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.

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).

[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.

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.

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.

Assessment of the stability of cell-surface components of microorganisms by MALDI-TOF-MS following preservation on lenticule discs.

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.

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.

Proteome analysis of serovars Typhimurium and Pullorum of Salmonella enterica subspecies I.

BACKGROUND: Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella. RESULTS: In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars. CONCLUSION: Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods.

New approaches to identification of bacterial pathogens by surface enhanced laser desorption/ionization time of flight mass spectrometry in concert with artificial neural networks, with special reference to Neisseria gonorrhoeae.

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) has been applied in large numbers of oncological studies but the microbiological field has not been extensively explored to date. This paper describes the application of SELDI-TOF MS in concert with a multi-layer perceptron artificial neural network (ANN) with a back propagation algorithm for the identification of Neisseria gonorrhoeae. N. gonorrhoeae, the aetiological agent of gonorrhoea, is the second most common sexually transmitted disease in the UK and USA. Analysis of over 350 strains of N. gonorrhoeae and closely related species by SELDI-TOF MS facilitated the design of an ANN model and revealed 20 ion peak descriptors of positive, negative and secondary nature that were paramount for the identification of the pathogen. The model performed with over 96 % efficiency when based on these 20 ion peak descriptors and exhibited a sensitivity of 95.7 % and a specificity of 97.1 %, with an area under the curve value of 0.996. The technology has the potential to link several ANN models for a comprehensive rapid identification platform for clinically important pathogens.

Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth.

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.

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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proteomics: a new era in anaerobic microbiology.

Genome sequence data provide a framework for predicting potential microbial activities; however, the proteome content of the cell dictates its response to its environment. Microbiology is witnessing a major initiative to elucidate the nature of the proteome of large numbers of species. The tool driving the proteomic revolution is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. During the analysis process, proteins are ionized and separated on the basis of their mass-to-charge ratios, which results in a characteristic mass-spectral profile. Because of the dynamic nature of the cell and the large number of external parameters that could influence its mass-spectral profile, considerable work was needed initially to optimize sample analysis and obtain consistent and reproducible results. For many anaerobes that grow poorly or are nonreactive in most diagnostic systems, proteome analysis is likely to have a major impact on microbial diagnosis and the delineation of centers of diversity associated with infections.

Gastrointestinal microflora studies in late-onset autism.

Some cases of late-onset (regressive) autism may involve abnormal flora because oral vancomycin, which is poorly absorbed, may lead to significant improvement in these children. Fecal flora of children with regressive autism was compared with that of control children, and clostridial counts were higher. The number of clostridial species found in the stools of children with autism was greater than in the stools of control children. Children with autism had 9 species of Clostridium not found in controls, whereas controls yielded only 3 species not found in children with autism. In all, there were 25 different clostridial species found. In gastric and duodenal specimens, the most striking finding was total absence of non-spore-forming anaerobes and microaerophilic bacteria from control children and significant numbers of such bacteria from children with autism. These studies demonstrate significant alterations in the upper and lower intestinal flora of children with late-onset autism and may provide insights into the nature of this disorder.

Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases.

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 <3.0 as the criterion for rejection. Cluster analysis using a nearest neighbour algorithm also enabled subsets of data to be compared. This was achieved using the bespoke MicrobeLynx trade mark software. Columbia blood agar was used to standardise all procedures for the database, since it permitted the culture of most human pathogens and also produced spectra with a broad range of mass ions. In some instances, alternative media such as CLED were used in specific studies with greater success. Following standardisation of the procedure, a database was developed comprising ca. 3500 spectra with multiple strain entries for most species. The results to date show unequivocally that as the number of strains per species increased, so too did the success of species matching. The technique demonstrated unique mass spectral profiles for each genus/species, with the variation in mass ions among strains/species being dependent on the intra-specific diversity. The success of identification against the database for wild-type strains ranged between 33 and 100%; the lower percentage results being generally associated with poor representation of some species within the database. These findings provide a new dimension for the rapid and high throughput characterisation of human pathogens with potentially broad applications across the field of microbiology.