Forschung

Louis Pasteur (1822 – 1895) the founder of microbiology refutes the theory of abiogenesis.

Due to convincing experiments Louis Pasteur could demonstrate the central role of microorganisms in metabolic processes as well as in the transmission of infectious diseases. Thus, he is to be seen as the founder of microbiology who successfully provided experimental evidence against the famous chemists of his time, Berzelius and Liebig, who still believed in the ancient theory of life (e.g. fermentation) being generated solely from abiotic material.

Robert Koch (1843-1910) –postulates for identifying microbial pathogens as causative agents of infectious diseases.

In succession of Pasteur’s ideas Robert Koch unambiguously demonstrated that a bacterial pathogen, Bacillus anthracis, is the causative agent of anthrax and consequently together with Henle coined the Koch-Henle’s postulates to identify a microbial pathogen as the causative agent of an infectious disease.

Paul Ehrlich (1854-1915) –„magic bullets“ as the first antimicrobial chemotherapeutics

In the following decades medical microbiologists focussed on the development of laboratory techniques for the isolation and identification of microorganisms as a major field of research. Among these researchers Paul Ehrlich, a physician and microbiologist, convincingly argued that specific chemical dyes which selectively bind to microbial cells to make them visible under a microscope could be modified to make them safely applicable for an in vivo use to combat the respective microorganism within the infected person. The idea of a selective antimicrobial chemotherapy by „magic bullets“ (a term coined by Ehrlich) was born. The first such magic bullet was salvarsan, an arsenic-containing compound which was used to treat sleeping sickness caused by Trypanosoma brucei.

Sir Alexander Fleming (1881 – 1955) and Gerhard Domagk (1895 -1864) – the founders of modern antibacterial therapy

In 1928 a fortuitous detection of the Scottish physician Alexander Fleming that the mould Penicillum notatum contaminating a culture of Staphylococcus aureus overgrows the bacteria led him to conclude the secretion of an „antibiotic“ compound by the mould capable of lysing the cells. This antibiotic was later called Penicillin. The first human application followed in 1941.

Following the ideas of Paul Ehrlich the German pathologist and industrial researcher Gerhard Domagk in 1932 developed a chemical dye, sulfachrysoidin, the prodrug of sulfanilamid the first sulphonamide to treat bacterial infections which for the first time was successfully used as a human therapeutic in 1935.

Edward Penley Abraham (1913 - 1999) and Ernest Boris Chain (1906 - 1979) – detection of the first penicillin-destroying enzyme as resistance mechanism

Antibiotic resistance in bacteria was a known problem before the introduction of the first antibiotic into therapy: In his early laboratory experiments Fleming already noticed that a proportion of clinical isolates of Staphylococcus aureus were able to grow in the presence otherwise inhibitory concentrations of penicillin. Pioneering work of Edward Abraham and Ernest Chain revealed the basic resistance mechanism of penicillin resistance in S. aureus – the production of a β-lactamase of the serine-protease family which hydrolyzes the antibiotic.

Selman Waksman (1888 – 1973) –discovering new antibiotics in nature

As one promissing strategy to combat antibiotic resistance scientists like Selman Waksman intensively searched for new antibiotics in nature. Inspired by findings of soil bacterial communities (predominantly composed of the genus streptomyces) as a rich source of antibacterial compounds many researchers from academic institutions as well as from industry isolated many thousand new antibacterial compounds. The natural role of antibiotics was supposed to be „microbial weapons“ in the competition for nutrients against other bacteria in the same ecosystem. From 1950 to 1970 most of the antibiotic classes which are in therapeutic use today or were introduced into clinical use until 2000 were detected from streptomycetes.

Antibiotic resistance – the „Hare and Tortoise“ game between humans and bacteria

From the beginning of the antibiotic era it was soon evident that the introduction of a new antibiotic was rapidly followed by the development of antibiotic resistance in at least some pathogens and in most cases by spreading of the genetic resistance traits to others. But it was not before the 1960s when molecular genetic research began to impact medical microbiology and antibiotic research to elucidate the molecular genetic mechanisms underlying the development and transfer of resistance traits between bacterial cells.

Antibiotic resistance – the phenomenon and three variations on a theme

Beside the enzymatic inactivation of antibiotics (1) bacteria have evolved mechanism to extrude and exclude antibiotics from the cell by increased active efflux (via membrane-penetrating molecular pumps) or decreased influx (via diminished formation of outer membrane-spanning pores- the porins), respectively (2), or to prevent the antibiotic from binding to the target site either by enzymatic or mutational alteration of the target or by hyper-production of a protecting structure (3). For mechanism (1) usually additional genetic material is acquired form other cells, while many mechanisms of type (2) and (3) are based upon genetic alteration of the microbes own genetic material.

Fluoroquinolones – the modern pharmaceutical chemist’s answer to antibiotic resistance

While an early approach to overcome antibiotic resistance was the chemical modification of natural antibiotic structures of already in-use antibiotics, the easy adaptibility of bacteria to these compounds required other strategies. The introduction in the mid 1980ies of first representatives of the purely synthetic fluoroquinolones which showed excellent bactericidal activity against a broad range of bacteria at very low concentrations was a major step ahead to a world without resistance. The early success lasting more than a decade made fluoroquinolones the new „magic bullets“ which seemed to be unaffected by mechanisms of clinical resistance.

Fluoroquinolone resistance - nature’s answer to pharmaceutical chemists

During the first 15 years of clinical use transferable resistance was not detectable and bacteria displaying clinically relevant fluoroquinolone resistance levels were rarely isolated from patients under therapy. This led to the optimistic view that fluoroquinolones can be used unrestricted not only in human therapy, but also in veterinary medicine. Unmindful use combined with an apparent lack of mechanisms for enzymatic degradation resulted in an accumulation of fluoroquinolones in environmental sites and, thus, is supposed to increase selective power for bacteria expressing fluoroquinolone resistance genes. Since the end of the last millennium first reports on transferable genes encoding fluoroquinolone resistance were reported.

The Heisigs‘ group approach to uncover the molecular basis of fluoroquinolone resistant clinical isolates

Fluoroquinolone resistance – from mechanisms to processes: 

Starting in 1988 early research in our laboratory to isolate highly fluoroquinolone (fq) -resistant mutants of normally very susceptible Escherichia coli strains revealed

(1) At least three selection steps are necessary to obtain high-level resistance to fq

(2) Gyrase-associated fq resistance is not transferable

(3) At least two mutations very close to each other affecting the putative binding pocket of the target enzyme DNA gyrase – a bacterial type II topoisomerase – are required, but not sufficient for high-level fq resistance.

(4) The presence of the same mutations in highly resistant clinical isolates and in the laboratory mutants indicated the significance of target alterations.

(5) Introducing the double mutation into a naturally susceptible – wildtype (WT) isolate did not result in high-level fq resistance indicating the presence of a second drug target.

(6) A genetic test based on the dominance of a fq susceptible allele in merodiploid cells carrying another fq resistant allele was developed and optimized to detect DNA gyrase-associated fq resistance mechanisms in a broad range of isolates of gram-negative bacteria.

(7) DNA sequence analysis of homologous genes encoding another type II-topoisomerase, i.e. topoisomerase IV (topo IV), led to the first detection of topo IV mutations associated with high fq resistance. These affect amino acids homologous those involved in DNA gyrase-mediated fq resistance.

(8) Sequence analysis and adoption of the genetic dominance test allowed for the identification of topo IV as a wide-spread secondary target of fq in most Gram-negative bacteria which was exploited for the development of new generations of fq antibiotics by the industry.

(9) Studying the intracellular accumulation of fq in susceptible and resistant bacteria revealed the involvement of mutations in the multiple antibiotic resistance (mar)-Operon leading to inactivation of the repressor MarR subsequently resulting in increased expression of transcriptional activator MarA which in turn induces mar-regulated genes such as acrAB-tolC encoding the multiple drug resistance (MDR) efflux pump AcrAB-TolC in E.coli.

(10) Extending the research to different clinically relevant enterobacteria, Pseudomonas aeruginosa and Acinetobacter baumannii led to the identification of species-specific combinations of known fq resistance mutations.

(11) The detection of at least four mutations acquired in only three selection steps raised the question of the underlying mechanism. As a potential mechanism the role of a transiently increased mutator activity of DNA-polymerase due to the transient reduction in proofreading activity by its ε subunit (dnaQ gene product) was investigated. Temporarily increased mutation rates during the process of fq resistance development were detectable in in-vitro mutants, but could not be demonstrated for clinical strains.

(12) Creating a set of isogenic mutants differing by combinations of chromosomal mutations in type II topoisomerase genes and mar with transferable resistance genes either qnr, encoding topoisomerase protection proteins (Qnr), aac(6‘)Ib-cr, encoding a fq modifying (= inactivating) enzym AAC6‘-Ib-cr or oqxAB, qepA encoding efflux pumps QepAB and QepA, respectively, revealed the dominating impact of chromosomal mutation on clinically fq resistance.

(13) A strong reduction in fitness determined as growth rate is observed for laboratory mutants carrying topo II gene mutations combined with marR mutations in comparison with clinical isolates displaying the same fq resistance mutations. This points to additional mutations acquired during the selection process which are required for the compensation of a fitness burden imposed by the fq resistance mutations.

(14) Gene replacement experiments combined with DNA-binding studies of isolated proteins identified mar mutations as the genetic cause for both reduced fitness and its compensation.

(15) Application of next-generation sequencing (NGS) technique helped to identify novel quinolone resistance stabilizing (qrs) mutations in yet unexplored gene loci associated with basic metabolic pathways in different laboratory mutants showing varying levels of fq resistance.

(16) As proof of principle a qrs mutation inactivating the tricarbonic acid (TCA) cycle, a central pathway not only in bacteria, and associated with a fitness burden and a slight increase in fq resistance, was complemented for by the wildtype gene resulting in fitness restoration.

(17) Current research focusses on identifying additional qrs mutations encoding potential novel drug targets by introducing into fq susceptible and resistant bacteria different combinations of qrs mutations with known fq resistance mutations.