Tag Archives: peptidoglycan

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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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