Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Antibiotic sensitivity testing of anaerobic bacteria – Workshop arranged by ESGARAB W4a and isoelectric points. This might be part of a broader Genetic background of carbapenem and programme of genetic control that alters the physiology to metronidazole resistance: detection of genes and protect and prevent metronidazole activation. Such alterations may also increase the virulence. mechanisms of expression M.E. Hedberg (Stockholm, S) W4b Antimicrobial resistance is increasingly common among anaer- Unexpected resistance mechanisms behind obic Gram-negative bacteria, especially for the Bacteroides known antibiotic resistance in anaerobic fragilis group. At present resistance has been described in pathogen and their detection this group of bacteria to almost all of the regularly used anti- L. Dubreuil, J. Behra, L. Calvet (Lille, F) anaerobic agents. Carbapenems and 5-nitroimidazoles are two of the most active antimicrobial agents against the B. fragilis Despite resistance to all b-lactams (including imipenem) by group, but isolates resistant to these drugs seem to appear production of a carbapenemase (cfiA gene), Bacteroides fragilis more frequently, although they still are quite rare. Metroni- group strains may be resistant to the combination of b-lactams dazole is often used as empiric therapy for anaerobic infections with b-lactamase inhibitor among imipenem susceptible and susceptibility testing is not always routinely performed. strains. If a silent carbapenemase could be involved, in other Strains resistant to carbapenems have been found to produce cases the lack of porin in combination with the production of metallo-beta-lactamase, an enzyme that hydrolyses various the chromosomal cephalosporinase (cep A gene) is responsible beta-lactam antibiotics and exhibits resistance to beta-lactamase for the resistance to co-amoxiclav among B. fragilis (lack of the inhibitors. B. fragilis metallo-beta-lactamase is encoded by the 45 kDa Outer Membrane Protein) or in B. thetaiotaomicron (lack cfiA gene. Several diverse types of insertion sequence (IS) of the 67 kDa OMP). In the last two years we were able to elements in the upstream region of cfiA in carbapenem- isolate metronidazole-resistant strains of B. fragilis (MIC from resistant B. fragilis strains have been found. The IS elements 16 to 256 mg/L). Their detection could be easily done using a most likely provide a promoter to express the cfiA. It has been disk-diffusion test if the incubation is prolonged for at least reported that about 2–4% of the B. fragilis strains carry the cfiA 48 hours. For one of these trains, the high-level metronidazole gene regardless of whether they express it. Also an IS-less resistance was associated with the presence of two copies of activation mechanism of cfiA in B. fragilis strains has been the nim A gene on the chromosome (Marchandin H. et al.). detected. The 5-nitroimidazole molecule is a prodrug whose Decreased susceptibility to metronidazole (MIC 8 and 16 mg/ activation depends up on the reduction of the nitro group in L) is still increasing in France and UK (Brazier et al.)as the absence of oxygen. Decreased uptake and/or alteration in demonstrated by antibiotic surveys. With exception of the reduction are believed to be responsible for metronidazole reference agar dilution method (NCCLS M11 A6), the detection resistance. Five nim genes (A–E) have been described in the B. remains problematic. Detection by disk-diffusion method is fragilis group that confer reduced susceptibility to 5-nitroimi- very difficult using 4 lg metronidazole disks and should be dazole compounds. nim-Positive strains have been shown to be improved by using a 10 lg potency disk, Rosco disks or the E more prone to induction than nim-negative isolates. A nimF test method. Resistance to metronidazole is widespread as nim gene has been detected in a metronidazole susceptible strain. genes are nowadays found in Prevotella and Veillonella species. Isolates lacking nim genes but still highly resistant to metroni- Intrinsic unknown resistance could be presumed from pub- dazole have also been demonstrated which probably means lished studies such as low-level resistance to vancomycin, that other resistance mechanisms are involved. Studies of linezolid and ramoplanin for C. ramosum, to vancomycin protein expression patterns in metronidazole susceptible and and daptomycin for C. innocuum and to teicoplanin and metronidazole resistant B. fragilis strains have revealed a ramoplanin for C. clostridioforme, respectively. With exception complex shift in the resistant strain resulting in both down- of C. innocuum (Leclercq et al.) most mechanisms had not been and up-regulation of proteins with different molecular weight investigated. History of medical microbiology in Denmark S8 The physiologist and epidemiologist Peter Panum (1820–85), the Danish medical microbiologists in the 19th bacteriologist Carl Salomonsen (1847–1927), and his assistant century: Panum, Salomonsen & Gram Christian Gram (1853–1938). Panum has gained international reputation for his epidemiological description of the measles H.J. Kolmos (Odense, DK) epidemic in the Faroe Islands in 1846, but he also made significant Three persons played a key role in the development of medical experimental contributions to microbiology. In 1855–56, during microbiology in Denmark in the second half of the 19th century: his appointment as professor at the University of Kiel, he 1 Abstracts performed a series of laboratory experiments with dogs, in order Hospital. It was extremely difficult to produce and to purify to characterize the so-called ‘putrescent poison’, a substance penicillin in sufficient amounts to be used to treat humans. It claimed to be the causative agent of blood-poisoning. The dogs was not until 1938 that Florey, Abraham and Chain succeeded were given i.v. infusions of rotten meat juice in different in isolating and purifying Penicillin in pure and stable form modifications, and observed for symptoms and signs. His thus allowing further studies of its unique activity, extremely observations on the biological effects of putrescent poison bear low toxicity, and high clinical efficiency. It was found that striking similarities with present-day endotoxins, e.g. the char- penicillin was a true wonder drug in treating infected acteristic symptoms and signs, the delay in onset of symptoms, wounds. The production in large scale was attempted in and the preserved biological activity after long-term boiling. England, but in 1941 It became necessary to move the entire Panum at first rejected the possibility that the putrescent poison project to USA to avoid the harassment caused by the German could be of bacterial origin, but later changed his opinion. From air raids. In USA the production of penicillin was considered 1864 Panum served as professor of physiology at the University of millitary importance The War Production Board in cooper- of Copenhagen, and in his institute he established the first ation with 22 medical companies united in the efforts to laboratory facilities for bacteriological studies in Denmark. produce penicillin in sufficient amounts for use by the allied Salomonsen was the first to perform microbiological examina- millitary forces and for civil use. All informations about the tions on an infected patient. They took place in 1873 at the production and purification processes were considered as war Municipality Hospital in Copenhagen, where he treated a patient secrets, and the penicillium strains used for production were with pyaemia. In pus aspirated from an inflamed knee joint he kept under strict custody. However, today it may not be was able to demonstrate streptococci, and he could also isolate the widely known, that Denmark was the only place outside streptococci from a rabbit that had been inoculated with pus from England and USA where the production of penicillin was the patient. In his thesis, which he wrote under the supervision of taken up during World War II. This fact is even more Panum from 1876–77, he described one of the earliest methods for remarkable because it took place during the German occupa- making pure cultures of bacteria, using capillary glass tubes. tion of Denmark, when exchange of information with the free From 1883, Salomonsen held Europe’s first chair in bacteriology world was practically impossible, and resources of nearly at the University of Copenhagen, and he played a leading role in every kind became increasingly scarce. The attempts to the foundation the State Serum Institute in 1902. Salomonsens produce penicillin in Denmark were started during the assistant, Gram, invented the Gram-stain during a short visit to spring of 1943 at the University Institute of General Pathol- Friedla¨nder’s laboratory in Berlin in 1884. He is probably the most ogy, and as early as September 1944 the first patient was famous of all Danish microbiologists, which is somewhat para- treated with Danish penicillin. This remarkable achievement doxical, since he left microbiology immediately after his inven- was made by the head of the institute professor K. A. Jensen. tion, and never worked with Gram staining afterwards. He came from the Danish Statens Serum Institute where he worked as a world-known scientist within the field of tuberculosis. He had experiences in standardization of biolo- S9 gical products and production of tuberculin and BCG vaccine, Serum treatment of diphtheria: a controlled and he was deeply engaged in research on antituberculous clinical trial performed in Copenhagen in 1898 chemotherapy. He was thus well equipped to start the A. Hro´bjartsson (Copenhagen, DK) research on penicillin when he in 1941 took over the chair in general pathology. The late owner of the Leo Pharmaceu- In 1898, the Danish Nobel laureate Johannes Fibiger published a tical Industry: Knud Abildgaard Elling initiated this research controlled trial of the effect of serum treatment on diphtheria. by providing him with a reprint obtained from Sweden of Fibiger was one of the first to discuss random allocation as a Florey, Abraham and Chain’s article about penicillin.