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Campylobacter jejuni isolates (n = 234) associated with gastroenteritis and the Guillain-Barré syndrome (GBS) in the island of Curaçao, Netherlands Antilles, and collected from March 1999 to March 2000 were investigated by a range of molecular typing techniques. Data obtained by pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), automated ribotyping, and sequence analysis of the short variable region of the flagellin gene (flaA) were analyzed separately and in combination. Similar groupings were obtained by all methods, with the data obtained by MLST and AFLP analysis exhibiting the highest degree of congruency. MLST identified 29 sequence types, which were assigned to 10 major clonal complexes. PFGE, AFLP analysis, and ribotyping identified 10, 9, and 8 of these clonal groups, respectively; however, these three techniques permitted subdivision of the clonal groups into more different types. Members of seven clonal groups comprising 107 isolates were obtained from November 1999 to February 2000, and no distinguishing characteristics were identified for two GBS-associated strains. The sequence type 41 (ST-41), ST-508, and ST-657 clonal complexes and their corresponding AFLP types have been rare or absent in the Campylobacter data sets described to date. We conclude that several clonal complexes of C. jejuni are associated with human disease in Curaçao, and some of these have not been reported elsewhere. Furthermore, given the observation that C. jejuni-associated diseases appear to be more severe from November to February, it can be speculated that this may be due to the presence of virulent clones with a limited span of circulation.

Risk assessment is increasingly used as a scientific process to assess the potential for adverse health effects to occur and as a basis for management of unacceptable risks. For each risk assessment activity, the purpose of the assessment should be clearly stated. For Listeria monocytogenes, the purpose of risk assessment may be providing information on the relative contribution of listeriosis to infectious diseases. For control purposes, the emphasis may be put on factors contributing to the risk of occurrence in a food or to inform risk managers that they should be setting food safety objectives. For an adequate risk assessment of L. monocytogenes, sound scientific data are necessary. This especially applies both to exposure assessment and hazard characterisation. Surveillance data indicates that cold storage to prolongs product shelf-life has opened an ecological window for the growth of L. monocytogenes. Assessment of dose-response relationship is often regarded as a key element in risk characterisation. Due to the large variability of the current assessed dose-response data, their contribution to assessing risks is low. The use of epidemiological data on incidence rate, types of food involved in listeriosis, etc. may be good alternatives. The use of performance standards or criteria, such as inactivation by heat or by fermentation, combined with processes that prevent outgrowth of the organism should be reconsidered. Presently, performance standards can simply be assessed since mathematical tools for their calculations are becoming increasingly available. Copyright (C) 2000 Elsevier Science B.V.

Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment. © 2009 SGM.

Background: Legionella, the causative agent for Legionnaires' disease, is ubiquitous in both natural and man-made aquatic environments. The distribution of Legionella genotypes within clinical strains is significantly different from that found in environmental strains. Developing novel genotypic methods that offer the ability to distinguish clinical from environmental strains could help to focus on more relevant (virulent) Legionella species in control efforts. Mixed-genome microarray data can be used to perform a comparative-genome analysis of strain collections, and advanced statistical approaches, such as the Random Forest algorithm are available to process these data. Methods: Microarray analysis was performed on a collection of 222 Legionella pneumophila strains, which included patient-derived strains from notified cases in the Netherlands in the period 2002-2006 and the environmental strains that were collected during the source investigation for those patients within the Dutch National Legionella Outbreak Detection Programme. The Random Forest algorithm combined with a logistic regression model was used to select predictive markers and to construct a predictive model that could discriminate between strains from different origin: clinical or environmental. Results: Four genetic markers were selected that correctly predicted 96% of the clinical strains and 66% of the environmental strains collected within the Dutch National Legionella Outbreak Detection Programme. Conclusions: The Random Forest algorithm is well suited for the development of prediction models that use mixed-genome microarray data to discriminate between Legionella strains from different origin. The identification of these predictive genetic markers could offer the possibility to identify virulence factors within the Legionella genome, which in the future may be implemented in the daily practice of controlling Legionella in the public health environment. © 2012 Euser et al.

We aimed to identify transcription signal sequences from Streptococcus gordonii strain CH1 by random chromosomal cloning. Five genomic fragments from a Sau3A digest, which constitutively activated transcription of a promoterless spectinomycin resistance gene in this strain, were isolated and characterized. Additionally, one promoter fragment was isolated that was specifically activated under iron-limiting conditions. A sequence motif with similarity to the consensus for Fur-binding regulatory DNA sequences (Fur box) in Escherichia coli was detected within the putative promoter region. The open reading frame downstream of this region possibly encodes a transmembrane protein involved in iron uptake.

Invasive group A streptococcal (GAS) disease reemerged in The Netherlands in the late 1980s. To seek an explanation for this resurgence, the genetic compositions of 22 M1 and 19 M28 GAS strains isolated in The Netherlands between 1960s and the mid-1990s were analyzed by using a mixed-genome DNA microarray. During this four-decade period, M1 and especially M28 strains acquired prophages on at least eight occasions. All prophages carried a superantigen (speA2, speC, speK) or a streptodornase (sdaD2, sdn), both associated with invasive GAS disease. Invasive and noninvasive GAS strains did not differ in prophage acquisition, suggesting that there was an overall increase in the pathogenicity of M1 and M28 strains over the last four decades rather than emergence of hypervirulent subclones. The increased overall pathogenic potential may have contributed to the reemergence of invasive GAS disease in The Netherlands. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

S-proteins are proteins which form a regular structure (S-layer) on the outside of the cell walls of many bacteria. Two S-protein-encoding genes are located in opposite directions on a 6.0-kb segment of the chromosome of Lactobacillus acidophilus ATCC 4356 bacteria. Inversion of this chromosomal segment occurs through recombination between two regions with identical sequences, thereby interchanging the expressed and the silent genes. In this study, we show that the region involved in recombination also has a function in efficient S-protein production. Two promoter sequences are present in the S-protein gene expression site, although only the most downstream promoter (P-1) is used to direct mRNA synthesis. S-protein mRNA directed by this promoter has a half-life of 15 min. Its untranslated leader can form a stable secondary structure in which the 5' end is base paired, whereas the ribosome- binding site is exposed. Truncation of this leader sequence results in a reduction in protein production, as shown by reporter gene analysis of Lactobacillus casei. The results obtained indicate that the untranslated leader sequence of S-protein mRNA is involved in efficient S-protein production.

Group A streptococci (GAS), or Streptococcus pyogenes, are associated with a remarkable variety of diseases, ranging from superficial infections to life-threatening diseases such as toxic-shock-like syndrome (TSS). GAS strains belonging to M types M1 and M3 are associated with TSS. This study aims to obtain insight into the gene profiles underlying different M types and disease manifestations. Genomic differences between 76 clinically well characterized GAS strains collected in The Netherlands were examined using a mixed-genome microarray. Inter-M-type genomic differences clearly outweighed intra-M-type genome variation. Phages were major contributors to observed genome diversification. We identified four novel genes, including two genes encoding fibronectin-binding-like proteins, which are highly specific to a subset of M types and thus may contribute to M-type-associated disease manifestations. All M12 strains were characterized by the unique absence of the citrate lyase complex and reduced growth under hypoxic, nutrient-deprived conditions. Furthermore, six virulence factors, including genes encoding a complement-inhibiting protein (sic), an exotoxin (speA), iron(III) binding factor, collagen binding factor (cpa), and fibrinogen binding factor (prt2-like), were unique to M1 and/or M3 strains. These virulence factors may contribute to the potential of these strains to cause TSS. Finally, in contrast to M-type-specific virulence profiles, we did not identify a common virulence profile among strains associated with TSS irrespective of their M type. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies.

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important. Index descriptors: Agrobacterium tumefaciens; T-DNA; Virulence proteins; DNA-transformation; Aspergillus awamori; Filamentous fungi © 2004 Elsevier Inc. All rights reserved.

The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with biotin. Tagged proteins were visualised through streptavidin probing of Western blots. Seven biotinylated proteins of Y. pestis were identified including two porins and the putative virulence factor catalase peroxidase. Crown Copyright © 2006.