The genus Burkholderia consists of a number of very diverse species, both in terms of lifestyle (... more The genus Burkholderia consists of a number of very diverse species, both in terms of lifestyle (which varies from category B pathogens to apathogenic soil bacteria and plant colonizers) and their genetic contents. We have used 56 publicly available genomes to explore the genomic diversity within this genus, including genome sequences that are not completely finished, but are available from the NCBI database. Defining the pan- and core genomes of species results in insights in the conserved and variable fraction of genomes, and can verify (or question) historic, taxonomic groupings. We find only several hundred genes that are conserved across all Burkholderia genomes, whilst there are more than 40,000 gene families in the Burkholderia pan-genome. A BLAST matrix visualizes the fraction of conserved genes in pairwise comparisons. A BLAST atlas shows which genes are actually conserved in a number of genomes, located and visualized with reference to a chosen genome. Genomic islands are common in many Burkholderia genomes, and most of these can be readily visualized by DNA structural properties of the chromosome. Trees that are based on relatedness of gene family content yield different results depending on what genes are analyzed. Some of the differences can be explained by errors in incomplete genome sequences, but, as our data illustrate, the outcome of phylogenetic trees depends on the type of genes that are analyzed.
Background: Zika virus (ZIKV) is an emerging human pathogen. Since its arrival in the Western hem... more Background: Zika virus (ZIKV) is an emerging human pathogen. Since its arrival in the Western hemisphere, from Africa via Asia, it has become a serious threat to pregnant women, causing microcephaly and other neuropathies in developing fetuses. The mechanisms behind these teratogenic effects are unknown, although epidemiological evidence suggests that microcephaly is not associated with the original, African lineage of ZIKV. The sequences of 196 published ZIKV genomes were used to assess whether recently proposed mechanistic explanations for microcephaly are supported by molecular level changes that may have increased its virulence since the virus left Africa. For this we performed phylogenetic, recombination, adaptive evolution and tetramer frequency analyses, and compared protein sequences for the presence of protease cleavage sites, Pfam domains, glycosylation sites, signal peptides, trans-membrane protein domains, and phosphorylation sites.
Colonization of body epithelial surfaces with a highly specific microbial community is a fundamen... more Colonization of body epithelial surfaces with a highly specific microbial community is a fundamental feature of all animals, yet the underlying mechanisms by which these communities are selected and maintained are not well understood. Here, we show that sensory and ganglion neurons in the ectodermal epithelium of the model organism hydra (a member of the animal phylum Cnidaria) secrete neuropeptides with antibacterial activity that may shape the microbiome on the body surface. In particular, a specific neuropeptide, which we call NDA-1, contributes to the reduction of Gram-positive bacteria during early development and thus to a spatial distribution of the main colonizer, the Gram-negative Curvibacter sp., along the body axis. Our findings warrant further research to test whether neuropeptides secreted by nerve cells contribute to the spatial structure of microbial communities in other organisms.
Significance and Impact of the Study: We have analysed five resistance genes in Staphylococcus au... more Significance and Impact of the Study: We have analysed five resistance genes in Staphylococcus aureus that are positioned between the replication elements of rolling-circle plasmids. For three of these genes, evidence was obtained indicative of recent mobilization. The historical use of the antibiotics to which the genes produce resistance could be related to the number of mutations collected in these genes. However, two other resistance genes have not collected any mutations over time, and the reasons for this are discussed. The analyses presented provide insights into the spread and evolution of antibiotic resistance genes. Abstract The qacC and lnuA genes of Staphylococcus species were recently proposed to comprise a mobile element when residing on rolling-circle plasmids. Here we present other examples of resistance genes on staphylococcal rolling-circle plasmids, including fosB producing resistance to fosfomycin, cat resulting in resistance to chloramphenicol and cadB for resistance to the toxic heavy metal cadmium. For three of these genes (qacC, lnuA and fosB), evidence was obtained that the genes have spread between different plasmid backgrounds. The lack of mutations in qacC suggests that the spread occurred relatively recently, while the build up of mutations in lnuA and fosB suggests their mobilization occurred in the more distant past. These observations can be explained by the use of the respective antibiotics over time. However, the cat and cadB genes sequences analysed had not collected any mutations, an observation that is not completely understood but possible explanations are discussed.
We present here the complete genome sequences of three mumps virus (MuV) strains isolated from pa... more We present here the complete genome sequences of three mumps virus (MuV) strains isolated from patients who tested positive for the mumps virus during a mumps outbreak in Springdale, AR (USA), in 2016. The virus genomes, se-quenced with Oxford Nanopore technology, belong to genotype G and have an average length of 15,342 nucleotides (nt).
The aim of this study was to determine if commercially available strains of probiotic bacteria be... more The aim of this study was to determine if commercially available strains of probiotic bacteria belonging to the species Ent. faecalis and E. coli were able to survive passage through the human stomach and colonise the gastrointestinal tract (GIT). Survival of bacteria following exposure to gastric pH levels was assessed using a dynamic in vitro model resembling conditions in the stomach as well as the SHIME® model more closely mimicking the upper GIT environment (stomach and small intestine) during fed conditions. Viability of both Ent. faecalis DSM 16431 and E. coli DSM 17257 decreased during acid exposure. However, subsequent exposure to simulated small intestine conditions resulted in no further decrease. A human volunteer took a single dose of probiotic Ent. faecalis, after which live bacteria were determined for 6 days in faeces by means of targeted cultivation and identification of colonies using species- and strain-specific PCR primers. Detection of the strain’s DNA by PCR in stool samples was positive for 4 days. This duration of colonisation was much shorter compared to previously determined human colonisation by E. coli DSM 17257. In conclusion, probiotic E. coli and Ent. faecalis are susceptible to gastric pH, which reduces their viability with several logs. However, sufficient numbers survive to colonise the gut, so that the bacteria are detected in the stool for several days (Ent. faecalis) and multiple weeks (E. coli) following a single dose.
Background: Guidelines for the control of hospital-acquired MRSA include decolonization measures ... more Background: Guidelines for the control of hospital-acquired MRSA include decolonization measures to end MRSA carrier status in colonized and infected patients. Successful decolonization typically requires up to 22 days of treatment, which is longer than the average hospital length of stay (LOS). Incomplete decolonization is therefore common, with long-term MRSA carriage as a consequence. To overcome this, we developed an integrated MRSA Management (IMM) by extending MRSA decolonization to the outpatient and domestic setting. The protocol makes use of polyhexanide-based products, in view of reported qac-mediated resistance to chlorhexidine in S. aureus and MRSA.
A century ago, Alfred Nissle discovered that intentional intake of particular strains of Escheric... more A century ago, Alfred Nissle discovered that intentional intake of particular strains of Escherichia coli could treat patients suffering from infectious diseases. Since then, one of these strains became the most frequently used probiotic E. coli in research and was applied to a variety of human conditions. Here, properties of that E. coli Nissle 1917 strain are compared with other commercially available E. coli probiotic strains, with emphasis on their human applications. A literature search formed the basis of a summary of research findings reported for the probiotics Mutaflor, Symbioflor 2, and Colinfant. The closest relatives of the strains in these products are presented, and their genetic content, including the presence of virulence, genes is discussed. A similarity to pathogenic strains causing urinary tract infections is noticeable. Historic trends in research of probiotics treatment for particular human conditions are identified. The future of probiotic E. coli may lay in what Alfred Nissle originally discovered: to treat gastrointestinal infections, which nowadays are often caused by antibiotic-resistant pathogens.
Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfe... more Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. Most qac gene products belong to the Small Multidrug Resistant (SMR) protein family, and are often encoded by rolling-circle (RC) replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ, and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO) and the Single-Strand replication Origin (SSO). The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance). The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations.
The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escheri... more The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli.
In want of a sensitive protocol, culture-dependent methods were compared to detect thermophilic C... more In want of a sensitive protocol, culture-dependent methods were compared to detect thermophilic Campylobacter species from untreated urban effluents. We evaluated various combinations of selective media, with and without an enrichment step, as well as an extra filtration step. Culture-independent real-time quantitative PCR was also included and all detected isolates underwent antimicrobial susceptibility testing. All tested water samples contained Campylobacter DNA, but only 64% were positive after culture. Although enrichment using Preston broth resulted in better recovery of potentially stressed Campylobacter than Bolton or Campyfood broth (CFB), there was no significant increase in efficiency compared to direct plating. The type of selective agar media used, on the other hand, had a significant effect, with CASA plates performing superior to mCCDA or CFA plates. Inclusion of an enrichment step increased the ratio of C. coli versus C. jejuni being isolated. Resistances against all antimicrobials tested were observed in C. coli, but fewer resistances were found in C. jejuni isolates. Whether this difference was the result of selection during the enrichment step could not be determined. The presence of Campylobacter in urban effluents can be considered as a valuable proxy for Campylobacter populations present in urban environments.
We determined whether different methods to isolate Campylobacter (including the ISO standard 1027... more We determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006-1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA-SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla-genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla-genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla-genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared.
Aims: To identify the optimal method for detection of thermophilic Campylo-bacter at various stag... more Aims: To identify the optimal method for detection of thermophilic Campylo-bacter at various stages in the food chain, three culture-dependent (direct plat-ing, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38). Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest num-ber of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples. Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensi-tive and powerful evaluation tool. Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distrib-uted, which suggests this is still a significant risk for consumers.
Bacterial pathogens are being sequenced at an increasing rate. To many microbiologists, it appear... more Bacterial pathogens are being sequenced at an increasing rate. To many microbiologists, it appears that there simply is not enough time to digest all the information suddenly available. In this chapter we present several tools for comparison of sequenced pathogenic genomes, and discuss differences between pathogens and non-pathogens. The presented tools allow comparison of large numbers of genomes in a hypothesis-driven manner. Visualization of the results is very important for clear presentation of the results and various ways of graphical representation are introduced.
The role of a dprA ortholog (Cj0634) in Campylobacter jejuni transformation was phenotypically as... more The role of a dprA ortholog (Cj0634) in Campylobacter jejuni transformation was phenotypically assessed using two strains. C. jejuni strain 11168 was naturally competent for transformation by chromosomal DNA, while efficiency decreased 100-fold in a Cj0634::aphA mutant, whereas C. jejuni strain 480 was not naturally competent. C. jejuni strain 480 but not 11168 could be elec-tro-transformed by shuttle plasmid pRY111, an effect completely abolished by Cj0634 interruption. Complementation of the Cj0634 mutation in C. jejuni strain 480 in trans with vectors containing the dprA homologs from C. jejuni, Helicobacter pylori, or Haemophilus influenzae, completely (for Cj0634) or partially (H. pylori > H. influenzae) restored electro-transformation. Thus, C. jejuni expresses a DprA ortholog that functionally most closely resembles that of H. pylori and is involved in DNA transformation.
Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (H... more Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a non-saturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulo-cytes. An oxidative burst coincided with elevation of intracellular Ca 2 , which was likely because of passive influx of Ca 2 through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a non-lytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the 2 integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.
For most bacteria it is essential to secrete particular proteins, and there are several methods a... more For most bacteria it is essential to secrete particular proteins, and there are several methods available for predicting which proteins will be secreted and how. This genome update deals with characterization of secretion systems in bacterial genomes.
Genotyping methods have been developed and applied to differentiate Campylobacter jejuni isolates... more Genotyping methods have been developed and applied to differentiate Campylobacter jejuni isolates over the last two decades. Although a wealth of information was generated, the data are disappointingly complex and do not support simple models of transmission. Several observations have apparently weakened the value and complicated the interpretation of genotyping methods. For several methods, instability of genotype has been demonstrated, due to recombinations with or without transformation. C. jejuni is partly non-clonal, as demonstrated by multi-locus sequence typing, which is explained by a natural abilitycompetence to take up DNA. Plasticity of the genome, allowing for
The genus Burkholderia consists of a number of very diverse species, both in terms of lifestyle (... more The genus Burkholderia consists of a number of very diverse species, both in terms of lifestyle (which varies from category B pathogens to apathogenic soil bacteria and plant colonizers) and their genetic contents. We have used 56 publicly available genomes to explore the genomic diversity within this genus, including genome sequences that are not completely finished, but are available from the NCBI database. Defining the pan- and core genomes of species results in insights in the conserved and variable fraction of genomes, and can verify (or question) historic, taxonomic groupings. We find only several hundred genes that are conserved across all Burkholderia genomes, whilst there are more than 40,000 gene families in the Burkholderia pan-genome. A BLAST matrix visualizes the fraction of conserved genes in pairwise comparisons. A BLAST atlas shows which genes are actually conserved in a number of genomes, located and visualized with reference to a chosen genome. Genomic islands are common in many Burkholderia genomes, and most of these can be readily visualized by DNA structural properties of the chromosome. Trees that are based on relatedness of gene family content yield different results depending on what genes are analyzed. Some of the differences can be explained by errors in incomplete genome sequences, but, as our data illustrate, the outcome of phylogenetic trees depends on the type of genes that are analyzed.
Background: Zika virus (ZIKV) is an emerging human pathogen. Since its arrival in the Western hem... more Background: Zika virus (ZIKV) is an emerging human pathogen. Since its arrival in the Western hemisphere, from Africa via Asia, it has become a serious threat to pregnant women, causing microcephaly and other neuropathies in developing fetuses. The mechanisms behind these teratogenic effects are unknown, although epidemiological evidence suggests that microcephaly is not associated with the original, African lineage of ZIKV. The sequences of 196 published ZIKV genomes were used to assess whether recently proposed mechanistic explanations for microcephaly are supported by molecular level changes that may have increased its virulence since the virus left Africa. For this we performed phylogenetic, recombination, adaptive evolution and tetramer frequency analyses, and compared protein sequences for the presence of protease cleavage sites, Pfam domains, glycosylation sites, signal peptides, trans-membrane protein domains, and phosphorylation sites.
Colonization of body epithelial surfaces with a highly specific microbial community is a fundamen... more Colonization of body epithelial surfaces with a highly specific microbial community is a fundamental feature of all animals, yet the underlying mechanisms by which these communities are selected and maintained are not well understood. Here, we show that sensory and ganglion neurons in the ectodermal epithelium of the model organism hydra (a member of the animal phylum Cnidaria) secrete neuropeptides with antibacterial activity that may shape the microbiome on the body surface. In particular, a specific neuropeptide, which we call NDA-1, contributes to the reduction of Gram-positive bacteria during early development and thus to a spatial distribution of the main colonizer, the Gram-negative Curvibacter sp., along the body axis. Our findings warrant further research to test whether neuropeptides secreted by nerve cells contribute to the spatial structure of microbial communities in other organisms.
Significance and Impact of the Study: We have analysed five resistance genes in Staphylococcus au... more Significance and Impact of the Study: We have analysed five resistance genes in Staphylococcus aureus that are positioned between the replication elements of rolling-circle plasmids. For three of these genes, evidence was obtained indicative of recent mobilization. The historical use of the antibiotics to which the genes produce resistance could be related to the number of mutations collected in these genes. However, two other resistance genes have not collected any mutations over time, and the reasons for this are discussed. The analyses presented provide insights into the spread and evolution of antibiotic resistance genes. Abstract The qacC and lnuA genes of Staphylococcus species were recently proposed to comprise a mobile element when residing on rolling-circle plasmids. Here we present other examples of resistance genes on staphylococcal rolling-circle plasmids, including fosB producing resistance to fosfomycin, cat resulting in resistance to chloramphenicol and cadB for resistance to the toxic heavy metal cadmium. For three of these genes (qacC, lnuA and fosB), evidence was obtained that the genes have spread between different plasmid backgrounds. The lack of mutations in qacC suggests that the spread occurred relatively recently, while the build up of mutations in lnuA and fosB suggests their mobilization occurred in the more distant past. These observations can be explained by the use of the respective antibiotics over time. However, the cat and cadB genes sequences analysed had not collected any mutations, an observation that is not completely understood but possible explanations are discussed.
We present here the complete genome sequences of three mumps virus (MuV) strains isolated from pa... more We present here the complete genome sequences of three mumps virus (MuV) strains isolated from patients who tested positive for the mumps virus during a mumps outbreak in Springdale, AR (USA), in 2016. The virus genomes, se-quenced with Oxford Nanopore technology, belong to genotype G and have an average length of 15,342 nucleotides (nt).
The aim of this study was to determine if commercially available strains of probiotic bacteria be... more The aim of this study was to determine if commercially available strains of probiotic bacteria belonging to the species Ent. faecalis and E. coli were able to survive passage through the human stomach and colonise the gastrointestinal tract (GIT). Survival of bacteria following exposure to gastric pH levels was assessed using a dynamic in vitro model resembling conditions in the stomach as well as the SHIME® model more closely mimicking the upper GIT environment (stomach and small intestine) during fed conditions. Viability of both Ent. faecalis DSM 16431 and E. coli DSM 17257 decreased during acid exposure. However, subsequent exposure to simulated small intestine conditions resulted in no further decrease. A human volunteer took a single dose of probiotic Ent. faecalis, after which live bacteria were determined for 6 days in faeces by means of targeted cultivation and identification of colonies using species- and strain-specific PCR primers. Detection of the strain’s DNA by PCR in stool samples was positive for 4 days. This duration of colonisation was much shorter compared to previously determined human colonisation by E. coli DSM 17257. In conclusion, probiotic E. coli and Ent. faecalis are susceptible to gastric pH, which reduces their viability with several logs. However, sufficient numbers survive to colonise the gut, so that the bacteria are detected in the stool for several days (Ent. faecalis) and multiple weeks (E. coli) following a single dose.
Background: Guidelines for the control of hospital-acquired MRSA include decolonization measures ... more Background: Guidelines for the control of hospital-acquired MRSA include decolonization measures to end MRSA carrier status in colonized and infected patients. Successful decolonization typically requires up to 22 days of treatment, which is longer than the average hospital length of stay (LOS). Incomplete decolonization is therefore common, with long-term MRSA carriage as a consequence. To overcome this, we developed an integrated MRSA Management (IMM) by extending MRSA decolonization to the outpatient and domestic setting. The protocol makes use of polyhexanide-based products, in view of reported qac-mediated resistance to chlorhexidine in S. aureus and MRSA.
A century ago, Alfred Nissle discovered that intentional intake of particular strains of Escheric... more A century ago, Alfred Nissle discovered that intentional intake of particular strains of Escherichia coli could treat patients suffering from infectious diseases. Since then, one of these strains became the most frequently used probiotic E. coli in research and was applied to a variety of human conditions. Here, properties of that E. coli Nissle 1917 strain are compared with other commercially available E. coli probiotic strains, with emphasis on their human applications. A literature search formed the basis of a summary of research findings reported for the probiotics Mutaflor, Symbioflor 2, and Colinfant. The closest relatives of the strains in these products are presented, and their genetic content, including the presence of virulence, genes is discussed. A similarity to pathogenic strains causing urinary tract infections is noticeable. Historic trends in research of probiotics treatment for particular human conditions are identified. The future of probiotic E. coli may lay in what Alfred Nissle originally discovered: to treat gastrointestinal infections, which nowadays are often caused by antibiotic-resistant pathogens.
Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfe... more Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. Most qac gene products belong to the Small Multidrug Resistant (SMR) protein family, and are often encoded by rolling-circle (RC) replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ, and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO) and the Single-Strand replication Origin (SSO). The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance). The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations.
The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escheri... more The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli.
In want of a sensitive protocol, culture-dependent methods were compared to detect thermophilic C... more In want of a sensitive protocol, culture-dependent methods were compared to detect thermophilic Campylobacter species from untreated urban effluents. We evaluated various combinations of selective media, with and without an enrichment step, as well as an extra filtration step. Culture-independent real-time quantitative PCR was also included and all detected isolates underwent antimicrobial susceptibility testing. All tested water samples contained Campylobacter DNA, but only 64% were positive after culture. Although enrichment using Preston broth resulted in better recovery of potentially stressed Campylobacter than Bolton or Campyfood broth (CFB), there was no significant increase in efficiency compared to direct plating. The type of selective agar media used, on the other hand, had a significant effect, with CASA plates performing superior to mCCDA or CFA plates. Inclusion of an enrichment step increased the ratio of C. coli versus C. jejuni being isolated. Resistances against all antimicrobials tested were observed in C. coli, but fewer resistances were found in C. jejuni isolates. Whether this difference was the result of selection during the enrichment step could not be determined. The presence of Campylobacter in urban effluents can be considered as a valuable proxy for Campylobacter populations present in urban environments.
We determined whether different methods to isolate Campylobacter (including the ISO standard 1027... more We determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006-1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA-SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla-genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla-genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla-genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared.
Aims: To identify the optimal method for detection of thermophilic Campylo-bacter at various stag... more Aims: To identify the optimal method for detection of thermophilic Campylo-bacter at various stages in the food chain, three culture-dependent (direct plat-ing, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38). Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest num-ber of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples. Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensi-tive and powerful evaluation tool. Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distrib-uted, which suggests this is still a significant risk for consumers.
Bacterial pathogens are being sequenced at an increasing rate. To many microbiologists, it appear... more Bacterial pathogens are being sequenced at an increasing rate. To many microbiologists, it appears that there simply is not enough time to digest all the information suddenly available. In this chapter we present several tools for comparison of sequenced pathogenic genomes, and discuss differences between pathogens and non-pathogens. The presented tools allow comparison of large numbers of genomes in a hypothesis-driven manner. Visualization of the results is very important for clear presentation of the results and various ways of graphical representation are introduced.
The role of a dprA ortholog (Cj0634) in Campylobacter jejuni transformation was phenotypically as... more The role of a dprA ortholog (Cj0634) in Campylobacter jejuni transformation was phenotypically assessed using two strains. C. jejuni strain 11168 was naturally competent for transformation by chromosomal DNA, while efficiency decreased 100-fold in a Cj0634::aphA mutant, whereas C. jejuni strain 480 was not naturally competent. C. jejuni strain 480 but not 11168 could be elec-tro-transformed by shuttle plasmid pRY111, an effect completely abolished by Cj0634 interruption. Complementation of the Cj0634 mutation in C. jejuni strain 480 in trans with vectors containing the dprA homologs from C. jejuni, Helicobacter pylori, or Haemophilus influenzae, completely (for Cj0634) or partially (H. pylori > H. influenzae) restored electro-transformation. Thus, C. jejuni expresses a DprA ortholog that functionally most closely resembles that of H. pylori and is involved in DNA transformation.
Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (H... more Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a non-saturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulo-cytes. An oxidative burst coincided with elevation of intracellular Ca 2 , which was likely because of passive influx of Ca 2 through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a non-lytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the 2 integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.
For most bacteria it is essential to secrete particular proteins, and there are several methods a... more For most bacteria it is essential to secrete particular proteins, and there are several methods available for predicting which proteins will be secreted and how. This genome update deals with characterization of secretion systems in bacterial genomes.
Genotyping methods have been developed and applied to differentiate Campylobacter jejuni isolates... more Genotyping methods have been developed and applied to differentiate Campylobacter jejuni isolates over the last two decades. Although a wealth of information was generated, the data are disappointingly complex and do not support simple models of transmission. Several observations have apparently weakened the value and complicated the interpretation of genotyping methods. For several methods, instability of genotype has been demonstrated, due to recombinations with or without transformation. C. jejuni is partly non-clonal, as demonstrated by multi-locus sequence typing, which is explained by a natural abilitycompetence to take up DNA. Plasticity of the genome, allowing for
When most people hear the word “bacteria” they think of food poisoning, infections, and acute, d... more When most people hear the word “bacteria” they think of food poisoning, infections, and acute, debilitating, or fatal diseases. Yet, while bacterial pathogens certainly cause their share of misery in the world, they are only a tiny portion of a vast universe of prokaryotes—the most basic of life forms. Without them, nothing else could live or grow on Planet Earth. This book introduces this diverse, microscopic world and explains the fundamental microbiological concepts needed to explore the life and behavior of bacteria. The book’s clear, jargon-free language informs the reader, without the need of a background in microbiology, about a wide variety of bacterial subjects.
This book is part of the series Computing for Comparative Microbial Genomics. The book is written... more This book is part of the series Computing for Comparative Microbial Genomics. The book is written for microbiologists needing an introduction to genomics as well as for bioinformaticists who need to be introduced to microbiology. First, a brief overview of molecular biology and of the concept of sequences as biological information are given. There are four main parts: Introduction, Comparative Genomics, Transcriptomics and Proteomics, and Microbial Communities. "It is a very well-written review of genomics and proteomics of microbes, and makes convincing arguments for the practicality of applying bioinformatics to the study of communities of these species. The references are well chosen. The writing style is superb. … There is an amazing amount of interesting material… The book is probably more suitable as an introduction to contemporary applications of bioinformatics and microbiology for computational scientists." (Anthony J. Duben, ACM Computing Reviews,
'An unusual job' is the first work of fication by Trudy Wassenaar.
Sue Swanson, a self-employed sci... more 'An unusual job' is the first work of fication by Trudy Wassenaar. Sue Swanson, a self-employed scientific consultant living in Germany, is frustrated with her life. For lack of work, she seeks an occupation to keep herself busy, and applies for an unusual job at a pharmaceutical company. When she discovers that some of the medicines produced on site appear to be faulty, she loses the job, but the director demands her services back in an even more unusual manner. Not knowing whether she can trust him, she decides to accept his request to investigate under cover. Soon, more irregularities become apparent, and when the first victim presents itself, Sue is determined to get to the bottom of it.
Sue Swanson, a self-employed scientific consultant living in Germany, is happy to work on her late... more Sue Swanson, a self-employed scientific consultant living in Germany, is happy to work on her latest project: she received an invitation to write a book about laboratory methods in forensic investigations. However, two recent local murder cases draw her attention, as there seems to be something wrong with the DNA evidence related to those cases. Reluctantly, she starts her own investigations, while her marriage gets seriously shaken up and the impact of the murders increases steadily. Eventually, her life becomes frustratingly complicated. This is the second Sue Swanson novel, where science meets the world of crime. The first is entitled An unusual job.
... Thus, your DNA finger-print doesn't reveal your genes. Fig. 7.1 Variation in the len... more ... Thus, your DNA finger-print doesn't reveal your genes. Fig. 7.1 Variation in the length of genomes for Archaea, Bacteria, and Eukaryotes (the latter grouped in four kingdoms) plotted on a logarithmic scale ... Fern Moss Arabidopsis Plants S. pombe Bulgaria Scutellospora Fungi ...
... At the time enzymes were known to consist of proteins, and proteins were still assumed to be ... more ... At the time enzymes were known to consist of proteins, and proteins were still assumed to be the basis of genetic material. However, a few years later, Oswald Avery, Maclyn McCarty, and Colin MacLeod demonstrated experimentally that DNA was the material of inheritance. ...
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Papers by Trudy Wassenaar
Viability of both Ent. faecalis DSM 16431 and E. coli DSM 17257 decreased during acid exposure. However, subsequent exposure to simulated small intestine conditions resulted in no further decrease. A human volunteer took a single dose of probiotic Ent. faecalis, after which live bacteria were determined for 6 days in faeces by means of targeted cultivation and identification of colonies using species- and strain-specific PCR primers. Detection of the strain’s DNA by PCR in stool samples was positive for 4 days. This duration of colonisation was much shorter compared to previously determined human colonisation by E. coli DSM 17257.
In conclusion, probiotic E. coli and Ent. faecalis are susceptible to gastric pH, which reduces their viability with several logs. However, sufficient numbers survive to colonise the gut, so that the bacteria are detected in the stool for several days (Ent. faecalis) and multiple weeks (E. coli) following a single dose.
Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest num-ber of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples.
Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensi-tive and powerful evaluation tool.
Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distrib-uted, which suggests this is still a significant risk for consumers.
Viability of both Ent. faecalis DSM 16431 and E. coli DSM 17257 decreased during acid exposure. However, subsequent exposure to simulated small intestine conditions resulted in no further decrease. A human volunteer took a single dose of probiotic Ent. faecalis, after which live bacteria were determined for 6 days in faeces by means of targeted cultivation and identification of colonies using species- and strain-specific PCR primers. Detection of the strain’s DNA by PCR in stool samples was positive for 4 days. This duration of colonisation was much shorter compared to previously determined human colonisation by E. coli DSM 17257.
In conclusion, probiotic E. coli and Ent. faecalis are susceptible to gastric pH, which reduces their viability with several logs. However, sufficient numbers survive to colonise the gut, so that the bacteria are detected in the stool for several days (Ent. faecalis) and multiple weeks (E. coli) following a single dose.
Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest num-ber of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples.
Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensi-tive and powerful evaluation tool.
Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distrib-uted, which suggests this is still a significant risk for consumers.
Sue Swanson, a self-employed scientific consultant living in Germany, is frustrated with her life. For lack of work, she seeks an occupation to keep herself busy, and applies for an unusual job at a pharmaceutical company. When she discovers that some of the medicines produced on site appear to be faulty, she loses the job, but the director demands her services back in an even more unusual manner. Not knowing whether she can trust him, she decides to accept his request to investigate under cover. Soon, more irregularities become apparent, and when the first victim presents itself, Sue is determined to get to the bottom of it.
This is the second Sue Swanson novel, where science meets the world of crime. The first is entitled An unusual job.