Molecular Typing of Multiple Isolates Is Essential to Diagnose Cutibacterium acnes Orthopedic Device-related Infection.

Cutibacterium acnes orthopedic device-related infections (ODRIs) range from obvious infections to solely culture-based diagnoses. Multilocus sequence typing of multiple isolates from the same procedure revealed that most cases with normal C-reactive protein levels that were classified as C. acnes ODRI would be considered contaminations when accounting for genotypic data.

In-Host Adaptation of Salmonella enterica Serotype Dublin during Prosthetic Hip Joint Infection.

Genome degradation has been central to the adaptation of Salmonella enterica serotypes to their hosts throughout evolution. We witnessed the patho-adaptation of a strain of Salmonella Dublin (a cattle-adapted serotype) to a human host during the course of a recurrent prosthetic hip joint infection evolving over several years.

Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members.

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria.

Selection of Resistance to Clarithromycin in Mycobacterium abscessus Subspecies.

Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm(41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm(41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl, erm(41), whiB, rpIV, and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm(41) T28 sequevar and strain M. abscessus subsp. bolletii, which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm(41) C28 sequevar and M. abscessus subsp. massiliense, not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV, encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm(41) C28 and M. abscessus subsp. massiliense, whereas it is less common for M. abscessus subsp. abscessus erm(41) T28 sequevar and M. abscessus subsp. bolletii, where other mechanisms may be responsible for failure.

Genome-wide mosaicism within Mycobacterium abscessus: evolutionary and epidemiological implications.

BACKGROUND: In mycobacteria, conjugation differs from the canonical Hfr model, but is still poorly understood. Here, we quantified this evolutionary processe in a natural mycobacterial population, taking advantage of a large clinical strain collection of the emerging pathogen Mycobacterium abscessus (MAB). RESULTS: Multilocus sequence typing confirmed the existence of three M. abscessus subspecies, and unravelled extensive allelic exchange between them. Furthermore, an asymmetrical gene flow occurring between these main lineages was detected, resulting in highly admixed strains. Intriguingly, these mosaic strains were significantly associated with cystic fibrosis patients with lung infections or chronic colonization. Genome sequencing of those hybrid strains confirmed that half of their genomic content was remodelled in large genomic blocks, leading to original tri-modal 'patchwork' architecture. One of these hybrid strains acquired a locus conferring inducible macrolide resistance, and a large genomic insertion from a slowly growing pathogenic mycobacteria, suggesting an adaptive gene transfer. This atypical genomic architecture of the highly recombinogenic strains is consistent with the distributive conjugal transfer (DCT) observed in M. smegmatis. Intriguingly, no known DCT function was found in M. abscessus chromosome, however, a p-RAW-like genetic element was detected in one of the highly admixed strains. CONCLUSION: Taken together, our results strongly suggest that MAB evolution is sporadically punctuated by dramatic genome wide remodelling events. These findings might have far reaching epidemiological consequences for emerging mycobacterial pathogens survey in the context of increasing numbers of rapidly growing mycobacteria and M. tuberculosis co-infections.

Mycobacterium lutetiense sp. nov., Mycobacterium montmartrense sp. nov. and Mycobacterium arcueilense sp. nov., members of a novel group of non-pigmented rapidly growing mycobacteria recovered from a water distribution system.

From our recent survey of non-pigmented rapidly growing mycobacteria in the Parisian water system, three groups of isolates (taxons 1-3) corresponding to possible novel species were selected for taxonomic study. The three taxa each formed creamy white, rough colonies, had an optimal growth temperature of 30 °C, hydrolyzed Tween 80, were catalase-positive at 22 °C and expressed arylsulfatase activity. All three were susceptible to amikacin, ciprofloxacin and tigecycline. The three taxa produced specific sets of mycolic acids, including one family that has never previously been described, as determined by thin layer chromatography and nuclear magnetic resonance. The partial rpoB sequences (723 bp) showed 4-6 % divergence from each other and more than 5 % differences from the most similar species. Partial 16S rRNA gene sequences showed 99 % identity within each species. The most similar sequences for 16S rRNA genes (98-99 % identity over 1444-1461 bp) were found in the Mycobacterium fortuitum group, Mycobacterium septicum and Mycobacterium farcinogenes. The three taxa formed a new clade (bootstrap value, 99 %) on trees reconstructed from concatenated partial 16S rRNA, hsp65 and rpoB sequences. The above results led us to propose three novel species for the three groups of isolates, namely Mycobacterium lutetiense sp. nov. [type strain 071T=ParisRGMnew_1T (CIP 110656T=DSM 46713T)], Mycobacterium montmartrense sp. nov. [type strain 196T=ParisRGMnew_2T (CIP 110655T=DSM 46714T)] and Mycobacteriu marcueilense sp. nov. [type strain of 269T=ParisRGMnew_3T (CIP 110654T=DSM 46715T)].

Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus.

Mycobacterium abscessus is an emerging pathogen that is increasingly recognized as a relevant cause of human lung infection in cystic fibrosis patients. This highly antibiotic-resistant mycobacterium is an exception within the rapidly growing mycobacteria, which are mainly saprophytic and non-pathogenic organisms. M. abscessus manifests as either a smooth (S) or a rough (R) colony morphotype, which is of clinical importance as R morphotypes are associated with more severe and persistent infections. To better understand the molecular mechanisms behind the S/R alterations, we analysed S and R variants of three isogenic M. abscessus S/R pairs using an unbiased approach involving genome and transcriptome analyses, transcriptional fusions and integrating constructs. This revealed different small insertions, deletions (indels) or single nucleotide polymorphisms within the non-ribosomal peptide synthase gene cluster mps1-mps2-gap or mmpl4b in the three R variants, consistent with the transcriptional differences identified within this genomic locus that is implicated in the synthesis and transport of Glyco-Peptido-Lipids (GPL). In contrast to previous reports, the identification of clearly defined genetic lesions responsible for the loss of GPL-production or transport makes a frequent switching back-and-forth between smooth and rough morphologies in M. abscessus highly unlikely, which is important for our understanding of persistent M. abscessus infections.

Protein folds as synapomorphies of the tree of life.

Several studies showed that folds (topology of protein secondary structures) distribution in proteomes may be a global proxy to build phylogeny. Then, some folds should be synapomorphies (derived characters exclusively shared among taxa). However, previous studies used methods that did not allow synapomorphy identification, which requires congruence analysis of folds as individual characters. Here, we map SCOP folds onto a sample of 210 species across the tree of life (TOL). Congruence is assessed using retention index of each fold for the TOL, and principal component analysis for deeper branches. Using a bicluster mapping approach, we define synapomorphic blocks of folds (SBF) sharing similar presence/absence patterns. Among the 1232 folds, 20% are universally present in our TOL, whereas 54% are reliable synapomorphies. These results are similar with CATH and ECOD databases. Eukaryotes are characterized by a large number of them, and several SBFs clearly support nested eukaryotic clades (divergence times from 1100 to 380 mya). Although clearly separated, the three superkingdoms reveal a strong mosaic pattern. This pattern is consistent with the dual origin of eukaryotes and witness secondary endosymbiosis in their phothosynthetic clades. Our study unveils direct analysis of folds synapomorphies as key characters to unravel evolutionary history of species.

Shared Pathogenomic Patterns Characterize a New Phylotype, Revealing Transition toward Host-Adaptation Long before Speciation of Mycobacterium tuberculosis.

Tuberculosis remains one of the deadliest infectious diseases of humanity. To better understand the evolutionary history of host-adaptation of tubercle bacilli (MTB), we sought for mycobacterial species that were more closely related to MTB than the previously used comparator species Mycobacterium marinum and Mycobacterium kansasii. Our phylogenomic approach revealed some recently sequenced opportunistic mycobacterial pathogens, Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense, to constitute a common clade with MTB, hereafter called MTB-associated phylotype (MTBAP), from which MTB have emerged. Multivariate and clustering analyses of genomic functional content revealed that the MTBAP lineage forms a clearly distinct cluster of species that share common genomic characteristics, such as loss of core genes, shift in dN/dS ratios, and massive expansion of toxin-antitoxin systems. Consistently, analysis of predicted horizontal gene transfer regions suggests that putative functions acquired by MTBAP members were markedly associated with changes in microbial ecology, for example adaption to intracellular stress resistance. Our study thus considerably deepens our view on MTB evolutionary history, unveiling a decisive shift that promoted conversion to host-adaptation among ancestral founders of the MTBAP lineage long before Mycobacterium tuberculosis has adapted to the human host.

Mycobacterium bolletii Lung Disease in Cystic Fibrosis.

BACKGROUND: The cystic fibrosis (CF) pathogen, Mycobacterium abscessus complex, covers three subspecies: M. abscessus, M. massiliense, and M. bolletii. There are no clinical outcome data concerning M. bolletii. Our aim was to characterize M. bolletii lung infections in patients with CF. METHODS: We included patients with M. bolletii lung infection recorded between 1994 and 2012 in France. Data were collected from the CF registry, medical records, and questionnaires submitted to the CF primary physician. Strains were typed by multilocus sequence typing analysis. RESULTS: Fifteen cases were identified in nine CF centers. Nine patients (60%) presented with nontuberculous mycobacterial pulmonary disease. Follow-up of 13 patients showed a trend to more rapid decline in FEV1 in the first year of colonization (-9.4%; SD 19.3) in comparison with noninfected control subjects (-2.3%; SD 12.1) (P = .16). Twelve patients were treated, and 11 received oral macrolides. Treatment-induced eradication occurred in five patients (41.7%). Four patients died (26.7%), including one patient with fatal nontuberculous mycobacterial pulmonary disease. Inducible macrolide resistance was demonstrated in all strains. Patients always harbored unique strains. CONCLUSIONS: Our study reports the largest study cohort of CF patients infected with M. bolletii. M. bolletii infection affects both children and young adults, is most often symptomatic, and may be fatal. Macrolide-based therapies have poor effectiveness. There is no evidence of patient-to-patient transmission.

Comparison of the RGM medium and the mycobacterial growth indicator tube automated system for isolation of non-tuberculous mycobacteria from sputum samples of cystic fibrosis patients in Belgium.

PURPOSE: Pulmonary infections due to non-tuberculous mycobacteria (NTM) are an emerging issue in the cystic fibrosis (CF) population. Due to bacterial and fungal overgrowth, isolation of mycobacteria from the sputum samples of these patients remains challenging. RGM medium, a novel agar-based culture medium was evaluated for the isolation of NTM from sputum samples of CF patients. METHODOLOGY: Sputum samples were inoculated onto RGM medium and conventional Mycobacterial Growth Indicator Tube (MGIT™, Becton Dickinson, USA). Agar plates were incubated at 35 °C and growth was recorded once a week during 42 days. We compared the yield of the two media. RESULTS: 217 samples were obtained from 124 CF patients. 20 samples (13 patients) had a positive culture for NTM. 79/217 (36.4%) MGIT had to be discontinued due to contamination compared to 18/217 (8.3%) for RGM. We reported equivalent NTM detection performances for RGM and MGIT (P = 0.579): these media enabled the isolation of 15 and 12 NTM strains respectively. CONCLUSION: RGM medium increases the proportion of interpretable results and the number of NTM cultured. Taking into account the non-inferiority compared to conventional methods and ease of use of RGM medium, we estimate that this test can replace current approaches for the screening of NTM among people with CF. Additionally, RGM provides semi-quantitative results (number of colonies) and information on the morphology of colonies, which may be clinically relevant information.

Genomic history of the seventh pandemic of cholera in Africa.

The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa.

Mycobacterial Pan-Genome Analysis Suggests Important Role of Plasmids in the Radiation of Type VII Secretion Systems.

In mycobacteria, various type VII secretion systems corresponding to different ESX (ESAT-6 secretory) types, are contributing to pathogenicity, iron acquisition, and/or conjugation. In addition to the known chromosomal ESX loci, the existence of plasmid-encoded ESX systems was recently reported. To investigate the potential role of ESX-encoding plasmids on mycobacterial evolution, we analyzed a large representative collection of mycobacterial genomes, including both chromosomal and plasmid-borne sequences. Data obtained for chromosomal ESX loci confirmed the previous five classical ESX types and identified a novel mycobacterial ESX-4-like type, termed ESX-4-bis. Moreover, analysis of the plasmid-encoded ESX loci showed extensive diversification, with at least seven new ESX profiles, identified. Three of them (ESX-P clusters 1-3) were found in multiple plasmids, while four corresponded to singletons. Our phylogenetic and gene-order-analyses revealed two main groups of ESX types: 1) ancestral types, including ESX-4 and ESX-4-like systems from mycobacterial and non-mycobacterial actinobacteria and 2) mycobacteria-specific ESX systems, including ESX-1-2-3-5 systems and the plasmid-encoded ESX types. Synteny analysis revealed that ESX-P systems are part of phylogenetic groups that derived from a common ancestor, which diversified and resulted in the different ESX types through extensive gene rearrangements. A converging body of evidence, derived from composition bias-, phylogenetic-, and synteny analyses points to a scenario in which ESX-encoding plasmids have been a major driving force for acquisition and diversification of type VII systems in mycobacteria, which likely played (and possibly still play) important roles in the adaptation to new environments and hosts during evolution of mycobacterial pathogenesis.

Genome-wide transcriptome analyses of silicon metabolism in Phaeodactylum tricornutum reveal the multilevel regulation of silicic acid transporters.

BACKGROUND: Diatoms are largely responsible for production of biogenic silica in the global ocean. However, in surface seawater, Si(OH)(4) can be a major limiting factor for diatom productivity. Analyzing at the global scale the genes networks involved in Si transport and metabolism is critical in order to elucidate Si biomineralization, and to understand diatoms contribution to biogeochemical cycles. METHODOLOGY/PRINCIPAL FINDINGS: Using whole genome expression analyses we evaluated the transcriptional response to Si availability for the model species Phaeodactylum tricornutum. Among the differentially regulated genes we found genes involved in glutamine-nitrogen pathways, encoding putative extracellular matrix components, or involved in iron regulation. Some of these compounds may be good candidates for intracellular intermediates involved in silicic acid storage and/or intracellular transport, which are very important processes that remain mysterious in diatoms. Expression analyses and localization studies gave the first picture of the spatial distribution of a silicic acid transporter in a diatom model species, and support the existence of transcriptional and post-transcriptional regulations. CONCLUSIONS/SIGNIFICANCE: Our global analyses revealed that about one fourth of the differentially expressed genes are organized in clusters, underlying a possible evolution of P. tricornutum genome, and perhaps other pennate diatoms, toward a better optimization of its response to variable environmental stimuli. High fitness and adaptation of diatoms to various Si levels in marine environments might arise in part by global regulations from gene (expression level) to genomic (organization in clusters, dosage compensation by gene duplication), and by post-transcriptional regulation and spatial distribution of SIT proteins.

New tools for labeling silica in living diatoms.

Silicon biomineralization is a widespread mechanism found in several kingdoms that concerns both unicellular and multicellular organisms. As a result of genomic and molecular tools, diatoms have emerged as a good model for biomineralization studies and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited. Here, new probes were developed specifically to label the pre-existing or the newly synthesized silica frustule of several diatoms species. It is shown that the LysoTracker Yellow HCK-123, which can be used to visualize silica frustules with common filter sets, presents an enhanced signal-to-noise ratio and allows details of the frustules to be imaged without of the use of ionophores. It is also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells. The coupling of labeling procedures might help to address the challenging question of the process of frustule exocytosis.

The N terminus of the HasA protein and the SecB chaperone cooperate in the efficient targeting and secretion of HasA via the ATP-binding cassette transporter.

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.

The SecB chaperone is bifunctional in Serratia marcescens: SecB is involved in the Sec pathway and required for HasA secretion by the ABC transporter.

HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in Escherichia coli, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the secB gene from S. marcescens. We show that S. marcescens SecB is 93% identical to E. coli SecB and complements the secretion defects of a secB mutant of E. coli for both the Sec and ABC pathways of HasA secretion. In S. marcescens, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that S. marcescens SecB is the genuine chaperone for HasA secretion in S. marcescens. These results also demonstrate that S. marcescens SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of secB point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.

Antifolding activity of the SecB chaperone is essential for secretion of HasA, a quickly folding ABC pathway substrate.

We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s(-1)) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s(-1)) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.