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.

Wohlfahrtiimonas chitiniclastica: current insights into an emerging human pathogen.

Since the first description of Wohlfahrtiimonas chitiniclastica in 2008, a number of well described case reports demonstrating its pathogenic role in humans have been published. Infections may be closely linked to flies, such as Wohlfahrtia magnifica, Lucilia sericata, Chrysomya megacephala or Musca domestica. These insects are potent vectors for the distribution of W. chitiniclastica causing local or systemic infections originating from wounds infested with fly larvae. However, other potential sources of transmission of W. chitiniclastica have been described such as soil or chicken meat. Infections in humans reported to date comprise wound infections, cellulitis, osteomyelitis and sepsis. This review summarizes all the literature available up to now and gives the current knowledge about this emerging human pathogen. Additionally, four patients with proven W. chitiniclastica infections treated at Dresden University Hospital between 2013 and 2015, are included. Special focus was placed on microbiological identification and antibiotic susceptibility testing of the pathogen.

Mercury-induced fragmentation of n-decane and n-undecane in positive mode ion mobility spectrometry.

Ion mobility spectrometry is a well-known technique for trace gas analysis. Using soft ionization techniques, fragmentation of analytes is normally not observed, with the consequence that analyte spectra of single substances are quite simple, i.e. showing in general only one peak. If the concentration is high enough, an extra cluster peak involving two analyte molecules can often be observed. When investigating n-alkanes, different results regarding the number of peaks in the spectra have been obtained in the past using this spectrometric technique. Here we present results obtained when analyzing n-alkanes (n-hexane to n-undecane) with a pulsed electron source, which show no fragmentation or clustering at all. However, when investigating a mixture of mercury and an n-alkane, a situation quite typical in the oil and gas industry, a strong fragmentation and cluster formation involving these fragments has been observed exclusively for n-decane and n-undecane.

[Vibrio infections from food and sea water. Introducing the "VibrioNet"]. / Vibrio-Infektionen durch Lebensmittel und Meerwasser. Das Netzwerk "VibrioNet" stellt sich vor.

Vibrio is a genus of bacteria present in surface and coastal waters as well as in marine organisms worldwide. In many countries, pathogenic Vibrio species are a main cause of bacterial diarrhea, which may result from comsumption of contaminated seafood and fish products or from drinking contaminated water. Vibrio infections may also gain in importance in our regions due to global warming and the increase in the world trade of seafood. The research network "VibrioNet" studies pathogenic Vibrios in the marine environment and in seafood consumed by humans as a potential, new emerging zoonotic agent. An assessment of the risk arising from pathogenic non-cholera-vibrios in central Europe is the target of a multidisciplinary research effort. The research network will be strengthened by cooperations with international partners from countries in which Vibrio infections play a major role (Bangladesh, Chile, India, Thailand, and Vietnam).

Construction of recombinant Shiga-like toxin-IIv (SLT-IIv) and its use in monitoring the SLT-IIv antibody status of pigs.

We constructed and purified recombinant B-subunits of the SLT-IIv as well as tested their usefulness in an immunoblot assay. The slt-IIvB gene amplified by PCR was ligated into the fusion vector pGEX-2T, and expressed in E. coli K 12 laboratory strains. Deletion of the signal sequence was necessary for optimal expression. High quantities of the fusion protein could be purified by affinity chromatography and subsequently used as antigen for immunoblot analysis with serum samples from diseased pigs and healthy controls. IgG antibodies against SLT-IIv were detected in the sera of 11 of 52 (21.15%) healthy pigs. By contrast, only in 1 of 28 (3.57%) serum samples of pigs with edema disease caused by SLT-IIv-producing E. coli we could demonstrate SLT-IIv-specific antibodies. During an outbreak of edema disease, sera from 10 pigs were taken at 4, 20, and 40 days after disease onset to investigate the immune response elicited by SLT-IIv. Immunoblot analysis with the recombinant SLT-IIv fusion protein revealed that the number of IgG-positive serum samples increased within this period of 40 days from one on day 4, to seven on day 20, to ten on day 40; the number of IgM-positive samples also increased from one after 4 days to eight after 20 days. Forty days after disease onset, IgM reactivity was no longer detectable. Since all animals seroconverted in the follow-up sera, the antigenicity of SLT-IIv during infection of pigs seems to differ from that of SLT-II in human hemolytic uremic syndrome where only a minority of patients are known to mount an immune response. The recombinant SLT-IIvB described here may be a possible candidate for vaccination trials.

CD4+ T cell mediated intestinal immunity: chronic inflammation versus immune regulation.

BACKGROUND: Several studies have suggested that chronic inflammatory bowel disease may be a consequence of antigen specific recognition by appropriate T cells which expand and induce immunopathology. AIMS: We wished to investigate whether autoreactive CD4+ T cells can initiate the disease on recognition of enterocyte specific antigens directly and if induction of mucosal tolerance occurs. METHODS: Transgenic mice (VILLIN-HA) were generated that showed specific expression of haemagglutinin from influenza virus A exclusively in enterocytes of the intestinal epithelium. To investigate the impact of enterocyte specific haemagglutinin expression in an autoimmune environment, we mated VILLIN-HA mice with T cell receptor (TCR)-HA mice expressing an alpha/beta-TCR, which recognises an MHC class II restricted epitope of haemagglutinin, and analysed the HA specific T cells for induction of autoimmunity or tolerance. RESULTS: In VILLIN-HAxTCR-HA mice, incomplete central deletion of HA specific lymphocytes occurred. Peripheral HA specific lymphocytes showed an activated phenotype and increased infiltration into the intestinal mucosa, but not into other organs of double transgenic mice. Enterocyte specific lamina propria lymphocytes showed a dose dependent proliferative response on antigen stimulation whereas the proliferative capacity of intraepithelial lymphocytes was reduced. Mucosal lymphocytes from VILLIN-HAxTCR-HA mice secreted lower amounts of interferon gamma and interleukin (IL)-2 but higher levels of tumour necrosis factor alpha, monocyte chemoattractant protein 1, and IL-6. Mucosal immune reactions were accompanied by broad changes in the gene expression profile with expression of proinflammatory genes, but strikingly also a remarkable set of genes discussed in the context of peripheral induction of regulatory T cells, including IL-10, Nrp-1, and Foxp3. CONCLUSIONS: Enterocyte specific antigen expression is sufficient to trigger a specific CD4+ T cell response leading to mucosal infiltration. In our model, progression to overt clinical disease was counteracted most likely by induction of regulatory T cells.

Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology.

We used the plasmid vector pGEX-2T for the expression of recombinant subunits of Shiga-like toxin II (SLT-II). The 5' terminus of the genes that code for either the SLT-IIA or SLT-IIB subunits was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The subunit genes were constructed synthetically by polymerase chain reaction, with appropriate restriction sites to permit in-frame downstream insertion of the genes. The resulting plasmids containing the A and B subunit genes were designated pFG1 and pFG2, respectively. Induction of Escherichia coli laboratory strains harboring pFG1 with isopropyl-beta-D-thiogalactopyranoside (IPTG) yielding only small quantities of SLT-IIA fusion proteins. Since IPTG induction was lethal for cells harboring pFG2, we constructed the recombinant plasmid pFG4, which contained a subgenic fragment of slt-IIB but without the 5' signal sequence. With this construct we were able to express very large quantities of a 33.5-kDa fusion protein, which was purified by affinity chromatography on immobilized glutathione and used as an antigen in immunoblot analysis. Rabbit serum against native SLT-II, as well as all of 12 serum samples with high neutralizing activity against SLT-II, reacted with SLT-IIB purified from an E. coli pFG4 expression system, whereas only 3 of 208 human serum samples with low neutralization titers and none of 54 serum samples with no SLT-II-neutralizing capability reacted. Failure of specific reactivity with the SLT-IIB fusion protein in the majority of human serum samples with low neutralizing activity suggests that serum factors other than immunoglobulins may be responsible for neutralizing activity in these cases. The immunoblot assay with recombinant SLT-IIB as the antigen can be recommended for use in a diagnostic setting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.

Infection of primary human gingival fibroblasts by Porphyromonas gingivalis and Prevotella intermedia.

Adhesion and penetration of clinical isolates of Porphyromonas gingivalis and Prevotella intermedia in human gingival fibroblast monolayers were studied by transmission electron microscopy (TEM). Fibroblasts were cultured from biopsies of human healthy gingiva. Porphyromonas gingivalis and Prevotella intermedia were isolated from patients with periodontitis. Fibroblasts were incubated with microorganisms in an antibiotic-free medium for 24 h. Then cultures were washed to remove nonadherent bacteria. Consecutively, infected cultures were grown for another 24 h. Thereafter, the treated monolayers were prepared for TEM investigations. Internalized Porphyromonas gingivalis and Prevotella intermedia were visible after 24 h of incubation. Prevotella intermedia showed only division in cytoplasm of fibroblasts after 24 h and 48 h incubations. Infected fibroblasts revealed various morphological alterations such as extensive vacuolization and breakdown of mitochondria. These findings demonstrate that Porphyromonas gingivalis and Prevotella intermedia may invade human gingival fibroblasts and thus may damage these cells directly or due to the release of microbial cytotoxic components.

Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-.

We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-like toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics.

Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome.

Shiga-like toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-like toxin I and Shiga-like toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit toxin genes revealed that all 17 isolates produced Shiga-like toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.

[Bacterial pathogens, resistance patterns and treatment options in community acquired pediatric urinary tract infection]. / Bakterielle Erreger, Resistenzentwicklung und Behandlungsoptionen beim ambulant erworbenen Harnwegsinfekt im Kindesalter.

BACKGROUND: Epidemiology and resistance patterns of bacterial pathogens in pediatric UTI show large interregional variability and rates of bacterial resistances are changing due to different antibiotic treatment. We intended to evaluate data from northern Germany. PATIENTS AND METHODS: In 100 children (53 female, 47 male, mean age 4.4 +/- 4.2 years) with community acquired UTI, who presented in the emergency department of our medical school from 2000 - 2002, urine cultures were performed. Inclusion criteria were: acute voiding symptoms, significant bacteriuria with growth of at least 10 (5) colony-forming units/ml urine, leukocyturia > 50/ micro l. Exclusion criteria were underlying renal diseases, anatomic abnormalities of the urinary tract, age < 2 months and recurrent UTI. RESULTS: Patients presented with a mean rectal temperature of 38.6 +/- 1.3 degrees C, mean CRP of 66 +/- 68 mg/dl, mean WBC 13 500 +/- 5 600/ micro l and mean urinary leukocytes of 425 +/- 363/ micro l. In urine cultures E. coli was found in 47 % of the cases, Enterococcus faecalis 23 %, Proteus mirabilis 8 %, Klebsiella oxytoca 4 %, Pseudomonas aeruginosa 5 % and others 13 %. In 76 % one and in 24 % two different bacterial species (60 % Enterococcus faecalis) were cultured. Mean resistance rates were in all bacteria (in E. coli): Ampicillin 53 % (69 %), Ampicillin and Sulbactam 51 % (61 %), Cefalosporin 1 (st) generation (Cefaclor) 48 % (24 %), Cefalosporin 2 (nd) generation (Cefuroxim) 40 % (3 %), Cefalosporin 3 (rd) generation (Cefuroxim) 33 % (0 %), Tobramycin 30 % (2 %), Ciprofloxacine 0 %, Cotrimoxazole 40 % (42 %), Nitrofurantoin 12 % (0 %). CONCLUSION: The resistance rates to Ampicillin (+/- Sulbactam) did not increase as compared to previous analyses (1990 - 1995), however, resistance rates to Cotrimoxazole and 1 (st) generation Cefalosporines increased about 20 %. We conclude that the policies for treatment of UTI in children should be re-evaluated every 5 years according to local resistance rates.

Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes.

In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.

Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.

In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.

The role of the eaeA gene in diarrhea and neurological complications in a gnotobiotic piglet model of enterohemorrhagic Escherichia coli infection.

We reported previously that mutation of the chromosomal gene eaeA from enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 prevented bacterial attachment in vivo. Attachment was restored when the EHEC or enteropathogenic E. coli (EPEC) eaeA gene was introduced into the mutant on a plasmid. In this communication we have compared in gnotobiotic piglets the pathogenicities of wild-type O157:H7 strain 86-24 and its eaeA mutant UMD619 with those of the two plasmid-complemented strains expressing IntiminO157 (EHEC) and IntiminO127 (EPEC). 86-24 colonized the surface and glandular epithelium of the large intestine and induced diarrhea, while UMD619 did not colonize any intestinal site and induced little or no diarrhea. Surprisingly, strain UMD619 expressing IntiminO127 behaved in pigs more like EPEC than EHEC strains; it colonized the distal half of the small intestine and the surface of the large intestine, inducing serious diarrhea. In contrast, strain UMD619 expressing IntiminO157 colonized the colon extremely poorly, inducing little or no diarrhea. While only the two strains causing extensive attachment--86-24 and UMD619 expressing IntiminO127--induced diarrhea, neurological symptoms attributed to Shiga-like toxin II occurred equally in all four groups of animals. The intimate bacterial attachment and mucosal damage were not a prerequisite for Shiga-like toxin II translocation from the gut lumen into the circulation. IntiminO127 appears not only to facilitate intimate attachment to cells but also to influence the site of intestinal colonization and other characteristics of EPEC infection.

Construction and characterization of an isogenic slt-ii deletion mutant of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga-like toxins (SLT), potent protein synthesis inhibitors. To further dissect the role of SLT-II in the course of disease, we have constructed E. coli TUV86-2, an isogenic SLT-II-negative mutant of EHEC strain 86-24. The slt-ii gene was inactivated by suicide vector mutagenesis. We also isolated derivatives of strain 86-24 that were cured of the phage carrying the toxin genes.