Peter Giesbrecht

The rapid differentiation and identification of pathogenic bacteria using Fourier transform infrared spectroscopic and multivariate statistical analysis

Abstract A novel, spectroscopic approach to the differentiation and identification of pathogenic bacteria was elaborated. The method essentially takes advantage of the Fourier transform infrared spectra of intact microorganisms. Such bacterial ir-spectra could be recorded within minutes with ample sensitivity and excellent reproducibility when applying a highly standardized experimental procedure prior to ir-measurements. The comparison of the fingerprint-like bacterial ir-pattern yielded, on the basis of the first and/or second derivatives of selected spectral regions, the differentiation and identification of a variety of bacterial species when applying cross-correlation and correspondence analysis techniques. Some examples, comprising selected series of clinically relevant gram-positive and gram-negative bacteria, are quoted to demonstrate the possible range of application of the new techniques.

On the Morphogenesis of the Cell Wall of Staphylococci

Publisher Summary For several reasons, there is an increasing interest in studies concerning the morphogenesis of bacterial cell walls. Apparently, Escherichia coli is at the center of interest, but data on several gram-positive bacterial cell walls have also been published, and some excellent reviews on this subject exist. However, the morphogenesis of one of the more complicated and even rather exceptional cell-wall systems—that of staphylococci—deserves to be considered. This chapter discusses the morphogenesis of the staphylococcal cell wall under normal conditions and under the influence of some antibiotics, mainly chloramphenicol and penicillin. For all the studies discussed in the chapter, laboratory strains of Staphylococcus aureus were used, which proved to be especially sensitive to antibiotics and are therefore widely used as test strains for testing sensitivity to antibiotics. These strains are well known as S . aureus SG 511 Berlin and S . aureus SG 511 Hoechst. Much of the data presented in this chapter have not been published before. Several schematic drawings are presented, not only to summarize findings and to make results as clear as possible but also to initiate more precise questions and more sophisticated experiments that could lead to further progress in knowledge on this interesting subject.

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with14C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. “attack from the inside”). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but “stabilized protoplasts” were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.

Inhibition of wall autolysis of staphylococci by sodium polyanethole sulfonate "liquoid".

SummaryLiquoid (polyanethole sulfonate) was neither capable of influencing the growth nor the viability of staphylococci. But liquoid induced a suppression of the activity of different autolytic wall systems of normally growing staphylococci, i.e., autolysins which participate in cross wall separation as well as autolysins which are responsible for cell wall turnover. Additionally, the lysostaphin-induced wall disintegration of staphylococci was inhibited by liquoid.However, no indication could be found for a direct inhibition of lytic wall enzymes by liquoid; rather an interaction of liquoid with the target structure for the autolytic wall enzymes, the cell wall itself, was postulated. On the basis of the experimental data with the teichoic acid- mutant S. aureus 52A5 the sites of wall teichoic acid were supposed to be an important target for the binding of liquoid to the staphylococcal cell wall.

Elektronenmikroskopische Untersuchungen ber die Entwicklung der ?Chromatophoren? von Rhodospirillum molischianum Giesberger

SummaryThe chromatophores of Rhodospirillum molischianum originate de novo from the cytoplasmic membrane. Their laminar membranes arise by invagination of the cytoplasmic membrane and these have a circular shape. 5–15 of these membranes are piled up to form the chromatophores. Probably, the membranes of the chromatophores remain always connected with the cytoplasmic membrane by a tubular stalk.

A special morphogenetic wall defect and the subsequent activity of "murosomes" as the very reason for penicillin-induced bacteriolysis in staphylococci.

The actual reason for the penicillin-induced bacteriolysis of staphylococci was shown to be the “punching” of one or a few minute holes into the peripheral cell wall at predictable sites. These perforations were the result of the lytic activity of novel, extraplasmatic vesicular structures, located exclusively within the bacterial wall material, which we have named “murosomes”.In untreated staphylococci the punching of holes into the peripheral wall is a normal process which follows cross wall completion and represents the first visible step of cell separation. Under penicillin, however, analogous holes are punched by the murosomes at sites of presumptive cell separation even if no sufficient cross wall material had been assembled before at this site (but had rather been deposited at other sites). Consequently, because of the internal pressure of the protoplast, lytic death is the inevitable result of this perforation of the protective peripheral wall.Hence, the real mechanism of penicillin-induced bacteriolysis in staphylococci is considered to be mainly the result of a special morphogenetic wall defect: bacteriolysis is taking place regularly when a cell separation process is no longer preceeded by sufficient cross wall assembly at the correct place. However, hypotheses which are based purely on some variations of overall biochemical processes like total wall enzyme activities or total wall synthesis are not regarded to be sufficient to explain this type of lytic death.

Zero order kinetics of cell wall turnover inStaphylococcus aureus

Cells exponentially grown from four strains ofS. aureus (SG 511, H, 52A5G, and248 PN-1) and uniformly labeled in their walls with3H-N-acetylglucosamine, were found to turn over their old walls at constant rates of up to 25% per generation. Wall turnover was not observed to follow first order kinetics, thus ruling out the implication that maintenance of normal wall thickness was achieved by a random distribution of new wall components in the old wall. Instead, wall turnover in all cases strictly followed zero order kinetics, indicating that newly synthesized wall material was placed layer by layer beneath the inner surface of the old cell wall. This finding correlates with evidence obtained from earlier electron microscopic investigations into the regeneration of the staphylococcal cell wall after chloramphenicol treatment. Based on the experimental data presented, a simplified model for wall turnover of the growing staphylococcal cell was proposed. The model also takes into account the finding, derived from additional experiments with strainSG 511, that the total cell wall turned over at a somewhat higher rate than the old portions of the wall. The rates of cell wall turnover found inS. aureus SG 511 are the highest reported to date for pathogenic bacteria. The medical implications of this finding were discussed.

Neither an enhancement of autolytic wall degradation nor an inhibition of the incorporation of cell wall material are pre-requisites for penicillin-induced bacteriolysis in staphylococci

In contrast to what has been postulated, penicillin G at its optimal lytic concentration of 0.1 μg per ml did not lead to a detectable activation of autolytic wall processes in staphylococci in terms of the release of uniformly labelled wall fragments from cells pretreated with the drug for 1 h. Rather a considerable inhibition of this release was observed. A similarly profound inhibition of the release of peptidoglycan fragments occurred when staphylococci pretreated for 1 h with 0.1 μg penicillin per ml acted as a source of crude autolysins on peptidoglycan isolated from labelled normal cells of the same strain. This clearly demonstrated that the overall inhibition of autolytic wall processes caused by penicillin was mainly due to a decreased total autolysin action rather than to an altered wall structure. Furthermore, no substantial penicillin-induced inhibition of the incorporation of 14C-N-acetylglucosamine into the staphylococcal wall could be observed before bacteriolysis started, i. e., approximately during the first 80 min of penicillin action. These results are not consistent with any of the models hitherto proposed for the action of penicillin.

Initial data for the comparison of murein and pseudomurein conformations

In order to compare possible conformations of murein and pseudomurein from Methanobacterium thermoautotrophicum with one another (especially with respect to the peptide moiety), X-ray diffraction data, density measurements, and conformational energy calculations were used. All results obtained indicated a similar certain layer-like arrangement and similar ringshaped peptide backbone conformations, thus pointing to similar construction principles for the two polymers.

A novel, “hidden” penicillin-induced death of staphylococci at high drug concentration, occurring earlier than murosome-mediated killing processes

In log-phase cells of staphylococci, cultivated under high, “non-lytic” concentrations of penicillin G, there occurred a novel killing process hitherto hidden behind seemingly bacteriostatic effects. Two events are essential for the apprearance of this “hidden death”: (i) the failure of the dividing cell to deposit enough fibrillar cross-wall material to be welded together, and (ii) a premature ripping up of incomplete cross walls along their splitting system. “Hidden death” started as early as 10–15 min after drug addition, already during the first division cycle. It was the consequence of a loss of cytoplasmic constituents which erupted through peripheral slit-like openings in the incomplete cross walls. The loss resulted either in more or less empty cells or in cell shrinkage. These destructions could be prevented by raising the external osmotic pressure. In contrast, the conventional “non-hidden death” occurred only much later and exclusively during the second division cycle and mainly in those dividing cells, whose nascent cross walls of the first division plane had been welded together. These welding processes at nascent cross walls, resulting in tough connecting bridges between presumptive individual cells, were considered as a morphogenetic tool which protects the cells, so that they can resist the otherwise fatal penicillin-induced damages for at least an additional generation time (“morphogenetic resistance system”). Such welded cells, in the virtual absence of underlying cross-wall material, lost cytoplasm and were killed via ejection through pore-like wall openings or via explosions in the second division plane and after liberation of their murosomes, as it was the case in the presence of low, “lytic” concentrations of penicillin. Bacteriolysis did not cause any of the hitherto known penicillin-induced killing processes.