The Even Earlier Discovery of Antibiotic Resistance

So about a month ago, I wrote about how amazing it was that penicillin resistance was discovered as early as 1940, two years before it went on general sale. But whilst researching that article, I realised that Sulphonamide drugs entered the market long before penicillin, with their discoverer, Gerhard Domagk, being nominated for a Nobel prize in 1939. He had been tasked by Bayer pharmaceuticals to test out a gargantuan number of dye molecules to see whether they could kill off bacteria, and in the process , stumbled across the first antibiotic.
You may recall from the previous instalment that Heinrich Hoerlein was the man to recruit Gerhard Domagk into Bayer. Heinrich Hoerlein was a talented chemist, who had specialised in developing dyes for wool. How did this dye maker end up working to create one of the most important pharmaceuticals that the world had seen up until that point ? Why was it that when Bayer decided to devise new treatments against bacterial disease, they focussed on the compounds used to colour clothes ?

To understand how this state of affairs occurred, we need to go back further in time to the 1800s, and look at bacteria. If we were to do this in this era, we would need to get a good microscope. Bacteria tend not to be visible to the naked eye. So let us look down our microscope, what would we see ?
We would probably see tiny transparent blobs. This could mean that we either have lots of bacteria, or are looking at some air bubbles. There are various legends of scientists proclaiming that they have found an entirely new type of bacteria, only to later realise that this bacteria was nothing more than a bubble of air in the wrong place at the wrong time.
This is where dyes become important. The odds are that you are wearing clothes which have undergone the dye process. A dye works by chemically binding to the surface of whatever you want coloured.
A number of scientists of that era began to work on dyes that bind to cells, allowing them to be more easily seen under a microscope. This allowed scientists to see that bacteria came in all sorts of shapes and sizes, and that different species were associated with different diseases.
One of the pioneers of this research was a man named Paul Ehrlich. His PhD had been dedicated to studying how aniline dyes, which had previously only been used for fabrics, could actually be used to colour cells. He also noticed that dyes he used would colour some cells differently to others. Some cells would take up a lot of the dye, whilst others would not and remain transparent. At the age of 24, using the new "staining" techniques, he had managed to discover a new type of cell, known as a Mast cell.
A lot of his scientific achievements  could be tracked down to the one simple question he asked himself when he saw this effect: Why did some cells take up more dye than others?
He theorised that cells took up specific nutrients, and that receptors on the surface of these cells played a crucial role in this process. Different cell types have different receptors on their surface. some of these receptors allow dye molecules to enter the cell, and some do not. The reason that different cells "stained" differently was due to the different receptors on their surfaces.
He suggested that toxins on the surface of the bacteria bound the receptors on the surface of the host during infection. In response, the host cell would secrete these receptors to flood the toxins on the surface of the bacteria, thus neutralising them. He called these secreted receptors antibodies.
 Whilst this is far from our modern understanding of antibodies, it was a crucial step in the right direction, and he would be credited as one of the founders of immunology as a result of it.
He also suspected that bacteria also had receptors which they used to ingest dye molecules. He noted that some dyes were taken up by bacterial cells, but not human cells. He suggested that this was because the dye molecules resembled nutrients that the bacteria eat. If he could manufacture the chemical structures of these dye molecules to include poison, then he could have a chemical that kills of bacterial cells, and leave human cells alone. He termed these chemicals "Magic Bullets".
In 1904 came his first breakthrough, with a compound, known as Trypan Red, due to it's colour, and sucess for treating mice infected with trypanosomes. Whilst this was useful as a proof of concept, Trypan Red only worked against the types of trypanosomes that infect mice, but not those which attack humans
It was while he was working on this problem that researchers at the Liverpool School of Tropical Hygiene, Anton Breinl and Harold Wolferstan Thomas, discovered that a compound known as Atoxyl, though to be non-toxic for humans, could kill off trypanosomes. From 1906, a number of expeditions to Africa took it with them to protect themselves from the Sleeping Sickness caused by these organisms. Robert Koch, one of the founders of microbiology, used it to treat patients on the shore of Lake Victoria. It became incredibly popular at the time.
Intrigued, Paul Ehrlich investigated this wonder drug, in addition to the other dye based drugs he was developing. During the course of his research, he noticed a worrying trend. After prolonged therapy with these drugs, the resistance of the trypanosomes to these chemicals increased, until they were completely resistant to the therapy. He coined the term "fastness" to describe this trait in bacteria. The fact that he had observed this "fastness" occurring in response to such a broad range of chemicals suggested to him that this was an inevitable event.
Ehrlich was unexpectedly energised by this discovery of resistant organisms. This was because of the finding that once an organism became resistant compound, it was also resistant to chemicals with the same shape and structure. This provided evidence for his fledgling theory of surface receptors which bind to specific chemicals based on their shape and structure.
But his discovery of resistance put him on a crash course with Robert Koch, who had not observed this effect, and thus disputed that it had ever occurred outside of the lab. The main differences were that Robert Koch used much higher doses of Atoxyl than Ehrlich. Either way, Atoxyl was fast falling from popularity. Patients treated with it would go blind due to its severe side effects. A study in 1910 would show that it merely halted progression of trypanosome disease, and that patients were no better off using it.
 Ehrlichs lab was still screening drugs to fight off pathogens, and it came across compound 606, a derivative of atoxyl that not only had less severe side effects, but had proven utility against syphilis.
This was marketed as Salversan, and became an important drug in the fight against syphilis, and was used up until the 1940's, when penicillin replaced it.
The discovery of these drugs, and the apt demonstration that dye molecules could make good antibiotics cemented his place in history. One of his assistants, Wilhelm Roehl, would go on to head a research department at Bayer. It was he who would recruit a Dye chemist, Heinrich Hoerlein, to find the next big drug.
So whilst Ehrlichs theory of "magic bullets" would live on, and laid the foundations for the discoveries of Domagk and Fleming, what happened to his theories on antibiotic resistance ?
There were a number of weaknesses in Ehrlichs theories that a number of researchers called into question. Ehrlichs theories of antibiotics and antibiotic resistances were too simplistic. He posited that for each compound, there was a single path to resistance through the mutation of a single receptor. But we know that bacteria and other pathogens can adapt to antibiotics in multiple ways. This made it difficult to replicate his results, and even more difficult for him to explain why they didn't replicate.  The rules he had set out for explaining antibiotic resistance did not always hold true. The disparity observed between his experiments of Atoxyl, and of Kochs experiments did not help his case.
 This apparent wooliness would mask the threat of antibiotic resistance as a merely theoretical phenomenon. It would for the next 30 years be regarded as a curiosity that would never pose a threat to people.
Over a hundred years after Ehrlich's initial observation of antibiotic resistance, we have a slightly different perspective on his discovery than his contemporaries.

References

Gradmann C. (2011). Magic bullets and moving targets: antibiotic resistance and experimental chemotherapy, 1900-1940, Dynamis, 31 (2) 305-321. DOI: 10.4321/S0211-95362011000200003

Titford M. (2010). Paul Ehrlich: Histological Staining, Immunology, Chemotherapy, Laboratory Medicine, 41 (8) 497-498. DOI: 10.1309/LMHJS86N5ICBIBWM

Casanova J.M. (1992). Bacteria and their dyes: Hans Christian Joachim Gram, Historia de La Immunologia, 11 (4) DOI:

Ehrlich P. Address in Pathology, ON CHEMIOTHERAPY: Delivered before the Seventeenth International Congress of Medicine., British medical journal, PMID: 20766753

Kaufmann S.H.E. (2008). Immunology's foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff, Nature Immunology, 9 (7) 705-712. DOI: 10.1038/ni0708-705