Two-site study on performances of a commercially available MALDI-TOF MS-based assay for the detection of colistin resistance in Escherichia coli.

Colistin is a last resort drug for the treatment of multiple drug-resistant (MDR) Gram-negative bacterial infections. Rapid methods to detect resistance are highly desirable. Here, we evaluated the performance of a commercially available MALDI-TOF MS-based assay for colistin resistance testing in Escherichia coli at two different sites. Ninety clinical E. coli isolates were provided by France and tested in Germany and UK using a MALDI-TOF MS-based colistin resistance assay. Lipid A molecules of the bacterial cell membrane were extracted using the MBT Lipid Xtract Kit™ (RUO; Bruker Daltonics, Germany). Spectra acquisition and evaluation were performed by the MBT HT LipidART Module of MBT Compass HT (RUO; Bruker Daltonics) on a MALDI Biotyper® sirius system (Bruker Daltonics) in negative ion mode. Phenotypic colistin resistance was determined by broth microdilution (MICRONAUT MIC-Strip Colistin, Bruker Daltonics) and used as a reference. Comparing the results of the MALDI-TOF MS-based colistin resistance assay with the data of the phenotypic reference method for the UK, sensitivity and specificity for the detection of colistin resistance were 97.1% (33/34) and 96.4% (53/55), respectively. Germany showed 97.1% (33/34) sensitivity and 100% (55/55) specificity for the detection of colistin resistance by MALDI-TOF MS. Applying the MBT Lipid Xtract™ Kit in combination with MALDI-TOF MS and dedicated software showed excellent performances for E. coli. Analytical and clinical validation studies must be performed to demonstrate the performance of the method as a diagnostic tool.

Improved DNA extraction and purification with magnetic nanoparticles for the detection of methicillin-resistant Staphylococcus aureus.

Molecular methods offer fast, safe and cost-efficient detection of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). These tests depend on a rapid extraction of bacterial DNA. The aim of this study was to compare an optimized DNA extraction and purification protocol for MRSA using magnetic nanoparticles with the original method. The purity of the extracted DNA was assessed by photometric measurements and the amount of DNA was determined by real-time PCR. Three MRSA reference strains (S. aureus ATCC® 70699, S. aureus ATCC® 43300; S. aureus ATCC® 33592) and eleven MRSA field strains, which include SCCmec elements of types I to XI, were used in this study. The optimized protocol can save approximately 20 min time compared to the original method and the DNA yield was higher with the new protocol. Therefore, this new protocol allows a faster DNA extraction from MRSA cultures.

Whole-genome sequencing of a large collection of Myroides odoratimimus and Myroides odoratus isolates and antimicrobial susceptibility studies.

The genus Myroides comprises several species of Gram-negative, non-motile, and non-fermenting bacteria, which have been regarded as non-pathogenic for decades. Multiple recent reports, however, underscore the pathogenic potential that Myroides sp. possesses for humans. These bacteria seem to be resistant to a wide range of antibiotics (including ß-lactams and aminoglycosides). Therefore, treatment options are limited. Knowledge of antimicrobial resistance, however, is based on only one meaningful comprehensive study and on data published from case reports. This lack of data motivated us to test 59 strains from our Myroides collection (43 M. odoratimimus and 16 M. odoratus) for resistance against 20 commonly used antibiotics. We also performed molecular analyses to reveal whether our bacteria harbor the genus-specific M. odoratimimus metallo-ß-lactamase (MUS-1) or the M. odoratus metallo ß-lactamase (TUS-1), and other ß-lactamases, which may provide an explanation for the extended antimicrobial resistance.

Plasmodium Detection and Differentiation by Direct-on-Blood PCR Nucleic Acid Lateral Flow Immunoassay: Development, Validation, and Evaluation.

Decreasing malaria transmission warrants the search for highly sensitive point-of-care diagnostics, especially in resource-limited settings. The direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA) is a simplified PCR-based technique with a lateral flow readout that does not require sample preparation. Two duplex db-PCR-NALFIAs were developed: a pan-Plasmodium/Plasmodium falciparum (pan/P. falciparum) and a pan-Plasmodium/Plasmodium vivax (pan/P. vivax) assay. Confirmed positive samples (n = 61) and negative controls (n = 40) were used for laboratory validations. A prospective field evaluation of the pan/P. falciparum assay was performed in Kenya (n = 300). In the laboratory validation, sensitivity and specificity of the pan/P. falciparum assay were 100% (95% CI, 94.1%-100%) and 100% (95% CI, 91.2%-100%), respectively. Sensitivity and specificity of the pan/P. vivax assay were 100% (95% CI, 94.1%-100%) and 97.5% (95% CI, 86.8%-99.9%), respectively. In Kenya, sensitivity of the pan/P. falciparum db-PCR-NALFIA was 97.2% (95% CI, 93.0%-99.2%) and specificity was 74.2% (95% CI, 67.0%-81.0%) compared with reference standard microscopy. When using real-time quantitative PCR as a reference standard, sensitivity was 84.5% (95% CI, 78.7%-89.3%) and specificity was 85.4% (95% CI, 77.1%-91.6%). Db-PCR-NALFIA is a sensitive, specific, and easy method for the detection and species differentiation of Plasmodium. This test is especially of interest for malaria control or elimination programs in low-transmission settings that require accurate detection of low parasite densities.

Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC.

The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all ß-lactam antibiotics. To avoid the use of ß-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA and mecC that can easily be integrated in routine laboratory diagnostics. The aim of this study was the development of such a rapid detection method for methicillin resistance based on nucleic acid lateral flow immunoassay (NALFIA) technology. In NALFIA, the target sequences are PCR-amplified, immobilized via antigen-antibody interaction and finally visualized as distinct black bars resulting from neutravidin-labeled carbon particles via biotin-neutravidin interaction. A screening of 60 defined strains (MRS and non-target bacteria) and 28 methicillin-resistant S. aureus (MRSA) isolates from clinical samples was performed with PCR-NALFIA in comparison to PCR with subsequent gel electrophoresis (PCR-GE) and real-time PCR. While all samples were correctly identified with all assays, PCR-NALFIA was superior with respect to limits of detection. Moreover, this assay allowed for differentiation between mecA and mecC by visualizing the two alleles at different positions on NALFIA test stripes. However, since this test system only targets the mecA and mecC genes, it does not allow to determine in which staphylococcal species the mec gene is included. Requiring only a fraction of the time needed for cultural methods (i.e. the gold standard), the PCR-NALFIA presented here is easy to handle and can be readily integrated into laboratory diagnostics.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.

The impact of zoonotic MRSA colonization and infection in Germany.

Methicillin-resistant Staphylococcus aureus (MRSA) causes colonization and infection both in animals and humans. In Germany, cases of MRSA colonization among humans, w+hich affect 0.5-1.5% of the general population and 1.0-2.5% of patients at hospital admission, are still mostly associated with previous healthcare contact and defined epidemic clonal lineages. However, MRSA is also distributed in livestock production in Germany, mostly without causing infections in the animals. These MRSA predominantly belong to the clonal complex (CC) 398, but also to CC9 and CC97. Zoonotic transmission of MRSA CC398 from livestock to humans occurs predominantly in people with occupational livestock contact. Spread of MRSA CC398 to household members of these persons is also frequently observed, but dissemination in the general population is limited so far However, especially in areas with intensive livestock husbandry, about 20-38% of MRSA CC398 cases among humans cannot be epidemiologically linked to direct livestock contact, indicating other transmission pathways. MRSA CC398 currently causes about 2% of all human MRSA infections (wound infections, pneumonia, sepsis) in Germany, but up to 10% in regions characterized by a high density of livestock-farming. The burden of MRSA in companion animals was demonstrated to range between 3.6-9.4% within wound samples obtained from dogs, cats and horses, respectively. In contrast to livestock and horses, MRSA distributed in pet animals are mostly associated with MRSA clonal lineages that are also prevalent in human healthcare facilities. Overall, zoonotic exchange of MRSA between humans and animals has relevant impact on the epidemiology of MRSA in Germany.

Assessing DNA damage and health risk using biomarkers.

Carcinogenesis is a multi-stage and prolonged process. At the present time, our knowledge of biological activities along the process is incomplete, therefore, a variety of experimental data are used to assess health risk from exposure to environmental chemicals. However, experimental approaches may not be adequate unless human data are available to support the assessment. In this brief review, benzene (CAS No. 71-43-2), a well-established human leukemogen, will be used as an example to illustrate the challenge in assessing toxicological mechanisms and cancer risk. Benzene has been shown to form DNA-adducts in experimental animals but the adducts have proved elusive of detection in human. Several toxic metabolites of benzene have been identified but the metabolite(s) responsible for the carcinogenic activities is unknown. Furthermore, the significant differences between rodents and human in response to benzene exposure are not understood. Therefore, the bone marrow specificity for the induction of leukemia in human by benzene remains to be elucidated. These complications illustrate the complexity of the assessment process and identify serious information gaps. These information gaps can be viewed as research opportunities to provide more precise data for assessment of toxicological effects and health risk.