Tedizolid susceptibility in linezolid- and vancomycin-resistant Enterococcus faecium isolates.

Vancomycin-resistant enterococci (VRE) are of ever-increasing importance, most notably in high-risk patient populations. Therapy options are often limited for these isolates, and apart from tigecycline and daptomycin, oxazolidinone linezolid is frequently administered. The broad usage of linezolid, however, has driven the emergence of linezolid-resistant VRE strains (LR-VRE), further shortening therapeutic options. Second-generation oxazolidinone tedizolid has the advantage of being active against a specific subset of LR-VRE, i.e. isolates expressing the plasmid-encoded chloramphenicol-florfenicol resistance (cfr) gene. Here we tested tedizolid activity in a collection of 30 LR Enterococcus faecium VRE (MIC range 32-256 mg/l) isolated between 2012 and 2015 from clinical and screening specimens. By pulsed field gel electrophoresis (PFGE) isolates were assigned to 16 clonal lineages. In three cases, linezolid-susceptible progenitor isolates of LR-VRE were isolated, thus demonstrating the de-novo emergence of the linezolid-resistant phenotype. PCR did not detect cfr, cfr(B) or novel oxazolidinone resistance gene optrA in LR-VRE. All isolates, however, carried mutations within the 23S rDNA. Compared to linezolid, tedizolid MICs were lower in all isolates (MIC range 2-32 mg/l), but remained above the FDA tedizolid breakpoint for E. faecalis at 0.5 mg/l. Thus, related to the predominant resistance mechanism, tedizolid is of limited value for treatment of most LR-VRE and represents a therapeutic option only for a limited subset of isolates.

Improved detection of bacterial central nervous system infections by use of a broad-range PCR assay.

A universal PCR assay for bacteria and fungi detected meningitis pathogens in 65% of 20 cerebrospinal fluid (CSF) samples from patients with suspected central nervous system (CNS) infections compared to a 35% detection rate by culture and/or microscopy methods. Thus, the PCR assay can improve the diagnosis rate of infective meningitis when standard methods provide a negative result.

Combined ramR mutation and presence of a Tn1721-associated tet(A) variant in a clinical isolate of Salmonella enterica serovar Hadar resistant to tigecycline.

A Salmonella enterica serovar Hadar strain resistant to tigecycline (MIC, 16 microg/ml) was isolated. Molecular characterization revealed the presence of a plasmid-borne tet(A) variant associated with Tn1721 mediating a rise of the MIC for tigecycline when transferred to Escherichia coli. Additionally, a truncating mutation in ramR was detected. Transformation with wild-type ramR but not with the mutated ramR lowered the MIC for tigecycline. Characterization of this Salmonella isolate implicates ramR in resistance to tigecycline.

ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to tigecycline.

Five Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline (MIC, 2 microg/ml) were analyzed. A gene homologous to ramR of Salmonella enterica was identified in Klebsiella pneumoniae. Sequencing of ramR in the nonsusceptible Klebsiella strains revealed deletions, insertions, and point mutations. Transformation of mutants with wild-type ramR genes, but not with mutant ramR genes, restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. Thus, this study reveals a molecular mechanism for tigecycline resistance in Klebsiella pneumoniae.

Rapid identification of the vanA/vanB resistance determinant in Enterococcus sp. from blood cultures using the Cepheid Xpert vanA/vanB cartridge system.

Especially in immunocompromised and intensive care patients VRE sepsis is associated with high mortality. The GeneXpert vanA/vanB assay is marketed for fast molecular surveillance of VRE colonization in peri-anal and rectal swabs. The aim of this study was to evaluate this assay for its usefulness for rapid identification of the vanAB determinant from positive blood cultures. During an evaluation phase, 33/34 blood cultures (negative = 13; vanA = 1; and vanB = 19) were correctly identified. In the validation phase 205/211 blood cultures were correctly identified (negative, n = 160; vanA, n = 2; vanB, n = 43). Sensitivity and specificity calculated from valid tests was 100% (95% CI: 90.2-100%) and 100% (95% CI: 97.1-100%), respectively. The error rate was 2.8%. The Xpert vanA/vanB cartridge is a reliable tool in the rapid molecular identification of the vanA and vanB determinant from positive blood cultures with moderate inhibition rates (2.8%) and high PPV and NPV. However, additional methods for species identification are required.

[The first COVID-19 hotspot in a retirement home in Hamburg]. / Der erste COVID-19-Hotspot in einer Hamburger Senioreneinrichtung: Präventionskonzept, Letalität und Obduktionsbefunde.

Background: Coronavirus disease 2019 (COVID-19), a disease caused by the new coronavirus (SARS-CoV-2), is a particular threat to old people. At the end of March 2020, the first and so far largest outbreak of the disease occurred in a retirement home in Hamburg. Methods: Analysis of procedures in dealing with a residential unit affected by SARS-CoV­2, accommodating a risk group of 60 seniors with dementia is presented as well as a detailed presentation of post-mortem examination results of all 8 deceased tested positive for SARS-CoV­2. Results: Out of 60 residents, 39 were infected by SARS-CoV­2. Due to preventive procedures it was possible to stop further spreading of the infection to other residential areas. In all 8 fatal cases, the autopsy diagnosis was death due to COVID-19. Autopsies revealed all COVID-19 patients to have a fatal (broncho)pneumonia and signs of relevant pre-existing cardiac, renal and pulmonary conditions in all cases. In 75% (n = 6) of the cases a fresh venous thrombosis was found. In 66.7% (n = 4) of the cases thrombotic events were combined with peripheral pulmonary artery thromboembolisms. Conclusion: The cohort of SARS-CoV­2 infected residents of a nursing home is characteristic for clinical and epidemiological features of the new coronavirus disease. Due to a centralized evaluation of all fatalities at the Institute of Legal Medicine in Hamburg, a detailed examination of all deceased positive for SARS-CoV­2 was possible. Thereby, increased case fatality rates of approximately 20% could in all cases be assigned to a relevant number of pre-existing comorbidities of multiple organ systems, which was consistent with the clinical data available.

Expression and functional characterization of gfpmut3.1 and its unstable variants in Staphylococcus epidermidis.

Reporter gene systems are an invaluable tool for investigation of gene transcription activity in eukaryotes and prokaryotes. In order to analyze the temporal and spatial resolution of gene expression patterns in situ and for quantitatively investigating gene expression, the green fluorescent protein (GFP) appears to be especially useful. GFP has been broadly used in various bacterial species, however, there is only limited knowledge about key biological properties in S. epidermidis. Here, the crucial influence of different ribosomal binding sites (RBS) on gfpmut3.1 translation initiation in S. epidermidis 1457 is demonstrated. Only by using the RBS of the delta-hemolysin promoter, after 24 hours a strong fluorescence signal was obtained. The half-life of GFPmut3.1 in S. epidermidis 1457 was significantly shorter than in E. coli (7 h vs. 24 h). GFPmut3.1 derivatives with shorter half-lives (GFP(AAV) and GFP(ASV)) did not reach sufficient quantitative protein levels, and the resulting low fluorescence limits their use as reporter genes in S. epidermidis. This work provides fundamental insights into gfpmut3.1 expression in S. epidermidis and describes the crucial determinants of its biological behavior in this species. In general, this study underlines the need to accurately characterize key biological properties of this transcription marker in gram-positive hosts.

Modulation of Rho GTPases by type III secretion system translocated effectors of Yersinia.

Pathogenic species of the bacterial genus Yersinia subdue the immune system to proliferate and spread within the host organism. For this purpose yersiniae employ a type III secretion apparatus which governs injection of six effector proteins ( Y ersinia outer proteins; Yops) into host cells. Yops control various regulatory and signalling proteins in a unique and highly specific manner. YopE, YopT, and YpkA/YopO modulate the activity of Rho GTP-binding proteins, whereas YopH dephosphorylates phospho-tyrosine residues in focal adhesion proteins. Furthermore, YopP/YopJ and YopM affect cell survival/apoptosis and cell proliferation, respectively. In this review the focus will be on the biochemistry and cellular effects of YopT, YopE, YopO/YpkA, and YopH.

Modulation of Rho GTPases and the actin cytoskeleton by YopT of Yersinia.

Pathogenic Yersinia species evade the innate cellular immune response by injecting antihost effector proteins (Yersinia outer proteins, Yops) into host cells through a type III secretion (TTS) apparatus. One of the six effector Yops, YopT, inactivates the small GTPase RhoA by removing the geranylgeranylated C-terminal cysteine. This cleavage results in release of RhoA from the cell membrane and subsequently in blockage of stress fiber formation. Thus YopT impairs cellular functions associated with cytoskeleton rearrangements.

Tropheryma whipplei in children with diarrhoea in rural Ghana.

Tropheryma whipplei has been hypothesized to be able to cause diarrhoea, but data from young children are scarce. In this hospital-based case-control study 534 stool samples of children aged between 2 months and 15 years from rural Ghana were analysed for the presence of T. whipplei. Overall stool prevalence of T. whipplei was high (27.5%). Although there was no difference in T. whipplei carriage overall between cases and controls, cases aged between 0 and 12 months carried T. whipplei in their stool twice as often as controls without diarrhoea. The results from this study may support the hypothesis that T. whipplei can cause diarrhoea in first-time infection.

Cooperative effects of interferon-gamma on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells.

The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-D3) or human recombinant interferon-gamma (IFN-gamma) on the induction of NADPH oxidase was studied in premonocytic U937 cells. Differentiation with the combination of either RA (1 microM) or 1,25-D3 (10 nM) with IFN-gamma (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O2-) generation in response to stimulation with phorbol myristate acetate (PMA, 100 nM). Induction of NADPH oxidase activity was preceded by increases in mRNA levels of p47-phox, p67-phox and gp91-phox, which encode three subunits of the enzyme, and immunoblot analysis of the p47-phox and p67-phox proteins revealed that the increases in mRNA levels were equally reflected by increases in protein levels. In contrast, RA, 1,25-D3 or IFN-gamma alone did not induce NADPH oxidase activity which correlated with their failure to increase p67-phox and gp91-phox mRNA levels. The mRNA of p21 rac1, a GTP-binding protein that regulates NADPH oxidase activity in macrophages, was constitutively expressed in undifferentiated cells and was not affected by differentiation. These data indicate that induction of a functional NADPH oxidase in premonocytic U937 cells requires the cooperative actions of IFN-gamma plus RA or 1,25-D3 and is reflected in the increased expression of p67-phox and gp91-phox.

1,25-(OH)2 vitamin D-3 enhances Paf-stimulated calcium mobilization in U937 cells by expression of specific Paf binding sites.

The effect of 1,25-(OH)2 vitamin D-3 (10 nM, 72 h) on cytosolic free Ca2+ concentration ([Ca2+]i) in U937 cells before and after stimulation with Paf, LTD4 and ADP was investigated. 1,25-(OH)2 vitamin D-3 increased basal [Ca2+]i from 98 +/- 1 nM to 121 +/- 5 nM (P less than 0.01) and the Paf (10 nM) stimulated increase in [Ca2+]i from 143 +/- 15 to 406 +/- 44 nM (P less than 0.01). These vitamin D-3 effects were time-related and occurred after 24 h (basal [Ca2+]i) and 12 h (Paf stimulated Ca2(+)-mobilization) but not after 3 h. In comparison, vitamin D-3 failed to modulate Ca2(+)-mobilization in response to ADP (1-40 microM) and increased it only in response to low leukotriene D4 concentrations (0.1-1 nM). The total binding of [3H]Paf (2.8 nM) was not significantly different in untreated vs. vitamin D-3-treated cells. However, the Paf receptor antagonist Web 2086 (1 microM) inhibited [3H]Paf binding only in vitamin D-3-treated cells. The specific binding reached a plateau of 28 +/- 3 fmol per 2.5.10(6) cells between 1.4 and 2.8 nM [3H]Paf. The Paf receptor antagonist Web 2086 (1-1000 nM) also inhibited the Paf-mediated Ca2(+)-mobilization in vitamin D-3-treated cells (IC50 = 191 +/- 55 M). These data suggest that the enhanced Ca2(+)-mobilization in vitamin D-3-treated U937 cells in response to Paf is mediated by an expression of putative Paf receptors.

Involvement of the GTPase Rho in the cellular uptake of low density lipoprotein by human skin fibroblasts.

Members of the Rho subfamily of small GTPases have been implicated in the regulation of endocytosis of ligand/receptor complexes localised to clathrin-coated pits. In this paper, we investigated the role of Rho A in the post-receptor regulation of cellular uptake and metabolism of native low density lipoprotein (LDL) by primary human skin fibroblasts. Incubations of cells with the selective Rho GTPase inhibitor C3-transferase (C3T) upregulated the binding, lysosomal degradation and cell accumulation of labelled LDL. The rate of internalisation of surface-bound LDL was also higher in C3T-treated cells. Single cell injections with C3T and dominant active V14Rho confirmed the negative regulation of LDL uptake by Rho. While cells injected with C3T internalised more 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL, diI-LDL internalisation was dramatically suppressed in cells injected with the constitutively active V14Rho. The negative regulation of LDL uptake by Rho appeared to be independent of changes in the actin cytoskeleton. An increasing number of naturally occurring toxins and serum factors have been shown to influence Rho GTPase signalling cascades. The herein described post-translational regulation of LDL internalisation may modulate cell events occurring subsequent to cellular lipoprotein uptake.

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells.

We investigated the effect of cellular cholesterol content on platelet activating factor (PAF)-stimulated Ca2+ mobilization in the human monocytic cell line U937. When cholesterol auxotroph U937 cells were depleted of cellular cholesterol by a 48-h incubation in delipidated medium, a 40% reduction in the PAF (100 nM)-stimulated increase in cytosolic Ca2+ concentration was seen. Ca2+ mobilization following stimulation with LTD4 (10 nM) or ATP (10 microM) was not affected. Addition of LDL (100 micrograms/ml, 24 h) to the delipidated medium completely recovered cellular cholesterol content and PAF-induced Ca2+ mobilization. These two LDL effects had very similar time- and dose-dependences. Partial recoveries of PAF-induced Ca2+ mobilization, seen after addition of pure cholesterol dissolved in ethanol (30 micrograms/ml, 24 h) or acetyl-LDL (100 micrograms/ml, 24 h), were associated with partial recoveries of cellular cholesterol content. Our results indicate that cellular cholesterol influences PAF-stimulated events in monocytic cells.

Docosahexaenoic acid inhibits PAF and LTD4 stimulated [Ca2+]i-increase in differentiated monocytic U937 cells.

We investigated the effects of different polyunsaturated fatty acids (PUFAs) of the n-6 and n-3 family on the PAF and LTD4 stimulated increase in cytosolic free Ca(2+)-concentration [Ca2+]i in retinoic acid (RA) differentiated, human monocytic U937 cells. Docosahexaenoic acid (10 microM DHA) reduced the PAF induced increase in [Ca2+]i from 455 +/- 25 nM to 319 +/- 24 nM (P less than 0.01). DHA also significantly attenuated the LTD4 induced increase in [Ca2+]. However [Ca2+]i-increase stimulated by f-MLP, ATP, or ionophore A 23187 was not affected by DHA. Other PUFAs like eicosapentaenoic acid (EPA), alpha-linolenic acid (LnA), arachidonic acid (AA) or gamma-linoleic acid (LA) were ineffective. Cellular differentiation as assessed by nitrobluetetrazolium reduction and enhanced expression of specific PAF binding sites in RA treated cells were not altered by DHA. Fatty acid composition in cellular phospholipids revealed effective incorporation of each PUFA. The DHA-effect on [Ca2+]i was time dependent and occurred at 48 h, whereas the DHA-content in phospholipids reached a plateau already at 24 h. The antioxidant vitamin E, the lipoxygenase inhibitor NDGA and the cytochrome P-450 inhibitor SKF 525A completely prevented the DHA induced reduction of PAF stimulated [Ca2+]i-increase. In contrast, the cyclooxygenase inhibitor indomethacin had no effect. Our results indicate that DHA selectively reduces intracellular [Ca2+]i-increases induced by PAF and LTD4 in RA-treated U937 cells, presumably involving an oxidative modification of DHA.

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C.

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.

Arachidonic acid increases activation of NADPH oxidase in monocytic U937 cells by accelerated translocation of p47-phox and co-stimulation of protein kinase C.

Arachidonic acid (AA) has been implicated as an important amphiphilic co-factor in the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neutrophils and reconstituted cell-free systems. To assess the role of AA in the activation of O2- generation in monocytic cells, we studied pre-monocytic U937 cells differentiated with 1,25-(OH)2-vitamin D3 plus interferon-gamma (IFN-gamma). AA dose-dependently enhanced phorbol myristate acetate (PMA)-stimulated O2- generation, with a maximum increase of 4,5-fold, through: (1) a more than 50% reduction of the lag-phase, defined as the time between addition of PMA and detection of O2-; and (2) a more than 60% increase in the constant rate of O2- generation. Reduction of the lag phase was associated with increased protein kinase C (PKC)-independent translocation of the cytosolic subunit of NADPH oxidase p47-phox to the cell membrane, whereas increased generation of O2- correlated with enhanced activation of PKC. The data indicate that AA increases activation of NADPH oxidase by accelerating its assembly and by co-stimulating PKC in monocytic U937 cells.

Lovastatin inhibits receptor-stimulated Ca(2+)-influx in retinoic acid differentiated U937 and HL-60 cells.

Lovastatin was used to study the role of isoprenylated proteins on stimulus-induced increase of cytosolic Ca2+ in retinoic acid-differentiated U937 and HL-60 cells. Preincubation of the cells with lovastatin for 11-24 h reduced the Ca(2+)-influx induced by PAF of FMLP. The maximal decrease was 60% in U937 cells and 40% in HL-60 cells. The ID50s of lovastatin in U937 and HL-60 cells were 5 microM and 15 microM, respectively. Lovastatin did not inhibit Ca(2+)-discharge from intracellular stores. Addition of mevalonate to lovastatin-treated cells completely reversed the inhibition of PAF- and FMLP-stimulated Ca(2+)-mobilization. Immunoreactivity of ras-like proteins was decreased in membranes and increased in the cytosol of U937 cells by 1 day treatment with lovastatin. We conclude that isoprenylated proteins are involved in the regulation of receptor-stimulated Ca(2+)-entry of differentiated HL-60 and U937 cells.

Distinct patterns of differentiation induced in the monocytic cell line Mono Mac 6.

The human Mono Mac 6 cell line exhibits many characteristics of mature blood monocytes including expression of the CD14 molecule and production of cytokines, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor. To determine whether these cells can be further differentiated, we treated the cells for up to 3 days with either prostaglandin E2 (PGE2; 10(-5) or 10(-6) M), lipopolysaccharide (LPS; 10-20 ng/ml), or tetradecanoylphorbol-13-acetate (TPA; 10-50 ng/ml). All three reagents reduced proliferation and expression of the early myelomonocytic antigen CD33, and all increased phagocytosis of staphylococci and constitutive expression of mRNA for the macrophage colony-stimulating factor (M-CSF) receptor. By contrast, with respect to CD23 (Fc epsilon RII) expression, CD14 expression, and production of O2-, the three reagents induced distinct responses. Expression of CD23 (Fc epsilon RII) on Mono Mac 6 cells (36%) was not increased by LPS and TPA but was increased by PGE2 treatment to 48%, with a 50% increase of fluorescence intensity. The CD14 antibody My4 stained more than 75% of untreated Mono Mac 6 cells with a specific mean fluorescence intensity of 87.5 channels. This staining was increased more than twofold by both PGE2 and LPS. Staining with the CD14 antibody UCHM1 (6%) was increased to 43% by PGE2 and to 43% by LPS. This increase in CD14 cell surface expression was accompanied by a rise in soluble CD14 and enhancement of CD14 mRNA. By contrast, TPA treatment resulted in a twofold decrease of CD14 cell surface staining with no significant change in sCD14, while CD14 mRNA was transiently down-regulated. Secretion of O2- (stimulated by TPA) was already detectable in untreated Mono Mac 6 cells (6.1 mmol/10(6) cells/30 min), and this response was enhanced 10-fold by pretreatment with LPS but not with PGE2 or TPA. The kinetics of M-CSF receptor mRNA, CD14 expression, and O2- production revealed that these monocytic features started to increase at 6-24 h and were maximal at 2 days. These data suggest that the three reagents induce maturation of the Mono Mac 6 cells to different levels or into different branches of the monocyte system with the notable differences that PGE2 enhances CD23 expression, LPS enhances O2- secretion, and TPA down-regulates CD14.

[Are we being threatened by multiresistant strains of Staphylococcus aureus?]. / Prävention und Therapie von Staphylokokkeninfektionen... bevor sich MRSA & Co. in lhrer Praxis niederlassen.

Multiresistant strains of Staphylococcus aureus (MRSA) are characterized by their virulence and clinical resistance to all known beta-lactam antibiotics. Furthermore, the representatives of most other classes of antibiotics are also proving to be no longer effective. While infections with the usual S. aureus strains can mostly be readily managed with penicillinase-resistant penicillins, the rescue antibiotic vancomycin, as also teicoplanin, in combination with, for example fosfomycin, are required in the treatment of MRSA and S. epidermidis infections. Since infections with S. aureus/MRSA are usually of endogenous origin, decolonization with mupirocin-containing nasal ointment applied as a prophylactic measure in patients with high colonization rates and/or immunosuppression, is of major importance. The best protection against further spread of highly resistant germs, such as MRSA is, in particular, profession hygiene management.