Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. Traditionally, tick-borne pathogen detection has been carried out using PCR-based methods that rely in known sequences for specific primers design. This approach matches with the view of a 'single-pathogen' epidemiology. Recent results, however, have stressed the importance of coinfections in pathogen ecology and evolution with impact in pathogen transmission and disease severity. New approaches, including high-throughput technologies, were then used to detect multiple pathogens, but they all need a priori information on the pathogens to search. Thus, those approaches are biased, limited and conceal the complexity of pathogen ecology. Currently, next generation sequencing (NGS) is applied to tick-borne pathogen detection as well as to study the interactions between pathogenic and non-pathogenic microorganisms associated to ticks, the pathobiome. The use of NGS technologies have surfaced two major points: (i) ticks are associated to complex microbial communities and (ii) the relation between pathogens and microbiota is bidirectional. Notably, a new challenge emerges from NGS experiments, data analysis. Discovering associations among a high number of microorganisms is not trivial and therefore most current NGS studies report lists of microorganisms without further insights. An alternative to this is the combination of NGS with analytical tools such as network analysis to unravel the structure of microbial communities associated to ticks in different ecosystems.
- MeSH
- Bacteria isolation & purification MeSH
- Host-Pathogen Interactions MeSH
- Ticks microbiology MeSH
- Coinfection microbiology MeSH
- Humans MeSH
- Microbial Interactions MeSH
- Microbiota * MeSH
- Tick-Borne Diseases diagnosis microbiology MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The aim of the review is to characterize Enterobacter sakazakii alias Cronobacter sp. as one of most dangerous microbial contaminants in foods. The microbe is a pathogen causing a serious disease in premature and newborn infants, rarely in adults. Despite a low occurrence of the infections, a high mortality (up to 80 %) has been reported. A survey of methods of its detection is given. Cultivation, chromogenic/fluorogenic media, biochemical kits and molecular genetic methods are promising in Enterobacter sakazakii screening in foods.
- MeSH
- Cronobacter sakazakii pathogenicity MeSH
- Molecular Diagnostic Techniques methods MeSH
- Financing, Organized MeSH
- Food Contamination legislation & jurisprudence MeSH
- Culture Techniques methods MeSH
- Polymerase Chain Reaction methods MeSH
- Food Microbiology legislation & jurisprudence MeSH
- Reagent Kits, Diagnostic utilization MeSH
Bakteriální zoonózy představují i v současnosti závažný medicínský problém. K méně častým, ale závažným zoonózám patří maltská horečka způsobená bakterií Brucella melitensis. Předkládaná kazuistika popisuje případ relapsu importované maltské horečky u mladého muže. Diagnóza byla vedle čtyř sérologických testů verifikována přímým průkazem původce v hemokultuře. Izolát Brucella melitensis byl identifikován metodou polymerázové řetězové reakce a metodou hmotností spektrometrie MALDI-TOF.
Bacterial zoonoses still represent a serious medical problem. One of the less frequent but severe zoonoses is brucellosis caused by the bacterium Brucella melitensis. The presented case report describes relapsing imported brucellosis in a young male. In addition to four serological tests, the diagnosis was confirmed by direct detection of the pathogen in blood culture. The isolate of Brucella melitensis was identified using the MALDI-TOF BioTyper method and subsequently also by PCR.
- Keywords
- MALDI-TOF, alimentární infekce, zoonóza,
- MeSH
- Brucella melitensis isolation & purification MeSH
- Brucellosis diagnosis microbiology MeSH
- Travel MeSH
- Humans MeSH
- Young Adult MeSH
- Check Tag
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Publication type
- Case Reports MeSH
Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 10⁶ cells mL⁻¹ in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 10⁴ cells mL⁻¹, was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG₆). However, when the EG₆ based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples. The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19 nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk.
- MeSH
- Enterobacteriaceae isolation & purification pathogenicity MeSH
- Antibodies, Immobilized MeSH
- Infant MeSH
- Humans MeSH
- Limit of Detection MeSH
- Milk microbiology MeSH
- Infant Formula MeSH
- Polyhydroxyethyl Methacrylate MeSH
- Food Microbiology methods statistics & numerical data MeSH
- Surface Plasmon Resonance methods statistics & numerical data MeSH
- Antibodies, Bacterial MeSH
- Gold MeSH
- Animals MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
334 s.
Aphanomyces astaci, the causal agent of the crayfish plague, has recently been confirmed to infect also freshwater-inhabiting crabs. We experimentally tested the resistance of freshwater shrimps, another important decapod group inhabiting freshwaters, to this pathogen. We exposed individuals of two Asian shrimp species, Macrobrachium dayanum and Neocaridina davidi, to zoospores of the pathogen strain isolated from Procambarus clarkii, a known A. astaci carrier likely to get into contact with shrimps. The shrimps were kept in separate vessels up to seven weeks; exuviae and randomly chosen individuals were sampled throughout the experiment. Shrimp bodies and exuviae were tested for A. astaci presence by a species-specific quantitative PCR. The results were compared with amounts of A. astaci DNA in an inert substrate to distinguish potential pathogen growth in live specimens from persisting spores or environmental DNA attached to their surface. In contrast to susceptible crayfish Astacus astacus, we did not observe mortality of shrimps. The amount of detected pathogen DNA was decreasing steadily in the inert substrate, but it was still detectable several weeks after zoospore addition, which should be considered in studies relying on molecular detection of A. astaci. Probably due to moulting, the amount of A. astaci DNA was decreasing in N. davidi even faster than in the inert substrate. In contrast, high pathogen DNA levels were detected in some non-moulting individuals of M. dayanum, suggesting that A. astaci growth may be possible in tissues of this species. Further experiments are needed to test for the potential of long-term A. astaci persistence in freshwater shrimp populations.
Receptor adenylate cyclases (RACs) on the surface of trypanosomatids are important players in the host-parasite interface. They detect still unidentified environmental signals that affect the parasites' responses to host immune challenge, coordination of social motility, and regulation of cell division. A lesser known class of oxygen-sensing adenylate cyclases (OACs) related to RACs has been lost in trypanosomes and expanded mostly in Leishmania species and related insect-dwelling trypanosomatids. In this work, we have undertaken a large-scale phylogenetic analysis of both classes of adenylate cyclases (ACs) in trypanosomatids and the free-living Bodo saltans. We observe that the expanded RAC repertoire in trypanosomatids with a two-host life cycle is not only associated with an extracellular lifestyle within the vertebrate host, but also with a complex path through the insect vector involving several life cycle stages. In Trypanosoma brucei, RACs are split into two major clades, which significantly differ in their expression profiles in the mammalian host and the insect vector. RACs of the closely related Trypanosoma congolense are intermingled within these two clades, supporting early RAC diversification. Subspecies of T. brucei that have lost the capacity to infect insects exhibit high numbers of pseudogenized RACs, suggesting many of these proteins have become redundant upon the acquisition of a single-host life cycle. OACs appear to be an innovation occurring after the expansion of RACs in trypanosomatids. Endosymbiont-harboring trypanosomatids exhibit a diversification of OACs, whereas these proteins are pseudogenized in Leishmania subgenus Viannia. This analysis sheds light on how ACs have evolved to allow diverse trypanosomatids to occupy multifarious niches and assume various lifestyles.
- MeSH
- Adenylyl Cyclases genetics MeSH
- Gene Duplication MeSH
- Phylogeny * MeSH
- Genome, Protozoan MeSH
- Host-Pathogen Interactions genetics MeSH
- Evolution, Molecular * MeSH
- Trypanosomatina enzymology genetics MeSH
- Up-Regulation MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The development of integrated, fast and affordable platforms for pathogen detection is an emerging area where a multidisciplinary approach is necessary for designing microsystems employing miniaturized devices; these new technologies promise a significant advancement of the current state of analytical testing leading to improved healthcare. In this work, the development of a lab-on-chip microsystem platform for the genetic analysis of Salmonella in milk samples is presented. The heart of the platform is an acoustic detection biochip, integrated with a microfluidic module. This detection platform is combined with a micro-processor, which, alongside with magnetic beads technology and a DNA micro-amplification module, are responsible for performing sample pre-treatment, bacteria lysis, nucleic acid purification and amplification. Automated, multiscale manipulation of fluids in complex microchannel networks is combined with novel sensing principles developed by some of the partners. This system is expected to have a significant impact in food-pathogen detection by providing for the first time an integrated detection test for Salmonella screening in a very short time. Finally, thanks to the low cost and compact technologies involved, the proposed set-up is expected to provide a competitive analytical platform for direct application in field settings.
- MeSH
- DNA, Bacterial analysis MeSH
- Lab-On-A-Chip Devices microbiology MeSH
- Milk microbiology MeSH
- Food Microbiology methods MeSH
- Salmonella genetics isolation & purification MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.
- MeSH
- Bacterial Proteins genetics MeSH
- Burkholderia genetics isolation & purification pathogenicity MeSH
- Cystic Fibrosis complications microbiology MeSH
- Child MeSH
- Virulence Factors genetics metabolism physiology MeSH
- Genome, Bacterial MeSH
- Burkholderia Infections complications MeSH
- Humans MeSH
- Communicable Diseases, Emerging MeSH
- Opportunistic Infections complications microbiology MeSH
- Quorum Sensing MeSH
- Gene Expression Regulation, Bacterial MeSH
- Gene Expression Profiling MeSH
- Virulence genetics MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Stále častější používání katétrů a umělých implantátů, podávání antibiotik a vysoký počet imunokompromitovaných pacientů patří mezi hlavní příčiny rostoucího významu biofilmových infekcí. Jejich charakteristické rysy, především značná rezistence k antimikrobiálním látkám a tvorba dlouhodobě perzistujících ložisek, komplikují terapii. Proto je pro klinické lékaře znalost přítomnosti tohoto faktoru patogenity cenná a měla by mít vliv na postup při léčbě pacienta. K průkazu schopnosti tvořit biofilm u klinicky významných mikroorganismů je však potřeba dostatečně spolehlivý a citlivý postup, kterého lze použít i v podmínkách běžné mikrobiologické laboratoře. Pro průkaz tohoto faktoru virulence se v současnosti používá široké spektrum vyšetřovacích technik. Vizualizací biofilmu mikroskopickými technikami počínaje přes kultivační průkaz, detekci jeho složek, odhalování fyzikálně-chemických odlišností biofilmpozitivních mikroorganismů ve srovnání s jejich planktonickými formami až po průkaz genů zodpovědných za jeho tvorbu. Vzhledem k omezením jednotlivých metod lze nejlepších výsledků dosáhnout nejlépe jejich vzájemnou kombinací.
The increasing use of catheters, artificial implants and antimicrobials as well as high numbers of immunocompromised patients are major causes for concern over biofilm infections. These infections are characterized particularly by high resistance to antimicrobials and formation of persistent foci that may complicate therapy. Therefore, detection of biofilm formation is of high relevance to the clinician and his/her approach to the treatment. Reliable and sensitive methods for detection of this pathogenicity factor in clinically important organisms, suitable for use in routine microbiological laboratories, are needed for this purpose. Currently, a wide array of techniques are available for detection of this virulence factor, such as biofilm visualization by microscopy, culture detection, detection of particular components, detection of physical and chemical differences between biofilm-positive organisms and their planktonic forms and detection of genes responsible for biofilm formation. Since each of these methods has limitations, the best results can be achieved by combining different approaches.
