, and that there is a distinct group of dairy yeasts etc. There are two groups of beer yeasts: those used to make ale and those used to make speciality mosaic beers and the like. If we can picture yeast species as breeds of horse: the ale yeasts might be a Clydesdale, the wine yeasts a Shire and the bread yeasts an Andalusian. Each of these breeds is for a completely different use, has arisen in a different part of the world and has a different evolutionary genetic history.

How the wild yeast that gained their living from fermenting fallen fruit and the sap from plants evolved into the biotechnological strains which we use today is becoming clearer. Most domesticated strains possess duplicated (or triplicated or more) genes for the metabolism of maltose – the most common sugar found in wort. Many domesticated strains have also been found to have mutations which render their aquaporins non-functional. Aquaporins control the movement of water in and out of the cell and not having working ones protects you if you’re a yeast cell trying to make a living in very sugary environments such as grape juice, wort or molasses (i.e. environments of high osmotic stress). Other novel modifications of yeast on the road to domestication include the ability to digest sucrose and to manufacture the vitamin, biotin.

What Pontes et al. have been unable to do is point towards one single domestication event of S. cerevisiae. It’s beginning to look like we domesticated yeast multiple times, depending on the use to which we intended putting it and the geographic region where the activity occurred. The relationships between the nineteen yeast “breeds” and wild yeasts is still up for debate. What it does look like, though, is that the strains within each usage category are fairly ancient, going back thousands of years, and that yeast has gained and lost genes, as well as borrowed, begged or stolen these from other species in order to adapt to the conditions us humans have demanded of it. S. cerevisiae is indeed one of our most faithful friends!

Breathing Easy: Anaerobic Bacteria

Being animals who breathe oxygen – the technical term is “aerobic” – we quite understandably have an aero-centric view of life on this planet. We cannot envisage a world devoid of oxygen or oxygen-dependent organisms, even though the earth was just so for the first few hundred million years of life existing on it. It was only until photosynthesising bacteria evolved that oxygen became abundant. The question is often asked: “what would happen if all the oxygen were to run out?” One answer to that question is that, for many organisms, life would continue much as before. Why? Because a significant chunk of the microbial world has no need for oxygen, apart from that bound up in the water molecule.

First, let’s look at oxygen’s function in aerobic organisms. 

You remember that famous equation from school?

C₆H₁₂O₆+6O₂→6CO₂+6H₂O+energy

One molecule of glucose + 6 molecules of oxygen give 6 molecules of carbon dioxide, 6 molecules of water and energy.

Put at its simplest, our cells take an energy-rich molecule such as glucose and combine it with oxygen to release the energy necessary for life, with carbon dioxide and water as by-products. This is called aerobic respiration. The process of breaking up, for example, glucose to release the energy contained within its bonds is akin to burning it. You can take some grains of sugar, hold a lit match to them and see this combustive release of energy. Our cells cannot simply burn glucose: the energy released by this would be far too high and lethally destructive for the components that make up the cell. Instead, our cells release the energy from glucose in tiny packages. In that way, the explosive release of energy we see during combustion is broken up into more manageable packages. 

Thus, three stepwise processes, very gradually relieve glucose of its energy. These are:

glycolysis (the breakdown of the glucose molecule into two three-carbon molecules of pyruvate);

the tricarboxylic acid cycle (or Krebs cycle; the pyruvate is pulled apart bit by bit to release the energy stored in those covalent bonds and where most of the carbon dioxide is formed;

the electron transport chain and oxidative phosphorylation (the electrons harvested from the breakdown of glucose are mopped up by oxygen, producing water and a heck of a lot of energy in the form of ATP). Oxygen, acting is this role as chief-mopper-upper of electrons is called a terminal electron acceptor.

The difference between aerobic and anaerobic respiration is that in anaerobic bacteria the electron mopper-upper is something other than oxygen. Alternative electron acceptors include nitrate, sulphite, sulphate, carbon dioxide and sulphur itself. These compounds are less powerful electron mopper-uppers than oxygen – technically they are referred to as having smaller reduction potentials – and so generate less energy. Following on from this, anaerobes are less efficient growers than aerobes, who are essentially turbo-charged through their use of oxygen. Important groups of anaerobic bacteria are the methanogens, who produce methane from the reduction of carbon dioxide. Some of these guys live in the bellies of cattle and termites and are responsible for much of the methane behind the greenhouse effect causing global warming. Anaerobic decomposers, which produce eggy smelling hydrogen sulphide gas include members of the Desulfovibrio genus.

One very important genus of anaerobes from a medical and food safety perspective is Clostridium. Everyone knows about C. botulinum, the organism responsible for botulism and the producer of the botulinum toxin, BOTOX, used in cosmetic surgery. Foods from which oxygen is excluded such as tinned food and low- or no-oxygen modified-atmosphere-packaged products are the ones most at risk from growth of C. botulinum, which, like the rest of the genus, Clostridium, forms spores which can survive cooking. A swollen tin, or one that makes an audible hiss or pop upon opening could very well be contaminated with C. botulinum or one of its cousins, the gas producing the swelling or pop being as a result of the bacteria’s anaerobic respiration. C. tetani is the dude responsible for causing tetanus, against which, thankfully, we are all vaccinated. Spores of this critter find their way into the anaerobic or microaerobic environment of deep wounds and grow to dangerous levels, producing the tetanospasmin toxin responsible for the horrific symptoms of this lethal disease. C. perfringens causes a mild form of food poisoning, but is a scourge of certain industries which produce dried food such as the dairy and infant formula industries. Clostridiodes (formerly Clostridium) difficileis an organism responsible for nosocomial (i.e. hospital-based) infections. Targeting post-op patients, many of whom have weakened gut microflora as a result of antibiotic administration, this bug causes potentially lethal diarrhoea which can often escalate to full-scale colitis. Once it establishes itself in a hospital environment it can be very difficult to eliminate as it also forms resistant spores.

I have something grave and shameful for any worth-their-salt microbiologist to admit: I have only minimal experience in cultivating anaerobes. There was that second-year laboratory practical back in 1995 where we played around with anaerobic jars and a few dishes of C. butyricum, and I isolated a couple of plates of suspected anaerobes last year for the fun of it, like, but that’s about it. And now, I find myself, for the purposes of the project I am working on having to learn how to grow up and perform experiments on Clostridium. I’m a bit scared. These guys are supposed to be very fussy, not only in terms of an absolute need not to come into contact with oxygen (it is toxic for many anaerobes), but as regards diet. I’ve just spent a grand on specialised media, replete with electron soaker-uppers. Let’s hope these boyos are kind to me and don’t catch a draft or decide to go off their food!

Élie Metchnikoff

Some taxonomical names for microbes can be on the tongue-twisting side of difficult to utter. It takes a few run-throughs before you’re able to properly pronounce even some of the best-known bugs – Escherichia, Saccharomyces cerevisiae, Streptococcus thermophilus – not to mind some of the more extreme examples from the further reaches of taxonomy – Thermovenabulum ferriorganovorum, Acholeplasma multilocale, Roseimicrobium gellanilyticum. I will never forget the difficulty some of my fourth-year students had last term in pronouncing the name of a yeast they had isolated from a natural cider fermentation: Metschinikowia pulcherrima. It’s a tough one, alright, but today it’s not this yeast or its role in producing the delicious drink derived from apples that I will write about, but rather the man in whose honour it and dozens of other yeasts are named after – Élie Metchnikoff.

Before I get into Élie Metchnikoff’s biography, here’s a list of what the man discovered:

• Phagocytosis (where a cell eats another cell, especially important in the case of our phagocytotic immune cells – macrophages and monocytes)
• Important aspects of white blood cell chemotaxis (the way they are “attracted” to the “scent” of bacteria)
• The idea of probiotics as beneficial to human health.

It was for his discovery of phagocytosis that he won the Nobel Prize in Physiology/Medicine in 1908 (shared with Paul Ehrlich). 

Metchnikoff (spelt Metschnikow in the German from which the yeast genus name derives) was a Russian of Ukrainian Jewish and Moldovan origin, born in 1845. Not only his mixed-race origins, but the fact that he carried out his research all over the continent of Europe (from Odessa to Paris and Göttingen to Sicily) lend the status of grand European scientist to Metchnikoff and means all us Europeans can seek to lay an equal claim on his brilliance. His pattern of moving from country to country to pursue his research (he eventually settled in Louis Pasteur’s institute in Paris) will be all too familiar to modern research scientists. If he was modern in his mobility, there is something quaint about the manner in which he managed to be a giant in so many scientific subjects. He came from an age where so much was there to be discovered that people who studied “small stuff” (as Metchnikoff did) found themselves crossing areas of endeavour in a way that would be unimaginable nowadays. The man made hugely significant discoveries in such a number of fields that it leads us to wonder if he was an immunologist, microbiologist, biochemist or gerontologist (he invented this latter subject, by the way – being obsessed towards the latter part of his career with stalling and reversing the ageing process). With scientific specialisation so entrenched and strict nowadays it is almost unimaginable that a food microbiologist such as myself would dabble in, for example, plant microbiology, let alone move across the chasm into immunology or human physiology.

For a microbiologist, it is Metchnikoff’s work on human intestinal flora (fields that would nowadays be called microbiome studies and probiotics) that are of greatest interest. To quote from Ed Yong’s I Contain Multitudes (2016, Random House, UK):

On the one hand, Metchnikoff said that intestinal bacteria produce toxins that cause illness, senility, and ageing and were “the principal cause of the short duration of human life”. On the other hand he also believed that some microbes could prolong life. In this, he was inspired by Bulgarian peasants, who regularly drank soured milk and lived well past the age of 100. The two traits were connected, said Metchnikoff. The fermenting milk contained bacteria, including one that he called the Bulgarian bacillus. These made lactic acid, which killed the harmful life-shortening microbes in the peasants’ intestines. Metchnikoff was so convinced by this idea that he started regularly quaffing sour milk himself.

More than 100 years after Metchnikoff’s death (in 1916) we are still quaffing the soured milk known as yoghurt, produced by a cooperative fermentation of the aforementioned S. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus – named in honour of Metchnikoff’s Bulgarian peasants.

*As an aside, the aforementioned Paul Ehrlich also has his own genus named in his honour – Ehrlichia – a group of nasties transmitted by tick bites to vertebrates. Also, Lactobacillus delbrueckii is named in honour of the biophysicist, Max Delbrück. Both of these were Nobel winners, just like Metchnikoff, something which seems to come with the territory of having a bug named after you!

Biofilms

Bacteria are single-celled organism that are quite happy to, as the song goes, float on alright as individual cells. All the tools and necessary bits and bobs for a successful life are contained within their one tiny cell. They don’t need to mate with other cells to reproduce. They don’t display or have a requirement for social behaviour. Strictly speaking, they don’t need to co-operate or even be around other cells of their own species. They are self-contained, independent, autonomous – The Grizzly Adams of the microbial world. When we first come across bacteria in secondary school it is this single-cell status that is emphasised, probably for reasons of contrasting them with multicellular organisms. There’s no mention that they might co-operate or co-ordinate with other bugs. And sometimes, because of our reductive manner of doing science in order to make things simpler to understand, us microbiologists ignore or avoid considering something that has been known for years: many (if not most) species of bacteria do come together, do co-operate and do perform better as part of a collective.

This is where biofilms come in. It is reckoned that the majority of bacterial cells on the Earth exist as components of a biofilm, rather than as planktonic (free-floating) cells. So, what is a biofilm? Answer: a load of bacterial cells, not necessarily of the same species, encased in a goo composed of extracellular polymeric substances (EPS). EPS involves a complex array of proteins (e.g. lectins), polysaccharides (e.g. alginate and cellulose) and extracellular DNA and constitute 90% of biofilm mass. These substances, not only act as a glue to hold the biofilm together and anchor it onto your tooth or a door handle, but serve a myriad of other functions; to protect the bugs from e.g. sunlight and drought; to carry out “communal” enzyme activity; to serve as information superhighways between cells; to allow the exchange of electrons between cells.

Biofilm formation. From: Fulaz et al., 2019. https://doi.org/10.1016/j.tim.2019.07.004

Being part of a biofilm, rather than free-floating must hold competitive advantages for the bacteria involved, otherwise these bugs wouldn’t spend significant resources in terms of energy and metabolites to come up with the membership fees  – EPS and other secreted substances, including food, antibiotics and signalling molecules. Let’s look at some ways in which being a member of biofilm benefits bugs.

Protection

For bugs, being coddled in a biofilm is akin to being a soldier in a bomb-proof fort. The many compounds in an EPS afford protection from a wide array of insults which would otherwise damage them, the list of which is lengthy: sunlight, oxygen, free radicals, heavy metals, predation, antibiotics, detergents and other cleaning agents, heat, drought and pressure. The more species present in a biofilm, the richer the array of EPS compounds, and therefore the broader the range of insults the bugs are protected from.

Co-operation

Bug A might have a metabolic repertoire which allows it to use perhaps 20 sugars as an energy source. Bug B might be able to use 25 sugars. If there is an overlap of, say 15 sugars, between the pair of species, it means that together the species can metabolise 30 sugars. The species can then share the products of their non-overlapping sugars and tackle an environment together in a way they wouldn’t otherwise be able to do. Also, if cell X is going through a tough time, is running low on ATP or electrons, it can send a message out into the biofilm and one of its richer brethren will excrete a few molecules of glucose or some anions to help its neighbour out. Cell X might in the future receive a message that cell Y is coming unstuck and might stick out a flagella to keep it attached to the collective.

The effort to understand biofilms is not just led by academic curiosity. Biofilms are a significant problem in a variety of contexts. Stents, pacemakers and other prothesis need to be absolutely sterile before implantation. There have been instances, though, when these have been placed in a patient with the beginnings of a biofilm seeded on them. These biofilms can prove extremely recalcitrant to antibiotic treatment and can eventually cause septicaemia and death. In food processing plants, biofilms on stainless steel surfaces are enemy number one. Such biofilms are very difficult to remove using standard cleaning protocols. Cells from these can end up in the final product, where they cause spoilage and perhaps food poisoning in those consuming the product. In the biotechnology industry, where “good” bacteria, yeast, mammalian and even insect cells are used to produce anything from enzymes for washing powders to vaccines, the fouling of fermenters, filters and lines with biofilms can cause a dose of the horrors in production managers. Entire batches of valuable product can be dropped because of such contamination, which, once more is very difficult to eliminate. I’ve heard stories of plants being shut down for months and receiving deep clean after deep clean until the biofilms are eliminated.

The more we come to understand biofilms and how bacteria behave within them, the better we can combat them in medical and industrial contexts. With antibiotic resistance spreading among bacteria like wildfire identifying compounds which break up or weaken biofilms might be a fruitful approach. The tactic of separating a single individual from the herd in order to hunt it down may not just work for mammals. Bacteria already smarting from being turfed out of their warm and cosy biofilm are more susceptible to a variety of treatments.

Salmonella Hides Out Inside Vegetables

Food safety microbiologists have known for years that one of the most important routes for transmission of foodborne disease is the contamination of fruit and vegetables in the field with bacteria derived from human or animal faecal matter. Water for irrigation can be contaminated by agricultural runoff containing the faeces of sheep, pigs or cattle or by human sewage. Similarly, the water used for washing dirt, dust and soil off fruit and vegetables post-harvest may be unclean. And, speaking of dust, as previously discussed on this blog, wind-borne particles contaminated with faecal matter may contaminate even the highest-hanging fruit which is not at risk from contact with irrigation water. The principal food poisoning bugs associated with faecal contamination are Salmonella, E. coli, other enteric bacteria and viruses such as Norovirus.

Washing has always been regarded as one of the surest methods of removing contaminant bacteria and viruses from fruit and vegetables. It will generally reduce the count of bacteria on fruit and vegetable surfaces by a factor of more than 100, which, except in the case of heavy contamination, will lower the risk of foodborne infection to almost zero. It was always thought that the bacteria associated with plants were attached to their surfaces – that the insides of your carrots, potatoes, lettuces and onions were sterile – and by peeling vegetables such as turnips or parsnips you could eliminate all the bacteria. A recent paper by researchers at the University of Delaware has turned these assumptions on their head: bacteria can worm their way into plants.

The scientists who conducted this study wished to see if bacteria were capable of entering plants through their stomata – the pores found on leaves which control the exchange of gases between the plant and the atmosphere. They inoculated lettuce leaves with a strain of Salmonella enterica Typhimurium and measured the extent of stomatal aperture (how wide these pores open) and levels of contamination over seven days. Their findings may be a game-changer: Salmonella was able to override the normal response of stomata which normally shut up shop when they sense a nearby intruder. In a molecular sense, Salmonella was able to jack the pore open so that it could seek shelter inside the leaf’s tissue.

What does this mean for food safety? Since washing is no longer guaranteed to remove at least a proportion of the Salmonella (and possibly other species) which may be lurking within the fruit or vegetable), producers have to be extra stringent in ensuring that faecal matter does not come into contact with their produce. Water for irrigation must be close to if not equal to the standards for potable water. Process water must be fit for human consumption. The paper by the University of Delaware group offers another route: inhibition of the process whereby bacteria squeeze inside the stomata. This is achieved by applying another bacterium, Bacillus subtilis strain UD1022 to the leafy part of plants. This bug is a, in a nutshell, a gatekeeper – a stomata closer – and so protects plants from the likes of sneaky Salmonella.

Old School Tracking and Tracing: The Moore Swab

In the midst of the current COVID-19 pandemic we may be decrying our rotten luck to be afflicted as badly as we have by a mere virus. There were strains of thought up until January of this year that human beings had almost pulled themselves free from nature’s cruel attacks: within a few decades we would have cures for all the major diseases, aging would be reversible – we would be eternally young and disease free. But what is happening currently is not the new normal, it is a kind of return of the old normal, where outbreaks of viral or bacterial disease, albeit not on a global scale, were a constant threat. A recent review of a thingamabob known as the Moore Swab, which, in combatting typhoid and cholera has probably saved hundreds of thousands of lives, reminded me of how precarious life was regardless of your social status even as recently as the middle of the last century.

 

Constructing a Moore swab. (A and B) A length of gauze, 6 inches by 48 inches, is folded onto itself in a pleated pattern to form a pad. (C and D) The gauze pad is tied at the center with high-test fishing line. (E) The Moore swab may be suspended in flowing sewers or surface waters.
From Michael J. Sikorski and Myron M. Levinea, 2020

The Moore swab is named after its inventor, Brendan Moore who worked in Exeter’s (UK) Public Health Laboratory and who was charged with hunting down the sources of infection responsible for clusters of paratyphoid fever cases in North Devon between 1943 and 1945 and for a larger outbreak of 25 cases in 1946. The technique used by Moore and his colleagues to identify specific foci of infection, particularly individuals who were shedding the organism responsible for the disease – Salmonella enterica subspecies enterica serovar Paratyphi B – in the same manner as the infamous typhoid Mary, was sewage tracing. This involved sampling the public sewage system from where waste entered the water treatment plant, checking samples for growth of Salmonella, and then sampling upwards in ever more detail until the specific residence of the shedder was identified. This was a painstaking process, made more difficult by the obvious need to get lucky with your sample taking: if you dipped your sample jar into the sewer before or after your shedder’s daily motion your sample would come up negative. The Moore swab eliminated luck from the equation.

Brendan Moore came up with the idea of leaving a swab suspended in the sewage stream for 48 hours, trap-like. In this manner the microbiologist would have a better chance of capturing the tell-tale bacteria than during a single sampling session. Moore swabs are crafted using strips of cotton gauze (or cheese cloth) cut into 6-inch by 48-inch lengths and folded eight times until an 8-ply square pad is formed The 6- by 6-inch square-pad is tied by a string, twine, wire, or fishing line around the centre and often sterilized in an autoclave before being hung from a manhole, for example. After spending 48 hours in contact with the flowing sewage, the Moore swab is taken back to the lab, the bacteria are extracted in nutrient broth, and then volumes of this extract are plated on media which favour the growth of Salmonella. Results are forthcoming after two days.

The modus operandi of Brendan Moore and his team was fascinating:

When typhoidal Salmonella-positive points were discovered and mapped, additional swabs were deployed upstream of sewage flow of the positive point. This was repeated systematically until results showed that the swabs were positive downstream and negative upstream of an identifiable point of possible contamination, such as a block of houses sharing a common sewer. This general approach was also used in sampling rivers and streams until a pipe carrying wastewater with human faecal material was identified or septic tanks were observed to be located nearby. In all cases, a team of public health workers was then required to interview the people living at or near the suspected source and to obtain stool samples to confirm bacteriologically the silent excretion.

Of the many cases given as examples in the paper I found this one fascinating:

In one Louisiana town, Barrett et al. used sewer swabs to track V. cholerae O1 in a manner reminiscent of Moore’s tracing the source of S. Paratyphi B. They recovered V. cholerae O1 from a Moore swab cultured on 9 September 1978 from the intake line of a sewage treatment plant. Four days thereafter, a second Moore swab was placed into the sewage treatment intake line, and swabs were also inserted into lines of 17 pumping stations draining different sections of town. Swabs from two sites yielded V. cholerae O1: one from the treatment plant intake and the second from a pumping station serving 50 blocks of dwellings. They then placed swabs in the tributary lines and, in 4 days, traced the source of infection to a 2-block area amenable to a Moore-like house-to-house search for the infected person(s). Meanwhile, a resident from this small area was admitted to the hospital with cholera gravis, and the stool culture grew V. cholerae O1. The patient’s daughter, who had subclinical V. cholerae O1 infection and remained at home, was the presumed source of V. cholerae O1 in the sewage line following her parent’s hospitalization.

Examples of this old-school public health detective work give me great faith that our public health officers of today, with all the extra technology they have at their disposal, will be able to stomp out the SARS-CoV-2 virus in the fullness of time. If, without, computers, internet, Bluetooth and GPS and using “plate and wait” microbiology rather than the rapid PCR test which is used for detecting COVID-19 infection, Moore and colleagues could successfully track and trace deadly typhoid, paratyphoid and cholera, we must surely face promising odds in the elimination of SARS-CoV-2.

A Sheep in Wolf’s Clothing: Using Clostridium Spores to Fight Cancer

Everyone’s heard of Clostridium, right? It’s the genus in which lies the nasty food-borne bug that causes botulism and gives us the toxin (botulism toxin = botox) that freezes our nerves and as a side effect smooths wrinkles. Clostridium tetani causes – you guessed – tetanus, and there are some other cousins such as C. perfringens (food poisoning and gangrene) and C. difficile (diarrhoea) who are also far from stand-up guys. Other species of Clostridium do everything from causing spoilage in Swiss cheese to producing useful enzymes for the biotechnology industry. A mixed bunch, indeed! Two things make Clostridium special: they are anaerobic (i.e. they don’t “breath” oxygen) and form highly resistant spores.

The strategy behind Clostridial-directed enzyme pro-drug therapy. From Minton and Heeg, 2017.

This blog has talked of spores before. Because of their ability to withstand treatments that kill normal bacterial vegetative cells, making them ideal for industrial processes, as well as their ability to retain viability for years, if not decades, which simplifies their transport and storage, biotechnologists are always looking to use spores for various beneficial purposes. We’re already using spores as insecticidal treatments for crops and, experimentally, delivery vehicles for genetically engineered vaccines. The latest novel application for spores sees them as vehicles for treatments for solid tumours. It’s a very clever system.

Firstly, you genetically engineer your Clostridium species to be able to convert a harmless pro-drug into a an anti-tumour magic bullet. This involves the insertion of a few novel genes into the cell. Then you inject millions of spores into the tumour area, while at the same time giving the patient the pro-drug. The Clostridium spores germinate in the anaerobic environment of the tumour, but not in any other normal aerobic tissue. They begin to produce the enzymes (little molecular machines) engineered into them and convert the pro-drug to drug, which acts on the cells of the tumour, killing them in the same manner regular chemotherapy does. Unlike regular chemotherapy, though, there is very little in the way of side effects, because the pro-drug is only converted to drug in the tumour area: oxygen is lethal for Clostridium and it does not survive to produce drug in normal oxygenated areas of the body.

Now time for a couple of acronyms, so that you can impress your friends and blind them with science! This kind of anti-cancer strategy is called Clostridial-directed enzyme pro-drug therapy (CDEPT) and the enzymes are called pro-drug converting enzymes (PCEs). You may ask: what happens if the Clostridium spores (usually of the species C. sporogenes) escape the patient and become widespread in the environment? Could there be spores in the wild in a few years producing anti-cancer drugs willy-nilly and despoiling our soil and water? Scientists have introduced a break on this happening. The spores injected into patients have been engineered to have an absolute requirement for uracil – their machinery for its production have been disabled. Therefore, along with pro-drug, uracil is administered to patients. If the spores find their way outside the body, they fail to survive unless uracil is provided – which in nature it is not.

CDEPT looks like a promising strategy for some types of cancer. Trials in mice have seen animals fully cured of their cancers. It is now a matter to prove that this works for humans.

 

References

Green, E., Minton, N. and Heeg, D. (2017). Making Clostridia great again. Industrial Biotechnology. 13, 52e56.

Kubiak, A.M. and Minton, N. P. (2015). The potential of clostridial spores as therapeutic delivery vehicles in tumour therapy. Research in Microbiology. 166(4), 244-254.

Num, S.M. and Useh, N.M. (2014). Clostridium: pathogenic roles, industrial uses and medicinal prospects of natural products as ameliorative agents against pathogenic species. Jordan Journal of Biological Sciences. 7 (2014) 81e94.