Tag Archives: nitrate

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.

back pain and blue cheese

These are lambs kidneys - not eaten much in the UK but muched loved in France - rognons de veau
these are lambs kidneys – not eaten much in the UK but much loved in France – rognons de veau

Kirsty was admitted this morning. She is twenty eight  and had been unwell for the last two days. The first thing she noticed was a nasty burning pain when she passed urine. Almost as soon as she had finished peeing she needed to go again. Kirsty went to the doctor yesterday and was prescribed antibiotic tablets. She took one dose and was immediately sick. Then she developed pain in her back, on the left side, under her lower ribs. It started gradually but became almost unbearable. Then she started to feel feverish and shivery. Not ordinary shivery, but uncontrollably shivery – and then she vomited again and again. Her new husband, Sam, drove her up to the emergency department at nine thirty this morning. Their two year old daughter, Ellie, was in the back of the car. In the emergency department Ellie was sitting on Sam’s knee, looking very unconcerned when we talked to Kirsty, who was lying on the trolley.

“I think you have a serious kidney infection” I said, “and we’d better admit you and give you intravenous antibiotics”.

Kirsty was not looking well. She was pale, sweaty, febrile and a bit blotchy with wet hair stuck to her face.  She was clutching her painful back with one hand and holding a vomit bowl with the other. She was happy to come in and be looked after. So was Sam. Ellie did not look so sure.

its really important to take blood cultures before giving antibiotics - otherwise it will be difficult to find out the germs responsible for infection
its really important to take blood cultures before giving antibiotics – otherwise it will be difficult to find out the germs responsible for infection

We took a blood sample and blood cultures, gave her intravenous morphine, paracetamol, gentamicin, co-amoxyclav and cyclizine (an antiemetic). She had pyelonephritis – a bacterial infection of her kidney.

Young women get urinary tract infections much more commonly than young men. It’s to do with anatomy. The female urethra is very short, and germs can quite easily travel up to the bladder and then up the ureters to the kidney. Men get urine infections when they are older and have enlarged prostate glands.

Then they cannot empty their bladders completely and the stagnant urine is more likely to become infected. I was taught that urine is normally sterile in healthy people. It seems this is not the case. All of us have bacteria in our urine in small numbers – if you are interested read: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744036/ 

We use urine dipstick testing a lot on the acute medical unit. In theory it should be able to tell us which of our patients have a significant urinary tract infection. In practise it is not quite as useful as it should be. Kirsty’s urine tested positive for nitrites and leukocytes. In young women this is a good test, but we knew she had a urinary tract infection anyway. In elderly women, the tests are often positive even if a serious infection is not evident – perhaps because of the innocent commensal bacteria which are present.

Slide02

How do urine dipsticks work? The test for leukocytes is leukocyte esterase. In infected urine, the leukocytes are polymorphonuclear leukocytes, or neutrophils. I talked earlier (phlegm and horseradish) about how neutrophils, when they get excited by the presence of germs, make the enzyme myeloperoxidase which generates bleach. Esterase is another enzyme neutrophils make which breaks down peptide bonds, and specifically is useful in breaking down the peptidoglycan in bacterial cell walls. I guess its like getting stains out of clothes. Bleach works fine, but the proteolytic enzymes in washing powder can help too.

So what about nitrites? Bacteria like Eschericia coli, which are a common cause of urine infections are known as facultative anaerobes. This means that they can use oxygen to “burn” carbohydrates, protein and fats. But they can also use other “electron acceptors” to do this such as nitrate. Mammalian cells can only use oxygen. When I say mammalian cells, what I mean is mitochondria in mammalian cells – these small structures in our cells are responsible for all the energy generation from glucose, fats and protein. Think what happens when you eat a slice of toast. The amylase in our saliva starts to break down the starch in the toast to form glucose, a process which is finished by amylase in pancreatic secretions. This yields glucose, which is absorbed into the bloodstream. Glucose can be made into energy by the Kreb’s cycle, or citric acid cycle mainly happens in mitochondria. This is a complicated process, but essentially it means that glucose is turned into carbon dioxide and protons and electrons:

The mitochondria don’t get much energy from the Kreb’s cycle, but rely on the protons and electrons produced to make energy by combining them with oxygen. This happens in the electron transport chain:

Bacteria are more versatile than mammalian cells and can get energy out of these protons and electrons, even if oxygen is not available, by using nitrate instead of oxygen:

To do this they need to have the enzyme nitrate reductase. Human cells do not have nitrate reductase, so if nitrate is being turned into nitrite there must be bacteria present. So, if urine contains significant amounts of nitrite, the only way it can get there is if bacteria are using nitrate to “breathe” – a tell-tale sign that infection is present. The reason E.coli is called a facultative anaerobe is that it can survive by making energy if oxygen is present and also when it is not, by using molecules such as nitrate to “breathe”.

our mitochondria can only use oxygen to get energy from electrons and protons derived from glucose - some bacteria have nitrate reductase which can use nitrate as an electron acceptor - nitrite is a by-product- and appears in infected urine as an indicator of infection
our mitochondria can only use oxygen to get energy from electrons and protons derived from glucose – some bacteria have nitrate reductase which can use nitrate as an electron acceptor – nitrite is a by-product- and appears in infected urine as an indicator of infection

When I saw Kirsty later in the afternoon she was much better. The pain had not gone, but was eased by the morphine. Her temperature had come down, and she had stopped vomiting. Her infection was coming under control.

Both gentamicin and co-amoxyclav are very effective in treating urinary tract infections. They are both rapidly excreted by the kidneys and achieve much higher concentrations in the urine than in the bloodstream. Gentamicin has a half-life of about 2 hours in patients with normal renal function. So, lets say we give a person 250mg of gentamicin intravenously. The blood volume is about 5 litres, so the immediate concentration will be 50mg/litre of gentamicin. In two hours about 100ml of urine will be made, and half of the gentamicin previously given intravenously will be in that urine. That is 125mg in 100mls or 1250mg/litre – more than twenty times the concentration in blood. Amoxycillin has a half-life of more like one hour, so achieves even higher urine concentration in comparison to blood.

What do gentamicin and co-amoxyclav do? They are antibiotics that work in quite different ways.

Gentamicin is an aminoglycoside. That means it is a sugar with amine groups. Here is the structure – just three sugars with lots of NH2 groups:

gentamicin is a relatively small molecule with three sugar groups and lots of amine groups (in red) - an aminoglycoside
gentamicin is a relatively small molecule with three sugar groups and lots of amine groups (in red) – an aminoglycoside

It gets into the bacteria and binds strongly to its ribosomes. These are the really important and clever machines in bacteria which make proteins from DNA. Mammalian cells also have ribosomes to make our proteins – but they are not at all the same. They work in the same way but over the past 2 billion years have changed with evolution so they are a different shape and are larger than bacterial ribosomes. Gentamicin does not interfere with mammalian ribosomes. For an antibiotic to be useful it has to damage bacteria but not human cells. Luckily, ribosomes are so different between bacteria and mammalian cells that some chemicals such as gentamicin will selectively bind only to bacterial ribosomes.

mitochondrion - it has all the stuff inside that a bacterium has, but without a tough cell wall
mitochondrion – it has all the stuff inside that a bacterium has, but without a tough cell wall – redrawn from wikipedia – author kevinsong

Other antibiotics such as tetracyclines, macrolides (erythromycin and clarithromycin), chloramphenicol and clindamycin also work by interfering with bacterial ribosomal function. A bacterium with damaged ribosomes has major problems – it cannot make new proteins. That means it cannot divide and make new bacteria. It will be immobilised and suffer a slow and painful death. (Not really, I don’t think bacteria don’t feel pain – but then I don’t have evidence for that). If it is a bacterium which makes a protein toxin, such as staphylococcus causing toxic shock syndrome, turning off protein production with a ribosomal poison such as clindamycin is a good idea – rather than causing bacterial cell wall damage and leakage of more toxin with penicillin therapy.

Gentamicin can cause problems if it is given over prolonged periods, because it can accumulate and cause damage to ears and kidneys. The damage to hearing is probably due to damage to mitochondria. More specifically damage to mitochondrial ribosomes. We only gave Kirsty one dose of gentamicin – problems with this drug usually happen when patients with impaired renal function are given aminoglycosides for several days, or when aminoglycosides are given with other drugs such as vancomycin which can impair renal function.

Mitochondria are thought to derive originally from bacteria. Once upon a time, a long time ago there was a cell that survived well enough by getting energy from glycolysis – turning glucose into pyruvate. This cell did not need any oxygen. It scraped a living producing at most 2 ATP molecules per glucose molecule. Then it had a conversation with a bacterium which said “Hi doll, I could take that pyruvate you make and turn it into another 28 ATP molecules by combining it with oxygen – how about it?” Maybe this conversation happened at the time oxygen had begun to appear in the atmosphere (see great oxygenation event in last week’s blog). “With your looks and my talent we could do Broadway together”. This is technically known as endosymbiosis, where one type of cell engulfs another to work together to their mutual benefit. The result is eukaryotic cells – the cells we are made of. Our cells contain mitochondria that derive from bacteria. They can make lots of energy from glucose and oxygen. The bacteria are looked after and nurtured inside the cells which engulfed them. Like bacteria, these mitochondria have their own ribosomes, that, not surprisingly, are similar to the ribosomes of bacteria that are causing Kirsty’s pyelonephritis. Too much gentamicin can damage mitochondrial ribosomes and cause hearing loss – see:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1376819/pdf/9443888.pdf

We also gave Kirsty co-amoxyclav.

the basic penicillin molecule - the central beta lactam is in the pink circle
the basic penicillin molecule – the central beta lactam is in the pink circle

This is a combination of amoxicillin and clavulanic acid. Amoxycillin is a penicillin. Originally discovered by Alexander Fleming, the original penicillin, benzylpenicillin, has been modified by pharmaceutical companies to be more effective. Unlike benzylpenicillin, amoxycillin is rapidly absorbed by the stomach. It is also effective against gram negative organisms such as E.coli. Penicillins have a beta lactam group. This structure makes it difficult for bacteria to make a vital component of their cell wall – peptidoglycan.  This is a tough polymer made of special sugars and short peptide chains. The beta-lactam group in penicillins is the right shape to get stuck in the cell wall building enzymes and prevent cell walls being made. Our cells do not have cell walls – they just have thin, delicate plasma membranes made of phospholipid and cholesterol. Similarly, although mitochondria are similar to bacteria, they also do not have cell walls. Mammalian cells and their mitochondria are very cosseted and protected in a 5-star luxury apartment with all mod-cons.  They are looked after in a temperature-controlled environment. Oxygen is supplied free and waste carbon dioxide and other unwanted substances taken away continually. Acidity is tightly controlled – pH between 7.35 and 7.45, osmolarity not too high or low. The poor bacterium, in contrast, has to tolerate acid, alkali, high and low osmolarity and a whole host of chemical insults as well as having to find its own food. And then there is the danger of being chased by an angry green neutrophil. No wonder it feels happier with a thick, tough cell wall to protect it. The clavulanic acid is to inactivate beta lactamase – an enzyme some wily bacteria have started making to destroy beta lactam antibiotics. No doubt the bacteria will soon be making betalactamaseinhibitorase enzymes.

There are other ways to selectively attack bacteria without harming human cells. All cells need folate to manufacture nucleic acids. We get our folate from diet – particularly green, leafy vegetables (folate is related to the word foliage). Bacteria do not, in general, have a healthy diet. They instead make the large folate molecule themselves from much smaller molecules. Trimethoprim and sulphonamides prevent bacteria from making folate causing them to suffer and die a “thymineless death” (look it up in Wiki).

Fluoroquinolones such as ciprofloxacin are some of the newer antibiotics which have come into clinical use since I qualified. They were hailed as the new wonder drug, but we now use them relatively rarely because they particularly seem to promote C.difficile infections in frail, elderly people. They work by inhibiting DNA gyrase and Topoisomerase IV. I hope the illustrations will explain how they work:

bacterial DNA is circular
bacterial DNA is circular
DNA helicase pulls the DNA strands apart so that they can be replicated to make more DNA  when the bacterium divides - but it causes a problem, the DNA becomes supercoiled
DNA helicase pulls the DNA strands apart so that they can be replicated to make more DNA when the bacterium divides – but it causes a problem, the DNA becomes supercoiled
DNA gyrase sorts out this problem - cutting the DNA strand and rejoining it having removed the twist
DNA gyrase sorts out this problem – cutting the DNA strand and rejoining it having removed the twist – fluoroquinolones such as ciprofloxacin stop this enzyme working
having replicated the DNA - the two circular strands are interlinked!
having replicated the DNA – the two circular strands are interlinked!
but topoisomerase IV comes to the rescue and chops the chain and rejoins it to separate the circular strands
but topoisomerase IV comes to the rescue and chops the chain and rejoins it to separate the circular strands- fluoroquinolones also inhibit this enzyme
all sorted
all sorted

The food link this week is blue cheese.

blue stilton
blue stilton – the blue bits are penicillum mould

The reason it is blue is because of the growth of the mould penicillium, which is a grey/greenish blue colour.  The spores are blue, not the fungus itself.

volkornbrot past its sell by date - the blue mould is penicillium - not sure what the yellow stuff is - any ideas anyone?
volkornbrot past its sell by date – the blue mould is penicillium – not sure what the yellow stuff is – any ideas anyone?

The mould in Roquefort and Stilton is P. roquefortii, a close relative of P. notatum (now known as P. chrysogenum), the penicillium mould that Alexander Fleming found inhibiting the growth of staphylococci.

I’ll be in France next week so the next post will be in 2 weeks’ time