Shift in bacterial etiology from the CAPNETZ cohort in patients with community-acquired pneumonia: data over more than a decade.

To determine the most relevant pathogens for CAP in Germany, patients with radiologically confirmed pulmonary infiltrates and at least one clinical sign of lung infection were prospectively recruited within the CAPNETZ cohort from 2004 until 2016. In 990 out of 4.672 patients (21%) receiving complete diagnostics the most prominent change of pathogens was a decrease of S. pneumoniae (58% in 2004 to 37.5% in 2016; p ≤ 0.001, ρ = - 0.148) and an increase of H. influenzae (12.2% to 20.8%; p = 0.001, ρ = 0.104).

Prevalence and characterization of azole-resistant Aspergillus fumigatus in patients with cystic fibrosis: a prospective multicentre study in Germany.

Objectives: Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with cystic fibrosis (CF). The aim of this prospective multicentre study was to investigate the prevalence of azole-resistant A. fumigatus (ARAF) in respiratory secretions from CF patients across Germany and to characterize ARAF isolates by phenotypic and molecular methods. Methods: Twelve tertiary care centres from Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients were screened for ARAF by using azole-containing agar plates. Antifungal susceptibility testing of isolates was performed by broth microdilution according to EUCAST guidelines. Analysis of mutations mediating resistance was performed using PCR and sequencing of the cyp51A gene. Furthermore, genotyping by microsatellite PCR was performed. Results: Of a total of 2888 A. fumigatus isolates, 101 isolates from 51 CF patients were found to be azole resistant (prevalence per patient 5.3%). The Essen centre had the highest prevalence (9.1%) followed by Munich (7.8%), Münster (6.0%) and Hannover (5.2%). Most ARAF isolates (n = 89) carried the TR34/L98H mutation followed by eight G54E/R, one TR46/Y121F/T289A and one F219S mutation. In two isolates no mutation was found. Genotyping results showed no major clustering. Forty-five percent of CF patients with ARAF had previously received azole therapy. Conclusions: This is the first multicentre study analysing the prevalence of ARAF isolates in German CF patients. Because of a resistance rate of up to 9%, susceptibility testing of A. fumigatus isolates from CF patients receiving antifungal treatment should be part of standard diagnostic work-up.

Electron tomography and cryo-SEM characterization reveals novel ultrastructural features of host-parasite interaction during Chlamydia abortus infection.

Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.

Chlamydia trachomatis prevalence, genotype distribution and identification of the new Swedish variant in Southern Germany.

PURPOSE: In Germany, reliable data about the prevalence of urogenital Chlamydia trachomatis infections, causative genotypes, as well as corresponding clinical, demographic and behavioural information are sparse. We, therefore, performed a prospective prevalence study including 1,003 sexually active volunteers of a Southern German city. METHODS: Study participants completed a standardised questionnaire and provided first void urine samples for analysis. Our screening strategy included the performance of two nucleic acid amplification tests with different target genes, enabling the detection of the new Swedish variant of C. trachomatis (nvCT). Direct genotyping of positive specimens was performed by sequence analysis of the ompA gene. RESULTS AND CONCLUSION: The overall prevalence of C. trachomatis infection was 4.2 % in women and 4.6 % in men. A relatively high prevalence of 8.3 % was found in men older than 25 years. Never using condoms was an independent risk factor for infection. The most common symptom was discharge; however, 64.5 % of infected females and all of the infected men were asymptomatic, supporting the need for screening programmes. The most frequently encountered genotypes were E (46.5 %), F (20.9 %) and K (14.0 %). Since the nvCT was detected in one female student, this is one of the rare studies that reports on the molecular identification of nvCT apart from Sweden.

Performance of a novel microarray multiplex PCR for the detection of 23 respiratory pathogens (SYMP-ARI study).

Symptoms of acute febrile respiratory tract infection are often unspecific, but the rapid identification of pathogens allows optimised patient management. The objective of this study was to evaluate a novel multiplex polymerase chain reaction (PCR) suspension microarray which detects 19 viral and four atypical bacterial targets. A comprehensive set of sensitive monoplex real-time PCR assays was used for each pathogen as the gold standard. A panel of archived as well as 300 prospectively collected clinical samples was analysed by both methods. At least one target was detected in 165/300 (55 %) samples by monoplex PCR and in 140/300 (46 %) samples by multiplex PCR, respectively. The positivity rate was significantly higher in paediatric patients compared to adults [126/154 (82 %) vs. 39/146 (27 %) by monoplex and 114/154 (74 %) vs. 26/146 (18 %) by multiplex PCR, respectively]. Among all samples, 17/300 (5.6 %) were positive for atypical bacteria by monoplex and 8/300 (2.6 %) by multiplex PCR, respectively. Multiple detections were recorded in 35/300 (11.6 %) samples by monoplex and 26/300 (8.7 %) by multiplex PCR. For the most common pathogens, the sensitivity ranged from 57 to 93 % and the specificity ranged from 95 to 100 %. The overall concordance between both methods was 77 % [95 % confidence interval (CI) 72-81 %]. False-negative results by multiplex PCR were mainly due to the low target concentration. Compared to monoplex PCR, the novel microarray assay proved its principle but displayed overall lower sensitivities, potentially restricting its use to paediatric patients. For some targets, only small numbers of positive samples were available, requiring larger studies to firmly assess the sensitivity and specificity.

[Interdisciplinary management of a large Legionella outbreak in Germany]. / Interdisziplinäres Management eines länderübergreifenden Legionellenausbruchs.

Between December 2009 and the end of January 2010, the largest hitherto known outbreak of Legionella in Germany took place in the cities of Ulm and Neu-Ulm. Of a total of 64 patients involved, 60 patients had to be hospitalized, and 5 patients died from the infection. This event was caused by a wet cooling tower of a large air conditioning system in the city center of Ulm. The search for the source of the Legionella emission was extremely difficult, since these plants are neither notifiable nor subject to authorization in Germany. We report about the search for the source and the measures to control the outbreak. We also discuss communication and coordination during these investigations. Regulatory measures as proposed by the World Health Organization (WHO) and the European Network for Legionellosis (EWGLI) and already implemented in numerous other European countries would be desirable to prevent such outbreaks in the future.

Preliminary report: outbreak of Legionnaires disease in the cities of Ulm and Neu-Ulm in Germany, December 2009 - January 2010.

Currently an investigation is ongoing to explore and control an outbreak of Legionnaires disease, affecting 65 people as of 22 January 2010, in the cities of Ulm and Neu-Ulm, south-west Germany. A hitherto unidentified wet cooling system in these twin cities is considered as the most likely source of infection.

Kinetics of bidirectional active sodium fluxes in the toad bladder.

The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current I and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at deltapsi=0, 25, 50, 75 or 100 mV. Subsequent elimination of active Na+ transport with mucosal amiloride permitted calculation of the rates of active Na+ transport JaNa and active and passive influx leads to JaNa and leads to JpNa. The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence JaNa=Ja. Ja was abolished at deltapsi=E=96.3+/-1.9 (S.E.) mV. As deltapsi approached E, active efflux comes from Ja became demonstrable. At deltapsi=100 mV, comes from Ja exceeded leads to Ja, so that Ja was negative. Experimental values of leads to Ja agreed well with theoretical values predicted by a thermodynamic formulation: leads to Jaexp=0.985 leads to Jatheor (r=0.993). The dependence of leads to Ja on deltapsi is curvilinear.

Ion tracer flows and flux ratios in heterogeneous membranes.

Ion tracer flows and flux ratios at various electrical potentials were studied in heterogeneous membranes comprising parallel pathways of different intrinsic resistance. The total resistance to net flow exceeded the tracer exchange resistance, and the flux ratio was abnormal, as in exchange diffusion commonly attributed to a mobile carrier.

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder.

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations. With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.

Effects of antidiuretic hormone on kinetic and energetic determinants of active sodium transport in frog skin.

The effects of antidiuretic hormone (ADH) on the rate of transepithelial active Na transport JaNa and the rate of suprabasal O2 consumption of Jsbr were studied in paired hemiskins of frog. Within some 30 min following administration of ADH both JaNa and Jsbr increased to near-maximal levels and then remained stable for at least an hour. On symmetric perturbation of the transepithelial electrical potential delta psi at 6-min intervals, the dependence of JaNa and Jsbr on delta psi was near-linear, both in control and experimental hemi-skins. The stability and near-linearity of the system permitted systematic analysis of the parameters of linear non-equilibrium thermodynamic (NET) and electrical equivalent circuit (EC) formulations. ADH (100 mU/ml) stimulated two of the three NET phenomenological L coefficients, as well as A, the affinity (negative Gibbs free energy) of a metabolic reaction driving transport. Observations at partially depressed levels of transport indicated that the effects of kinetic and energetic factors are to some extent discrete. EC analysis showed stimulation of the amiloride-sensitive conductance Ka, but not of the apparent electromitive force of Na transport 'ENa'. Similar effects were produced by 10 mU/ml of ADH or by 10 mM dibutyryl cyclic AMP, although less marked effects on the L coefficients were noted with the lower concentration of hormone. It is suggested that, in contrast to EC analysis, the NET formulation distinguishes between kinetic and energetic determinants of transport, supporting a dual mechanism of action of ADH.

The thermodynamic degree of coupling between metabolism and sodium transport in frog skin.

The tightness of coupling between two processes is advantageously evaluated by the thermodynamic degree of coupling q, varying in absolute value from zero for uncoupled processes to unity for processes which are related stoichiometrically. Two methods for the determination of q in the active pathway in frog skin have been developed, employing amiloride to abolish active sodium transport. The values of q in 6 frog skins varied, but were always less than unity (mean 0.79 +/- 0.06 S.E. according to one method, 0.78 +/- 0.06 S.E. according to the other). This indicates that metabolism and sodium transport are incompletely coupled in this tissue even when passive transepithelial leakage pathways are taken into account.

Action of aldosterone on frog skin in the presence and absence of in vitro molting effects.

The molting which occurs in frog skin following exposure to high concentrations of aldosterone interferes with the interpretation of physiological measurements. Exposure of skins from frogs maintained in standard smooth tanks to 5 - 10(-7) M aldosterone caused within a few hours erratic responses in short-circuit current Io and conductance K followed by sustained stimulation of Io and K; 10(-8) M aldosterone caused only stimulation of Io and K. Storage of frogs in "rojgh tanks" eliminated in vitro molting on exposure to 5 - 10(-7) M aldosterone. IO and K were then superimposable for 3 h, after which Io increased far more rapidly than K. These results are consistent with an early effect on permeability of the active pathway and later effects on metabolism, either a direct effect on the pump or enhanced interaction between transport and metabolism.

A comparison of the effects of ouabain and 2-deoxy-D-glucose on the thermodynamic variables of the frog skin.

Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na-+ transport and oxygen consumption Jr to the electrical potential difference delta-psi and the affinity A (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na-+ transport. Ouabain, a specific inhibitor of the Na-+ pump, might be expected to diminish the dependence of Jr on delta-psi without affecting A, whereas 2-deoxy-D-glucose, a competitive inhibitor of glucose metabolism should be expected to diminish A. Both inhibitors were used at concentrations adequate to depress Na-+ transport (i.e. short-circuit current Io) to some 50% of control level. Measurements were made of Io and dJr/d(delat-psi), and the apparent value of the affinity Aapp was calculated according to the thermodynamic formulation. Ouabain depressed minus dJr/d(delta-psi) without affecting Aapp whereas 2-deoxy-D-glucose depressed Aapp without affecting minus dJr/d(delta-psi). The demonstration of these effects indicated the utility of the formalism.

Sodium transport and oxygen consumption in toad bladder. A thermodynamic approach.

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference delta phi. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (delta phi = 0) and when delta phi was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na/O2 was constant for each tissue, but varied among different tissues. Only when delta phi was varied did the ratio Na+/O2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)delta phi = 0[. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and intepreted in terms of a simple model of the transepithelial sodium transport system.

Energetics of sodium transport in frog skin. I. Oxygen consumption in the short-circuited state.

Sodium transport and oxygen consumption were studied simultaneously in the short-circuited frog skin. Sodium transport was evaluated from I(o)/F, where I(o) is the short-circuit current measured with standard Ringer's solution bathing each surface and F is the Faraday constant. Oxygen tension was measured polarographically. Under a variety of circumstances the rate of oxygen consumption from the outer solution exceeded that from the inner solution, the ratio being constant (0.57 +/- 0.09 SD). Both I(o) and the associated rate of oxygen consumption J(ro) declined nonlinearly with time, but the relationship between them was linear, suggesting that the basal oxygen consumption was constant. For each skin numerous experimental points were fitted by the best straight line. The intercept (J(ro))(Io=0) then gave the basal oxygen consumption, and the slope dNa/dO(2) gave an apparent stoichiometric ratio for a given skin. The basal oxygen consumption was about one-half the total oxygen consumption in a representative untreated short-circuited skin. Values of dNa/dO(2) in 10 skins varied significantly, ranging from 7.1 to 30.9 (as compared with Zerahn's and Leaf and Renshaw's values of about 18). KCN abolished both I(o) and J(ro). 2,4-dinitrophenol (DNP) depressed I(o) while increasing J(ro) four- to fivefold. Anti-diuretic hormone stimulated and ouabain depressed both I(o) and J(ro); in both cases apparent stoichiometric ratios were preserved.

Energetics of sodium transport in frog skin. II. The effects of electrical potential on oxygen consumption.

Studies were made of the dependence of the rate of oxygen consumption, J(r), on the electrical potential difference, Deltapsi, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Deltapsi. In fresh skins J(r) was a linear function of Deltapsi over a range of at least +/-70 mv. Treatment with aldosterone stimulated the short-circuit current, I(o), and the associated rate of oxygen consumption, J(ro), and increased their stability; linearity was then demonstrable over a range of +/-160 mv. Brief perturbations of Deltapsi (+/-30-200 mv) did not alter subsequent values of I(o). Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I(o); positive clamping induced the opposite effect. Changes in J(ro) were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I(o). The linearity of J(r) in Deltapsi indicates the validity of analysis in terms of linear nonequilibrium thermodynamics-brief perturbations of Deltapsi appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.

Genetic evidence suggests that Psoroptes isolates of different phenotypes, hosts and geographic origins are conspecific.

The second internal transcribed spacer of the rRNA gene was characterised in 15 Psoroptes isolates collected from the ears or bodies of rabbits, goats, sheep and cattle originating from four continents. Morphologically, the isolates were differentiated as Psoroptes cuniculi, Psoroptes ovis and Psoroptes cervinus. Genotypically, the isolates were highly homogeneous, except for the existence of different rDNA classes. In view of previous phenotypic data, a possible conspecificity of these species is proposed.

Molecular analyses suggest monospecificity of the genus Sarcoptes (Acari: Sarcoptidae).

To clarify the taxonomic status of mites of the genus Sarcoptes, the second internal transcribed spacer (ITS-2) of the rRNA gene, as well as phenotypic characters, were investigated in 23 isolates from nine host species in four continents. Phenotypic differences among isolates were observed, but the range of variation within each isolate precluded the differentiation of individual mites. Genotypically, there was no delimitation between distinct genotypic groups and no correlation with host species or geographic origin was evident. These results support the conspecificity of the mites investigated and confirm the view that the genus Sarcoptes consists of a single, heterogenous species.

Cleavage of tumor necrosis factor-alpha by Legionella exoprotease.

The role of the major secretory protein of Legionella pneumophila, a zinc protease, in Legionella infection is not known. Since an important step of the host reaction in Legionnaires' disease is the production of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages, we studied the interaction of Legionella protease and U-937 cells with respect to TNF-alpha. The Legionella protease was purified by fractionated precipitation, gel filtration and hydrophobic interaction chromatography. The purified enzyme was added to U-937 cells, a promyelocytic cell line. In the supernatants of PMA-treated U-937 cells we found low concentrations of TNF-alpha after incubation with protease. Therefore we pursued the hypothesis of direct enzymatic degradation of TNF-alpha by Legionella protease. Enzymatic cleavage of TNF-alpha was proven by SDS-PAGE, ELISA and TNF-alpha bioassay with L-929 cells. The degradation of TNF-alpha by the Legionella protease was shown in all three systems. Enzymatic degradation of TNF-alpha might be important for the pathogenesis of Legionnaires' disease.