Tag Archives: nitric oxide

tuberculosis and edam

Robbie was really happy to be diagnosed with pulmonary tuberculosis. Before he arrived he was convinced he had lung cancer.

Robbie is in his late 50’s, he was born in Glasgow and left school at fifteen with no qualifications. His father was a shipbuilder on the Clyde. Robbie thought he would do the same, but in the seventies the shipyards closed* and there were no prospects for young men like him, so he came south.

He got various unskilled jobs, mainly in the china clay industry, got married and had two daughters. But he drank much too much and was not faithful to his wife, who left him. He says he does not entirely regret it – he had a great time.

Or as George Best famously said “I spent a lot of money on booze, birds and fast cars, the rest I just squandered”

Nationaal Archief Fotocollectie Anefo
Nationaal Archief Fotocollectie Anefo

Then, about three months ago, he developed a cough that kept him up at night. He had terrible sweats and would wake up in the morning with his bedclothes really wet. He took the sheets off his bed to hang them up to dry during the day. He knew something was seriously wrong when he started coughing up blood and was losing a lot of weight. He had always been “wiry” but now was becoming skeletal. Robbie became increasingly sure he had lung cancer, but was too frightened to get help. He still talked to his daughters every Sunday on the ‘phone. Last Sunday one of them was so worried when he told her what was happening that she went to find him and brought him in to our hospital. He had a chest Xray and within a couple of hours we told him that we thought he might have TB.

pulmonary TB usually affects the upper part of the lungs
pulmonary TB usually affects the upper part of the lungs

We put him in a side-room and got him to cough up some blood-stained sputum which we sent to the lab. Within a couple more hours we got a phone call to say that the sample was “teeming” with AFBs – acid-fast bacilli.

I have previously talked about bacterial cell walls and  the Gram stain, which is a process which involves staining a biological sampe with a blue and red stain which have different affinities for the various components of a bacterial cell wall. Gram-positive bacteria stain bluish-purple with this stain because their cell wall is mainly peptidoglycan. Gram-negative have a thin layer of peptidoglycan, but a thick covering layer of lipopolysaccharide (LPS), which, like peptidoglycan, helps protect the germ from damage. As its name suggests, LPS is made of chains of sugar molecules and lipids – with carbon chains about 16-20 carbon atoms – similar to those in triglycerides in butter and olive oil. Gram-negative bacteria look red under the microscope after the Gram staining process

Image from Centers for Disease Control and Prevention
Tuberculosis bacilli stained with auramine and counterstained with acridine orange – viewed under uv light the mycobacteria shine out –  Image from US Centers for Disease Control and Prevention –

Tuberculosis is caused by the bacterium mycobacterium tuberculosis. It has a quite different cell wall structure which makes it very difficult for our white cells to destroy.  The secret weapon is mycolic acid. This a long molecule composed mainly of hydrocarbon chains – up to 54 carbon atoms long.

Hydrocarbon chains are common in all sorts of lengths in nature, most of which are useful to us. One carbon atom, surrounded by four hydrogen atoms is methane – a gas which I rely on to cook my dinner. Three or four carbons and we get the gases propane and butane, also useful in cooking the barbecue. Octane, a liquid with eight carbon atoms gets me to work in the morning. When we get to 16-20 carbon atoms there are endless things I can get from the supermarket, mainly to eat, with this size of hydrocarbon chain. These are mainly liquids or soft solids such as olive oil, butter, meat fat, and cooking oil. With longer hydrocarbon chains we are talking about waxes. Waxes have huge numbers of use in nature and are used extensively by humans, but mainly for protection rather than as a food. We use waxes a lot for protection – shoe and furniture polish for instance.

wax is used to protect shoes, furniture and a whole lot of other things
wax is used to protect shoes, furniture and a whole lot of other things

The wax in our ears is a mixture of squalene, a hydrocarbon of 26 carbon atoms mixed with cholesterol and dead skin cells, also designed to protect us from microbial infection.  Plants produce waxes to protect the surface of their leaves, and sheep produce waxes to protect their wool (lanolin, chemically similar to earwax).  Waxes are protective because they are difficult to break down chemically. The tuberculosis bacteria use this property of wax to protect them from the nasty chemicals white blood cells use to try to destroy them. We can detect mycobacteria by using a special stain which is taken up by the waxy coating. The original stain was called Ziehl–Neelsen, or ZN, but we now use a better one called auramine. This is a fluorescent dye which sticks to the wax, even when the specimen is flooded with acid and alcohol, which will remove it from just about any other bacteria which do not have mycolic acid in their cell wall. The specimen is then viewed under ultraviolet light and the TB germs light up.

Pulmonary TB is transmitted from one human to another mainly by coughing – producing a fine spray of droplets which contain the tuberculosis bacteria. Robbie mixed with a number of alcoholics and drug addicts, who are more prone to develop this disease. When the bacteria are detected by the cells lining the lung (see asthma and pineapple below), neutrophils will have a go at engulfing them and soaking them with lysozyme and bleach. TB germs will laugh at that (no, not really, TB germs don’t have much of a sense of humour). Their waxy coat makes them impervious to hypochlorite. Then the professionals are called in –macrophages. They will also engulf the tubercle bacilli. They will then call in help from T-lymphocytes, walling off the tubercle germs in a special structure called a granuloma.

cartoon of a tuberculous granuloma - the centre is caveating - cheese-like - made from dead macrophages
cartoon of a tuberculous granuloma – the centre is caseating – cheese-like – made from dead macrophages

The TB germs can only grow very slowly, because they have to make the very long hydrocarbon chains to protect the new bacterium after cell division. Macrophages and lymphocytes use all the weapons at their disposal to try to kill them, many of which I think we don’t properly understand. We do know that nitric oxide, made by neutrophils is important. This is a gas which can penetrate the waxy coat and damage the TB germs. Another important molecule is thought to be granulysin made by cytotoxic T-cells. We don’t know how either of these molecules really do the damage and kill TB.  Usually the human wins, but in about one in ten, as in Robbie’s case, TB wins. Robbie’s smoking and previous exposure to clay dust will have caused lung damage, which makes him more susceptible. His heavy drinking and poor diet will also make it worse. Some think vitamin D, which is made in the skin from cholesterol and sunlight might be important in defence against TB. Robbie likes dim, smoky rooms and avoids sunlight most of the time. Before effective drug therapy, sanitoriums would encourage patients to sit in balconies in the sun as part of their therapy, making lots of vitamin D.

Even when the human wins, it is not always a total victory. Sometimes there is an impasse, where the TB germs cannot be killed, but only constrained in the granuloma. In this siege scenario macrophages have another trick up their sleeve – calcification. Macrophages are very closely related to cells which make bone – osteoblasts. When the granuloma cannot be sterilised the macrophages start laying down insoluble calcium salts and imprison the TB germs. Often we see white spots on the chest xrays of elderly people who were exposed to TB when they were young – this is due to the TB calcium prisons which allowed their lungs to solve the problem of what to do with indestructible organisms.

It is quite likely that Robbie will recover from his TB, as long as he takes his drug therapy regularly, even though his lungs will end up even more damaged than they were. There are some very drug-resistant strains of TB emerging in India and Africa, but at present most TB in the UK is sensitive to rifampicin, isoniazid and ethambutol. He has promised his daughter that he will stop smoking and drinking so much, and eat better food.

This week’s food link is Edam cheese.

Edam cheese is case in wax to protect it from drying out and from spoilage. I think it is too bland, but my son likes it
Edam cheese is case in wax to protect it from drying out and from spoilage. I think it is too bland, but my son likes it

This is traditionally encased in a wax coating to protect it from drying out and from microbial spoilage. I do not really understand why micro-organisms have not yet worked out how to easily break down wax and use its energy for their benefit. It has the same calorific value as fat but is clearly unappetising for all the normal germs in my kitchen which are happy to feed on shorter chain hydrocarbons.

*There is a very powerful speech by Jimmy Reid, a Glasgow shipyard worker who was elected Rector of Glasgow University.

“The greatest speech since President Lincoln‘s Gettysburg Address

– required reading:

http://www.gla.ac.uk/media/media_167194_en.pdf

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diarrhoea and kidney bean salad

Chris is a self-employed builder. He takes a tablet of omeprazole and some gaviscon every evening to prevent heartburn, but otherwise has been healthy most of his life, and is looking forward to retiring in a couple of years – he is now sixty three.  But last week was awful. On Monday morning he went to work as usual – his company is converting a large house into apartments. When he arrived at work he felt suddenly unwell and vomited up his breakfast of toast and jam.

Slide4Then he started to get really bad cramping pains in his belly, and had to rush to the toilet to produce large amounts of diarrhoea. Luckily they had put a new bathroom into the conversion a few days before. He ‘phoned his wife, who came and brought him home.  He continued to feel really unwell with completely liquid diarrhoea every two or three hours, including through the night.  Sometimes the diarrhoea contained dark red blood.  His wife wanted to call the doctor, but Chris hates making a fuss.

“It’s just a tummy bug” he told her, “I’ll be fine soon”.

After two days the doctor was called and found that Chris was really not very well. His pulse was fast and his blood pressure was a bit on the low side. He strongly suggested that it would be best for him to go to hospital for a drip, but Chris was not at all keen.  He agreed to drink some oral rehydration made up from sachets the doctor had left him, and gave a blood sample. When the GP ‘phoned a few hours later, Chris’s wife told him that he had not been able to keep the salt solution down.

“I’m calling an ambulance – just persuade him that he has to go” the GP said.

When he arrived at the emergency department we had the results of the blood sample – his plasma creatinine was over twice the upper limit of normal – 260 micromoles/litre. His haemoglobin was a bit low- 105g/l and his CRP was very high at 180.

The ED department doctors had persuaded him that he had to be admitted and wrote “infective gastroenteritis and AKI” on the clerking form. AKI means acute kidney injury. I don’t like the blanket use of this recently fashionable term for any sudden increase in creatinine or drop in glomerular filtration rate.  In this case Chris’s kidneys are not injured – they are doing their job very well, under difficult circumstances. By stopping urine output they are saving his life – if they had carried on producing the normal amount of urine despite severe dehydration Chris would most likely be dead.  When an athlete runs 100metres and is very short of breath we don’t say he has acute lung injury – his respiratory system is responding appropriately to physiological demand – as were Chris’s kidneys.

He was put into a side-room on the acute medicine unit and his intravenous fluid was continued.  His pulse, blood pressure and urine output were monitored.  A couple of days later the nausea and abdominal pains had settled, his diarrhoea had reduced to twice a day, and he was starting to feel hungry again.   The microbiology lab ‘phoned to tell us that they had found Campylobacter in the pot of blood-stained, watery liquid labelled as a stool sample we sent when he was admitted.

Where did the Campylobacter come from, and why did his wife not get it?

Campylobacter are helical, like H pylori - it helps them swim through the mucus layer and invade into the intestinal epithelium By De Wood, Pooley, USDA, ARS, EMU. [Public domain], via Wikimedia Commons
Campylobacter are helical, like H pylori – it helps them swim through the mucus layer and invade into the intestinal epithelium
By De Wood, Pooley, USDA, ARS, EMU. [Public domain], via Wikimedia Commons
Campylobacter jejuni is a spiral-shaped bacterium and a relative of Helicobacter pylori which I have talked about previously (vomiting blood and sweetbreads). In the UK raw or undercooked chicken is the most likely source. It does not cause disease in the chickens, but is a commensal organism – amazingly about 60% of chickens reared in this country contain campylobacter in their intestines. Chris likes to cook, and a couple of days before he became ill had prepared one of his favourite meals – fried chicken and rice. He admits he might not have been as careful as he should about washing his hands and cleaning the chopping board after jointing the chicken – he will be in the future.

Most chickens bought in the UK contain campylobacter
Most chickens bought in the UK contain campylobacter

Chris was particularly at risk because he was taking omeprazole, which reduces stomach acidity. The low pH in our stomach is important in killing nasty, pathogenic germs in food we eat and stopping them getting into our intestines. Nitrate secreted into saliva is also important – it is converted to nitrite on the surface of our tongue by bacteria which normally live there. This nitrite is swallowed and reacts with stomach acid to produce nitric oxide – which is toxic to many germs including campylobacter.

Chickens do not get ill from campylobacter - it is a commensal in their intestines
Chickens do not get ill from campylobacter – it is a commensal in their intestines

Stomach acid also has other uses. In many parts of the world people are lactose intolerant and get a lot of their calcium from eating bones rather than dairy products. I am told that when chicken is eaten in Africa and China, the smaller bones are crunched and swallowed. Stomach acid is necessary to dissolve bones and release the calcium.

The next question is why do some bacteria, if they get past the stomach’s defences, cause vomiting and diarrhoea, when our colon is full of bacteria all the time? Why do our resident colonic bacteria not stimulate an inflammatory response?

There are two main reasons – invasiveness and bacterial toxins. Like the stomach, the lining of the small and large bowel is covered with a layer of mucus, secreted by goblet cells. Our resident bacteria are well-behaved citizens and do not try to invade across the mucus layer into the gut epithelial cells. Mild incursions are allowed, but if they get as far as the base of the epithelial cells, they will trigger an alarm.  These epithelial cells have toll-like receptors on the basal and lateral surfaces which, when triggered, will cause the police, in the form of neutrophils, to be called to the scene. The neutrophils will hunt down and digest these wrong-doers, causing a bit of havoc in the process. That is what inflammation is – the action of our immune system to destroy threatening pathogens. Campylobacter is not well-behaved, but attacks gut epithelial cells to produce inflammation. The result is leaking out of large amounts of fluid from the gut wall and stimulation of intestinal smooth muscle. Together these caused Chris’s abdominal cramps and diarrhoea. The inflammation can sometimes be so severe, as with Chris, to cause bleeding from the inflamed gut mucosa – bloody diarrhoea is known as dysentery. Some unfortunate patients with ulcerative colitis have inflammation and diarrhoea when there is no obvious pathogen – either something has gone wrong with the police force (immune system), or there is a pathogen which we have not yet found.

Many germs which cause gastroenteritis, including campylobacter, produce a bacterial toxin. I warn our students to be careful of using the word toxin, unless describing something nasty made by a bacterium. At least we should know the chemical name of the toxin. Alternative medicine therapists use the word toxin all the time, but I’m not usually sure what they are talking about, and you have probably guessed I’m not a fan.

The most dramatic effect of a toxin-producing organism is seen in cholera. This organism makes a toxin that interferes with the normal mechanism for absorbing water from the gut. Typically, patients with cholera produce more than ten litres a day of very watery diarrhoea, and if not given lots of oral or intravenous fluids will die from dehydration. There is an interesting story about sporadic cholera outbreaks happening in the 1970s, apparently at random in communities with good sanitary facilities. It was found that these occurred under the flight-path of aeroplanes flying from cholera-endemic areas. The planes were discharging the contents of their sewage waste tanks in mid-air at 30,000 feet to reduce weight. The cholera organisms were surviving the drop and causing mini-outbreaks of disease. The airplanes don’t do that any more.

Another toxin-producing bacterium we commonly see in hospitals is Clostridium difficile diarrhoea. It used to be very common in frail, elderly patients given broad-spectrum antibiotics. C difficile would frequently kill these patients in a thoroughly depressing, undignified manner. It is called C. difficile because, when originally identified as the cause of pseudomembranous colitis, it was very difficult to grow.  This was because it is an obligate anaerobe. That means even very small amounts of oxygen inhibit its growth, but now we know this we can easily grow it in the lab. We can also identify the toxin, and detect C.difficile DNA using rapid PCR testing. We now see much less C.difficile diarrhoea – due to a combination of obsessional hand-washing and more careful use of antibiotics. It is worth knowing that the alcohol hand rubs do not kill C.difficile spores – when the lab are trying to isolate C.difficile, the first stage is to flood the specimen with absolute alcohol to kill off all the other bacteria. Washing with soap and water works by removing spores from the hands, not by killing them. Clostridium is closely related to anthrax, another nasty organism which makes almost indestructible spores. Clostridium botulinum also produces a very unpleasant toxin which can cause death by paralysis – interestingly nitrite is added to help preserve meat and prevent the growth of C.botulinum organisms.

salami contains nitrite which helps kill C. botulinum spores
salami contains nitrite which helps kill C. botulinum spores (and makes it pink)

This week’s food link is red kidney beans. They are fine if they are cooked in chilli-con-carne. But if they are eaten uncooked they can produce a nasty illness which resembles bacterial gastroenteritis – vomiting, diarrhoea and abdominal pain. It usually happens only 2-3 hours after eating raw or undercooked beans. Bacterial gastroenteritis typically causes symptoms between one and two days after eating infected food. Red kidney beans contain a toxin known as phytohaemaglutinin. This is destroyed by boiling the beans for at least 10 minutes. Undercooking (such as in a slow cooker which does not reach boiling point) can actually increase the amount of toxin.

Red kidney beans contain a toxin, phytohaemaglutinin which causes diarrhoea and vomiting if the beans are not cooked for long enough or at a high enough temperature.
Red kidney beans contain a toxin, phytohaemaglutinin which causes diarrhoea and vomiting if the beans are not cooked for long enough or at a high enough temperature.

Phytohaemaglutinin is one of a family of proteins, called lectins,  produced by plants which selectively bind sugar molecules. The notorious spy-poison ricin, derived from castor oil seeds is a lectin. These proteins have many uses in medical research and in the past were used to identify the sugar molecules on red blood cells allowing us to type blood into A,B and O groups. Binding sugar molecules on the intestinal epithelial cells triggers increased secretion of fluid and smooth muscle contraction in a similar way to invasive bacterial or viral pathogens, but the illness following eating raw kidney beans normally only lasts a few hours, rather than days. But I would still encourage you to cook your beans well.