High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei.

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei.

Phage and MLVA typing of Salmonella enteritidis isolated from layers and humans in Belgium from 2000-2010, a period in which vaccination of laying hens was introduced.

The aim of the study was to characterize isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from humans and layer farms in Belgium collected during 2000-2010. Three periods were compared, namely (i) before implementation of vaccination (2000-2004), (ii) during voluntary vaccination (2005-2006) and (iii) during implementation of the national control program (NCP) for Salmonella including mandatory vaccination against S. Enteritidis (2007-2010). The characteristics compared across time periods were distributions of phage type and multiple-locus variable number tandem-repeat assay (MLVA). While PT4 and PT21 were predominantly isolated in Belgium in layers and humans before 2007, a significant reduction of those PTs was observed in both populations in the period 2007-2010. The relative proportion of PT4b, PT21c and PT6c was found to have increased considerably in the layer population since 2007. In the human population, PT8, PT1 and the group of 'other' PTs were more frequently isolated compared to the previous periods. When comparing the proportion of the predominant MLVA types Q2 and U2, no significant difference was found between the layer and human population in the three periods and between periods within each category (layer and human). A significant difference in isolate distribution among MLVA clusters I and II was found between human and layer isolates recovered during Period 3 and in the human population between Period 1 and 3. Results suggest that the association between S. Enteritidis in layers and the occurrence of the pathogen in humans changed since implementation of the NCP in 2007.

B. anthracis in a wool-processing factory: seroprevalence and occupational risk.

In a Belgian wool-processing factory, living anthrax spores were found in raw goat hair and air dust, but confirmed anthrax cases had never been reported. Anthrax vaccines are not licensed nor recommended in Belgium. We conducted a B. anthracis seroprevalence study to investigate risk factors associated with positive serology and advise on protective measures. Overall 12·1% (8/66) employees were seropositive; 30% of persons processing raw goat hair and 20% of persons sorting raw goat hair were seropositive compared to 3% in less exposed jobs [adjusted prevalence ratio (aPR) 44·4, P=0·001; aPR 14·5, P=0·016, respectively). The number of masks used per day was protective (aPR 0·3, P=0·015). Results suggest a dose-response association for those processing raw goat hair. Host-related factors probably played a role as antibody response varied from person to person within an exposure group. Workers exposed to raw goat hair should be offered higher protection against anthrax and have access to anthrax vaccines.

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century.

Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the S19, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio- and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a "One Health" approach to be successful.

A non-invasive fluorescent staining procedure allows Confocal Laser Scanning Microscopy based imaging of Mycobacterium in multispecies biofilms colonizing and degrading polycyclic aromatic hydrocarbons.

To study the micro scale interactions of Mycobacterium with bacteria belonging to other genera by means of Confocal Laser Scanning Microscopy (CLSM), a procedure was developed to non-invasively and fluorescently stain Mycobacterium without compromising the signal produced by commonly used fluorescent reporter genes. The procedure makes use of the commercial non-specific nucleic acid stain Syto62 and was optimized to efficiently stain Mycobacterium cells in suspensions and biofilms. The staining procedure was found non-invasive towards overall cell viability, biofilm architecture and fluorescence signals emitted by other organisms expressing the fluorescent reporter genes gfp and dsRed. The procedure was successfully applied to visualize the comportment of the PAH-degrading Mycobacterium sp. VM552 in triple species biofilms containing, in addition to strain VM552, the GFP labeled PAH-degrading Sphingomonas sp. LH128-GFP and DsRed-labeled Pseudomonas putida OUS82(RF), and colonizing a glass substrate coated with phenanthrene crystals in flow chambers. CLSM imaging and subsequent appropriate image processing of the biofilms show that the comportment of strain Mycobacterium sp. VM552 was largely affected by the presence of the other organisms. The data support the value of the staining procedure to study ecological questions about micro scale behavior and niche occupation of Mycobacterium in multi-species systems.

Virulence-associated traits in avian Escherichia coli: comparison between isolates from colibacillosis-affected and clinically healthy layer flocks.

Colibacillosis appears to be of increasing importance in layer flocks. The aim of this study was to determine characteristics of avian pathogenic Escherichia coli associated with the occurrence of colibacillosis outbreaks at flock level. Forty E. coli strains originating from layers from healthy flocks ('control isolates'), consisting of 25 caecal and 15 extra-intestinal isolates, were compared with 40 strains isolated from layers originating from colibacillosis-affected flocks ('outbreak isolates'), consisting of 20 caecal and 20 extra-intestinal isolates. The examined characteristics were adhesins, invasivity in T84 cell culture, serum resistance, iron uptake, colicin production, and toxinogenicity. The following traits were significantly more often detected in the outbreak isolates than in the control isolates: tsh, iss, iucA, iutA, irp2, fyuA, iroC, cvaC, colicin and colicin V production. A comparison of the extra-intestinal outbreak isolates and the caecal control isolates yielded the same results as when the caecal isolates, extra-intestinal isolates and total number of isolates of the outbreak and the control group were compared. When comparing the caecal and extra-intestinal isolates within the control and within the outbreak group, no significant differences were detected. The O78 and O2 groups showed significant differences with other O-types and NT strains for prevalence of most of the same characteristics. The combination of type 1 fimbriae, tsh, serum resistance, iss, traT, iucA, fyuA, iroC and colicin or colicin V production was significantly more often present in extra-intestinal outbreak isolates than in extra-intestinal control isolates. Only the combination of serum resistance, fyuA and colicin production was present in all outbreak isolates, with a significantly lower prevalence in the control isolates. None of the characteristics or combinations examined were exclusive to the outbreak isolates.

Use of the pAL5000 replicon in PAH-degrading mycobacteria: application for strain labelling and promoter probing.

Three environmental Mycobacterium strains (LB501T, LB307T and VM552) able to degrade anthracene, phenanthrene or pyrene, respectively, were successfully electroporated with pAL5000-based plasmids containing the green fluorescent protein (gfp) gene of Aequoria victoria under the control of the hsp60 promoter of Mycobacterium bovis following a slightly modified standard procedure. Transformants showed irregular gfp expression profiles. Four plasmid derivatives were constructed that contained gene promoters isolated from, and adapted to, gene expression in polycyclic aromatic hydrocarbon (PAH)-degrading mycobacteria. One derivative directed strong and homogeneous expression of GFP, allowing dual analysis of both GFP- and PAH-derived fluorescence as assessed by confocal laser scanning microscopy. The results reported here demonstrate the suitability of the pAL5000 replicon for the development of recombinant DNA-based studies in PAH-degrading Mycobacterium spp.

A PCR test to identify bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment.

A PCR test based on the 16S rRNA gene was set up that could identify any of the five species of the 'Bacillus subtilis group' (B. subtilis, B. pumilus, B. atrophaeus, B. lichenijormis and B. amyloliquefaciens). The test was directly applicable to single colonies and showed excellent specificity. In the mixed population context of wastewater analysis, direct detection of the target Bacillus species by PCR on either crude or purified DNA extracts had poor sensitivity. When assayed on cell suspensions derived from enriched wastewater samples, sensitivity was increased. Using a simple calibration method, it was possible to estimate the proportion of the target organisms. This method was found suitable for easy monitoring of a wastewater bioaugmentation experiment carried out with a mixture of sporulated Bacillus strains.

A transcriptional luxAB reporter fusion responding to fluorene in Sphingomonas sp. LB126 and its initial characterisation for whole-cell bioreporter purposes.

The promoter probe mini-Tn5-luxAB-tet was used to create a luxAB transcriptional fusion responding to fluorene in the fluorene utilising bacterium Sphingomonas sp. LB126. The mutant strain, named L-132, was impaired in fluorene utilisation and strongly emitted light upon addition of fluorene to the growth medium. L-132 was initially characterised and examined for its potential use as a whole-cell biosensor in the perspective of quantifying fluorene in environmental samples. Activity of the reporter gene as a response to fluorene was detectable after 30 min and was optimal after 4 h. A linear response to fluorene concentrations within the water solubility range was achieved, with a detection limit of 200 microg per litre. Besides fluorene, L-132 weakly responded to the polycyclic aromatic hydrocarbons phenanthrene and dibenzothiophene, whereas strong responses were obtained with 9-fluorenone, 9-hydroxyfluorene, phthalic acid and protocatechuic acid. The latter four compounds are metabolites formed in course of fluorene degradation, which suggested that a fluorene metabolite rather than fluorene itself was the true inducer of the luxAB fusion in L-132.

Fluorene degradation by Sphingomonas sp. LB126 proceeds through protocatechuic acid: a genetic analysis.

Sphingomonas sp. LB126 is able to utilize fluorene as sole source of carbon and energy. In the present study, a mutagenic vector was constructed and a "plasmid rescue" strategy was set up to isolate a 16.5-kb DNA fragment containing genes required for fluorene degradation. A 14.5-kb portion of the cloned DNA was sequenced revealing thirteen open reading frames. Two encoded hypothetical proteins (FldE and FldY) similar to transcriptional regulators and one (ORF360) located on an IS-like element (ISSsp126) encoded a putative transposase. Three other putative proteins (FldB, FldU and FldV) displayed strong similarity with enzymes of the protocatechuate 4,5-degradation pathway utilized by Sphingomonaspaucimobilis SYK-6 for the degradation of lignin breakdown products. The remaining hypothetical proteins displayed only limited similarity with enzyme sequences available from databases. Suicide plasmid-directed mutagenesis and genetic complementations showed that integrity of the protocatechuate catabolic pathway was an absolute requirement for fluorene degradation to proceed. These findings were further supported by the analysis of metabolites in bacterial culture supernatants obtained from appropriate mutants. The results presented here demonstrated the suitability of the genetic tool constructed and supplied the first genetic evidence for the participation of a protocatechuate 4,5-degradation pathway in a bacterial fluorene degradation pathway.

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers.

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

Corynebacterium simulans sp. nov., a non-lipophilic, fermentative Corynebacterium.

Three coryneform strains isolated from clinical samples were analysed. These strains fitted the biochemical profile of Corynebacterium striatum by conventional methods. However, according to recently described identification tests for fermenting corynebacteria, the strains behaved rather like Corynebacterium minutissimum. The three isolates could be distinguished from C. minutissimum by a positive nitrate and nitrite reductase test and by not fermenting maltose; from C. striatum by their inability to acidify ethylene glycol and to grow at 20 degrees C. Genetic studies based on 16S rRNA showed that the three strains were in fact different from C. minutissimum and C. striatum (96.9 and 98% similarity, respectively) and from other corynebacteria. They represent a new species for which the name Corynebacterium simulans sp. nov. is proposed. The type strain is DSM 44415T (= UCL 553T = Co 553T).

Status of YopM and YopN in the Yersinia Yop virulon: YopM of Y.enterocolitica is internalized inside the cytosol of PU5-1.8 macrophages by the YopB, D, N delivery apparatus.

The Yersinia Yop virulon is an anti-host system made up of four elements: (i) a type III secretion system called Ysc; (ii) a system designed to deliver bacterial proteins into eukaryotic target cells (YopB, YopD); (iii) a control element (YopN); and (iv) a set of intracellularly delivered proteins designed to disarm these cells or disrupt their communications (YopE, YopH and possibly others). YopM, another Yop protein, binds thrombin and is thus presumed to act as an extracellular effector. Here, we analyzed YopM from Y.enterocolitica and we wondered whether it could also be delivered inside eukaryotic cells. To answer this question we applied the Yop-Cya reporter strategy. Hybrids made of 141 or 100 N-terminal residues of YopM fused to Cya were delivered inside PU5-1.8 macrophages by recombinant Y.enterocolitica strains. YopB and YopD were required as translocators. Leakage of the reporters into the macrophage culture supernatant during the bacterial infection increased strongly when YopN was missing, showing that YopN is involved in the control of delivery of YopM inside eukaryotic cells. YopN itself was not delivered into the macrophages. In conclusion, YopM is translocated inside the eukaryotic cells and its physiopathological role should be revised or completed.

Differential secretion of interleukin-8 by human epithelial cell lines upon entry of virulent or nonvirulent Yersinia enterocolitica.

Epithelial cells of the intestinal mucosa are among the first cells encountered by invasive pathogens. Bacterial invasion of the mucosa gives rise to an inflammatory response, characterized by the influx of polymorphonuclear leukocytes. The chemotactic stimulus responsible for this accumulation is unknown, but several in vitro studies have demonstrated that epithelial cells secrete the chemokine interleukin-8 (IL-8), a potent chemoattractant of polymorphonuclear leukocytes, upon bacterial entry. In this study we analyzed the secretion of IL-8 by human intestinal (T84) and cervical (HeLa) epithelial cell lines in response to infection with the enteric pathogen Yersinia enterocolitica. IL-8 was secreted by T84 and HeLa cells in response to invasion by Y. enterocolitica. Virulent Y. enterocolitica induced a significantly lower level of IL-8 secretion than nonvirulent Y. enterocolitica. Subsequent analysis employing a mutant defective in Yop secretion and various yop mutants showed that the reduced secretion of IL-8 is due to the presence of Yop proteins. Our data suggest that YopB and YopD are required for the suppressive effect.

Customized secretion chaperones in pathogenic bacteria.

Pathogenic yersiniae secrete about a dozen anti-host proteins, the Yops, by a pathway which does not involve cleavage of a classical signal peptide. The Yop secretory apparatus, called Ysc, for Yop secretion, is the archetype of type III secretion systems (which serve for the secretion of virulence proteins by several animal and plant pathogens) and is related to the flagellar assembly apparatus. The Yop secretion signal is N-terminal but has not been defined to date. Apart from the Ysc machinery, secretion of at least four Yops requires cytoplasmic proteins called Syc (for specific Yop chaperone). Each Syc protein binds to its cognate Yop. Unlike most cytoplasmic chaperones, these proteins do not have an ATP-binding domain, and are presumably devoid of ATPase activity. They share a few common properties: an acidic pl, a size in the range of 15-20 kDa, and a putative amphipathic alpha-helix in the C-terminal portion. They were recently shown to have counterparts in other pathogenic bacteria, where they appear to have a similar function.

Individual chaperones required for Yop secretion by Yersinia.

Pathogenic yersiniae secrete anti-host proteins called Yops, by a recently discovered Sec-independent pathway. The Yops do not have a classical signal peptide at their N terminus and they are not processed during membrane translocation. The secretion domain is nevertheless contained in their N-terminal part but these domains do not resemble each other in the different Yops. We have previously shown that YopE secretion requires SycE, a 15-kDa acidic protein acting as a specific cytosolic chaperone. Here we show that the gene downstream from yopH encodes a 16-kDa acidic protein that binds to hybrid proteins made of the N-terminal part of YopH and either the bacterial alkaline phosphatase or the cholera toxin B subunit. Loss of this protein by mutagenesis led to accumulation of YopH in the cytoplasm and to a severe and selective reduction of YopH secretion. This protein thus behaves like the counterpart of SycE and we called it SycH. We also engineered a mutation in lcrH, the gene upstream from yopB and yopD, known to encode a 19-kDa acidic protein. Although this mutation was nonpolar, the mutant no longer secreted YopB and YopD. The product of lcrH could be immunoprecipitated together with cytoplasmic YopD. lcrH therefore seems to encode a YopD-specific chaperone, which we called SycD. Determination of the dependence of YopB on SycD requires further investigation. SycE, SycH, and SycD appear to be members of a new family of cytosolic chaperones required for Yop secretion.

Identification of DNA sequences recognized by VirF, the transcriptional activator of the Yersinia yop regulon.

Pathogenic bacteria of the genus Yersinia harbor a 70-kb plasmid required for virulence. The plasmid-encoded virulence proteins of yersiniae are positively regulated at the transcriptional level by the product of the virF gene, the key activator of the system. virF encodes a DNA-binding protein related to the AraC family of transcriptional activators. The VirF protein from Yersinia enterocolitica is a 30-kDa protein that forms dimers in vitro and that specifically binds to the promoter region of VirF-regulated genes. In this work, we determined the sequences of eight VirF-binding sites from four different genes, by DNase I or hydroxyl radical footprinting. The protected regions, about 40 bases long, were aligned, and a number of conserved residues were identified. A 13-bp sequence resembling TTTTaGYcTtTat (in which nucleotides conserved in > or = 60% of the sequences are in uppercase letters and y indicates C or T) appeared, either isolated or as an inverted repeat in each of the eight sites.

YscN, the putative energizer of the Yersinia Yop secretion machinery.

Pathogenic yersiniae secrete a set of 11 antihost proteins called Yops. Yop secretion appears as the archetype of the type III secretion pathway. Several components of this machinery are encoded by the virA (lcrA) and virC (lcrC) loci of the 70-kb pYV plasmid. In this paper, we describe yscN, another gene involved in this pathway. It is the first gene of the virB locus. It encodes a 47.8-kDa protein similar to the catalytic subunits of F0F1 and related ATPases, as well as to products of other genes presumed to be involved in a type III secretion pathway. YscN contains the two consensus nucleotide-binding motifs (boxes A and B) described by Walker et al. (J. E. Walker, M. Saraste, M. J. Runswick, and N. J. Gay, EMBO J. 1:945-951, 1982). We engineered a pYV mutant encoding a modified YscN protein lacking box A. This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. We conclude that YscN is a component of the Yop secretion machinery using ATP. We hypothesize that it is either the energizer of this machinery or a part of it.