Natural Infections of Angiostrongylus vasorum and Crenosoma vulpis in Dogs in Germany (2007–2009)

In order to assess the occurrence and regional geographical distribution of Angiostrongylus vasorum and Crenosoma vulpis in Germany, faecal samples of 810 dogs with clinical symptoms of respiratory and circulatory disease, bleeding disorder and/or neurological signs were collected from September 2007 to March 2009. The zinc chloride/sodium chloride flotation and Baermann funnel technique were used to examine the samples for presence of lungworm larvae. Infections with lungworms were diagnosed in 105 (13.0%) of the examined dogs. A. vasorum and C. vulpis were found in 60 (7.4%) and 49 (6.0%) faecal samples, respectively. 33 A. vasorum- and 12 C. vulpis-positive dogs were located in Baden-Württemberg, 13 and 12 in North Rhine-Westphalia, 3 and 4 in Bavaria, 1 and 7 in Rhineland-Palatinate, 7 and 4 in Saarland, 1 and 2 in Saxony, respectively. In Brandenburg only 2 dogs with A. vasorum and in Hesse a total of 5 dogs with C. vulpis were detected. In Mecklenburg-Western Pomerania, Lower Saxony and Thuringia only 1 dog with C. vulpis was detected at a time. 4 dogs were coinfected with A. vasorum and C. vulpis. These surprisingly high prevalence rates indicate that both parasites are endemic in Germany.     

Emergence of daptomycin resistance following vancomycin-unresponsive Staphylococcus aureus bacteraemia in a daptomycin-na?ve patient—a review of the literature

A patient developed a daptomycin-resistant methicillin-resistant Staphylococcus aureus (MRSA) infection, despite being daptomycin-na?ve, in the setting of persistent bacteraemia secondary to vertebral osteomyelitis. Modified population analysis profiling of sequential MRSA blood culture isolates revealed transition from a vancomycin-susceptible phenotype to a vancomycin-intermediate S. aureus (VISA) phenotype through a vancomycin-heteroresistant S. aureus (hVISA) intermediary. Increased cell wall thickening, determined by transmission electron microscopy, correlated with the emergence of daptomycin resistance. This case supports the current hypothesis that MRSA with reduced glycopeptide susceptibility are less susceptible to daptomycin because of a thickened cell wall. This may have significance for the use of daptomycin in salvage therapy. Other predictors of daptomycin resistance include bacteraemic persistence and the presence of high inoculum infections. As resistance may appear de novo and be unstable in vivo, all isolates should have daptomycin susceptibility testing performed. The optimal antibiotic option for salvage therapy of these daptomycin-resistant infections is unknown. However, these findings emphasise the importance of optimising management, including the consideration of early surgical intervention to avoid the emergence of daptomycin resistance, especially in high inoculum infections.     

Expression of sodium channel α- and β-subunits in the nervous system of themyelin-deficient rat

Summary Using subtype-specific riboprobes and a non-isotopein situ hybridization technique, the pattern of expression of the mRNAs for voltage dependent sodium channel -subunits I, II, III and NaG, and the ₁-subumt were compared inmyelin-deficient rats and unaffected male littermates. Tissues examined included the hippocampus, cerebellum, spinal cord and dorsal root ganglia. Previous studies have demonstrated that the expression of sodium channel - and ₁-subunits follows a distinct temporal and spatial pattern during development, characterized in part by greater expression of -subunit III and its mRNA during development than in the adult. We examined animals of 20–22 days of age, a time when, according to earlier reports, the unaffected animals should nearly have reached an adult expression pattern. Normal male littermates were indeed found to express a sodium channel subunit mRNA pattern generally consistent with previous reports on adult rats.Myelin-deficient animals exhibited an expression pattern identical to the unaffected littermates, indicating that myelination is not required for the progression from the embryonic to the adult expression pattern of sodium channel subunits.     

Role for the fibrinogen-binding proteins Coagulase and Efb in the Staphylococcus aureus–Candida interaction

Staphylococcus aureus and Candida species are increasingly coisolated from implant-associated polymicrobial infections creating an incremental health care problem. Synergistic effects between both genera seem to facilitate the formation of mixed S. aureus–Candida biofilms, which is thought to play a critical role in coinfections with these microorganisms. To identify and characterize S. aureus factors involved in the interaction with Candida species, we affinity-panned an S. aureus phage display library against Candida biofilms in the presence or absence of fibrinogen. Repeatedly isolated clones contained DNA fragments encoding portions of the S. aureus fibrinogen-binding proteins coagulase or Efb. The coagulase binds to prothrombin in a 1:1 ratio thereby inducing a conformational change and non-proteolytic activation of prothrombin, which in turn cleaves fibrinogen to fibrin. Efb has been known to inhibit opsonization. To study the role of coagulase and Efb in the S. aureus–Candida cross-kingdom interaction, we performed flow-cytometric phagocytosis assays. Preincubation with coagulase reduced the phagocytosis of Candida yeasts by granulocytes significantly and dose-dependently. By using confocal laser scanning microscopy, we demonstrated that the coagulase mediated the formation of fibrin surrounding the candidal cells. Furthermore, the addition of Efb significantly protected the yeasts against phagocytosis by granulocytes in a dose-dependent and saturable fashion. In conclusion, the inhibition of phagocytosis of Candida cells by coagulase and Efb via two distinct mechanisms suggests that S. aureus might be beneficial for Candida to persist as it helps Candida to circumvent the host immune system.     

Antigenicity of tissues and organs from GGTA1/CMAH/β4GalNT2 triple gene knockout pigs

Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts. To overcome biological incompatibility between pigs and humans, one strategy is to remove the major antigens [Gal, Neu5Gc, and Sd(a)] present on pig cells and tissues. Triple gene (GGTA1, CMAH, and β4GalNT2) knockout (TKO) pigs were produced in our laboratory by CRISPR-Cas9 targeting. To investigate the antigenicity reduction in the TKO pigs, the expression levels of these three xenoantigens in the cornea, heart, liver, spleen, lung, kidney, and pancreas tissues were examined. The level of human IgG/IgM binding to those tissues was also investigated, with wildtype pig tissues as control. The results showed that αGal, Neu5Gc, and Sd(a) were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs. Compared to wildtype pigs, the liver, spleen, and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding, whereas corneas, heart, lung, and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding. These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.     

Histamine release “in vivo” and “in vitro” induced by hypnotics in normal and atopic patients

The in vitro test of histamine release induced in the leukocytes of atopic subjects selected according to specific criteria would seem to be much more accurate to study the histamine releasing characteristics of intravenous agents than the in vivo study in patients who have to be anaesthetised. Moreover, different concentrations of the test drug may be used, and thus the threshold for histamine release may be compared. It is the test which we recommend for investigating new drugs. However, it is lengthy and expensive; it can therefore not be recommended as a routine preoperative investigation.     

Efficacy of Emodepside plus Praziquantel Tablets (Profender? Tablets for Dogs) against Mature and Immature Adult Ancylostoma caninum and Uncinaria stenocephala Infections in Dogs

This paper reports the efficacy of a novel flavoured tablet formulation of emodepside plus praziquantel (Profender? tablets for dogs) against mature and immature adult hookworms (Ancylostoma caninum and Uncinaria stenocephala) in dogs. The tablets were used at the minimum recommended dose of 1 mg emodepside and 5 mg praziquantel per kg body weight. Four randomised, blinded and controlled laboratory studies demonstrated >95% efficacy against mature and immature adult stages of U. stenocephala and four randomised, blinded and controlled laboratory studies demonstrated >98% efficacy against mature and immature adult stages of A. caninum. No side effects of the treatment were observed. It is concluded that the emodepside plus praziquantel tablet is an effective and safe treatment against mature and immature hookworms.     

Efficacy of Emodepside plus Praziquantel Tablets (Profender? Tablets for Dogs) against Mature and Immature Infections with Toxocara canis and Toxascaris leonina in Dogs

The efficacy of emodepside plus praziquantel tablets (Profender? tablets for dogs) against mature adult, immature adult and larval stages of Toxocara canis and Toxascaris leonina was evaluated in ten randomised, blinded and placebo-controlled dose confirmation studies in naturally or experimentally infected dogs. The tablets were used at the proposed minimum dose of 1 mg emodepside and 5 mg praziquantel per kg body weight. Efficacy was calculated based on worm counts after necropsy. Five studies demonstrated >99% efficacy against mature adult, >92% efficacy against immature adult, >98% efficacy against L4 and >94% efficacy against L3 larval stages of T. canis. Another five studies demonstrated >99% efficacy against mature and immature adult and >95% efficacy against L4 larval stages of T. leonina. No side effects of the treatment were observed. Emodepside plus praziquantel tablets thus provide a comprehensive new treatment option for ascarid infections in the dog.     

Effects of the PPAR-β agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination

Background  

Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-β seems to play an important role in the regulation of central inflammation. In addition, PPAR-β agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-β agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-γ and LPS.     

Evaluation of the capability of the VITEK 2 system to detect extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates, in particular with the coproduction of AmpC enzymes

A total of 317 Klebsiella pneumoniae and 291 Escherichia coli nonduplicate isolates were tested by the VITEK 2 system to evaluate its capability to detect extended-spectrum β-lactamases (ESBLs) among putative ESBL-producing isolates, in particular those with the coproduction of AmpC enzymes. β-lactamases produced by the test isolates had been characterised. The sensitivity and specificity for ESBLs were 98.9% and 98.5%, respectively. Ninety of the isolates were AmpC (CMY-2, CMY-8 or DHA-1) and ESBL (SHV and/or CTX-M) coproducers, and 74 isolates (82.2%) of them were flagged as ESBL producers. Our study indicates that the VITEK 2 system is an acceptable tool for ESBL detection among K. pneumoniae and E. coli isolates for laboratories where both imported AmpC and ESBLs are prevalent.     

Staphylococcus aureus nasal carriage is associated with serum 25-hydroxyvitamin D levels, gender and smoking status. The Troms? Staph and Skin Study

Vitamin D induces the expression of antimicrobial peptides with activity against Staphylococcus aureus. Thus, we studied the association between serum 25-hydroxyvitamin D (25(OH)D) and S. aureus nasal colonization and carriage. Nasal swabs, blood samples and clinical data from 2,115 women and 1,674 men, aged 30–87 years, were collected in the Troms? Staph and Skin Study 2007–08, as part of the population-based sixth Troms? Study. Multivariate logistic regression analyses were stratified by recognized risk factors for S. aureus carriage: sex, age and smoking. In non-smoking men, we observed a 6.6% and 6.7% decrease in the probability of S. aureus colonization and carriage, respectively, by each 5 nmol/l increase in serum 25(OH)D concentration (P < 0.001 and P = 0.001), and serum 25(OH)D > 59 nmol/l and ≥75 nmol/l as thresholds for ~30% and ~50% reduction in S. aureus colonization and carriage. In non-smoking men aged 44–60 years, the odds ratio for S. aureus colonization was 0.44 (95% confidence interval, 0.28−0.69) in the top tertile of serum 25(OH)D versus the bottom tertile. In women and smokers there were no such associations. Our study supports that serum vitamin D is a determinant of S. aureus colonization and carriage.     

Host–pathogen interactions in epidermolysis bullosa patients colonized with Staphylococcus aureus

Patients with the genetic blistering disease epidermolysis bullosa (EB) often have chronic wounds that can become colonized by different bacteria, especially the opportunistic pathogen Staphylococcus aureus. We therefore determined the S. aureus colonization rates in EB patients from the Netherlands by collecting swabs from their anterior nares, throats and wounds. Within a period of ∼2 years, more than 90% of the sampled chronic wounds of EB patients were found to be colonized by S. aureus. Molecular typing revealed that EB patients were not colonized by a single S. aureus type. Rather the S. aureus population structure in the sampled EB patients mirrored the local S. aureus population structure within the Netherlands. Furthermore, multiple types of S. aureus were found in close proximity to each other within individual chronic wounds, indicating that these S. aureus types are not mutually exclusive. Over time, strong fluctuations in the S. aureus types sampled from individual EB patients were observed. This high exposure to different S. aureus types is apparently reflected by high plasma levels of antistaphylococcal IgG's, especially in patients carrying multiple S. aureus types. It remains to be determined to what extent this strong immune response protects EB patients against serious staphylococcal infections. Lastly, further research is needed to define the impact of staphylococcal colonization of chronic wounds on the development, exacerbation and healing of such wounds in patients with EB.     

Characterization of IncA/C conjugative plasmid harboring blaTEM-52 and blaCTX-M-15 extended-spectrum β-lactamases in clinical isolates of Escherichia coli in Tunisia

To characterize the extended-spectrum β-lactamases (ESBLs) as well as their genetic environment in different isolates of Escherichia coli from patients with repeated urinary tract infections, large multidrug resistance (MDR) plasmids have been found. Definitive evidence for the presence of an A/C incompatibility complex (IncA/C) plasmid in the MDR isolates was provided by the probing of plasmids extracted from the clinical isolates. Conjugation experiments showed that bla genes were transferred by conjugation from the ten E. coli clinical isolates to E. coli XL1-Blue recipient. A comparative restriction fragment length polymorphism (RFLP) analysis of these plasmids showed that they are genetically similar, while the overall similarity of these plasmids supports the likelihood of recent movements among these E. coli isolates. Polymerase chain reaction (PCR) amplification and sequencing of the amplicons showed that the IncA/C plasmids harbor two ESBLs, identified as TEM-52 and CTX-M-15. Analysis of the plasmid DNA surrounding the bla _CTX-M-15 gene in the clinical isolates under study revealed a partially truncated fragment of ISEcp1 tnpA transposase. This result indicates the variety of genetic events that have enabled associations between ISEcp1 sequences and bla _CTX-M-15 genes in these clinical isolates.     

The complete genomes of Staphylococcus aureus bacteriophages 80 and 80α—Implications for the specificity of SaPI mobilization

Staphylococcus aureus pathogenicity islands (SaPIs) are mobile elements that are induced by a helper bacteriophage to excise and replicate and to be encapsidated in phage-like particles smaller than those of the helper, leading to high-frequency transfer. SaPI mobilization is helper phage specific; only certain SaPIs can be mobilized by a particular helper phage. Staphylococcal phage 80α can mobilize every SaPI tested thus far, including SaPI1, SaPI2 and SaPIbov1. Phage 80, on the other hand, cannot mobilize SaPI1, and ?11 mobilizes only SaPIbov1. In order to better understand the relationship between SaPIs and their helper phages, the genomes of phages 80 and 80α were sequenced, compared with other staphylococcal phage genomes, and analyzed for unique features that may be involved in SaPI mobilization.     

Rifampicin–fosfomycin coating for cementless endoprostheses: Antimicrobial effects against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)

New strategies to decrease infection rates in cementless arthroplasty are needed, especially in the context of the growing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections. The purpose of this study was to investigate the antimicrobial activity of a rifampicin–fosfomycin coating against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA in a rabbit infection prophylaxis model. Uncoated or rifampicin–fosfomycin-coated K-wires were inserted into the intramedullary canal of the tibia in rabbits and contaminated with an inoculation dose of 10⁵ or 10⁶ colony-forming units of MSSA EDCC 5055 in study 1 and MRSA T6625930 in study 2, respectively. After 28 days the animals were killed and clinical, histological and microbiological assessment, including pulse-field gel electrophoresis, was conducted. Positive culture growth in agar plate testing and/or clinical signs and/or histological signs were defined positive for infection. Statistical evaluation was performed using Fisher’s exact test. Both studies showed a statistically significant reduction of infection rates for rifampicin–fosfomycin-coated implants compared to uncoated K-wires (P = 0.015). In both studies none of the 12 animals that were treated with a rifampicin–fosfomycin-coated implant showed clinical signs of infection or a positive agar plate testing result. In both studies, one animal of the coating group showed the presence of sporadic bacteria with concomitant inflammatory signs in histology. The control groups in both studies exhibited an infection rate of 100% with clear clinical signs of infection and positive culture growth in all animals. In summary, the rifampicin–fosfomycin-coating showed excellent antimicrobial activity against both MSSA and MRSA, and therefore warrants further clinical testing.     

Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells – An in vivo proteomics perspective

Staphylococcus aureus is a versatile pathogen that can be a commensal but also cause a wide range of different infections. This broad disease spectrum is a reflection of the complex regulation of a large collection of virulence factors that together with metabolic fitness allow adaptation to different niches. The alternative sigma factor SigB is one of the global regulators mediating this adaptation. However, even if SigB contributes to expression of many virulence factors its importance for successful infection greatly varies with the strain and the infection setting analyzed. We have recently established a proteomics workflow that combines high efficiency cell sorting with sensitive mass spectrometry and allows monitoring of global proteome adaptations with roughly one million bacterial cells. Thus, we can now approach the adaptation of pathogens to the intracellular milieu. In the current study this proteomics workflow was used in conjunction with qRT-PCR and confocal fluorescence microscopy to comparatively analyze the adaptation of the S. aureus wild type strain HG001 and its isogenic sigB mutant to the intracellular milieu of human S9 bronchial epithelial cells. The study revealed fast and transient activation of SigB following internalization by human host cells and the requirement of SigB for intracellular growth. Loss of SigB triggered proteome changes reflecting the different residual growth rates of wild type and sigB mutant, respectively, the resistance to methicillin, adaptation to oxidative stress and protein quality control mechanisms.     

Description of macrolide-resistant and potential virulent clones of Streptococcus pyogenes causing asymptomatic colonization during 2000–2006 in the Lisbon area

The asymptomatic oropharyngeal colonization rate by Streptococcus pyogenes was 10.7% in children (901 among 8,405 children 0–16 years old) and 3.3% in adults (37 among 1,126 households of children) in the Lisbon area during 2000–2006. Macrolide-resistant S. pyogenes from children (n = 149) was variable with time: 9.8–10.7% in 2000–2002, 28.1% in 2003, 19.6–2.7% in 2004–2005 and 14.6% in 2006. Eight lineages (97.3% of isolates) were identified based on at least 80% similarity of PFGE patterns, T types, emm types and multilocus sequence types (ST). The elevated frequency of macrolide resistance was associated with M phenotype lineages I (emm12/ST36) and V (emm4, emm75/ST39 and a novel emmstMrp6 type) and with one cMLS_B lineage IV (emm28/ST52) known to be associated with upper respiratory tract and invasive infections. Significant associations (p < 0.05) between emm type/virulence genotype were found, such as emm1/speA ⁺ ssa ^-, emm4/ssa ⁺ prtF1 ⁺, emm12/speA ^- ssa ^-. The high prevalence (>20%) of speC, prtF1 or ssa was probably caused either by clonal dissemination (speC), or to horizontal gene transfer events (prtF1 and ssa). This report contributes to a better understanding of the molecular epidemiology and evolution of macrolide-resistant S. pyogenes causing symptom-free oropharyngeal colonization. These colonizing strains carry macrolide resistance and virulence genes capable of being transferred to other bacterial species sharing the same niche.     

Clinical and biological significance of GSK-3β inactivation in breast cancer—an immunohistochemical study

Glycogen synthase kinase 3β, recently found to be functionally abnormal in various types of human disease, is negatively regulated by the PI3K/Akt signaling pathway. As Akt is constitutively activated in a subset of breast cancer, we hypothesized that glycogen synthase kinase 3β is inappropriately inactivated in these cases. In this study, we aimed to assess (1) the overall frequency of glycogen synthase kinase 3β inactivation in breast cancer; (2) whether there is an association between Akt activation and glycogen synthase kinase 3β inactivation; and (3) whether there is a correlation between glycogen synthase kinase 3β inactivation and various pathologic and clinical parameters. The phosphorylated form of glycogen synthase kinase 3β (pGSK-3β) and Akt (pAkt) were used as surrogate markers for glycogen synthase kinase 3β inactivation and Akt activation, respectively. Immunohistochemistry applied to paraffin-embedded tissues was used to assess 72 consecutive invasive mammary carcinomas, of which 50 were estrogen receptor positive. Overall, pGSK-3β and pAkt were positive in 34 (47.2%) and 35 (48.6%) cases, respectively. These 2 markers were significantly correlated with each other in the overall group and in the estrogen receptor–positive subgroup (P = .01 and .003, Spearman, respectively). Importantly, pGSK-3β, but not pAkt, significantly correlated a worse clinical outcome in this cohort (P = .004, log rank). In summary, evidence of glycogen synthase kinase 3β inactivation was found in approximately half of the invasive mammary carcinomas. Our data suggest that this abnormality is likely attributed to Akt activation and that glycogen synthase kinase 3β inactivation confers a worse clinical outcome.