Spotlight - not a Greek tragedy / Dorit Farnes

The antibiotic resistance epidemic of bacteria is not a decree from heaven, says Professor Natan Tzitari, who invented a kit for the immediate detection of resistance to antibiotics that are coming into use these very days.

In the article before you, we interviewed Professor Natan Tzitri. To our great regret, Professor Tzitri passed away on September 16, the eve of Rosh Hashanah, shortly before the publication of the article about him, and he is 91 years old. Professor Tzitri was born in 1921 in Lodz, Poland named Natan Tzitrinowski. Although his father came from a family of Hassidim, Natan's parents were both Zionists, taught Hebrew and educated their children, Natan and Miriam on the knees of the Zionist idea. Thus, in 1936, 15-year-old Natan immigrated to Israel as part of the youth immigration, and was the only one who survived. The rest of his family perished in the Holocaust.

In Israel, Natan studied at Ben Shemen, and in 1942 he volunteered for the British Army, and served in it until 1946. During the War of Independence, he served as an officer in the IDF. When he was released from the army, he dreamed of becoming a farmer, but in the end he turned to academia and studied general sciences and parasitology. He was a respected scientist and teacher, and continued to teach even after he retired. Although his first studies did focus on the Leishmania parasite (the cause of the disease known as the "Jericho rose"), he soon moved on to investigate antibiotic resistance, and in particular the enzyme penicillinase (beta-lactamase), which breaks down penicillin and gives bacteria resistance to this antibiotic. His articles were published in leading journals, including Nature.

Natan Tzitari left behind two children, six grandchildren and three great-grandchildren.

The antibiotic resistance epidemic of bacteria is not a decree from heaven, says Professor Natan Tzitari who invented a kit for the immediate detection of resistance to antibiotics that are coming into use these very days.

Recently, there have been many publications about terrible bacteria that have developed resistance to antibiotics. In the previous issue of Scientific American Israel, for example, we published an article about the growing resistance to the disease gonorrhea [see "Return of the gonorrhea", by Marin McKenna, August-September 2012]. One of the problems described there is that the doctors do not know that they are dealing with a resistant bacteria because the quick and cheap tests accepted in clinics do not detect resistance of bacteria to antibiotics.

However, a revolutionary new kit for the immediate detection (within 15 minutes) of antibiotic-resistant bacteria will soon enter the market, invented by 91-year-old Professor Emeritus Natan Tzitari, from the Faculty of Medicine of the Hebrew University, and his late wife, Naomi, in a makeshift laboratory in their Jerusalem apartment. The development took only about two years, and the idea raises the question: "How come this wasn't thought of before?" Well, there was someone who thought of it first: Professor Tzitari himself, who as a pure researcher hoped that someone else would put the idea into practice. To understand the sequence of events, it is worth going back and reviewing the greatest achievement of Western medicine: the discovery of antibiotics.

The wonders of penicillin

The story of Tzitari, who took an active part in the history of microbiology, begins in London in 1961. He then participated in a conference where the great antibiotic researchers of those days were present: John K. Sheehan, an organic chemist from the Massachusetts Institute of Technology (MIT), who discovered how to synthesize penicillin; Edward P. Abraham, a biochemist from the University of Oxford, one of the penicillin researchers, and Ernest B. Chain, a biochemist who shared the Nobel Prize in Medicine with Alexander Fleming and Howard Florey for the discovery and development of penicillin.

The penicillin molecule, produced by the mold Penicillium, causes fatal damage to the bacterial wall. Fleming discovered the substance as early as 1928 and its medical implications were clarified by Cheyenne and Fleury in 1939 - just in time to save the lives of many Allied soldiers in World War II. The molecule has two rings, one pentagonal and one square, known as beta-lactam (see figure).

The beta-lactamase enzyme breaks down the square ring at the heart of the penicillin molecules, a model of which is shown here. (The black spheres represent carbon atoms, the white spheres - hydrogen, the red spheres - oxygen, the blue spheres - nitrogen and the yellow sphere - sulfur. Different chemical groups can appear instead of the pink sphere.)

In fact, penicillin-resistant bacteria appeared already almost on the first day of its use. The resistance is due to an enzyme called beta-lactamase, which is able to break down the penicillin molecule and is found in many bacteria. In the late 50s, Cheyne discovered that it was possible to change the structure of the side chain of atoms attached to the beta-lactam ring and easily produce new types of antibiotics that the enzyme was unable to break down. This is how a new family of antibiotics began to emerge, the beta-lactam family.

Chain's discovery opened up a whole world of possibilities for chemists in which they acted freely without imagining that the "celebration" might end - and soon. And so, at the meeting in London in 1961, twenty-one years after penicillin went on the market, and long before antibiotic resistance had reached alarming proportions, chemists celebrated the victory of penicillin and their own victory. The feeling was that for every biochemical "bunny" that would pull the bacterium out of its hat, the chemists would be able to synthesize a new, more effective weapon.

But unlike the other participants of the conference, Tzitari, who researched the action of the beta-lactamase enzyme, did not participate in the celebrations, and even asked what would happen when the bacteria developed complete resistance to the existing drugs. It was clear to him that the drugs that will be widely used are the broad-spectrum antibiotics, which will be effective for most diseases. And precisely this efficiency will be in their hands and will cause a widespread outbreak of resistance in many bacterial strains. Sheehan dismissed this bleak vision, saying: "Why don't we trust the adaptability of the chemist instead of trusting the adaptability of the bacterium?" Unfortunately, time has proven that the bacterium's ability to survive is indeed dozens of times greater than the chemists' ability to invent.

Page 122

In 1990 Tzitari again tried to shake up the world of bacteriologists and again without much success. At a conference intended to mark the 50th anniversary of the discovery of the first beta-lactamase by Avraham and Chain, Tzitri called on those present to develop a strategy against the development of resistance in bacteria. "I am already old," he said and did not imagine that quite a few years after that he would answer his own call.

One of the things that motivated him to act was the book "Penicillin, Triumph and Tragedy" (published by Oxford University, 2007) written by Robert Budd, a historian of science from Queen Mary University in London. The book discusses the drug's initial victory over infectious diseases and the development of "superbugs" that are resistant not only to the first miracle drug discovered by Fleming, but also to almost every type of antibiotic known to us today. Tzitari was disturbed by the link between antibiotics and a Greek tragedy and went to meet Bud. He came to explain why, in his opinion, this is not a matter of predetermined fate, nor "the will of the gods". If we learn from our mistakes, he claimed, we can win in a few sets even if not the whole battle. On page 122 of Budd's book, Tzitari found the key, and it was precisely in the quote from Sheehan's answer to Tzitari in 1961 that underestimated the adaptability of the bacterium. "The key" said Tzitari. "Is that we must understand that the bacterium is not a simple chemical system. It is a living creature that will do anything to live.” The bacterium develops more and more mutations that change the beta-lactamase enzyme and allow it, in the end, to overcome any new antibiotic from the beta-lactam family.

What is so special about this enzyme that makes it central to the development of resistance in so many bacteria? Bacteria can also develop other resistance mechanisms, but in most cases the mutations involved will also harm the bacteria itself. But in the case of beta-lactamase, "the bacterium came up with a brilliant solution," says Tzitri. The enzyme has no other role in the bacterium, and if there are no antibiotics in the environment, it is not needed at all, and the bacterium does not even bother to synthesize it, as Tzitari showed in his studies. And since the enzyme is not essential, the bacterium is free to modify it. Even if most of the mutations damage the activity of the enzyme, they will not damage the entire bacterium and at some point one mutation will surely emerge that will make the bacterium resistant. This is an example of the evolutionary mechanism of natural selection. In any bacterial population there is a significant number of bacteria with beta-lactamase mutations. In fact, bacteria with some resistance to penicillin existed even before it was discovered. The use of penicillin and cephalosporin on their derivatives allowed bacteria that happened to be resistant to multiply at the expense of their neighbors that did not survive the antibiotic treatment. Over time, continued and widespread use of antibiotics, and ever-increasing doses of them, allowed only the most resistant bacteria to multiply.

But Tzitari realized that the advantage of the bacteria is also their weak point and that the bacteria will never give up its universal weapon. In other words, it is the resistance enzyme, present in all multi-resistant bacteria, that will betray them in our hands. If so, said Tzitri, all that needs to be done is to check if the patient's samples contain beta-lactamase. Tzitari presented the idea already at a conference in 1990, but apparently the way to translate it into practical language was not found. Because of this, about three years ago, he felt he had no choice but to develop such a kit himself, with the cooperation of Naomi, his wife. Thanks to Naomi, who passed away on the eve of the completion of the task that until then was considered hopeless, we have in our hands a kit that allows for the first time the discovery of durability in place.

Assessment and doomsday weapon

The idea of ​​an assessment is astonishingly simple and is based on the simple iodometric method known since the 70s to identify resistance in cultures, but until now no one thought of using it for rapid identification: when beta-lactamase breaks the square ring and breaks down beta-lactam antibiotics, a substance capable of binding iodine is created. Every chemist knows that iodine and starch form a blue conjugate, therefore, as long as the antibiotic is not decomposed the solution is blue. However, if the bacterium succeeds in breaking down the beta-lactam, the breakdown product will compete with the starch and the color will disappear. The kit therefore consists of a transparent cover on which are dark spots of iodine-starch, and in front of them are colored spots, each of which contains a different beta-lactam. A streak from the sample intended for the laboratory is brought into direct contact with each colored stain. If there are antibiotic-resistant bacteria in the sample, they will break down the antibiotic in that particular colored spot and the blue spot on the lid will disappear.

The effectiveness of the kit has been verified at the Hadassah and Shaare Zedek medical centers in Jerusalem, and it is expected to revolutionize the quality of care that many patients will receive. Patients will not be treated with the wrong antibiotics during the days of waiting for the results of the microbiological laboratory, and patients infected with particularly resistant bacteria will be immediately placed in isolation. In extreme cases such patients are currently treated with colistin, the "last resort" antibiotic also known as the "doomsday weapon". Since colistin does not belong to the beta-lactam family, it is currently effective against most gram-negative bacteria, but causes severe side effects, so it is advisable to avoid using it unnecessarily. Tzitari's evaluation also checks for resistance to colistin, but the mechanism has not yet been approved for publication.

The kit passed the commercialization stage by the Hebrew University's implementation company. It is in the final stages of production at Bio-Connection, a company that specializes in the production of tests for the detection of bacterial resistance, and will be released on the market very soon. But Tzitari is not optimistic about the distant future, according to him, the bacteria lived as the age of the oldest rocks on the planet and man is a negligible episode in their lives. But at least we can postpone the victory of the bacteria. "This is not a Greek tragedy, and the situation is not dictated by the will of the gods," if we learn from our mistakes, we can tip the scales in our favor for many years to come.

on the notebook

Dr. Dorit Ferns is the scientific-operational editor of Scientific American Israel, a biology lecturer and a practitioner of Chinese medicine.