The Project assigment of Nicholas, Peter & Jonatan, VRG Spring 1997

1. Introduction

In 1929 Alexander Flemming discovered Penicillin, the first antibiotic. Since it was put into medical use in the 40:ies it has cured millions from bacterial infections and prolonged our life expectancy with an estimated 10 years. Diseases that once killed more people than all the wars in the world, have become rare or even extinct.

However, the past 40 years of living in safety from bacteria have come to an end. So called "Killer bugs", resistant to most known antibiotics, are infecting people leaving doctors all over the world helpless.

This project will investigate the causes for the development of antibiotic resistant bacteria (ARB), and how it has evolved into multiple antibiotic resistant bacteria (MARB).The mechanism of antibiotics and the basic biology of bacteria will also be explained, in order to enable the reader to fully grasp this issue. In the end, a number of possible solutions to the problem of MARB will be discussed.

These topics will be dealt with from a global point of view, although statistics from individual countries will sometimes be shown when their information is judged to be relevant and transferable to the rest of the world.

2.1. Bacteria

Bacteria are small micro-organisms which can be found almost everywhere in nature. There are plenty of different species of bacteria, however the only bacteria of interest here are the once causing disease, pathogens.

2.1.1 Construction

A bacterium consists of only one cell (unicellular) making the bacterium quite a simple organism. The interior of the cell contains one single chromosome,

the plasmids, the ribosomes and all around them the cytoplasm. The two first ones carry the genetic information about the bacterium and the ribosomes create proteins, needed for bacterial growth, from amino acids.

The interior parts are protected by a cell membrane a the cell wall, which can be multiple (gram-negative)or single layered (gram-positive).

The cell wall consists of peptidoglycan, built of polymers of sugar molecules and amino acids. Transpeptidation is the process when the polymers link together in transpeptide bonds forming the peptidoglycan cell wall.

The production of autolysins, enzymes which degrade peptidoglycan, makes it possible for the cell to reshape during cell division and growth. The transpeptidation and the production of autolysin are balanced, so the cell wall stays stable.

All over the exterior shield, small fibres are attached. These fibres help the bacterium move around and adhere to the inside of the body.

2.1.2 Reproduction and toxicity

Bacteria need nutrients for survival and different bacteria have different methods of acquiring this. Some bacteria collect sugar and oxygen from the surrounding environment, others have special enzymes which can break down tissue and organisms and utilize the nutriments.

Under extraordinary circumstances bacteria duplicate every 20 minutes, thus one germ can have 16 million descendants in 24 hours. Usually, though, one duplication takes12-24 hours due to temperature and rival organisms.

It is this ability to multiply at avalanche speed combined with their toxin-production capabilities that make bacteria dangerous to humans. Toxin is a strong poison which the bacteria secrete to injure the cells of the infected organs. Toxins are the principal cause for actual disease when bacteria invade our body.

2.1.3 Bacterial species

There is a vast amount of different species of pathogens, and the following table will only contain five of the most important ones and the diseases they cause.

Pathogen Disease

Enterococcus Inflamation of the urinary tract, pneumonia

Haempophilus influenzae Blood-poisoning, inflammation of the ear, meningits

Staphylococcus aureus Blood-poisoning, food poisoning, suppuration

Streptococcus haemolyticus Blood-poisoning, scarlet fever, suppuration, tonsillitis

Streptococcus pneumoniae Blood-poisoning, inflammation of the ear, pneumonia

2.2. Antibiotics

Antibiotics have existed in nature for millions of years, but they have only been known to man for a century. Antibiotics are chemicals that are produced by micro-organisms. However, many of the antibiotics in medical use are synthetical versions of naturally occurring dito.

In this part we are going to look at theantibiosis, the antibiotic function.

2.2.1 The antibiotic function

An adequate antibiotic neutralises the pathogen while leaving the infected body unhurt. Antibiotics have different ways of achieving this.

One of the methods to neutralise pathogens is to prevent the cell wall of the bacteria to grow properly,by interfering with the transpeptiation or the cell wall synthesis.

When antibiotics, for instance beta-lactams, reach the cell it combines with the transpaptidase, an enzyme catalyzing the transpeptidation process, preventing the process from occurring. Instead of being a strong mesh, the newly-formed cell wall is just a spaghetti of loose polymers.

The antibiotics also interfere with the autolysin production, causing the cell to overproduce the enzyme. The autolysins dissolve the peptidoglycan in the cell wall and since the transpeptiation cannot continue, the cell wall becomes so debilitated that water can penetrate it and the bacteria dies.

A second method of killing bacteria is to attack its ribosomes. The antibiotics attach to subunits of theribosomes and prevent them from producing protein. This inhibits the growth of the bacterium.

2.2.2 Antimicrobial agents

According to Staffan Arvidsson penicillin, the first antibiotic, is also the best one since it has no side effects. Most newer antibiotics are actually only variations of the original penicillin molecule.

Penicillin neutralises bacteria both by dissolving the cell wall and by interfering with the ribosomes, making them highly effective. Penicillin can be used on pathogens such as staphylococci, streptococci and S. pneumoniae, however today 90%of these bacteria are resistant to penicillin.

Tetracyclines is another antimicrobial agent and has, like penicillin, a broad spectrum which means that it is effective against numerous different pathogens. Tetracyclines are used all over the world, though it can be dangerous to humans under certain circumstances.

Quinolones are one of the newer antibiotics and it eliminates bacteria differently from the two above. The quinolone molecule attracts an enzyme needed for bacterial DNA replication, in effect prohibiting bacterial cell division.

Vancomycin is a popular quinolone because quite few bacteria have become resistant to it. Therefore, physicians often use it as a last resort when nothing else helps.

2.3. The development of resistance

Charles Darwin once wrote in his book On the origin of species that there is a 'natural selection' in nature among species; the survival of the fittest. Quite often natural selection is caused by mutations resulting in a beneficial property for an individual. That individual will then be more fit than his peers, and his descendants will in time dominate the population.

This is actually what has happened in the world's bacterial populations.

This section will study how resistance arised, its functions and how it spreads in our society.

2.3.1 What is resistance?

When bacteria, pathogens or non-phantogens, multiply, there is a chance a mutation will occur. These mutations are actually a change in the DNA and are of three different kinds: Point mutations, insertions and deletions. Point mutations is a minor mistake in DNA causing small changes in an enzyme or structural protein. The two latter mutations can result in destruction of a structural protein or enzyme activity.

Mutations in bacteria are rare, spontaneous mutations with beneficial result only occurring in one bacterium in a million or in a hundred million bacteria. Originally, resistance arised from such a mutation.

2.3.2 The resistance mechanism

ARB have four methods of resisting antibiotic substances:

The first form of resistance is the production of an enzyme called Beta-Lactamase. This enzyme can destroy apart of the antibiotic's structure, the beta-lactam ring, thereby preventing it from binding to its target, making it ineffective.
Within the bacterial cell, cell membrane or cytoplasm a substitute protein can be developed. The modified protein has a few similarities to beta-lactams, therefore it can survive beta-lactam antibiotic attacks and continue cell-wall construction.
Gram-negative bacteria have porins in the outer membrane. Porins are small water-filled channels where the antibiotic usually gets into the cell. These porins can mutate into constricting intake of antibiotics to non-lethal levels.
The fourth mechanism relies on the presence of pumps. They remove all antibiotics from the bacterium cell cytoplasm before it can bind to their target.

2.3.3 The development of resistance

The impression may have been conveyed to You that the explosive outbreak of the MARB is the result of natural evolution solely.

This is not the case. In this case, like many before it, human interference has disrupted the natural way of things. Like Science&Technology, Business Week magazine puts it, 40 years of "widespread and inappropriate use of antibiotics has helped disease causing bacteria develop resistance".

When a patient carrying pneumonia with only few resistant bacteria is treated with antibiotics, all susceptible pneumonia bacteria will be killed and those resistant to the antibiotic used will survive. In such a competition-free environment, surviving bacteria can multiply very quickly, soon rebuilding the entire colony. This is what is called 'selection'.

2.3.4 Sharing resistance

A colony of penicillin-resistant germs are not that dangerous, since there are about 99 other antibiotics to kill them with. The real killers are the so called multiple antibiotic resistant bacteria (MARB), who by sharing resistance genes with other microbes have acquired resistance to several of our medicines. One such bacteria of special interest is the staphylococcus, which will be examined later.

Next the process of sharing genes will be studied.

In resistant bacteria plasmids as well as the chromosome carry the resistance gene. However, unlike chromosomes, plasmids can be transferred between microbes.

This is possible through four processes; conjugation, transposons, transduction and transformation. The two latter more or less work together; one bacterium let out DNA in the surrounding environment (transduction) and another bacteria picks up and incorporate it into its chromosome (transformation). Transposons on the other hand serve as a bridge for DNA in the cell or between different cells.

Not very different from transposons are conjugation tubes. Plasmids are transferred between bacteria through these tubes, making it possible for microbes of different species (!)to share DNA-regulated properties, such as antibiotic resistance.

2.3.5 Noscomial infections

There are certain places around us where the concentrations of MARB are especially high. One such place is day nurseries, where the kids' frequent body contact allows bacteria to spread and flourish easily, often without actually causing disease.

Another more important place is our hospitals, which may be the location where you are at the greatest risk of being infected by MARB.

A Rockefeller University workshop estimated that 5% of all American hospital-patients acquire an infection while being treated. Of these so called noscomial infections, the same workshop attributed 50-60% to MARB.

Another man, professor Jacques Acar, estimates that: "In hospitals alone, […] one million bacterial infections occur every day [globally], and most of these are drug-resistant".

The worst source of contagion within hospitals are the intensive care units where the risk of infection can be as high as 25-70%. Karolinska Institutet Senior Master Staffan Arvidson explains this by pointing out that patients are given massive doses of antibiotics when going into surgery as a preventive measure.

Another reason for the abundance of MARB in hospitals is the fact that hospitals are frequently visited by people infected by phantogens, people taking antibiotics and people with impaired immune defenses.

Also, the patients in geriatric care often carry a virtual zoo of non-phantogenic bacteria. In this variety of microbes, antibiotic resistance sometimes arises and then it can spread to more dangerous bacteria in other wards.

2.3.6 VRS - a true killer bacteria

Staphylococcus aureus bacteria is a very dangerous microbe which currently, according to RAF (Referensgruppen för antibiotikafrågor) is resistant to all other antibiotics but vancomycin. Vancomycin is often a last resort and doctors dread the moment when this drug becomes ineffective.

Exactly that may happen any day. Scientists fear that the less dangerous, but vancomycin-resistant, enterococcus bacteria (VRE) will transfer its genes to staph, creating a killer bacteria that none of our drugs can touch.

Staffan Arvidson tells us that this transfer is possible; it has been made in laboratories.

The resulting VRS from the experiments are kept locked up inside mountain vaults, he adds.

2.4. How to solve the problem of MARB

Will humanity be wiped out by bacteria untouchableby antibiotics? If not, how will humanity fight back when conventionalmedicine will not do? In this section present and future meansto defend ourselves against resistant bacteria will be lookedat.

2.4.1 Short term solutions

First of all we need to start producing new antibiotics again. "Again", because in the late 80:iesthe medical industry noticed how apothecaries were bulging with different antibiotics and how bacterial diseases seemed to be under control. This resulted in a down-scaling of new research, however new development has increased recently.

Oxazolidinones is a new class of antibiotics that is being developed by Pharmacia & Upjohn. Their effect is a restriction of protein synthesis in bacteria, growth-stifling.

A similar drug, called Synercid, is being developed by Roune-Poulec Rorer. Synercid is not yet available on the market, but it has been tried on 95 patients when all other options, including Vancomycin, failed. The results were positive, with 70 % of the patients showing improvement or reversal of their infection.

However, giving people new drugs is only a temporary solution, since bacteria will soon evolve resist new drugs as well. Nonetheless, on the short term, it is necessary or doctors will soon be watching helplessly as people die from once treatable diseases.

2.4.2 Long term solutions

As earlier stated, most new antibiotics are simply modifications of older ones. Switch one carbon atom here, an oxygen atom there and the bacteria will not recognize the antibiotic molecule.

Instead of restructuring blindly like this, Staffan Arvidson proposes we should study the mechanisms that make bacteria resistant to certain antibiotics. If we can understand why Staphylococcus became resistant to Tetracyclines only a few years after its introduction, and why Streptococcus Haemolyticus after 40 years still are susceptible to Penicillin, we could find new and more effective approaches to kill other bacteria, he says.

A few such new approaches are being looked at by medical companies. A list of the most promising will follow.

Natural antibacterial proteins such as those found in mothers milk, sharks, frogs and cows fight infections in a different way from antibiotics. Many proteins are cationic, positively charged molecules which can attach to the surface of the often negatively charged bacterial cells. They then force their way inside-in effect bursting the cell apart.
Another protein, found in mothers milk, seems to inhibit bacterial growth by absorbing iron, an element bacteria need to convert nutrients to energy. For bacteria to develop resistance they would have to build a new metabolism, which is a lot more than a single gene adaptation and probably impossible.
Several companies are currently looking at the genetic structure of these proteins for later synthesization.
Genetic sabotage .A lot of effort is put into finding the specific genes that make bacteria dangerous. These "virulence" genes activate processes only used when bacteria invade and infect the body, for example when a bug attaches to the surface of the lining of the lung. When these genes are identified, chemicals will be found that can destroy them.
Disarming bacteria in this way is actually preferable to killing them, since the latter results in a Darwinian survival of the fittest, in this case the resistant bugs.
Vaccines is another promising area, potentially superior to any drugs, since by using the body's own immune system to prevent disease, vaccines do not have resistance problems.
Companies have already begun inoculating volunteers with dead staphylococcus in order to harvest the antibodies these people produce. The antibodies can then be used to treat patients suffering from serious infections.
Other solutions being looked at are topical remedies like nose or ear drops, protecting like a vaccine by stimulating immune cells on mucous membranes, such nasal passages, to prevent infectious bacteria from entering the body to begin with.

However, pharmaceutical research is a lengthy and costly business. According to Pharmacia & Upjohn, the total cost of a new product is approximately $500 million over an average development period of 10-15 years. Established companies are reluctant to invest such sums in the insecure markets of either antibiotics that may be useless in five years or in untried domains like protein agents. Development of the above drugs are often left to smaller companies, eager for market shares, but having a harder time raising the money it takes.

The real solution to ARB may not involve more drugs at all, but indeed the opposite. Decades of excessive and improper use of antibiotics is what gave us ARB to begin with.

Says microbiology Professor Richard Lacey: "[Bacteria] don't want to become resistant-they do so in the face of needless over-prescribing by doctors."

No matter how hard it is to recommend a mother not to give her sick child a penicillin, it must be done if we are to reverse this process. Patients, too, must do their part by not demanding antibiotics whenever they feel ill and by not taking pills only seven days of a ten day course.

Likewise, prescriptions made on a "just in case" basis, often before it is ascertained that they will not be treating viral infections (to which antibiotics have no effect) must stop.

Sweden has come a relatively long way in the process of cutting down on antibiotics use and monitoring of MARB-outbreaks. The main reason for this, says Arvidson, is that the recommendations from socialstyrelsen are generally followed here.

"In America, doctors get sued if they refuse people antibiotics", he adds.

Even though it seems that fighting ARB by decreasing our massive consumption of antibiotics (see illustration)has its advantages over new research, more research is surely needed. But upon asking Arvidson about this, an unexpected answer is received.

"Millions of children die every year from different [bacterial] infections. Why? They have a bad standard of living, they have bad food, bad clothes, bad housing.

"There are huge problems in this world and, as a scientist, you should not believe that basic research will solve them all."

3. Conclusion

This project has dealt with the antibiotic resistance in bacteria and solutions to the current problem world-wide.

Section one, Bacteria, described the structure and composition of bacteria and how they infect our bodies with disease.

The second section explained how antibiotics work when they cure illness.

Section three was about the biological development of resistance and how the resistant bacteria so rapidly have colonized our society. It was found that the arising of resistance is perfectly natural but that mankind's massive use of antibiotics has distinguished the resistant bacteria and eliminated its competitors, thus helping them thus speeding their development.

Section four brought up some promising prospects a solution to the problem of MARB, but also found that none of these are of much unless we change our antibiotic consuming-habits.

It is evident that any solution must include both new research and a change in behavior. We must not forget that our bodies are adjusted to a life without antibiotics, vaccines or toothpaste. Our immune defense needs a little exercise, just like the muscles in our legs, even though we have cars and bicycles to take us everywhere and medicine to keep us healthy. Otherwise, we stand helpless when all cars break down or when antibiotics stop working.