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The chapter is on development and application of matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF) to identification and and classification of bacteria.

In 2016, a new species name Cutibacterium acnes was coined for the well-documented species, Propionibacterium acnes, one of the most successful and clinically important skin commensals. The nomenclatural changes were brought about through creation of the genus Cutibacterium, when a group of propionibacteria isolates from the skin were transferred from the genus Propionibacterium and placed in the phylum Actinobacteria. Almost simultaneously, the discovery of two novel species of Cutibacterium occurred and the proposal of three subspecies of C. acnes were reported. These dramatic changes that occurred in a long-established taxon made it challenging for the non-specialist to correlate the huge volume of hitherto published work with current findings. In this review, we aim to correlate the eco-specificity and pathophysiological properties of these newly circumscribed taxa. We envisage that this information will shed light on the pathogenic potential of new isolates and enable better assessment of their clinical importance in the foreseeable future. Currently, five species are recognized within the genus: Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium modestum (previously, “Propionibacterium humerusii”), and Cutibacterium namnetense. These reside in different niches reflecting their uniqueness in their genetic makeup. Their pathogenicity includes acne inflammation, sarcoidosis, progressive macular hypomelanosis, prostate cancer, and infections (bone, lumbar disc, and heart). This is also the case for the three newly described subspecies of C. acnes, which are C. acnes subspecies acnes (C. acnes type I), subspecies defendens (C. acnes type II), and subspecies elongatum (C. acnes type III). C. acnes subspecies acnes is related to inflamed acne and sarcoidosis, while subspecies defendens to prostate cancer and subspecies elongatum to progressive macular hypomelanosis. Because the current nomenclature is based upon polyphasic analyses of the biochemical and pathogenic characteristics and comparative genomics, it provides a sound basis studying the pathophysiological roles of these species.

Purpose: Staphylococcus aureus is a highly successful human pathogen responsible for wide range of infections. In this study, we provide insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin susceptible and methicillin resistant Staphylococcus aureus (MSSA; MRSA) recovered from non-healthcare environments. Experiment design: Three environmental MSSA and three environmental MRSA were selected for proteomic profiling using iTRAQ MS/MS. Gene Ontology (GO) Annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Annotation were applied to interpret the functions of the proteins detected. Results: 792 proteins were identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA revealed that 8 of out 792 proteins were up-regulated and 156 were down-regulated. Proteins that had differences in abundance were predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 were involved in pathogenesis, antimicrobial resistance，stress response, mismatch repair and cell wall synthesis. Twenty-two proteins associated with pathogenicity, including spa, sbi, clfA and dlt were up-regulated in MRSA. Moreover, the up-regulated pathogenic protein entC2 in MSSA was determined to be a super antigen potentially capable of triggering toxic shock syndrome in the host. Conclusions: Enhanced pathogenicity, antimicrobial resistance and stress response were observed in MRSA compared to MSSA.

Don Whitley Scientific Limited announced the sad news of the death of its founder and chairman, Don Whitley on February 28, 2019. We mourn the loss of this great scientific entrepreneur who was born in London in June 1929. With his family’s relocation to Leeds in 1940, Don attended Leeds’s prestigious Morley Grammar School where he envisioned a career as a medical doctor. Family’s opposition led him to medical technology, working for a decade at Leeds Maternity Hospital and Killingbeck Hospital before moving to industry.

Introducción: Rhodococcus equi es un cocobacilo grampositivo que provoca compromiso pulmonar en pacientes inmunodeprimidos. Métodos: En el presente trabajo se analizaron de manera retrospectiva los hallazgos epidemiológicos, clínicos, microbiológicos, imagenológicos, inmunológicos y la evolución de 13 pacientes con SIDA y enfermedad por R. equi. Resultados: Entre enero de 1994 y diciembre de 2012, 13 pacientes internados en la División de VIH/SIDA del hospital de referencia para Enfermedades Infecciosas de la ciudad de Buenos Aires egresaron con diagnóstico de enfermedad por R. equi. Todos eran varones y la mediana de edad fue 27 años. La mediana de linfocitos T CD4+ fue de 11 céls/μl Doce pacientes presentaron enfermedad pulmonar con aislamiento del microorganismo del esputo o del lavado bronco-alveolar; en el restante se recibió post mortem el cultivo positivo de líquido cefalorraquídeo. Las manifestaciones clínicas más frecuentes fueron fiebre, hemoptisis y pérdida de peso. La imagen radiológica predominante fue la consolidación con cavitación. Nueve pacientes fallecieron, con una mediana de supervivencia de 5,5 meses. En todos ellos el cultivo persistió positivo hasta la última internación. Los cuatro restantes abandonaron los controles y no pudieron ser evaluados en el tiempo. Conclusión: El curso insidioso de la enfermedad por R. equi y las dificultades en la identificación del microorganismo, contribuyen al retardo en el diagnóstico y a la elevada mortalidad de esta infección oportunista en esta población de pacientes.

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.

The genus Bacillus is a phylogenetically incoherent taxon with members of the group lacking a common evolutionary history. Comprising aerobic and anaerobic spore-forming bacteria, no characteristics are known that can distinguish species of this genus from other similar endospore-forming genera. With the availability of complete genomic data from over 30 different species from this group, we have constructed detailed phylogenetic trees to determine the relationships among Bacillus and other closely related taxa. Additionally, we have performed comparative genomic analysis for the determination of molecular markers, in the form of conserved signature indels (CSIs), to assist in the understanding of relationships among species of the genus Bacillus in molecular terms. Based on the analysis, we report here the identification of 11 and 6 CSIs that clearly differentiate a ‘ Bacillus subtilis clade’ and a ‘ Bacillus cereus clade’, respectively, from all other species of the genus Bacillus . No molecular markers were identified that supported a larger clade within this genus. The subtilis and the cereus clades were also the largest observed monophyletic groupings among species from the genus Bacillus in the phylogenetic trees based on 16S rRNA gene sequences and those based upon concatenated sequences for 20 conserved proteins. Thus, the relationships observed among these groups of species through CSIs are independently well supported by phylogenetic analysis. The molecular markers identified in this study provide a reliable means for the reorganization of the currently polyphyletic genus Bacillus into a more evolutionarily consistent set of groups. It is recommended that the genus Bacillus sensu stricto should comprise only the monophyletic subtilis clade that is demarcated by the identified CSIs, with B. subtilis as its type species. Members of the adjoining cereus clade (referred to as the Cereus clade of bacilli), although they are distinct from the subtilis clade, will also retain the Bacillus genus name as they contain several clinically important species, and their transfer into a new genus could have serious consequences. However, all other species that are currently part of the genus Bacillus and not part of these two clades should be eventually transferred to other genera. We also propose that all novel species of the genus Bacillus must meet minimal requirements, foremost among which is that the branching of the prospective species with the Bacillus sensu stricto clade or the Cereus clade of bacilli should be strongly supported by 16S rRNA gene sequence trees or trees based upon concatenated protein sequences. Additionally, the presence of one or more of the CSIs that are specific for these clades may be used to confirm molecularly the placement of the species into these clades. The identified CSIs, in addition to their usefulness for taxonomic and diagnostic purposes, also provide novel probes for genetic and biochemical studies of these bacteria.

Objective: The current increasing interest in the application of mass spectrometry, in particular MALDI-TOF MS, to identification of bacteria and fungi calls for a need to utilise this technology for identification of other infectious agents such as viruses. The aim of the present study was to develop a rapid and reliable mass spectrometry-based proteomic method for identification of Escherichia coli phages.\ud Methods: The approach was based on rapid in-solution tryptic digestion of suspensions of plaque-purified bacteriophage followed by mass spectral analysis. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography – tandem mass spectrometry (LC-MS) were used to analyse the tryptic digests. Processing of tandem mass spectrometry data and interpretation of results were achieved using Mascot software and the Swiss-Prot database.\ud Results: Five bacteriophage species (Enterobacteria phages P2, T4, T5, T7 and Lambda) isolated in E. coli cultures were identified. The viral proteins were identified from a complex mixture of host bacterial proteins. In addition, using a single ion monitoring method, a Lambda prophage derived protein was also identified.\ud Conclusion: The data obtained demonstrate that LC-MS/MS can be used for accurate identification of E.coli- specific bacteriophages in both lytic and lysogenic cycles\ud Keywords: Bacteriophage virus; Mass-spectrometry; Liquid chromatography; MALDI; LC-MS/MS; Lytic; Lysogenic; Enterobacteria; E.coli; Phage; Viruses

Salmonella enterica is one of the most extensively studied bacterial species in terms of physiology, genetics, cell culture and development. As a very diverse group, the serovars of S. enterica display a spectrum of host specificities ranging from a broad host range to strictly host-adapted variants. This study utilized a classic proteomic approach combining 2D gel electrophoresis and mass spectrometry for the comparative analysis of the proteomes of serovars Typhimurium, Enteritidis, Choleraesuis, Pullorum and Dublin. The comparative analysis revealed species-specific protein factors with no significant change in expression amongst all isolates, as well as proteins with fluctuating expression levels between serovars and strains. Examples include an isoform of SodA specific for serovar Typhimurium, the third isoform of the lysine arginine ornithine (LAO)-binding amino acid transporter specific for serovar Pullorum, and the enzyme GabD found to be unique to serovar Choleraesuis. Overall the study demonstrated the importance of using multiple isolates when characterizing the expression patterns of bacteria in order to account for the intrinsic diversity of a bacterial population and revealed several factors with potential roles in host adaptation and pathogenicity of the serovars of S. enterica. © 2007 Health Protection Agency.

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/ CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip® arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/ MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.

Propionibacterium acnes, a commensal of human skin, is also an opportunistic pathogen of common acne and certain infectious diseases. However, it is still not obvious which strain is pathogenic for a certain infectious disease, and investigations to characterize pathogenic strains using molecular typing methods such as MLST using several housekeeping genes have been undertaken. However, to date, such analysis has focused mainly on strains isolated from Europeans, and it is unclear whether the clonal distribution in other parts of the world is similar. Here, we analysed 50 strains of P. acnes from healthy humans and patients with atopic dermatitis (AD) in Japan and utilized MLST of seven housekeeping genes to study their clonal patterns. The MLST successfully typed the strains into five types, IA, IB1, IB2, II and III. Strains that belonged to types IA, IB and II were common on the human skin of both populations (Europe and Japan), but this study demonstrated what we believe to be the first association of type III strains with human skin, existing on the skin of both the AD and non-AD population. These results indicate the global existence of type III strains on human skin.

Strains (n = 99) of Staphylococcus aureus isolated from a large number of clinical sources and tested for methicillin sensitivity were analysed by MALDI-TOF-MS using the Weak Cation Exchange (CM10) ProteinChip Array (designated SELDI-TOF-MS). The profile data generated was analysed using Artificial Neural Network (ANN) Analysis modelling techniques. Seven key ions identified by the ANNs that were predictive of MRSA and MSSA were validated by incorporation into a model. This model exhibited an area under the ROC curve value of 0.9147 indicating the potential application of this approach for rapidly characterising MRSA and MSSA isolates. Nearly all strains (n = 97) were correctly assigned to the correct group, with only two aberrant MSSA strains being misclassified. However, approximately 21% of the strains appeared to be in a process of transition as resistance to methicillin was being acquired.

The present article gives an overview of recent taxonomic changes among the Gram-negative, anaerobic rods, briefly highlighting areas where the biology and ecology have a bearing on recent nomenclatorial changes. The focus is among the genera Bacteroides, Prevotella, Porphyromonas, Leptotrichia, Dysgonomonas, Fusobacterium and the Synergistes group and additionally demonstrates the value of conserved indels and group-specific proteins for identifying and circumscribing many of these taxa and the Bacteroidetes-Chlorobi species in general.

Strains representing the species Campylobacter coli, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, and Staphylococcus aureus were randomly selected to assess the consistency of cells preserved on lenticule discs to those archived in traditional freeze-dried ampoules. Each matched pair was cultured using identical conditions and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to profile the surface-associated molecules of the cells. In addition, the cytosolic/membrane-bound proteins of C. coli and S. aureus strains were further analysed by surface-enhanced laser desorption/ionization time-of-flight MS. The mass spectral profiles in all cases showed a high degree of concordance between cells preserved by both methods and suggest that the properties of cells preserved on lenticule disc are consistent with those archived by the traditional method of freeze-drying.

Minute 2. Record of attendance. Members present were H. N. Shah (Chairman), I. Olsen (Secretary), K. Bernard, S. M. Finegold, S. E. Gharbia, M. Sakamoto (instead of T. Mitsuoka), A. C. R. Tanner and K. Ueno. Deep-felt sorrow was expressed over the recent deaths of members Daria N. Love and Hannele Jousimies-Somer and Martin A. Claydon, an invited guest at the last meeting in Manchester in 2000. Their immense contributions to taxonomy over the years were gratefully acknowledged.

Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the use of comparative sequencing by MALDI-TOF MS for automated high-throughput microbial DNA sequence analysis. Data derived from the public multilocus sequence typing (MLST) database ( http://pubmlst.org/neisseria ) established a reference set of expected peak patterns. A model pathogen, Neisseria meningitidis , was used to validate the technology and explore its applicability as an alternative to dideoxy sequencing. One hundred N. meningitidis samples were typed by comparing MALDI-TOF MS fingerprints of the standard MLST loci to reference sequences available in the public MLST database. Identification results can be obtained in 2 working days. Results were in concordance with classical dideoxy sequencing with 98% correct automatic identification. Sequence types (STs) of 89 samples were represented in the database, seven samples revealed new STs, including three new alleles, and four samples contained mixed populations of multiple STs. The approach shows interlaboratory reproducibility and allows for the exchange of mass spectrometric fingerprints to study the geographic spread of epidemic N. meningitidis strains or other microbes of clinical importance.

Introduction: Putative virulence factors of Enterococcus faecalis have been proposed by several workers and, by analogy, these have been linked to strains of endodontic origin. However, their distribution within the cell population is unknown. In the present study, isolates were taken from the dental root canals of two defined human populations, Lithuanian and Finnish, and examined for a range of virulence properties. In addition, surface-associated molecules and intracellular proteins were compared using matrix-assisted laser desorption-ionization/mass spectrometry (MALDI-TOF-MS) and ProteinChipTM capture/MS (SELDI-TOF-MS), respectively. Methods: Twenty-three Lithuanian and 35 Finnish dental root canal isolates were included. The esp, gelE, ace and efaA genes were detected by polymerase chain reaction, and cytolysin and gelatinase phenotypes were determined by hydrolysis of horse blood agar and gelatine agar, respectively. Protein extracts and surface-associated molecules of whole cells were analysed by SELDI-TOF-MS and MALDI-TOF-MS, respectively. Results: Presence of esp (n = 15), cytolysin (n = 9), ace (n = 55) and efaA (n = 58) was not statistically different in the two samples, whereas gelE and gelatinase production was detected more frequently in the Finnish material (chi-squared, P

Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov. are described. These emanate from the three known phylotypes of P. acnes, designated types I, II and III. Electron microscopy confirmed the filamentous cell shape of type III, showing a striking difference from types I/II, which were short rods. Biochemical tests indicated that, in types I/II, either the pyruvate, l-pyrrolidonyl arylamidase or d-ribose 2 test was positive, whereas all of these were negative among type III strains. Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, which profile mainly their ribosomal proteins, were different between these two groups. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) spectra of all phylotypes revealed a specific protein biomarker that was overexpressed in type III strains compared with types I/II only when grown aerobically. Reference strains had high whole-genome similarity between types I (>91 %) and II (>75 %), but a considerably lower level of 72 % similarity with type III. recA and gyrB sequence dendrograms confirmed the distant relatedness of type III, indicating the presence of two distinct centres of variation within the species P. acnes. On the other hand, cellular fatty acid profiles and 16S rRNA gene sequence relatedness (>99.3 %) circumscribed the species. Thus, we propose two subspecies, Propionibacterium acnes subsp. acnes subsp. nov. for types I/II and Propionibacterium acnes subsp. elongatum subsp. nov. for type III. The type strain of Propionibacterium acnes subsp. acnes is NCTC 737T ( = ATCC 6919T = JCM 6425T = DSM 1897T = CCUG 1794T), while the type strain of Propionibacterium acnes subsp. elongatum is K124T ( = NCTC 13655T = JCM 18919T).

Clostridium difficile is one of the leading causes of antibiotic-associated diarrhea in health care facilities worldwide. Here, we report the genome sequence of C. difficile strain G46, ribotype 027, isolated from an outbreak in Glamorgan, Wales, in 2006.

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.

AIM To investigate the prevalence of Porphyromonas gingivalis in root canals of infected teeth with periapical abscesses and to investigate the antimicrobial susceptibility of this species to some frequently prescribed antibiotics. METHODOLOGY Samples were obtained from 70 root canals of abscessed teeth. Microbial sampling, isolation and bacterial identification were accomplished using appropriate culture methods for anaerobic species. The antimicrobial susceptibility of the 20 strains of P. gingivalis isolated was determined by using the E-test. The antimicrobial agents tested were amoxicillin, amoxicillin + clavulanate, azythromycin, benzylpenicillin, cephaclor, clindamycin, erythromycin, metronidazole and tetracycline. RESULTS A total of 352 individual strains, belonging to 69 different species, were isolated. Eighty three percent of the strains were strict anaerobes and 47.5% of the isolated bacteria were Gram-negative. Porphyromonas gingivalis was found in 20 root canals and was most frequently found in symptomatic cases. Statistically, the presence of P. gingivalis was related to purulent exudates and pain on palpation (both P < 0.05). All P. gingivalis strains were sensitive to amoxicillin, amoxicillin + clavulanate, cephaclor, clindamycin, benzylpenicyllin, metronidazole and tetracycline. The lowest range of minimum inhibitory concentration (MIC) (0.026-0.125 microg mL(-1)) was observed against amoxicillin + clavulanate and clindamycin. The lowest MIC 90 was observed against clindamycin (0.064 microg mL(-1)). One strain was resistant to erythromycin and eight strains were resistant to azythromycin. CONCLUSION Porphyromonas gingivalis pathogen is isolated with frequency from root canals of infected teeth with periapical abscesses. Amoxicillin, as well as amoxicillin-clavulanic acid and benzylpenicillin were effective against P. gingivalis.

The purpose of this investigation was to quantify the concentration of endotoxin in necrotic root canals and investigate the possible relationship between the concentration of endotoxin and endodontic signs and symptoms. Samples were collected from root canals of 50 patients requiring endodontic treatment due to necrosis of the pulpal tissue. Anaerobic techniques were used to determine the number of c.f.u. in each sample. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the concentration of endotoxin in each sample. The presence of c.f.u. was detected by culture in all samples (range 10(2)-5x10(6)). In samples from cases of patients with spontaneous pain, the mean c.f.u. was 1.43x10(6) while in asymptomatic cases it was 9.1x10(4). Endotoxin was present in all the samples studied [range 2390.0-22100.0 endotoxin units (EU) ml-1]. The mean concentration of endotoxin in samples from patients with spontaneous pain was 18540.0 EU ml-1 while in asymptomatic cases it was 12030.0 EU ml-1. Asymptomatic cases generally had lower levels of endotoxin (i.e. a negative association). A positive association was found between endotoxin and symptomatic cases (e.g. spontaneous pain, tenderness to percussion, pain on palpation, swelling and purulent exudates). This study showed that endotoxin is present in high concentrations in root canals of symptomatic teeth. There was a positive correlation between the concentration of endotoxin in the root canal and the presence of endodontic signs and symptoms.

Motivation: Robust computer algorithms are required to interpret the vast amounts of proteomic data currently being produced and to generate generalized models which are applicable to 'real world' scenarios. One such scenario is the classification of bacterial species. These vary immensely, some remaining remarkably stable whereas others are extremely labile showing rapid mutation and change. Such variation makes clinical diagnosis difficult and pathogens may be easily misidentified. Results: We applied artificial neural networks (Neuroshell 2) in parallel with cluster analysis and principal components analysis to surface enhanced laser desorption/ionization (SELDI)-TOF mass spectrometry data with the aim of accurately identifying the bacterium Neisseria meningitidis from species within this genus and other closely related taxa. A subset of ions were identified that allowed for the consistent identification of species, classifying >97% of a separate validation subset of samples into their respective groups. Availability: Neuroshell 2 is commercially available from Ward Systems. Contact: graham.balls@ntu.ac.uk

A database of MALDI-TOF mass spectrometry (MS) profiles has been developed with the aim of establishing a high throughput system for the characterisation of microbes. Several parameters likely to affect the reproducibility of the mass spectrum of a taxon were exhaustively studied. These included such criteria as sample preparation, growth phase, culture conditions, sample storage, mass range of ions, reproducibility between instruments and the methodology prior to database entry. Replicates of 12 spectra per sample were analysed using a 96-well target plate containing central wells for peptide standards to correct against mass drift during analysis. The quality of the data was assessed statistically prior to database addition using root mean squared values of

The genus Bacillus comprises a complex group of species, many of which cannot be readily differentiated by phenotypic and genotypic methods. Here, we utilised two species that are genetically very closely related, Bacillus subtilis and Bacillus mojavensis as a model to ascertain the potential of a linear MALDITOF MS to differentiate them against a background of their sporulation cycle and ultrastructural changes. Previous studies indicated that MALDI-TOF MS ionises the high abundance/conserved intracellular proteins but also produces additional lower mass ions that, with the appropriate software, may help to delineate such closely related taxa. Cells were grown in liquid culture over 24 hours and samples taken intermittently to monitor growth and the presence of spores. Harvested cells were studied by electron microscopy to detect potential changes in ultra structure and its potential effect on reliable and reproducible mass spectra. Sporulation was clearly evident by 24 hours and correlated inversely with reliable identification scores obtained using MALDI-TOF MS. Thus, cells cultured for 4, 6, 8 and 24 hours had identification scores of 2.157, 2.204, 2.295 and < 2 respectively. Contrary to previous findings, these results demonstrated unequivocally that Bacillus species may be reliably identified using this approach prior to sporulation. Furthermore, the software ClinProTools 3.0 was used to tease out minor components of the mass spectrum that enabled unambiguous separation of both groups of strains.

Aims: This work aims to evaluate the potential of GeLC-MS/MS to delineate taxa which are beyond the resolution of 16S rRNA and MALDI-TOF-MS identification, simultaneously discerning biomarkers of pathogenicity. Methods and Results: 16S rRNA sequence analysis and MALDITOF- MS was performed to differentiate genetically closely related species from the family Enterobacteriaceae. In parallel GeLC-MS/ MS, using E. coli and related enteric bacteria as a model, was performed. Species specific peptides were identified and used to create an optimised identification database, against which a panel of test strains were analysed to determine the resolution of GeLCMS/ MS for bacterial identification and strain characterisation. The panel included amongst others pathogenic E. coli strains, of differing pathotype, including E. coli O104:H4. The test strains could be resolved to the species and pathotype (subspecies) in addition, specific features could be identified. Conclusions: Using an optimised genome database and proteome profiling, we identified biomarkers that were specific for the test species and characterised strain-specific virulence factors. Significance and Impact of the Study: This proof of concept study demonstrates that whole genome sequences and GeLC-MS/ MS have the potential to both identify and characterise pathogenic bacteria in a single assay, ushering in a new proteogenomic trend for microbial clinical diagnostics

The taxonomy of the genus Peptostreptococcus is currently under revision. Four well-characterised species, Peptostreptococcus asaccharolyticus, Pepto-streptococcus indolicus, Peptostreptococcus lacrimalis and Peptostreptococcus harei form a homogeneous group of species but differ so markedly from the type species of the genus, Peptostreptococcus anaerobius, that they warrant placement in a separate genus. We propose that these species, Peptostreptococcus asaccharolyticus, Peptostreptococcus indolicus, Peptostreptococcus lacrimalis andPeptostreptococcus harei be reclassified in a new genus, Schleiferella, as Schleiferella asaccharolytica comb. nov., Schleiferella indolica comb. nov., Schleiferella lacrimalis comb. nov. and Schleiferella harei comb. nov.

The ability of Porphyromonas gingivalis to biosynthesize tetrapyrroles de novo has been investigated. Extracts of the bacterium do not possess activity for 5- aminolevulinic-acid dehydratase or porphobilinogen deaminase, two key enzymes involved in the synthesis of uroporphyrinogen III. Similarly, it was not possible to detect any genetic evidence for these early enzymes with the use of degenerate polymerase chain reaction. However, the bacterium does appear to harbor some of the enzymes for cobalamin biosynthesis since cobyric acid, a pathway intermediate, was converted into cobinamide. Furthermore, degenerate polymerase chain reaction with primers to cbiP, which encodes cobyric-acid synthase, produced a fragment with a high degree of identity to Salmonella typhimurium cbiP. Indeed, the recently released genome sequence data confirmed the presence of cbiP together with 14 other genes of the cobalamin pathway. A number of these genes were cloned and functionally characterized. Although P. gingivalis harbors all the genes necessary to convert precorrin-2 into cobalamin, it is missing the genes for the synthesis of precorrin-2. Either the organism has a novel pathway for the synthesis of precorrin-2, or more likely, it has lost this early part of the pathway. The remainder of the pathway may be being maintained to act as a salvage route for corrin synthesis.

The genus Peptostreptococcus (Kluyver & van Niel 1936) as presently constituted is phylogenetically and phenotypically incoherent; biochemical, chemical and molecular data indicate that the genus comprises a collection of very heterogeneous species. The type species Peptostreptococcus anaerobius is only distantly related to other Peptostreptococcus species and as such cannot be considered a member of the same genus. We therefore propose that the genus Peptostreptococcus be restricted to P. anaerobius and emend the description accordingly.

Matrix-Assisted Laser Desorption:lonisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) as a tool for differentiating bacterial species was examined using reference strains representing gram-positive and gram-negative taxa. Initially, the effect of differences in medium composition on spectral profile was examined. The results indicated that growth on Columbia blood agar resulted in a larger spectrum of ionized residues and was therefore used for the cultivation of all strains in the rest of the study. The stability of the obtained mass spectral profiles against differences in batch and media processing suggested that no significant alterations to the profiles occurred in response to changes in media sources. The established conditions from these initial experiments were used to standardize subsequent experiments. The MALDI-TOF-MS profiles of 15 reference strains were compared and species characteristic markers were identified. The potential of using MALDI-TOF-MS as a tool for probing clonal diversity...