Design and application of a core genome multilocus sequence typing scheme for investigation of Legionnaires' disease incidents.

Sequence-based typing (SBT) for Legionella pneumophila (Lp) has dramatically improved Legionnaires' disease (LD) cluster investigation. Microbial whole genome sequencing (WGS) is a promising modality for investigation but sequence analysis methods are neither standardised, nor agreed. We sought to develop a WGS-based typing scheme for Lp using de novo assembly and a genome-wide gene-by-gene approach (core genome multilocus sequence typing, cgMLST). We analysed 17 publicly available Lp genomes covering the whole species variation to define a core genome (1,521 gene targets) which was validated using 21 additional published genomes. The genomes of 12 Lp strains implicated in three independent cases of paediatric humidifier-associated LD were subject to cgMLST together with three 'outgroup' strains. cgMLST was able to resolve clustered strains and clearly identify related and unrelated strains. Thus, a cgMLST scheme was readily achievable and provided high-resolution analysis of Lp strains. cgMLST appears to have satisfactory discriminatory power for LD cluster analysis and is advantageous over mapping followed by single nucleotide polymorphism (SNP) calling as it is portable and easier to standardise. cgMLST thus has the potential for becoming a gold standard tool for LD investigation. Humidifiers pose an ongoing risk as vehicles for LD and should be considered in cluster investigation and control efforts.

The innovative osmotic membrane bioreactor (OMBR) for reuse of wastewater.

An innovative osmotic membrane bioreactor (OMBR) is currently under development for the reclamation of wastewater, which combines activated sludge treatment and forward osmosis (FO) membrane separation with a RO post-treatment. The research focus is FO membrane fouling and performance using different activated sludge investigated both at laboratory scale (membrane area of 112cm2) and at on-site bench scale (flat sheet membrane area of 0.1 m2). FO performance on laboratory-scale (i) increased with temperature due to a decrease in viscosity and (ii) was independent of the type of activated sludge. Draw solution leakage increased with temperature and varied for different activated sludge. FO performance on bench-scale (i) increased with osmotic driving force, (ii) depended on the membrane orientation due to internal concentration polarization and (iii) was invariant to feed flow decrease and air injection at the feed and draw side. Draw solution leakage could not be evaluated on bench-scale due to experimental limitation. Membrane fouling was not found on laboratory scale and bench-scale, however, partially reversible fouling was found on laboratory scale for FO membranes facing the draw solution. Economic assessment indicated a minimum flux of 15L.m-2 h-1 at 0.5M NaCl for OMBR-RO to be cost effective, depending on the FO membrane price.

High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16S rRNA gene sequencing for species identification of nonfermenting bacteria.

Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>or=99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>or=97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.

EUREGIO MRSA-net Twente/Münsterland--a Dutch-German cross-border network for the prevention and control of infections caused by methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with increased mortality and morbidity and a leading cause of hospital-acquired infections. Community-acquired (CA)-MRSA are a growing concern worldwide. In the last 10 years, an increase in the MRSA rate from 2% to approximately 23% has been observed in Germany, while a rate under 5% has been recorded for many years in the Netherlands and Scandinavia. In the Netherlands in particular, MRSA rates have become very low in stationary care due to a consistent 'search and destroy' policy. The main focus in Germany lies on hospital-acquired MRSA, whereas the Netherlands focus on the control of the importation of MRSA cases from abroad and on CA-MRSA. As MRSA in hospitals and in the community can be a problem in cross-border health care, the European Union-funded EUREGIO MRSA-net project was established in the bordering regions Twente/Achterhoek, the Netherlands and Münsterland, Germany. The main aim of the project is the creation of a network of the major health care providers in the EUREGIO and the surveillance and prevention of MRSA infections. A spa-typing network was established in order to understand the regional and cross-border dissemination of epidemic and potentially highly virulent MRSA genotypes. As the reduction of differences in health care quality is an important prerequisite for cross-border health care, a transborder quality group comprising hospitals, general practitioners, public health authorities, laboratories, and insurerance companies has been established since 2005 equalising the quality criteria for the control of MRSA on both sides of the border.

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis.

Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45 degrees C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.

Ensuring backwards compatibility: traditional genotyping efforts in the era of whole genome sequencing.

When using next-generation whole genome sequencing (WGS), extraction of spa types from WGS data is essential for backwards compatibility with Sanger sequencing-based spa typing of methicillin-resistant Staphylococcus aureus (MRSA). We evaluated WGS-based spa typing with a 2×250 bp protocol in a diverse collection of 423 MRSA isolates using two pipelines that executed sequence quality-trimming and de novo assembly before spa typing. The SeqSphere(+) pipeline correctly typed 419 isolates (99.1%) whereas the CLCbio pipeline succeeded in 249 isolates (58.9%). In summary, WGS combined with an optimized de novo assembly enables nearly full compatibility with Sanger sequencing-based spa typing data.

State-wide surveillance of antibiotic resistance patterns and spa types of methicillin-resistant Staphylococcus aureus from blood cultures in North Rhine-Westphalia, 2011-2013.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of bacteraemia. We aimed to obtain a complete picture of severe MRSA infections by characterizing all MRSA isolates from bloodstream infections in the largest German federal state (North Rhine-Westphalia, 18 million inhabitants) using S. aureus protein A (spa) sequence-typing and antimicrobial susceptibility testing. MRSA isolates (n = 1952) were collected prospectively (2011-2013) and spa-typed. Among 181 different spa types, t003 (n = 746 isolates; 38.2%) and t032 (n = 594; 30.4%) were predominant. Analysis of the geographical occurrence of spa clonal complexes (spa-CCs) and spa types revealed divergent distribution between federal state districts for spa-CCs 003 (p < 0.001; including t003, p < 0.001 and t264, p < 0.001), 008 (p 0.021), 011 (p 0.002), 032 (p < 0.001; including t022, p 0.014 and t032, p < 0.001) and spa type t2807 (p < 0.001). MICs of antimicrobial substances were tested using broth microdilution. Of all isolates, 96% were resistant to fluoroquinolones, 78% to erythromycin, 70% to clindamycin, 4% to gentamicin, 2% to rifampicin, 0.4% to daptomycin, 0.1% to linezolid and 0% to vancomycin, respectively. Vancomycin MICs of 2 mg/L involved 0.5% of the isolates. In conclusion, the detection of regional molecular clusters added valuable information for epidemiological case tracing and allowed conclusions to be reached on the importance of newly emerging MRSA reservoirs, such as livestock (spa-CC011), for MRSA bacteraemia in some parts of the federal state. Susceptibility testing revealed broad resistance to substances used for oral treatment, but demonstrated that those antibiotics that are mostly applied for treatment of MRSA bacteraemia and important combination partners were highly susceptible.

Degradation of pharmaceuticals in UV (LP)/H2O2 reactors simulated by means of kinetic modeling and computational fluid dynamics (CFD).

UV/H2O2 treatment is a well-established technique to degrade organic micropollutants. A CFD model in combination with an advanced kinetic model is presented to predict the degradation of organic micropollutants in UV (LP)/H2O2 reactors, accounting for the hydraulics, fluence rate, complex (photo)chemical reactions in the water matrix and the interactions between these processes. The model incorporates compound degradation by means of direct UV photolysis, OH radical and carbonate radical reactions. Measurements of pharmaceutical degradations in pilot-scale UV/H2O2 reactors are presented under different operating conditions. A comparison between measured and modeled degradation for a group of 35 pharmaceuticals resulted in good model predictions for most of the compounds. The research also shows that the degradation of organic micropollutants can be dependent on temperature, which is relevant for full-scale installations that are operated at different temperatures over the year.

Invasion and destruction of mucosal plasma cells by Tropheryma whippelii.

Whipple's disease is a poorly understood systemic disorder associated with the bacillus, Tropheryma whippelii. An early stage of Whipple's disease is studied by using electron microscopy (ELMI) and immunohistochemistry. The diagnosis was confirmed by polymerase chain reaction-amplification and sequencing of the 16S ribosomal-RNA of the bacterium. By using ELMI, Tropheryma whippelii was found in plasma cells and macrophages in the jejunal mucosa. The immunoglobulin (Ig)A-positive plasma cells were focally destructed and their number significantly reduced. However, the bacilli in the plasma cells were morphologically intact. In contrast, the macrophages showed no signs of cell destruction, but contained bacilli in various stages of disintegration. A cytopathic effect of Tropheryma whippelii to IgA plasma cells may be the reason for the commonly found plasma cell reduction in the small intestine mucosa and an important pathogenic mechanism contributing to the evasion of the bacilli from local immune response.

Chronic recurrent multifocal osteomyelitis in children: diagnostic value of histopathology and microbial testing.

Chronic recurrent, unifocal or multifocal osteomyelitis (CRMO), an inflammatory disorder of unknown origin, involves different osseous sites and may be associated with palmoplantar pustulosis. Bacterial cultures of affected tissue were reported negative in nearly all cases. Radiological and magnetic resonance imaging features of CRMO have been described, but differential diagnosis remains difficult, including rheumatic diseases, bacterial osteomyelitis, and malignancy. Although definite diagnosis relies on histopathologic confirmation by biopsy, histopathologic criteria have not been defined. Because CRMO may be treated with nonsteroidal antiinflammatory drugs, but not antibiotics, distinguishing CRMO from bacterial osteomyelitis is of major importance. Histopathologic analysis of 12 patients with CRMO indicated a wide variation of reparative changes of bone, but chronic inflammation could not be found at all sites in the same biopsy. The inflammatory infiltrate was mostly scattered, consisting mainly of lymphocytes, plasma cells, histiocytes, and also few neutrophil granulocytes. Immunohistochemistry showed a predominance of CD3(+), CD45RO(+) T-cells, which were mainly CD8(+). In addition, CD20(+) B cells and CD68(+) macrophages were abundant in each biopsy specimen. Mild lymphocytic and granulocytic infiltrates were also detected in three synovial biopsy specimens obtained from adjacent joints. All bacterial and fungal cultures from native biopsy tissues were negative. Amplification of partial-length 16S ribosomal DNA by polymerase chain reaction (PCR) using broad-range eubacterial primers was below the detection limit in all patients. Because histopathologic features alone may not provide conclusive evidence, CRMO should be included in the differential diagnosis of chronic inflammatory bone lesions in children, and the definite diagnosis should be made by the clinical picture, x-ray studies, bone scan, bacterial culture, and histopathologic analysis in a multidisciplinary approach.

Genotyping of Shiga-like toxin genes in non-O157 Escherichia coli strains associated with haemolytic uraemic syndrome.

The pheno- and genotypes of Shiga-like toxins (SLTs) in non-O157 Escherichia coli strains from patients with haemolytic uraemic syndrome were determined. The clinical isolates investigated were from Italy and Germany and belonged to serotypes O22:H8, O26:H-, O26:H11, O91:H-, O111:H- and O128:H-; one isolate was non-typable. SLT genotypes were analysed by complete nucleotide sequence analysis of the B-subunit genes. The results showed that 14 strains possessed slt-I alone, two contained slt-II alone and five isolates harboured both slt-I and slt-II genes. In only two strains were slt-II-related genes found, together with either slt-I or slt-II. These findings indicate that variants of SLT-II are rarely found in non-O157 E. coli isolates from patients with haemolytic uraemic syndrome. Polymerase chain reaction (PCR) with Taq cycle sequencing was found to be a suitable method for classification of slt genotypes.

Standardized molecular typing.

Molecular typing methods are useful tools in molecular mycology. The results of these biotyping procedures may help to identify pathogenic strains in order to detect sources of nosocomial infection and for the investigation of epidemiological relationships. With respect to the facultative pathogen, Candida albicans, various methods such as pulse-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP), DNA fingerprinting methods and hybridization with repetitive DNA elements have been described as useful tools in molecular epidemiology. The previously described hybridization method with the Candida albicans specific CARE-2 probe and subsequent rehybridization with a molecular size marker is a standardized reproducible typing method for comparison of results obtained in different laboratories. In a larger epidemiological study conducted at the University Hospital of Würzburg analysing clinical C. albicans isolates, we were able to describe relationships between sequential patient isolates. These findings demonstrate that standardized molecular typing methods are a powerful tool in molecular mycology studies.

Prevalence of enteropathogenic bacteria in surgically treated chronic inflammatory bowel disease.

BACKGROUND/AIMS: In order to assess the potential impact of bacterial eradication on recurrence rates, the prevalence of various enteropathogenic bacteria and toxins in chronic inflammatory bowel diseases (CIBD) was prospectively examined. METHODOLOGY: Stool, sera and gut tissue samples from a total of 59 patients (33 males, 26 females; mean age: 42 years +/- 14; 21 Crohn's disease, 14 ulcerative colitis, 24 controls) were examined for the presence of enteropathogenic bacteria by culture, immunoblotting and PCR. RESULTS: Conventional cultures failed to detect obligate pathogenic bacteria. By PCR, mycobacteria were found in 85% of all groups, with mycobacterium paratuberculosis not detected. Yersinia species were observed in 63% of patients with Crohn's disease, in 46% of patients with ulcerative colitis, and in 36% of the control patients. Pathogenic E. coli were identified in stool samples of three patients with ulcerative colitis (21%) by amplifying the EAE-gene, one of whom exhibited shiga-like-toxin as well. CONCLUSIONS: We concluded that mycobacteria do not play a causative role in CIBD. Yersinia species seem to persist in intestinal tissue in CIBD patients without adequate immune response and might, thus, contribute to tissue destruction. E. coli infections contribute to the disease process in a small group of patients with ulcerative colitis and their eradication might eliminate the need for immediate surgical intervention.

Degradation of 40 selected pharmaceuticals by UV/H2O2.

The occurrence of pharmaceuticals in source waters is increasing. Although UV advanced oxidation is known to be an effective barrier against micropollutants, degradation rates are only available for limited amounts of pharmaceuticals. Therefore, the degradation of a large group of pharmaceuticals has been studied in this research for the UV/H2O2 process under different conditions, including pharmaceuticals of which the degradation by UV/H2O2 was never reported before (e.g., metformin, paroxetine, pindolol, sotalol, venlafaxine, etc.). Monochromatic low pressure (LP) and polychromatic medium pressure (MP) lamps were used for three different water matrices. In order to have well defined hydraulic conditions, all experiments were conducted in a collimated beam apparatus. Degradation rates for the pharmaceuticals were determined. For those compounds used in this research that are also reported in literature, measured degradation results are in good agreement with literature data. Pharmaceutical degradation for only photolysis with LP lamps is small, which is increased by using a MP lamp. Most of the pharmaceuticals are well removed when applying both UV (either LP or MP) and H2O2. However, differences in degradation rates between pharmaceuticals can be large. For example, ketoprofen, prednisolone, pindolol are very well removed by UV/H2O2, whereas metformin, cyclophosphamide, ifosfamide are very little removed by UV/H2O2.

Quantitative assessment of the efficacy of spiral-wound membrane cleaning procedures to remove biofilms.

Cleaning of high pressure RO/NF membranes is an important operational tool to control biofouling. Quantitative information on the efficacy of cleaning agents and protocols to remove biomass is scarce. Therefore, a laboratory cleaning test to assess the efficiency of cleaning procedures to remove attached biomass was developed. The major components of the test are (i) production of uniform biofilm samples, (ii) the quantification of the biomass concentrations with robust parameters and (iii) a simple test procedure with optimal exposure of the biofilm samples to the chemicals. The results showed that PVC-P is a suitable substratum for the production of uniform biofilm samples. ATP and carbohydrates (CH) as major components of the biofilm matrix for nucleotides (living bacterial cells) and extracellular polymeric substances EPS, respectively, were selected as robust biomass parameters. The removal of ATP and CH with the NaOH/Sodium Dodecyl Sulfate (SDS) mixture, selected as a standard treatment at pH 12.0, was reproducible. The resistance of the EPS matrix against chemical cleaning was demonstrated by a low CH removal (32.8 ± 6.0%) compared to the ATP removal (70.5 ± 15.1%). The inverse relationship of biomass removal with the CH to ATP ratio (µg/ng) of the biofilms demonstrated the influence of the biomass characteristics on cleaning. None of the 27 chemicals tested (analytical-grade and commercial brands) in single step or in double-step treatments were significantly more effective than NaOH/SDS. Oxidizing agents NaOCl and H(2)O(2), the latter in combination with SDS, both tested as common agents in biofilm control, showed a significantly higher efficiency (70%) to remove biofilms. In the test, simultaneously, the efficiency of agents to remove precipitated minerals such as Fe can be assessed. Validation tests with Cleaning in Place (CIP) in 8 and 2.5-inch RO membrane pilot plant experiments showed similar ranking of the cleaning efficiency of cleaning protocols as determined in the laboratory tests. Further studies with the laboratory test are required to study the effect of cleaning conditions such as duration, temperature, shear forces as well as chemical conditions (concentrations, alternative agents or mixtures and sequence of application) on the efficiency to remove attached biomass.

Formation and removal of genotoxic activity during UV/H(2)O(2)-GAC treatment of drinking water.

The objective of this study was to determine the genotoxic activity of water after UV/H(2)O(2) oxidation and GAC filtration. Pre-treated surface water from three locations was treated with UV/H(2)O(2) with medium pressure (MP) lamps and passed through granulated activated carbon (GAC). Samples taken before and after each treatment step were extracted and concentrated by solid phase extraction (SPE) and analyzed for genotoxicity using the Comet assay with HepG2 cells and the Ames II assay. The Comet assay showed no genotoxic response in any of the samples. In the Ames II, no genotoxic response was obtained with the TAMix (a mix of six strains), but the TA98 strain showed an increase in genotoxic activity after MP-UV/H(2)O(2) for all three locations. GAC post treatment effectively reduced the activities to control levels at two of the three locations and to below the level of the pre-treated water at one site. The results indicate that UV/H(2)O(2) treatment may lead to the formation of genotoxic by-products, which can be removed by subsequent GAC filtration.

Cross-border comparison of the admission prevalence and clonal structure of meticillin-resistant Staphylococcus aureus.

Since patient exchange between hospitals sharing a common catchment area might favour regional spread of meticillin-resistant Staphylococcus aureus (MRSA), the reliable detection of patients colonised at admission is crucial. Thus, hospitals in the Dutch-German border area EUREGIO MRSA-net aim at synchronising their local MRSA standards in order to prevent unidentified inter-hospital as well as cross-border spread. This assumes enhanced knowledge of MRSA prevalence and risk factors associated with MRSA carriage at admission. We conducted nasal MRSA screening of all inpatients admitted to 39 German hospitals (in the period 1 November to 30 November 2006) and to one Dutch hospital (in the period 1 July to 30 September 2007) in the EUREGIO MRSA-net. A total of 390 MRSA cases were detected among 25,540 patients screened. The admission prevalence was 1.6 MRSA/100 patients (6.5% of all S. aureus) in the German and 0.5 MRSA/100 patients (1.4% of all S. aureus) in the Dutch part of the border region. Overall, the predominating S. aureus protein A gene (spa) sequence types were t003, t032 and t011. One isolate (t044) carried Panton-Valentine leukocidin (PVL) encoding genes. Altogether, 79% and 67% of all MRSA patients in the German and Dutch regions respectively, were identifiable by the classical nosocomial risk factors assessed. In patients lacking all risk factors assessed, spa types t011 and t034 were predominant (P<0.001).