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The strains of Pseudomonas aeruginosa exhibit considerable differences in their genotypic and pathogenic properties. To clarify their evolutionary/taxonomic relationships, comprehensive phylogenomic and comparative genomic studies were conducted on the genome sequences of 212 P . aeruginosa strains covering their genetic diversity. In a phylogenomic tree based on 118 conserved proteins, the analysed strains formed two distinct clades. One of these clades, Clade-1, encompassing >70 % of the strains including the type strain DSM 50071T, represents the species P. aeruginosa sensu stricto. Clade-2, referred to in earlier work as the outlier group, with NCTC 13628T as its type strain, constitutes a novel species level lineage. The average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between the strains from Clade-1 and Clade-2 are in the range of 93.4–93.7, 95.1–95.3 and 52–53 %, respectively. The 16S rRNA gene of P. aeruginosa DSM 50071T also shows 98.3 % similarity to that of NCTC 13628T. These values are lower than the suggested cut-off values for species distinction, indicating that the Clade-2 strains (NCTC 13628T) constitute a new species. We also report the identification of 12 conserved signature indels in different proteins and 24 conserved signature proteins that are exclusively found in either Clade-1 or Clade-2, providing a reliable means for distinguishing these clades. Additionally, in contrast to swimming motility, twitching motility is only present in Clade-1 strains. Based on earlier work, the strains from these two clades also differ in their pathogenic mechanisms (presence/absence of Type III secretion system), production of biosurfactants, phenazines and siderophores, and several other genomic characteristics. Based on the evidence from different studies, we propose that the Clade-2 strains constitute a novel species for which the name Pseudomonas paraeruginosa is proposed. The type strain is NCTC 13628T (=PA7T=ATCC 9027T). The description of Pseudomonas aeruginosa is also emended to include information for different molecular markers specific for this species.

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.

Background Coagulase negative staphylococci (CoNS) are important reservoirs of antibiotic resistance genes and associated mobile genetic elements and are believed to contribute to the emergence of successful methicillin resistant Staphylococcus aureus (MRSA) clones. Although, these bacteria have been linked to various ecological niches, little is known about the dissemination and genetic diversity of antibiotic resistant CoNS in general public settings. Methods Four hundred seventy-nine samples were collected from different non-healthcare/general public settings in various locations (n = 355) and from the hands of volunteers (n = 124) in London UK between April 2013 and Nov 2014. Results Six hundred forty-three staphylococcal isolates belonging to 19 staphylococcal species were identified. Five hundred seventy-two (94%) isolates were resistant to at least one antibiotic, and only 34 isolates were fully susceptible. Sixty-eight (11%) mecA positive staphylococcal isolates were determined in this study. SCCmec types were fully determined for forty-six isolates. Thirteen staphylococci (19%) carried SCCmec V, followed by 8 isolates carrying SCCmec type I (2%), 5 SCCmec type IV (7%), 4 SCCmec type II (6%), 1 SCCmec type III (2%), 1 SCCmec type VI (2%), and 1 SCCmec type VIII (2%). In addition, three isolates harboured a new SCCmec type 1A, which carried combination of class A mec complex and ccr type 1. MLST typing revealed that all S. epidermidis strains possess new MLST types and were assigned the following new sequence types: ST599, ST600, ST600, ST600, ST601, ST602, ST602, ST603, ST604, ST605, ST606, ST607 and ST608. Conclusions The prevalence of antibiotic resistant staphylococci in general public settings demonstrates that antibiotics in the natural environments contribute to the selection of antibiotic resistant microorganisms. The finding of various SCCmec types in non-healthcare associated environments indicates the complexity of SCCmec. We also report on new MLST types that were assigned for all S. epidermidis isolates, which demonstrates the genetic variability of these isolates. Electronic supplementary material The online version of this article (10.1186/s13756-018-0367-4) contains supplementary material, which is available to authorized users.

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.

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.

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.

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.

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 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.

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.

Propionibacterium acnesis one of the dominant commensals on the human skin and also an opportunistic pathogen in relation to acne, sarcoidosis, prostate cancer, and various infections. Recent investigations using housekeeping and virulence genes have revealed that the species consists of three major evolutionary clades (types I, II, and III). In order to investigate protein expression differences between these phylogroups, proteomic profiles of 21 strains ofP. acneswere investigated. The proteins extracted from cells cultured under anaerobic and aerobic conditions were analysed using a SELDI-TOF mass spectrometer, high-resolution capillary gel electrophoresis, and LC-MS/ MS. The SELDI spectral profiles were visualised as a heat map and a dendrogram, which resulted in four proteomic groups. Strains belonging to type I were represented in the proteome Group A, while Group B contained type III strains. Groups C and D contained mixtures of types I and II. Each of these groups was not influenced by differences in culture conditions. Under anoxic growth conditions, a type IB strain yielded high expressions of some proteins, such as methylmalonyl-CoA epimerase and the Christie-Atkins-Munch-Petersen (CAMP) factor. The present study revealed good congruence between genomic and proteomic data suggesting that the microenvironment of each subtype may influence protein expression.

We conducted a retrospective study to update the bacteriology of 46 cases of anaerobic empyema that were originally studied between 1976 and 1993 at the Wadsworth Anaerobic Bacteriology Clinical Research Laboratory (Los Angeles). Anaerobic bacteriologic studies were completed for all 46 pleural fluid specimens, and aerobic bacteriologic studies were completed for 41 of these specimens. Thirty-seven clinical charts were available for review. A total of 161 anaerobic isolates (3.5 per patient) representing 64 species or groups were recovered. The most common isolates were as follows: Fusobacterium nucleatum (19); Prevotella oris-buccae group (13, 9 of which were P. oris); Bacteroides fragilis group (11, 4 of which were B. fragilis); pigmented Prevotella species (17, 8 of which were in the Prevotella intermedia-nigrescens group); Peptostreptococcus species (17, 9 of which were Peptostreptococcus micros); Eubacterium species (7); Lactobacillus species (8); Actinomyces species (7); and Clostridium species (7). Nineteen if the cases were of purely anaerobic etiology; of these, eight were caused by a single organism: F. nucleatum (five cases); B. fragilis (two cases); and Prevotella mangus (one case). Of the 45 aerobic isolates (1.1 per patient), viridans streptococci were most common (21 isolates), followed by group D nonenterococcal streptococcus (four isolates). Only nine gram-negative rods (six enteric and three nonenteric organisms) and one Staphylococcus aureus isolate were recovered. The susceptibility to penicillin of 64 isolates was examined with the use of the spiral gradient method; 21 (33%) of these isolates were beta-lactamase positive (MICs ranged from 1.1 toor = 54 micrograms/mL vsor = 0.27 micrograms/mL for beta-lactamase-negative strains).

The periodontal pocket provides a unique structural site for studies on host/bacterial interactions. The pocket is colonized by a complex but characteristic anaerobic bacterial flora. Many new taxa have been described that have now been supported by comparative rRNA sequence analysis. Within recent years, several molecular approaches have been used to describe both cultivable and noncultivable species, and new genotypes have been reported. Microbial activity rather than the mere presence of microorganism at a site must be a major factor in the process of disease development. Information on metabolic activities of species and identification of substrates are therefore essential to elucidate the complex interactions that are likely to occur in vivo. We have been using a variety of analytical procedures such as 13 C substrate-enrichment nuclear magnetic resonance, 14 C isotopic labeling experiments, enzyme assays, impedance measurements, and various chromatographic and electrophoretic procedures to study key species of this predominantly asaccharolytic flora. Results have so far indicated a major role for cationic and anionic acids as sources of energy, but the mechanisms of substrate processing may differ significantly between species. In this ecosystem, crevicular fluid is the likely source of nutrients for species. Components of this fluid appear to have a role in the selection of species in subgingival sites.

Cell free extracts from a wide variety of Gram-negative, anaerobic, non-sporeforming rods were screened for dehydrogenase enzymes of carbohydrate and nitrogen metabolism. Four enzymes, malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were found to be of diagnostic importance. The ‘Bacteroides fragilis’ group possessed all four enzymes whereas the ‘B. melaninogenicus-B. oralis’ group and the asaccharolytic, pigmented species were characterised by the presence of only malate and glutamate dehydrogenases. The saccharolytic and asaccharolytic pigmented species could be differentiated by the wide difference in pH optimum for malate dehydrogenase. Fusobacterium species and Leptotrichia buccalis both possessed only glutamate dehydrogenase, but there were differences in enzyme activity between both taxa. Other genera such as Anaerorhabdus, Megamonas, Mitsuokella. Rikenella, Sebaldella and Tissierella had characteristic enzyme profiles. These enzymatic data are of diagnostic value within the Bacteroidaceae and may serve as useful markers for studying the ecological interrelationships of these bacteria.Keywords: Bacteroidaceae; Dehydrogenases; Enzyme patterns.

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.

The growth response of Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola to peptides (supplied as trypticase) and amino acids (supplied as casamino acids) was measured over 24 hours by monitoring changes in alternating-current conductivity at 37 degrees C. All species utilized peptides preferentially over amino acids. These results are consistent with those obtained previously with conventional growth-response experiments. The duration of the linear growth response to trypticase of P. gingivalis was 9.7 hours, whereas that of both F. nucleatum and T. denticola was24 hours. By contrast, there was more uniformity in the utilization of amino acids from the casamino acid mixture, which previously has been shown to be a poor growth substrate. Furthermore, subtle differences in growth patterns, such as the ability of F. nucleatum to metabolize its storage glycopolymers before utilizing amino acids, were clearly evident. The present method provides an excellent means of studying bacterial growth kinetics and delineating bacterial/substrate specificities of both synthetic and natural substrates.

1 Classification 2 Bacteroides 3 Prevotella 4 Porphyromonas 5 The Current Status of Species that Previously Belonged to the Genus Bacteroides 6 Antibiotic Resistance 7 Molecular Analysis 8 Bacteroides, Prevotella, and Porphyromonas in the Normal Flora 9 Antimicrobial Susceptibility 10 Pathogenicity Keywords: Bacteroides, Prevotella, and Porphyromonas; gram-negative, nonsporing, anaerobic bacilli with rounded or pointed ends; isolation and classification of organisms-presenting difficulties; impact of rRNA sequence analysis; genus Bacteroides-small nonmotile gram-negative bacilli and coccobacilli; Bacteroides thetaiotaomicron; Bacteroides putredinis and Bacteroides microfusus; gram-negative anaerobic bacilli-in normal flora of gastrointestinal tract

A total of 31 strains of Prevotella intermedia were subjected to DNA-DNA hybridization and were characterized by performing physiological tests and by performing a multilocus enzyme analysis, using malate dehydrogenase and glutamate dehydrogenase. All of the strains assigned to P. intermedia fermented glucose and sucrose, hydrolyzed starch but not esculin, and produced indole, acetic, isobutyric, isovaleric, and succinic acids as metabolic end products. The results of DNA reassociation experiments performed with the reference probe permitted separation of the strains into two well-defined homology groups. In addition, strains with DNAs that hybridized with DNA from strain ATCC 25611T (T = type strain) had high levels of peptidase activity and cleaved lipid substrates (4-methylumbelliferyl laurate and 4-methylumbellifelyl elaidate). Multilocus enzyme electrophoresis revealed two electromorphic profiles, one characteristic of strain ATCC 25611T and the other characteristic of strain ATCC 33563T. We propose that a new species, Prevotella nigrescens, should be created for the genetically distinct group of strains that hybridized with strain ATCC 33563T. Strain ATCC 33563 is designated the type strain of P. nigrescens.

Summary 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 α-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.

Background Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches. Results In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion Using a multi-step digest approach the LPI™ technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI™ technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.

In order to survive in the host and initiate infection,Salmonella entericaneeds to undergo a transition between aerobic and anaerobic growth by modulating its central metabolic pathways. In this study, a comparative analysis of the proteome ofS. entericaserovar Typhimurium grown in the presence or absence of oxygen was performed. The most prominent changes in expression were measured in a semiquantitative manner using difference in-gel electrophoresis (DIGE) to reveal the main protein factors involved in the adaptive response to anaerobiosis. A total of 38 proteins were found to be induced anaerobically, while 42 were repressed. The proteins of interest were in-gel digested with trypsin and identified by MALDI TOF mass spectrometry using peptide mass fingerprinting. In the absence of oxygen, many fermentative enzymes catalysing reactions in the mixed-acid or arginine fermentations were overexpressed. In addition, the enzyme fumarate reductase, which is known to provide an alternative electron acceptor for the respiratory chains in the absence of oxygen, was shown to be induced. Increases in expression of several glycolytic and pentose phosphate pathway enzymes, as well as two malic enzymes, were detected, suggesting important roles for these in anaerobic metabolism. Substantial decreases in expression were observed for a large number of periplasmic transport proteins. The majority of these are involved in the uptake of amino acids and peptides, but permeases transporting iron, thiosulphate, glucose/galactose, glycerol 3-phosphate and dicarboxylic acids were also repressed. Decreases in expression were also observed for a superoxide dismutase, ATP synthase, inositol monophosphatase, and several chaperone and hypothetical proteins. The changes were monitored in two different isolates, and despite their very similar expression patterns, some variability in the adaptive response to anaerobiosis was also observed.

Human isolates of Fusobacterium nucleatum subspecies appear to colonize different niches in the oral cavity, which may be reflected in their nutritional properties. Consequently the utilization of nitrogenous substrates, their sources of energy (supplied here as amino acids) were compared between the 3 subspecies using the reference strain and fresh clinical isolates of each subspecies. All strains incorporated mainly acidic and basic amino acids but significant differences occurred between subspecies. Both reference and clinical isolates of F. nucleatum subspecies polymorphum utilized all amino acids in the medium but the levels of glutamate, arginine and cysteine were noticeably higher in the reference strain. By contrast, F. nucleatum subspecies fusiforme used a very restricted range of amino acids, of which only glutamate, arginine, histidine and cysteine were taken up at greater than 0.5 mM. F. nucleatum subspecies nucleatum utilized fewer amino acids than F. nucleatum subspecies polymorphum but higher concentrations were taken up by the former. Clinical isolates of F. nucleatum subspecies nucleatum incorporated polar and nonpolar neutral amino acids poorly but their levels increased steadily as a clinical isolate was subcultured over a period of 4 months, and was eventually similar to the reference strain. The effect of adding the key catabolic substrate, glutamate (10 mM), on the amino acid uptake profile of F. nucleatum subspecies nucleatum resulted in the complete suppression of the dibasic amino acids arginine, ornithine and histidine. Strains of this subspecies could grow on glutamate as a major source of carbon and energy but, morphologically, the cells appeared somewhat distended and had a tendency to clump.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.