Rapid screening for antibiotic resistance elements on the RNA transcript, protein and enzymatic activity level

Background The emerging threat posed by antibiotic resistance has affected public health systems all over the world. Surveillance of resistant bacteria in clinical settings and identifying them in mixed cultures is of paramount importance and can contribute to the control of their spreading. Culture- independent monitoring approaches are highly desirable, since they yield results much faster than traditional susceptibility testing. However, many rapid molecular methods like PCR only detect the sole presence of a potential resistance gene, do not provide information regarding efficient transcription, expression and functionality and, in addition, cannot assign resistance genes to species level in mixed cultures. Methods By using plasmid-encoded TEM β-lactamase mediated ampicillin resistances as a proof of principle system, we (1) developed a fluorescence in situ hybridization-test (FISH) capable to detect the respective mRNAs, (2) implemented an immunofluorescence test to identify the corresponding proteins and (3) compared these two microscopic tests with an established colorimetric nitrocefin assay to assess the enzymatic activity. Results All three methods proved to be suitable for the testing of antibiotic resistance, but only FISH and immunofluorescence were able to differentiate between susceptible and resistant bacteria on the single cell level and can be combined with simultaneous species identification. Conclusions Fluorescence in situ hybridization and immunofluorescence tests are promising techniques in susceptibility testing since they bridge the gap between the slow, but accurate and sound cultural methods and molecular detection methods like PCR with much less functional relevance

The Glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the "classical" species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH =2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC-glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis

Experimental Challenge of Atlantic Cod (Gadus morhua) with a Brucella pinnipedialis Strain from Hooded Seal (Cystophora cristata)

Publishe

Натечные карбонатные новообразования набережной реки Туры: особенности строения и процессы формирования

International audienc

Global burden of human brucellosis : a systematic review of disease frequency

BACKGROUND: This report presents a systematic review of scientific literature published between 1990-2010 relating to the frequency of human brucellosis, commissioned by WHO. The objectives were to identify high quality disease incidence data to complement existing knowledge of the global disease burden and, ultimately, to contribute towards the calculation of a Disability-Adjusted Life Years (DALY) estimate for brucellosis.METHODS/PRINCIPAL FINDINGS: Thirty three databases were searched, identifying 2,385 articles relating to human brucellosis. Based on strict screening criteria, 60 studies were selected for quality assessment, of which only 29 were of sufficient quality for data analysis. Data were only available from 15 countries in the regions of Northern Africa and Middle East, Western Europe, Central and South America, Sub-Saharan Africa, and Central Asia. Half of the studies presented incidence data, six of which were longitudinal prospective studies, and half presented seroprevalence data which were converted to incidence rates. Brucellosis incidence varied widely between, and within, countries. Although study biases cannot be ruled out, demographic, occupational, and socioeconomic factors likely play a role. Aggregated data at national or regional levels do not capture these complexities of disease dynamics and, consequently, at-risk populations or areas may be overlooked. In many brucellosis-endemic countries, health systems are weak and passively-acquired official data underestimate the true disease burden.CONCLUSIONS: High quality research is essential for an accurate assessment of disease burden, particularly in Eastern Europe, the Asia-Pacific, Central and South America and Africa where data are lacking. Providing formal epidemiological and statistical training to researchers is essential for improving study quality. An integrated approach to disease surveillance involving both human health and veterinary services would allow a better understand of disease dynamics at the animal-human interface, as well as a more cost-effective utilisation of resources

ЯКІВ ДМИТРОВИЧ ГРАХОВ – ДІЯЧ КУЛЬТУРИ ТА ОСВІТИ В КАТЕРИНОСЛАВСЬКІЙ ГУБЕРНІЇ

Розглянуто життєвий шлях, освітянську, наукову та музеєзнавчу діяльність директора Катеринославської гімназії та училищ краю Якова Дмитровича Грахова в середині ХІХ ст.The article covers life, scientific and museum activities of Yakiv Dmitrovych Grakhov, a director of Katerinoslav schools in the middle of the 19th centuary

MLVA-16 typing of 295 marine mammal <i>Brucella</i> isolates from different animal and geographic origins identifies 7 major groups within <i>Brucella ceti</i> and <i>Brucella pinnipedialis</i>

BackgroundSince 1994, Brucella strains have been isolated from a wide range of marine mammals. They are currently recognized as two new Brucella species, B. pinnipedialis for the pinniped isolates and B. ceti for the cetacean isolates in agreement with host preference and specific phenotypic and molecular markers. In order to investigate the genetic relationships within the marine mammal Brucella isolates and with reference to terrestrial mammal Brucella isolates, we applied in this study the Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) approach. A previously published assay comprising 16 loci (MLVA-16) that has been shown to be highly relevant and efficient for typing and clustering Brucella strains from animal and human origin was used.Results294 marine mammal Brucella strains collected in European waters from 173 animals and a human isolate from New Zealand presumably from marine origin were investigated by MLVA-16. Marine mammal Brucella isolates were shown to be different from the recognized terrestrial mammal Brucella species and biovars and corresponded to 3 major related groups, one specific of the B. ceti strains, one of the B. pinnipedialis strains and the last composed of the human isolate. In the B. ceti group, 3 subclusters were identified, distinguishing a cluster of dolphin, minke whale and porpoise isolates and two clusters mostly composed of dolphin isolates. These results were in accordance with published analyses using other phenotypic or molecular approaches, or different panels of VNTR loci. The B. pinnipedialis group could be similarly subdivided in 3 subclusters, one composed exclusively of isolates from hooded seals (Cystophora cristata) and the two others comprising other seal species isolates.ConclusionThe clustering analysis of a large collection of marine mammal Brucella isolates from European waters significantly strengthens the current view of the population structure of these two species, and their relative position with respect to the rest of the Brucella genus. MLVA-16 is confirmed as being a rapid, highly discriminatory and reproducible method to classify Brucella strains including the marine mammal isolates. The Brucella2009 MLVA-16 genotyping database available at http://mlva.u-psud.fr is providing a detailed coverage of all 9 currently recognized Brucella species

Analysis of RASFF notifications on food products contaminated with Listeria monocytogenes reveals options for improvement in the rapid alert system for food and feed

Tools for supranational communication of food safety risks like the European Rapid Alert System for Food and Feed (RASFF) play an increasingly important role in consumer protection along global supply chains. They allow for a coordinated response to emerging public health threats such as Listeria monocytogenes (Lm), the causative agent of the foodborne disease listeriosis. As a result of disease severity and the rising number of human listeriosis cases in Germany since 2011, an effective reporting system on Lm contamination in food products has become more crucial than ever to counteract this trend. Therefore, we analysed RASFF notifications on food products contaminated with Lm and distributed in Germany, 2001 to 2015, assessed trends in the reported data and addressed options for improvement in the current notification system. In RASFF Lm notifications concerning Germany from 2001 to 2015, there was often a discrepancy between country of origin and notifying country, indicating that the food safety risk was not always recognised at the earliest possible time point of the product's life span. In addition, in our dataset, most Lm notifications were driven by official controls when the respective product was already on the market. However, starting in 2005, there was an increasing trend for company's own checks. This trend of making food manufacturers accountable for the detection and notification of contaminated products in the production line is a first step into the right direction as it might help to reduce the number of contaminated food products that enter the market. Besides its function as a reporting tool, the RASFF may also facilitate the identification of risk factors associated with Lm contamination so that the problem can be tackled at its root. Unfortunately, information about packaging and food processing was only mentioned in a minority of Lm notifications. Hence, risk factors cannot be easily identified. In the future, a comprehensive database including additional metadata together with the RASFF notification should be established. Although a solid basis for the surveillance of Lm, there is still room for improvement in RASFF to speed-up the flow of information. This might help to identify food safety risks that can be harmful to European consumers much faster, more effectively prevent the spread of risk bearing food products and consequently reduce the burden of listeriosis

Translatability of WGS typing results can simplify data exchange for surveillance and control of Listeria monocytogenes

Where classical epidemiology has proven to be inadequate for surveillance and control of foodborne pathogens, molecular epidemiology, using genomic typing methods, can add value. However, the analysis of whole genome sequencing (WGS) data varies widely and is not yet fully harmonised. We used genomic data on 494 Listeria monocytogenes isolates from readyto- eat food products and food processing environments deposited in the strain collection of the German National Reference Laboratory to compare various procedures for WGS data analysis and to evaluate compatibility of results. Two different core genome multilocus sequence typing (cgMLST) schemes, different reference genomes in single nucleotide polymorphism (SNP) analysis and commercial as well as opensource software were compared. Correlation of allele distances from the different cgMLST approaches was high, ranging from 0.97 to 1, and unified thresholds yielded higher clustering concordance than schemespecific thresholds. The number of detected SNP differences could be increased up to a factor of 3.9 using a specific reference genome compared with a general one. Additionally, specific reference genomes improved comparability of SNP analysis results obtained using different software tools. The use of a closed or a draft specific reference genome did not make a difference. The harmonisation of WGS data analysis will finally guarantee seamless data exchange, but, in the meantime, knowledge on threshold values that lead to comparable clustering of isolates by different methods may improve communication between laboratories. We therefore established a translation code between commonly applied cgMLST and SNP methods based on optimised clustering concordances. This code can work as a first filter to identify WGS- based typing matches resulting from different methods, which opens up a new perspective for data exchange and thereby accelerates timecritical analyses, such as in outbreak investigations

Direct identification and molecular characterization of zoonotic hazards in raw milk by metagenomics using brucella as a model pathogen

Metagenomics is a valuable diagnostic tool for enhancing microbial food safety because (i) it enables the untargeted detection of pathogens, (ii) it is fast since primary isolation of micro-organisms is not required, and (iii) it has high discriminatory power allowing for a detailed molecular characterization of pathogens. For shotgun metagenomics, total nucleic acids (NAs) are isolated from complex samples such as foodstuff. Along with microbial NAs, high amounts of matrix NAs are extracted that might outcompete microbial NAs during next-generation sequencing and compromise sensitivity for the detection of low abundance micro-organisms. Sensitive laboratory methods are indispensable for detecting highly pathogenic foodborne bacteria like Brucella spp., because a low infectious dose is sufficient to cause human disease through the consumption of contaminated dairy or meat products. In our study, we applied shotgun metagenomic sequencing for the identification and characterization of Brucella spp. in artificially and naturally contaminated raw milk from various ruminant species. With the depletion of eukaryotic cells prior to DNA extraction, Brucella was detectable at 10 bacterial cells ml−1, while at the same time microbiological culture and isolation of the fastidious bacteria commonly failed. Moreover, we were able to retrieve the genotype of a Brucella isolate from a metagenomic dataset, indicating the potential of metagenomics for outbreak investigations using SNPs and core-genome multilocus sequence typing (cgMLST). To improve diagnostic applications, we developed a new bioinformatics approach for strain prediction based on SNPs to identify the correct species and define a certain strain with only low numbers of genus-specific reads per sample. This pipeline turned out to be more sensitive and specific than Mash Screen. In raw milk samples, we simultaneously detected numerous other zoonotic pathogens, antimicrobial resistance genes and virulence factors. Our study showed that metagenomics is a highly sensitive tool for biological risk assessment of foodstuffs, particularly when pathogen isolation is hazardous or challenging