Genome sequence analysis of Ebola virus in clinical samples from three British healthcare workers, August 2014 to March 2015.

We determined complete viral genome sequences from three British healthcare workers infected with Ebola virus (EBOV) in Sierra Leone, directly from clinical samples. These sequences closely resemble those previously observed in the current Ebola virus disease outbreak in West Africa, with glycoprotein and polymerase genes showing the most sequence variation. Our data indicate that current PCR diagnostic assays remain suitable for detection of EBOV in this epidemic and provide confidence for their continued use in diagnosis.

Possible contamination of organ preservation fluid with Bacillus cereus: the United Kingdom response.

We describe here the United Kingdom (UK) response following the recent international recall of an organ preservation fluid owing to potential Bacillus cereus contamination. This fluid is used for the transport of solid organs and pancreatic islet cells for transplant. We detail the response mechanisms, including the initial risk stratification, investigatory approaches, isolate analysis and communications to professional bodies. This report further lays out the potential need for enhanced surveillance in UK transplant patients.

An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting.

A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events.

Virulence Searcher: a tool for searching raw genome sequences from bacterial genomes for putative virulence factors.

There is often a delay between completion of a genome sequence and its publication, mainly because of the lengthy process of annotation. For most researchers, the raw sequence alone does not easily yield the rich information it contains. An online tool (Virulence Searcher) has been designed that enables scientists interested in bacterial pathogenesis to search sequences from unannotated bacterial genomes for putative genes encoding virulence factors. This will facilitate an immediate start on important research into bacterial disease without having to wait for the annotated sequence to be published.

Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum.

The rapid technique of pyrosequencing was used to examine 123 samples (in the form of DNA extracts and inactivated sputum) of Mycobacterium spp. Of 99 Mycobacterium tuberculosis samples investigated for single-nucleotide polymorphisms (SNPs), 68% of isoniazid-resistant isolates analysed had an AGC --> ACC mutation in katG at codon 315, resulting in the Ser --> Thr substitution associated previously with isoniazid resistance. Of the rifampicin-resistant isolates, 92% showed SNPs in rpoB at codons 516, 531 or 526. Inactivated sputum samples and DNA extracts could both be analysed by pyrosequencing, and the method was able to differentiate rapidly between the closely related species of the M. tuberculosis complex (M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti), except between M. tuberculosis, M. canetti and one of two M. africanum strains. This low-cost, high-throughput technique could be used as a rapid screen for drug resistance and as a replacement for some of the time-consuming tests used currently for species identification.

Isolation, purification and characterisation of 2-oxoglutarate reductase from Fusobacterium nucleatum.

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.

Lysis of erythrocytes by the secreted cysteine proteinase of Porphyromonas gingivalis W83.

The cysteine proteinase produced in the culture supernatant of Porphyromonas gingivalis was extensively purified. Haemagglutination type assays in which the enzyme was titrated against a fixed concentration of erythrocytes, showed that low levels of enzyme directly caused lysis of the red blood cells. However, using the same assay, the presence of stoichiometric amounts of the thiol blocking agent, 2,2'-dipyridyl disulphide (2-PDS) specifically inhibited the action of the enzyme or its haemagglutination with W83 cells or vesicles. In all cases, electron micrographs revealed that in the presence of 2-PDS the erythrocytes remained intact. Thiol activator free enzyme or aerated, inactivated enzyme had no effect on the red blood cells. These results show conclusively that the secreted cysteine proteinase of P. gingivalis causes lysis of erythrocytes and must now be regarded as a potent virulence determinant of P. gingivalis.

Recognition of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 by ribosomal RNA gene restriction patterns.

DNA from representative strains of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 was digested with restriction enzymes EcoRI and TaqI and the electrophoretically separated fragments hybridized with a 32P-16S rRNA gene probe from E. coli. The rRNA gene restriction patterns from DNA digested with either enzyme allowed the clustering of strains into the three subgroups. However, TaqI digested DNA yielded a wider distribution of taxonomically useful bands (ca 0.65 +/- 14.3 kbp) and the pattern produced was characteristic of each subgroup. The present method is a simple and reliable means of identifying the three subgroups of F. nucleatum and provides a useful method for further studies of the heterogeneity of F. nucleatum.

Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens.

Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens. These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.

Identification of Fusobacterium species by the electrophoretic migration of glutamate dehydrogenase and 2-oxoglutarate reductase in relation to their DNA base composition and peptidoglycan dibasic amino acids.

Rapid identification of Fusobacterium spp. is hampered by their inability to ferment carbohydrates and the availability of relatively few useful phenotypic characters. In an attempt to identify new diagnostic markers for species, we reported recently the potential utility of glutamate dehydrogenase (GDH) electrophoretic mobilities for distinguishing eight species of Fusobacterium. We have extended these observations to include all recognised members of the genus except F. prausnitzii and F. perfoetens, and our results show that they cluster into three broad electrophoretic groups. Some species, such as F. periodonticum, F. simiae and F. necrophorum, possessed GDH with similar electrophoretic mobilities. However, within such clusters, the electrophoretic migration of 2-oxoglutarate reductase (OGR) distinguished between species. Neither GDH or OGR mobility alone clearly differentiated all species, but their combined use provided unambiguous discrimination of all species except F. varium and F. mortiferum. The DNA base compositions of all species except F. naviforme (ATCC 25832) and F. sulci, were within the range 26-34 mol% G + C, suggesting the genus may be homogeneous. However, the peptidoglycan composition divided the genus into two major groups that contained either lanthionine or diaminopimelic acid; F. mortiferum peptidoglycan contained both dibasic amino acids.

Evidence for independent molecular identity and functional interaction of the haemagglutinin and cysteine proteinase (gingivain) of Porphyromonas gingivalis.

The sequence of events involved in haemagglutination and lysis of erythrocytes by washed cells, vesicles and the culture supernate of Porphyromonas gingivalis strain W83 was monitored by 51Cr release and transmission electronmicroscopy. All preparations, except capsular material and lipopolysaccharide, caused haemagglutination and, by a slow process of attachment and specific attack on the surface structures of the red blood cells, produced minute pores and eventual leakage of cellular contents. N-acetylglucosamine, N-acetylgalactosamine and several other sugars such as glucose and sucrose had no effect on haemagglutination. Antiserum raised against a cloned haemagglutinin of P. gingivalis strain 381 inhibited the activity of strain W83 cells, vesicles and supernate. The antiserum-neutralised supernate lost 70-80% of its hydrolytic activity towards alpha-N-benzoyl-L-arginine-4-nitroanilide but the residual activity behaved in a manner similar to the native supernate in that it was completely inhibited by the addition of 2,2'-dipyridyl disulphide and was fully restored upon addition of a low-Mr mercaptan. Binding of the antiserum to the haemagglutinin epitope of P. gingivalis still permitted titration of the active centre cysteinyl thiol group of the proteinase. Purified gingivain caused lysis of erythrocytes and was not neutralised by antiserum to the haemagglutinin. These results suggest that, although the haemagglutinin and gingivain are probably separate molecules, they are closely associated on the outer membrane of P. gingivalis and may be functionally related.

Assessment of the relative cytotoxicity of Porphyromonas gingivalis cells, products, and components on human epithelial cell lines.

Established human cell lines derived from a transitional cell carcinoma (J82), a squamous carcinoma (SCaBER), and a normal urothelium (HCV-29) were used to assess the relative cytotoxicity and tissue specificity of putative virulence determinants of P. gingivalis W83. Intact cells of W83 had no effect on any of the cell lines, whereas disrupted cells caused extensive cytotoxicity particularly to monolayers of HCV-29 and J82. The purified cysteine proteinase, gingivain, caused marked disruption of the basement membrane of the SCaBER monolayers but had no cytotoxic effects. Use of the thiol-inhibitor, 2,2'-dipyridyl disulphide revealed that the effects observed with the vesicles and the culture supernatant were due to the presence of the cysteine proteinase. The attachment of vesicles to the SCaBER cells was evident in electron micrographs. Short-chain volatile fatty acids added in concentrations equivalent to those present in the culture supernatant had no effect on any of the cell lines tested. Culture supernatants obtained from high speed centrifugation (150,000 x g) showed no cytotoxic effects. This was in marked contrast to the supernatant obtained by lower sedimentation (18,000 x g), which damaged all monolayers tested. These results suggest that these cell lines are potentially useful for assessing putative virulence determinants of P. gingivalis and other periodontal pathogens.

Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infections.

The occurrence and surface properties of prevotella intermedia and P. nigrescens in healthy sites and in periodontic and endodontic infections were studied among 73 strains, tentatively identified as P. intermedia. Fifteen strains were from necrotic root canal infections, 41 were from periodontal samples, and 17 isolates were obtained from healthy gingival sites. Identification of isolates as either P. intermedia or P. nigrescens was based on differences in malate and glutamate dehydrogenase electrophoretic mobilities which allowed unambiguous separation of P. intermedia and P. nigrescens. The majority of strains from periodontal samples were P. intermedia (29 of 41 strains). In endodontic samples only 4 out of 15 isolates were P. intermedia, while all except 1 of 17 strains from healthy gingival sites were identified as P. nigrescens. SDS-PAGE of whole cell proteins revealed 31 and 38 kDa proteins in P. nigrescens which were not detected in P. intermedia. Surface biotinylation of cells, followed by Western blotting and detection by alkaline phosphatase conjugated extravidin, showed strong staining of the 31 kDa protein in P. nigrescens indicating that this protein is located on the surface of the cell. Corresponding staining was not seen in P. intermedia. Fimbria-like projections were observed using electron microscopy of negatively-stained cells of P. nigrescens. The results show that P. intermedia and P. nigrescens may have different site specificities and surface properties and thus emphasize the need for accurate identification of these two species for the evaluation of their role in the pathogenesis of oral infections.

The use of a 16s rDNA directed PCR for the detection of endodontopathogenic bacteria.

The study evaluates a 16S rDNA directed polymerase chain reaction (PCR) to detect and differentiate bacteria in necrotic root canal samples. The examination focused on species that are fastidious concerning culture or are difficult to differentiate after culturing by biochemical methods. In the described PCR assay, a universal 16S rDNA directed forward primer in combination with a highly specific reversed one was used to amplify taxon specific gene fragments of 230 to 950 bp length. A similar PCR reaction using a universal 16S rDNA reversed primer was also established to demonstrate bacteria in root canal specimens in general. A first application of this method revealed the presence of Actinomycetales-species, Fusobacterium nucleatum, "Streptococcus milleri," and, presumably for the first time described in infected root canals, Bacteroides forsythus. The identity of amplificons was confirmed by generating sequence information and comparison to gene databanks.

Comparative proteomic analysis of Clostridium difficile isolates of varying virulence.

The soluble proteome of three Clostridium difficile strains of varying pathogenic potential, designated B-1, Tra 5/5 and 027 SM, were compared using differential in-gel electrophoresis in which the proteins of each strain were labelled with CyDyes. This enabled visual inspection of the 2D profiles of strains and identification of differentially expressed proteins using image analysis software. Unlabelled protein reference maps of the predominant proteins were then generated for each strain using 2D gel electrophoresis followed by protein sequencing of each spot using a Reflectron matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. Increased coverage of the proteome was achieved using 1D gel electrophoresis in a bottom-up approach using LC-MS/MS of 1 cm gel slices. A total of 888 different proteins were detected by comparative analysis of isolates grown in parallel for 64 h on blood agar plates. Of these, only 38 % were shared between all isolates. One hundred and ten proteins were identified as showing ≥2-fold difference in expression between strains. Differential expression was shown in a number of potential virulence and colonization factors. Toxin B was detected in the more virulent strains B-1 and 027 SM, but not in the lower virulent strain Tra 5/5, despite all strains possessing an intact pathogenicity locus. The S-layer protein (Cwp2) was identified in strains 027 SM and Tra 5/5 but not strain B-1, and differences in the post-translational modification of SlpA were noted for strain B-1. The variant S-layer profile of strain B-1 was confirmed by genomic comparison, which showed a 58 kb insertion in the S-layer operon of strain B-1. Differential post-translation modification events were also noted in flagellar proteins, thought to be due to differential glycosylation. This study highlights genomic and proteomic variation of different Clostridium difficile strains and suggests a number of factors may play a role in mediating the varying virulence of these different strains.