Unravelling the eco-specificity and pathophysiological properties of Cutibacterium species in the light of recent taxonomic changes.

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.

Comparative Proteomic Profiling of Methicillin-Susceptible and Resistant Staphylococcus aureus.

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin-susceptible and methicillin-resistant S. aureus (MSSA; MRSA) recovered from non-healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are 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 are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.

The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK.

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.

Dissecting the taxonomic heterogeneity within Propionibacterium acnes: proposal for Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov.

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).

Complete Genome Sequence of the Hypervirulent Bacterium Clostridium difficile Strain G46, Ribotype 027.

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.

[Rhodococcus equi infection in AIDS patients: retrospective analysis of 13 patients in Argentina]. / Infección por Rhodococcus equi en pacientes con SIDA: Análisis retrospectivo de 13 pacientes en Argentina.

INTRODUCTION: Rhodococcus equi is a gram positive coccoid rod that causes pulmonary infections in immunosuppressed patients. METHODS: We retrospectively analyzed epidemiological, clinical, microbiological, radiological, and immunological features as well as the outcomes of 13 AIDS patients with R. equi infection. RESULTS: Between January 1994 and December 2012, 13 patients attending the AIDS department of the Infectious Diseases reference hospital in Buenos Aires were diagnosed with R. equi infection. All were men, the median age was 27 years. At the time of diagnosis, the median of CD4+ T cell counts was 11 cells/µl Twelve patients presented pulmonary disease with isolation of the microorganism from sputum or bronchoalveolar lavage; in the other patient the diagnosis was postmortem with positive culture of cerebrospinal fluid. The most frequent clinical manifestations were fever, haemoptysis, and weight loss. The predominant radiological finding was lobe consolidation with cavitation. Nine patients died after a median survival of 5.5 months. In all of them, cultures persisted positive until the last admission. The other 4 patients did continue clinical follow-ups. CONCLUSION: The insidious course of R. equi disease and the difficulties in the isolation of the microorganism contribute to the delay in the diagnosis and to the high mortality rate of this opportunistic infection.

Infección por Rhodococcus equi en pacientes con SIDA: Análisis retrospectivo de 13 pacientes en Argentina / Rhodococcus equi infection in AIDS patients: Retrospective analysis of 13 patients in Argentina

Introduction: Rhodococcus equi is a gram positive coccoid rod that causes pulmonary infections in immunosuppressed patients. Methods: We retrospectively analyzed epidemiological, clinical, microbiological, radiological, and immunological features as well as the outcomes of 13 AIDS patients with R. equi infection. Results: Between January 1994 and December 2012, 13 patients attending the AIDS department of the Infectious Diseases reference hospital in Buenos Aires were diagnosed with R. equi infection. All were men, the median age was 27 years. At the time of diagnosis, the median of CD4+ T cell counts was 11 cells/μl Twelve patients presented pulmonary disease with isolation of the microorganism from sputum or bronchoalveolar lavage; in the other patient the diagnosis was postmortem with positive culture of cerebrospinal fluid. The most frequent clinical manifestations were fever, haemoptysis, and weight loss. The predominant radiological finding was lobe consolidation with cavitation. Nine patients died after a median survival of 5.5 months. In all of them, cultures persisted positive until the last admission. The other 4 patients did continue clinical follow-ups. Conclusion: The insidious course of R. equi disease and the difficulties in the isolation of the microorganism contribute to the delay in the diagnosis and to the high mortality rate of this opportunistic infection.

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.

Correlation between phylogroups and intracellular proteomes of Propionibacterium acnes and differences in the protein expression profiles between anaerobically and aerobically grown cells.

Propionibacterium acnes is 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 of P. acnes were 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.

Molecular signatures for Bacillus species: demarcation of the Bacillus subtilis and Bacillus cereus clades in molecular terms and proposal to limit the placement of new species into the genus Bacillus.

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.

Genetic diversity of Propionibacterium acnes strains isolated from human skin in Japan and comparison with their distribution in Europe.

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.

Developing an integrated proteo-genomic approach for the characterisation of biomarkers for the identification of Bacillus anthracis.

Bacillus anthracis is the causative agent of anthrax, an acute and often fatal disease in humans. Due to the high genomic relatedness within the Bacillus cereus group of species it is a challenge to identify B. anthracis consistently. Alternative strategies such as proteomics coupled with mass spectrometry (MS) provide a powerful approach for biomarker discovery. However, validating and evaluating these markers, particularly for genetically homogeneous species such as B. anthracis are challenging. The objective of this study is to develop a robust biomarker discovery and validation pipeline, using proteomic methodology combined with in silico and molecular approaches, to determine a biomarker list, using B. anthracis as a model. In this exploratory study we profiled the proteome of B. anthracis and genetically related species using GeLC-Liquid Chromatography MS/MS (GeLC-LC MS/MS), identifying peptides that could be used to detect B. anthracis. Peptides were filtered to remove low quality identifications. Using comparative bioinformatic approaches, matching and searching against genomic sequence data a shortlist of peptide biomarkers was determined and validated using DNA sequencing, against a panel of closely related strains, to determine marker specificity. Further validation was performed using MS quantitation methods to assess sensitivity and specificity. A biomarker discovery pipeline was successfully developed in this study, comprising four distinct stages: proteome profiling, comparative bioinformatic validation, DNA sequencing and MS validation. Using the pipeline, 5379 peptides specific for Bacillus species and 36 peptides specific for B. anthracis were identified and validated. The 36 peptides, representing 30 proteins were derived from over 15 different clusters of orthologous group categories, including proteins involved in transcription, energy production/conservation as well as multifunctional proteins. We demonstrated that the peptide biomarkers identified in this study could be detected in a complex background, in which 0.1 µg of protein extract from B. anthracis was spiked into 9.90 µg of B. cereus protein extracts. The integration of both stable non-redundant peptides with molecular methodology for marker discovery and validation, improves the robustness of identifying and characterising candidate biomarkers for the identification of bacteria such as B. anthracis.

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group.

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.

Tracing the transition of methicillin resistance in sub-populations of Staphylococcus aureus, using SELDI-TOF Mass Spectrometry and Artificial Neural Network Analysis.

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.

Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics.

BACKGROUND: Robust biomarkers are needed to improve microbial identification and diagnostics. Proteomics methods based on mass spectrometry can be used for the discovery of novel biomarkers through their high sensitivity and specificity. However, there has been a lack of a coherent pipeline connecting biomarker discovery with established approaches for evaluation and validation. We propose such a pipeline that uses in silico methods for refined biomarker discovery and confirmation. RESULTS: The pipeline has four main stages: Sample preparation, mass spectrometry analysis, database searching and biomarker validation. Using the pathogen Clostridium botulinum as a model, we show that the robustness of candidate biomarkers increases with each stage of the pipeline. This is enhanced by the concordance shown between various database search algorithms for peptide identification. Further validation was done by focusing on the peptides that are unique to C. botulinum strains and absent in phylogenetically related Clostridium species. From a list of 143 peptides, 8 candidate biomarkers were reliably identified as conserved across C. botulinum strains. To avoid discarding other unique peptides, a confidence scale has been implemented in the pipeline giving priority to unique peptides that are identified by a union of algorithms. CONCLUSIONS: This study demonstrates that implementing a coherent pipeline which includes intensive bioinformatics validation steps is vital for discovery of robust biomarkers. It also emphasises the importance of proteomics based methods in biomarker discovery.

Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique.

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.

Approaches to the study of the systematics of anaerobic, gram-negative, non-sporeforming rods: current status and perspectives.

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.

High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells.

Staphylococcus aureus remains an important human pathogen responsible for a high burden of disease in healthcare and community settings. The emergence of multidrug-resistant strains is of increasing concern world-wide. The identification of S. aureus is currently based upon phenotypic and genotypic methods. Here, an alternative approach involving mass spectral analysis of surface-associated proteins of intact bacterial cells by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated using 95 isolates obtained directly from a clinical laboratory at The Royal London Hospital and 39 isolates from the Staphylococcal Reference Unit, Health Protection Agency, London. Results obtained indicate that clinical isolates share many common mass ions with-type/reference strains which allowed their correct identification when searched against a comprehensive database that has been in the process of development for several years. The existing database contains more than 5000 profiles of various bacterial pathogens, but comprises mainly type or reference strains. The MicrobeLynx software successfully identified all isolates to the correct genus and all but four to the correct species. These were misidentified in the first instance due to contamination or low mass ion intensity but once the cultures were purified and re-analysed they were confirmed as S. aureus by both MALDI-TOF-MS and 16S rRNA sequence analysis. The high percentage of correct identifications coupled with the high speed and the minimal sample preparation required, indicate that MALDI-TOF-MS has the potential to perform high throughput identification of clinical isolates of S. aureus despite the inherent diversity of this species. The method is, however, only reproducible if variable parameters such as sample preparation, media, growth condition, etc. are standardised.