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Int J Med Microbiol [journal]
- Production of biofilm by Candida and non-Candida spp. isolates causing fungemia: Comparison of biomass production and metabolic activity and development of cut-off points. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Sep 1.
Biofilm production in Candida spp. can be studied by measuring the biomass produced after application of crystal violet stain or by measuring metabolic activity with XTT. Our study is the first in which crystal violet and XTT are compared to analyze the ability of clinically relevant Candida and non-Candida species to produce biofilm. We studied 577 isolates causing fungemia in 512 patients admitted from January 2007 to July 2013. Based on the biomass production measured by crystal violet and the metabolic activity measured by XTT, strains were divided into terciles to establish tentative cut-offs to classify isolates as being low, moderate, or high biofilm-forming and as having low, moderate, or high metabolic activity. Considerable variability in biofilm production and metabolic activity was found both between species and within species. C. tropicalis showed the highest biomass production, whereas C. glabrata showed the highest metabolic activity, and non-Candida species isolates showed the lowest metabolic activity (P<0.0023). The isolates were classified as low metabolic activity, moderate metabolic activity, and high metabolic activity according to their cut-offs by XTT (<0.097, 0.097-0.2, and >0.2) and as low biofilm-forming, moderate biofilm-forming, and high biofilm-forming according to their cut-offs by crystal violet (<0.44, 0.44-1.17, and >1.17). The overall categorical agreement between the procedures was 43.7%, which increased to >50% for C. albicans and C. parapsilosis. XTT and crystal violet are complementary procedures for the study of biofilm production.
- Influenza, a One Health paradigm-Novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 30.
Influenza virus is a paradigm for a pathogen that frequently crosses the species barrier from animals to humans, causing severe disease in the human population. This ranges from frequent epidemics to occasional pandemic outbreaks with millions of death. All previous pandemics in humans were caused by animal viruses or virus reassortants carrying animal virus genes, underlining that the fight against influenza requires a One Health approach integrating human and veterinary medicine. Furthermore, the fundamental question of what enables a flu pathogen to jump from animals to humans can only be tackled in a transdisciplinary approach between virologists, immunologists and cell biologists. To address this need the German FluResearchNet was established as a first nationwide influenza research network that virtually integrates all national expertise in the field of influenza to unravel viral and host determinants of pathogenicity and species transmission and to explore novel avenues of antiviral intervention. Here we focus on the various novel anti-flu approaches that were developed as part of the FluResearchNet activities.
- Capsular polysaccharide gene diversity of pneumococcal serotypes 6A, 6B, 6C, and 6D. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 17.
This study was performed to better understand the genetic diversity and evolutionary relatedness of pneumococcal serotypes 6A, 6B, 6C, and 6D. Multi-locus sequence typing (MLST) was performed for 160 serogroup 6 isolates from clinical specimens collected from children between 1991 and 2010. We identified 38 sequence types (STs) comprising five clonal complexes with 12 singletons. Although most STs were confined to a single serotype, some STs were shared by two serotypes, and one ST was shared by three serotypes. Many STs of serotype 6A showed genetic relatedness with those of serotype 6C or 6D in eBURST analysis. Five capsular polysaccharide (cps) genes - wchA, wciO, wciP, wzy, and wzx - were analysed in 74 isolates from our clinical samples and in 36 isolates from GenBank. There were several profiles and clades in each serotype on the analysis of the concatenated sequences of the five cps genes. Small genetic distances between serotypes 6A and 6B and between serotypes 6C and 6D were observed while serotype 6B with an indel sequence formed a distinct clade. When comparing the individual cps genes between the serotypes, there was also a high level of similarity in the wchA and wciO gene sequences between serotype 6C and serotype 6D. On the other hand, serotypes 6A and 6D had the most highly similar wzy and wzx gene sequences. The wzy sequences of serotype 6C were nearly identical (99.6%) to those of serotype 6A clade II strains. In conclusion, we revealed the diversity of the genetic background and cps sequences in each pneumococcal serotype of serogroup 6. Pneumococcal serotype diversity might be attributable to complex serial mutation and recombination events.
- Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 23.
L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.
- Bidirectional genomic exchange between Helicobacter pylori strains from a family in Coventry, United Kingdom. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 17.
The human gastric pathogen Helicobacter pylori is characterised by a high mutation rate and frequent recombination during mixed infection, which result in extensive genetic diversity and rapid allelic diversification. Mixed infections are believed to be much more common in regions with a high H. pylori prevalence than in industrialised countries. To better understand the genomic flexibility of H. pylori in a low prevalence region, we used 454 sequencing technology to investigate whole genome sequences of H. pylori strains isolated from members of three generations of a family living in Coventry, UK. The genomes of four H. pylori strains isolated from a grandfather, two of his sons and one grandson were sequenced. Three of these genomes showed a high overall sequence similarity, suggesting a recent common ancestor. The genomes differed by 316-336 SNPs, and recombination events (imports) resulted in 170-251 clusters of polymorphisms (CNPs). Imports were particularly frequent in genes encoding Helicobacter outer membrane proteins, suggesting an adaptation of the strains to their individual host. The fourth strain differed substantially from these three highly related strains but still shared long fragments of identical sequence, which most likely reflect imports from the highly related family variants. The data show extensive bidirectional exchange of DNA between the strains isolated from the family members, illustrating both the convergence and divergence effect that recombination can lead to. Detailed analysis of the distribution of SNPs and imports permits to draw up a complex scenario of the transmission history involving infection with at least two, and probably more separate strains. This complexity and the resulting high frequency of recombination were unexpected for an industrialised country where the prevalence of H. pylori infection has strongly declined in recent decades.
- Hypermutation in Burkholderia cepacia complex is mediated by DNA mismatch repair inactivation and is highly prevalent in cystic fibrosis chronic respiratory infection. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 28.
The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans -the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections.
- Subgrouping of ESBL-producing Escherichia coli from animal and human sources: An approach to quantify the distribution of ESBL types between different reservoirs. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 2.
Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of blaTEM, blaCTX, and blaSHV genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes blaCTX-M-1, blaCTX-M-15, or the combinations blaSHV-12+blaTEM or blaCTX-M-1+blaTEM. While the majority of animal isolates carried blaCTX-M-1 (37.5%) or the combination blaCTX-M-1+blaTEM (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried blaCTX-M-15 compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups blaCTX-M-1 genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, blaCTX-M-15 genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.
- A complementary tool for management of disseminated Histoplasma capsulatum var. capsulatum infections in AIDS patients. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 7.
In South America, disseminated histoplasmosis due to Histoplasma capsulatum var. capsulatum (H. capsulatum), is a severe and frequent opportunistic infection in AIDS patients. In areas outside the USA where specific-Histoplasma antigen detection is not available, the diagnosis is difficult. With the galactomannan antigen (GM) detection, a test commonly used for invasive aspergillosis diagnosis, there is a cross-reactivity with H. capsulatum that can be helpful for the diagnosis of histoplasmosis. The aim of this study was to evaluate the GM detection for the diagnosis of disseminated histoplasmosis in AIDS patients. The performance of the GM detection was evaluated with serum collected in French Guiana where H. capsulatum is highly endemic. Sera from AIDS patients with disseminated histoplasmosis occurring from 2002 to 2009 and from control HIV-positive patients without histoplasmosis were tested with the GM detection and Histoplasma-specific antibody detection (IEP). In 39 AIDS patients with proven disseminated histoplasmosis, the sensitivity of the Histoplasma IEP was only 35.9% and was linked to the TCD4+ lymphocyte level. For the GM detection, the sensitivity (Se) was 76.9% and specificity (Sp) was 100% with the recommended threshold for aspergillosis diagnosis (0.5). The test was more efficient with a threshold of 0.4 (Se: 0.82 [95% CI: 0.66-0.92], Sp: 1.00 [95% CI: 0.86-1.00], LR+: >10, LR-: 0.18). This study confirms that the GM detection can be a surrogate marker for the diagnosis of disseminated histoplasmosis in AIDS patients in endemic areas where Histoplasma EIA is not available.
- USA300 methicillin-resistant Staphylococcus aureus in Zurich, Switzerland between 2001 and 2013. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 19.
USA300 methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent MRSA in the United States of America (USA) and a global epidemic threat. We investigated the prevalence of USA300 at a tertiary care hospital in Zurich, Switzerland, where all MRSA strains have been collected and PFGE typed since 1992. These strains were retrospectively compared to the PFGE pattern of USA300 strain JE2. Isolates with a respective PFGE pattern were spa-typed and tested for the presence of the arginine catabolic mobile element (ACME) arc gene cluster and Panton-Valentine Leucocidin (PVL) genes. The first MRSA strain with a USA300 PFGE pattern was isolated in 2001 from a patient visiting from the USA. USA300 strains represented between 0% (in 2002) and 9.2% (in 2012) of all MRSA isolates in our hospital. We identified various USA300 subtypes based on either the PFGE pattern, the spa-type or absence of either the PVL genes or ACME arc gene cluster. All the USA300 strains including the variants (n=47) accounted for 5.6% of all MRSA isolates typed between 2001 and 2013 and reached a maximum of 14.5% in 2009. They predominantly caused skin and soft tissue infections (74.4%). In conclusion, even though USA300 has been present in our hospital for over twelve years it has not become the predominant MRSA clone like in the USA. However, in light of the global burden of USA300, care must be taken to further contain the spread of this lineage and of MRSA in general in our hospital.
- Human dendritic cell subsets display distinct interactions with the pathogenic mould Aspergillus fumigatus. [JOURNAL ARTICLE]
- Int J Med Microbiol 2014 Aug 18.
The mould Aspergillus fumigatus is primarily an opportunistic pathogen of immunocompromised patients. Once fungal spores have been inhaled they encounter cells of the innate immune system, which include dendritic cells (DCs). DCs are the key antigen-presenting cells of the immune system and distinct subtypes, which differ in terms of origin, morphology and function. This study has systematically compared the interactions between A. fumigatus and myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and monocyte-derived DCs (moDCs). Analyses were performed by time-lapse video microscopy, scanning electron microscopy, plating assays, flow cytometry, 25-plex ELISA and transwell assays. The three subsets of DCs displayed distinct responses to the fungus with mDCs and moDCs showing the greatest similarities. mDCs and moDCs both produced rough convolutions and occasionally phagocytic cups upon exposure to A. fumigatus whereas pDCs maintained a smooth appearance. Both mDCs and moDCs phagocytosed conidia and germ tubes, while pDCs did not phagocytose any fungi. Analysis of cytokine release and maturation markers revealed specific differences in pro- and anti-inflammatory patterns between the different DC subsets. These distinct characteristics between the DC subsets highlight their differences and suggest specific roles of moDCs, mDCs and pDCs during their interaction with A. fumigatus in vivo.