Strains In Germany Research Articles

Influence of Mutations and N-Glycosylation Sites in the Receptor-Binding Domain (RBD) and the Membrane Protein of SARS-CoV-2 Variants of Concern on Antibody Binding in ELISA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI- cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI- cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.

Molecular diversity for Entamoeba histolytica virulence factor genes isolated from patients with amoebic dysentery

Entamoebahistolytica is a parasitic protozoan that caused amoebiasis, a diseases which infects the liver and colon and spread all over the world. E.histolytica is causing the death of human cells and leading to develop invasion, and ulceration of the intestine. However, amoebic liver abscess(ALA) considers as major common extraintestinal disease caused by E.histolytica.The aims of this study, was to detect E. histolytica by polymerase chain reaction (PCR), and to differentiate it from other amoeba species in a stool samples of human. Study the pathogenicity of E. histolytica in human through determining the presence of virulence factors like cysteine protease, amoebapore and Gal/lectin. A total of 100 stool samples were collected from patients with amoebic dysentery and then examined microscopically to confirm the presence of E. histolytica and other Entamoeba species, 72 ( 72%) out of 100 examined samples with E.histolytica were positive by PCR and 28(28%) were negative. The positive samples with PCR have been submitted to detect the virulence factors of E.histolytica by PCR and the results showed that the total of 72(100%)were positive with cysteine proteases gene, shown 62(86.11%) were positive with amoebapore and 65(90.27) were positive with lectin. Gene sequence for cysteine protease, amoebapore and lectin in local E. histolytica human isolates showed the nucleotide alignment similarity and substitution mutations in all of cysteine protease, amoebapore and Gal/GalNAc lectin. Phylogenetic tree analysis based on cysteine protease gene partial sequence in local E.histolytica human isolates that used for genetic variation analysis showed genetic closed related to NCBI-BLAST E.histolytica Japan strain (AK421547.1) at total genetic changes (0.0060-0.0010%), while Phylogenetic tree analysis based on amoebapore B gene partial sequence in local E.histolytica human isolates that used for genetic variation analysis showed genetic closed related to NCBI-BLAST E.histolytica, Germany strain (X76904.1) at total genetic changes (0.0060-0.0010%). Phylogenetic tree analysis based on Gal/GalNAc lectin gene partial sequence in local E. histolytica human isolates that used for genetic variation analysis showed genetic closed related to NCBI-BLAST E.histolytica Bangladesh strain (AF501280.1) at total genetic changes (0.0060-0.0020.

Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae.

Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening about 10 000 genomes, over 400 000 metagenomes and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.

Almanya’da Yüksek Nitelikli Göçmenlerin Gündelik Hayat Deneyimleri ve Baş Etme Süreçleri

Almanya’da Turkiye kokenli gocmenlere yonelik olumsuz tutumlar, toplumda kurulan iliskileri zedelemekte ve yukselen gundelik hayat ayrimciliginin temelini teskil etmektedir. Turkiye-Almanya arasi goc dalgalarindan birini de akademik kaygilarla goc eden doktora ogrencileri olusturmaktadir. Calisma kapsaminda, Berlin’e doktora egitimleri icin gitmis ve sahip olduklari sosyal-kulturel sermaye ile yuksek nitelikli gocmen statusunde kabul edilebilecek 10 katilimci ile yari yapilandirilmis mulakat yapilarak, ogrencilerin gundelik hayat deneyimleri ve bu dogrultuda gecirdikleri bas etme surecleri nitel arastirma yontemlerinden icerik analizi ile degerlendirilmistir. Bu doktora ogrencileri, kendilerine ozgu goc amac ve kosullari ile Almanya’nin en buyuk azinligini olusturan diger gocmen kusaklardan farklilasmaktadirlar. Butun bu farklilasmaya ragmen gundelik hayat deneyimlerinin, algiladiklari kalip yargilarin azinlik toplumunun uyeleri ile benzerlikler gosterdigi gozlemlenmektedir. Ote yandan bas etme surecleri goz onune alinirsa onlardan ayrismaktadirlar. Bu aktorlerin deneyimleri, algiladiklari kalip yargilarla gelistirdikleri taktikleri ve bu baglamda kendisini gosteren aidiyet surecleri makalenin ana sorunsalini olusturmaktadir.

Genetic variation, phylogenetic relationship and spatial distribution of \u2018Candidatus Phytoplasma ulmi\u2019 strains in Germany

A recent survey in Germany revealed the wide presence of ‘Candidatus Phytoplasma ulmi’ in native elm stands. Accessions were studied for their genetic variability and phylogenetic relationship based on the conserved groEL and the variable imp gene. While the groEL sequences revealed a high intraspecific homology of more than 99%, the homology of the imp gene dropped to 71% between distantly related sequences. Twenty-nine groEL and 74 imp genotypes were distinguished based on polymorphic sites. Phylogenetic analysis of the groEL gene clustered all ‘Ca. P. ulmi’ strains and separated them from related phytoplasmas of the 16SrV group. The inferred phylogeny of the imp gene resulted in a different tree topology and separated the ‘Ca. P. ulmi’ genotypes into two clusters, one closely related to the flavescence dorée phytoplasma strain FD-D (16SrV-D), the other affiliated with the flavescence dorée phytoplasma strains FD-C and FD70 and the alder yellows phytoplasma (16SrV-C). In both phylograms, ‘Ca. P. ulmi’ genotypes from Scots elm trees formed a coherent cluster, while genotypes from European white elms and field elms grouped less strictly. The regional distribution pattern was congruent for some of the groEL and imp genotypes, but a strict linkage for all genotypes was not apparent.

Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4.

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors—active partitioning, toxin-antitoxin and conjugational transfer—are all involved in the prevalence of pESBL in the E. coli population.

Genetic Diversity and Spatial Segregation of Francisella tularensis Subspecies holarctica in Germany.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%.

The rise of mental health problems, inequality and the role of job strain in Germany

The prevalence of mental health problems among the working population has increased sharply since the turn of the millennium in Germany. Secondary analyses of the BIBB/BAuA employment surveys from 1999, 2006 and 2012 showed that, whereas in 1999 only about 4 to 6% of respondents reported mental health problems, this number had grown to 20% or higher in 2012. In this article, we investigate if this sharp increase in mental health problems, measured in terms of self-reported regular occurrence of four specific indicators during the preceding 12–month period, corresponds to a similar increase in social inequality; how different occupational social classes (European Socioeconomic Classification: ESeC) were affected between 1999 and 2012; and whether increasing job strain (mental strain, rigid work organization, ergonomic strain) can explain the increase in mental health problems among different occupational classes. We analyze job strain in relation to a) the increase in the prevalence of self-reported mental health problems and b) the differential development of these problems within different occupational social classes using a decomposition method. Results confirm an increasing trend of mental health problems. Inequality in mental health has also increased, but only slightly. Different occupational subgroups follow distinct trajectories, with particularly steep increases among social service occupations. Job strain, especially mental strain, increases the frequency of mental health problems, but explains only a minor part of the trend.

Mental Strain of Immigrants in the Working Context.

Inability to work due to reported mental strain and psychiatric disorders is rising in Germany these days. Meanwhile the country’s net migration is positive. While there is empirical evidence for a healthy migrant effect regarding the physical health in the beginning (mostly accompanied by a subsequent convergence effect), the mental health of migrants remains partly understudied. In order to evaluate the migrant’s share in the rise of reported mental strain in Germany, 4000 employees were surveyed by means of an online access panel. About 16 percent of them revealed a migration background. Their Copenhagen Burnout Inventory (CBI) score is slightly yet significantly above the German autochthonous’ one both using bi- and multivariate analysis, indicating that there is a specific vulnerability rather than a healthy migrant effect regarding mental strain at work.

Evidence for presumable feline origin of sporadic G6P[9] rotaviruses in humans

Species A rotaviruses are highly diverse and impose a substantial burden to human and animal health. Interspecies transmission between livestock, domestic animals and humans is commonly observed, but spread of animal-like rotaviruses within the human population is limited.During the continued monitoring of rotavirus strains in Germany, an unusual G6P[9] rotavirus strain was detected in feces of a child. The complete rotavirus coding sequences revealed a unique G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E2-H3 genotype constellation. The virus was phylogenetically related to feline G3P[9] strains and other human G6P[9] rotaviruses of presumable zoonotic origin. Analysis of primer binding sites of G6 specific genotyping revealed further evidence of a G6P[9] feline reservoir. Moreover, substantial deficits of conventional semi-nested PCR genotyping approaches in detecting contemporary G6P[9] were revealed. Rotavirus strain GER29-14 most likely resulted from a direct or recent interspecies transmission from a cat to human. Further studies could assess nucleic acid sequences and genotype constellations of feline rotavirus to confirm the likely feline origin of sporadic human G6P[9] strains.

Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination.

Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR) bacteria. They are self-transmittable via conjugation as carrying the required set of genes and cis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated from Escherichia coli O104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such ‘superspreader’ mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft, traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili) and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity.

Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains

BackgroundThe occurrence of the novel porcine circovirus type 3 (PCV3) was reported from the Americas, Asia and Europe. Although this virus was detected in association with various clinical syndromes in pigs, its role as possible swine pathogen remains unclear. PCV3 was detected with high prevalence in Polish farms, but to date no genome sequences were available from European PCV3 strains.MethodsWe collected 1060 serum samples from piglets at the age of 20–24 weeks from 53 farms distributed all over Germany. PCV3 DNA was detected using a real-time PCR and subsequently complete PCV3 genome sequences were obtained after multiply primed rolling circle amplification and sequencing of overlapping PCR products. Phylogenetic analysis was performed by neighbor-joining method and maximum likelihood method.ResultsWe obtained 15 complete PCV3 genome sequences as well as nine partial sequences including the putative ORFs 1, 2 and 3 from PCV3 viremic animals in German pig farms. Phylogenetic analysis of these German as well as 30 full genome sequences received from GenBank divided the PCV3 strains into two main groups and several subclusters. Furthermore, we were able to define group specific amino acid patterns in open reading frame 1 and 2.ConclusionPCV3 is distributed with high prevalence in German pig industry. Phylogenetic analysis revealed two clearly separated groups of PCV3 strains, which might be considered as PCV3 genotypes. Specific nucleotide and amino acid marker positions may serve for easy and fast intraspecies classification and genotyping of PCV3 strains. No correlation between PCV3 variants with their geographical origin was evident. We found the same diversity of PCV3 strains in Germany as in other countries. We hypothesize that PCV3 is not a newly emerging virus in the German pig population. Future studies will have to show, if PCV3 genotype specific biological properties are evident.

Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104:H4 outbreak strain and EHEC O157:H7

BackgroundShiga toxin (Stx) producing Escherichia coli (E. coli) (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative E. coli (EAEC). Pathotypes of E. coli evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of E. coli O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome.ResultsWe observed severe clinical symptoms, such as diarrhoea, dehydration and neurological disorders as well as attaching-and-effacing lesions (A/E) in the colon in STEC O157:H7 infected piglets. In contrast, STEC O104:H4 challenged animals exhibited only mild clinical symptoms including diarrhoea and dehydration and HUS-specific/severe histopathological, haematological and biochemical alterations were only inconsistently presented by individual piglets. A specific adherence phenotype of STEC O104:H4 could not be observed. Flow cytometric analyses of lymphocytes derived from infected animals revealed an increase of natural killer cells (NK cells) during the course of infection revealing a potential role of this subset in the anti-bacterial activity in STEC disease.ConclusionsUnexpectedly, E. coli O104:H4 infection caused only mild symptoms and minor changes in histology and blood parameters in piglets. Outcome of the infection trial does not reflect E. coli O104:H4 associated human disease as observed during the outbreak in 2011. The potential role of cells of the innate immune system for STEC related disease pathogenesis should be further elucidated.

Analysis of non-typeable Haemophilus influenzae in invasive disease reveals lack of the capsule locus

Among invasive Haemophilus influenzae, unencapsulated strains have largely surpassed the previously predominant serotype b (Hib) because of Hib vaccination. Isolates without the genomic capsule (cap) locus are designated non-typeable H. influenzae (NTHi). They are different from capsule-deficient variants that show deletion of the capsule transport gene bexA within the cap locus. The frequency of capsule-deficient variants in invasive disease is unknown. We analysed 783 unencapsulated invasive isolates collected over 5 years in Germany and found no single capsule-deficient isolate. Invasive unencapsulated strains in Germany were exclusively NTHi. A negative serotyping result by slide agglutination was therefore highly predictive for NTHi.

Cyp51A -Based Mechanisms of Aspergillus fumigatus Azole Drug Resistance Present in Clinical Samples from Germany

Since the mid-1990s, a steady increase in the occurrence of itraconazole-resistant Aspergillus fumigatus isolates has been observed in clinical contexts, leading to therapeutic failure in the treatment of aspergillosis. This increase has been predominantly linked to a single allele of the cyp51A gene, termed TR/L98H, which is thought to have arisen through the use of agricultural azoles. Here, we investigated the current epidemiology of triazole-resistant A. fumigatus and underlying cyp51A mutations in clinical samples in Germany. From a total of 527 samples, 17 (3.2%) showed elevated MIC0 values (the lowest concentrations with no visible growth) for at least one of the three substances (itraconazole, voriconazole, and posaconazole) tested. The highest prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among resistant isolates, the TR/L98H mutation in cyp51A was the most prevalent, but isolates with the G54W and M220I substitutions and the novel F219C substitution were also found. The isolate with the G54W substitution was highly resistant to both itraconazole and posaconazole, while all others showed high-level resistance only to itraconazole. For the remaining six isolates, no mutations in cyp51A were found, indicating the presence of other mechanisms. With the exception of the strains carrying the F219C and M220I substitutions, many itraconazole-resistant strains also showed cross-resistance to voriconazole and posaconazole with moderately increased MIC0 values. In conclusion, the prevalence of azole-resistant A. fumigatus in our clinical test set is lower than that previously reported for other countries. Although the TR/L98H mutation frequently occurs among triazole-resistant strains in Germany, it is not the only resistance mechanism present.

Low Rate of Macrolide Resistance in Mycoplasma pneumoniae Strains in Germany between 2009 and 2012

Mycoplasma pneumoniae is a common cause of a broad spectrum of human respiratory tract infections which can be followed by extrapulmonary complications. Especially in epidemics, up to 20% to 40% of all cases of community-acquired pneumonia have been attributed to these cell wall-less bacteria ([1][1

Outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany causes a paradigm shift with regard to human pathogenicity of STEC strains.

An outbreak that comprised 3,842 cases of human infections with enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 occurred in Germany in May 2011. The high proportion of adults affected in this outbreak and the unusually high number of patients that developed hemolytic uremic syndrome makes this outbreak the most dramatic since enterohemorrhagic E. coli (EHEC) strains were first identified as agents of human disease. The characteristics of the outbreak strain, the way it spread among humans, and the clinical signs resulting from EAHEC infections have changed the way Shiga toxin-producing E. coli strains are regarded as human pathogens in general. EAHEC O104:H4 is an emerging E. coli pathotype that is endemic in Central Africa and has spread to Europe and Asia. EAHEC strains have evolved from enteroaggregative E. coli by uptake of a Shiga toxin 2a (Stx2a)-encoding bacteriophage. Except for Stx2a, no other EHEC-specific virulence markers including the locus of enterocyte effacement are present in EAHEC strains. EAHEC O104:H4 colonizes humans through aggregative adherence fimbrial pili encoded by the enteroaggregative E. coli plasmid. The aggregative adherence fimbrial colonization mechanism substitutes for the locus of enterocyte effacement functions for bacterial adherence and delivery of Stx2a into the human intestine, resulting clinically in hemolytic uremic syndrome. Humans are the only known natural reservoir known for EAHEC. In contrast, Shiga toxin-producing E. coli and EHEC are associated with animals as natural hosts. Contaminated sprouted fenugreek seeds were suspected as the primary vehicle of transmission of the EAHEC O104:H4 outbreak strain in Germany. During the outbreak, secondary transmission (human to human and human to food) was important. Epidemiological investigations revealed fenugreek seeds as the source of entry of EAHEC O104:H4 into the food chain; however, microbiological analysis of seeds for this pathogen produced negative results. The survival of EAHEC in seeds and the frequency of human carriers of EAHEC should be investigated for a better understanding of EAHEC transmission routes.

Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens.

The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft(3)). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic system could successfully identify bacteria in <40 min with minimal operator intervention and perform strain identification, even from a mixed population with the target of a minority. We further demonstrated the ability to analyze the E. coli O157:H7 strain from a waste-water sample using enrichment followed by genotyping.

Similarity of Shiga Toxin–producingEscherichia coliO104:H4 Strains from Italy and Germany

Similarity of Shiga Toxin–producing<i>Escherichia coli</i>O104:H4 Strains from Italy and Germany

Polymerase chain reaction–based method for the typing of F18 fimbriae and distribution of F18 fimbrial subtypes among porcine Shiga toxin–encoding Escherichia coli in Germany

Edema disease is an enterotoxemic disorder of weaned piglets that represents a significant threat to pig husbandry worldwide. The causative Escherichia coli strains are highly adapted to the porcine host and characterized by the production of Shiga toxin type 2e (Stx2e) and adhesive F18 fimbria. The current study assessed the occurrence of F18 fimbrial subtypes in 241 porcine stx2e(+) fedA(+) E. coli strains in Germany, including 116 Shiga toxin-encoding E. coli (STEC) and 125 Shiga toxin E. coli/enterotoxigenic E. coli (STEC/ETEC) isolates. In addition, a novel multiplex polymerase chain reaction (PCR) was developed in order to improve the typing system in terms of costs, time, and discriminative power. Utilizing the novel F18 typing PCR, 93 E. coli strains (38.5%) tested positive for the F18ab fimbrial subtype and 147 strains (61.0%) for the F18ac fimbrial subtype, while 1 strain remained nontypeable. Six strains were classified as F18ac using the F18 typing PCR, but were classified as F18ab using the F18-restriction fragment length polymorphism assay. Nucleotide sequencing of the FedA gene revealed that 5 of these strains encoded F18ac fimbriae, while the FedA of 1 strain did not cluster with F18ab or with F18ac amino acid sequences. The F18 fimbrial subtype was significantly associated with the pathovar of the E. coli strains, as 73.2% of the STEC isolates harbored F18ab genes whereas 93.6% of the STEC/ETEC isolates proved F18ac positive. In conclusion, the novel F18 typing PCR allows a specific identification of the F18 fimbrial subtype. The genetic and phenotypic heterogeneity of F18 fimbriae in porcine E. coli strains should be considered in the development of new vaccines and diagnostic tools.