Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and gram-positive bacteria.

In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain. For the first time, both Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) isolates were tested with a label-free approach, and, in the presence of two groups of bactericidal antibiotic molecules, aminoglycosides and beta-lactams. Our results support the feasibility of label-free AST in less than 2 h and suggest that single cell auto-fluorescence adds value to the Susceptible/Resistant phenotyping over single-cell scattering alone, in particular for the mecA+ Staphylococcus (i.e., resistant) strains treated with oxacillin.

Species-targeted sorting and cultivation of commensal bacteria from the gut microbiome using flow cytometry under anaerobic conditions.

BACKGROUND: There is a growing interest in using gut commensal bacteria as "next generation" probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was to adapt flow cytometry and cell sorting to be able to detect, separate, isolate, and cultivate new strains of commensal species from fecal material. We focused on the extremely oxygen sensitive (EOS) species Faecalibacterium prausnitzii and the under-represented, health-associated keystone species Christensenella minuta as proof-of-concept. RESULTS: A BD Influx® cell sorter was equipped with a glovebox that covered the sorting area. This box was flushed with nitrogen to deplete oxygen in the enclosure. Anaerobic conditions were maintained during the whole process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii strains ATCC 27766, ATCC 27768, and DSM 17677. We then generated polyclonal antibodies against target species by immunizing rabbits with heat-inactivated bacteria. Two polyclonal antibodies were directed against F. prausnitzii type strains that belong to different phylogroups, whereas one was directed against C. minuta strain DSM 22607. The specificity of the antibodies was demonstrated by sorting and sequencing the stained bacterial fractions from fecal material. In addition, staining solutions including LIVE/DEAD™ BacLight™ Bacterial Viability staining and polyclonal antibodies did not severely impact bacterial viability while allowing discrimination between groups of strains. Finally, we combined these staining strategies as well as additional criteria based on bacterial shape for C. minuta and were able to detect, isolate, and cultivate new F. prausnitzii and C. minuta strains from healthy volunteer's fecal samples. CONCLUSIONS: Targeted cell-sorting under anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations, and can be used to sort EOS and/or under-represented strains of interest using specific antibodies, thus opening new avenues for culture experiments. Video abstract.

Draft Genome Sequence of Isolate POC01, a Novel Anaerobic Member of the Oscillospiraceae Family, Isolated from Human Feces.

We report the isolation, culture, and genome sequencing of isolate POC01, a strictly anaerobic bacterium isolated from a healthy donor, representing a previously uncultured member of the Oscillospiraceae family.

Assessment of Gram- and Viability-Staining Methods for Quantifying Bacterial Community Dynamics Using Flow Cytometry.

Over the past years, gut microbiota became a major field of interest with increasing reports suggesting its association with a large number of human diseases. In this context, there is a major interest to develop analysis tools allowing simple and cost-effective population pattern analysis of these complex ecosystems to follow changes over time. Whereas sequence-based metagenomics profiling is widely used for microbial ecosystems characterization, it still requires time and specific expertise for analysis. Flow cytometry overcomes these disadvantages, providing key information on communities within hours. In addition, it can potentially be used to select, isolate and cultivate specific bacteria of interest. In this study, we evaluated the culturability of strictly anaerobic bacteria that were stained with a classical Live/Dead staining, and then sorted using flow cytometry under anaerobic conditions. This sorting of "viable" fraction demonstrated that 10-80% of identified "viable" cells of pure cultures of strictly anaerobic bacteria were culturable. In addition, we tested the use of a combination of labeled vancomycin and Wheat Germ Agglutinin (WGA) lectin to discriminate Gram-positive from Gram-negative bacteria in complex ecosystems. After validation on both aerobic/anaerobic facultative and strictly anaerobic bacteria, the staining methods were applied on complex ecosystems, revealing differences between culture conditions and demonstrating that minor pH variations have strong impacts on microbial community structure, which was confirmed by 16S rRNA gene sequencing. This combination of staining methods makes it possible to follow-up evolutions of complex microbial communities, supporting its future use as a rapid analysis tool in various applications. The flow cytometry staining method that was developed has the potential to facilitate the analysis of complex ecosystems by highlighting changes in bacterial communities' dynamics. It is assumed to be applicable as an efficient and fast approach to improve the control of processes linked to a wide range of ecosystems or known communities of bacterial species in both research and industrial contexts.

The N-terminal domain of the R28 protein promotes emm28 group A Streptococcus adhesion to host cells via direct binding to three integrins.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28Nt, R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28Nt and both subdomains interacted directly with the laminin-binding α3ß1, α6ß1, and α6ß4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype.

Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110.

The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5' promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110ΔcovR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

Srr2, a multifaceted adhesin expressed by ST-17 hypervirulent Group B Streptococcus involved in binding to both fibrinogen and plasminogen.

The Group B Streptococcus (GBS) 'hypervirulent' ST-17 clone is strongly associated with invasive neonatal meningitis. Comparative genome analyses revealed that the serine-rich repeat (Srr) glycoprotein Srr2 is a cell wall-anchored protein specific for ST-17 strains, the non-ST-17 isolates expressing Srr1. Here, we unravel the binding capacity of GBS Srr proteins to relevant components of the host fibrinolysis pathway. We demonstrate that: (i) Srr2 binds plasminogen and plasmin whereas Srr1 does not; (ii) the ability of ST-17 strains to bind fibrinogen reflects a high level surface display of Srr2 combined with a higher affinity of Srr2 than Srr1 to bind this ligand; and (iii) Srr2 binding to host plasma proteins results in the formation of bacterial aggregates that are efficiently endocytosed by phagocytes. Importantly, we show that Srr2 increased bacterial survival to phagocytic killing and bacterial persistence in a murine model of meningitis. We conclude that Srr2 is a multifaceted adhesin used by the ST-17 clone to hijack ligands of the host coagulation system, thereby contributing to bacterial dissemination and invasiveness, and ultimately to meningitis.

Capsular switching in group B Streptococcus CC17 hypervirulent clone: a future challenge for polysaccharide vaccine development.

BACKGROUND: The capsular polysaccharide (CPS) is an important virulence factor and a vaccine target of the major neonatal pathogen group B Streptococcus (GBS). Population studies revealed no strong correlation between CPS type and multilocus sequence typing (MLST) cluster, with the remarkable exception of the worldwide spread of hypervirulent GBS CC17, which were all until recently CPS type III. METHODS: A total of 965 GBS strains from invasive infection isolated in France were CPS typed and the presence of the CC17-specific surface protein encoding gene hvgA gene was investigated. Three hvgA-positive GBS strains screened were surprisingly CPS type IV and thus further characterized by MLST typing, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. RESULTS: MLST and PFGE demonstrated a capsular switching from CPS type III to IV within the highly homogeneous GBS CC17. Sequence analysis revealed that this capsular switch was due to the exchange of a 35.5-kb DNA fragment containing the entire cps operon. CONCLUSIONS: This work shows that GBS CC17 hypervirulent strains have switched one of their main vaccine targets. Thus, continued surveillance of GBS population remains of the utmost importance during clinical trials of conjugate GBS vaccines.

Rga, a RofA-like regulator, is the major transcriptional activator of the PI-2a pilus in Streptococcus agalactiae.

Rapid adaptation to changing environments is key in determining the outcome of infections caused by the opportunistic human pathogen Streptococcus agalactiae. We previously demonstrated that the RofA-like protein (RALP) regulators RogB and Rga activate their downstream divergently transcribed genes, that is, the pilus operon PI-2a and the serine-rich repeat encoding gene srr1, respectively. Characterization of the Rga regulon by microarray revealed that the PI-2a pilus was strongly controlled by Rga, a result confirmed at the protein level. Complementation experiments showed that the expression of Rga, but not RogB, in the double ΔrogB/Δrga mutant, or in the clinical strain 2603V/R displaying frameshift mutations in rogB and rga genes, is sufficient to restore wild-type expression levels of PI-2a pilus and Srr1. Biofilm formation was impaired in the Δrga and Δrga/rogB mutants and restored on complementation with rga. Paradoxically, adherence to intestinal epithelial cells was unchanged in the Δrga mutant. Finally, the existence of several clinical isolates mutated in rga highlights the concept of strain-specific regulatory networks.

Group B Streptococcus surface proteins as major determinants for meningeal tropism.

Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism.

Selective antibody response to Streptococcus gallolyticus pilus proteins in colorectal cancer patients.

Streptococcus gallolyticus subsp. gallolyticus (previously called Streptococcus bovis biotype I) infections have long been associated with colorectal cancer (CRC). This work aimed to investigate the CRC-associated humoral immune response to four pilus proteins of this bacterium by newly developed ELISAs. Pilus proteins are interesting diagnostic targets as they are the building blocks of pilin-like structures that mediate bacterial virulence and are readily exposed to the host immune system upon infection. The presence of serum antibodies against these pilus proteins was evaluated in Dutch and American populations. These analyses showed that an immune response to these antigens was specific for clinical S. gallolyticus subsp. gallolyticus infections, but that increased serum antibody titers to multiple pilus proteins in single individuals were rarely observed. However, a multiplex approach based on antibody titers against any of these four antigens resulted in assay sensitivities between 16% and 43% for the detection of early-stage CRC. Together these findings underscore the potential of a multi-antigen approach to complement diagnosis of S. gallolyticus subsp. gallolyticus-associated CRC.

The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates.

Streptococcus agalactiae (group B streptococcus; GBS) is a normal constituent of the intestinal microflora and the major cause of human neonatal meningitis. A single clone, GBS ST-17, is strongly associated with a deadly form of the infection called late-onset disease (LOD), which is characterized by meningitis in infants after the first week of life. The pathophysiology of LOD remains poorly understood, but our epidemiological and histopathological results point to an oral route of infection. Here, we identify a novel ST-17-specific surface-anchored protein that we call hypervirulent GBS adhesin (HvgA), and demonstrate that its expression is required for GBS hypervirulence. GBS strains that express HvgA adhered more efficiently to intestinal epithelial cells, choroid plexus epithelial cells, and microvascular endothelial cells that constitute the blood-brain barrier (BBB), than did strains that do not express HvgA. Heterologous expression of HvgA in nonadhesive bacteria conferred the ability to adhere to intestinal barrier and BBB-constituting cells. In orally inoculated mice, HvgA was required for intestinal colonization and translocation across the intestinal barrier and the BBB, leading to meningitis. In conclusion, HvgA is a critical virulence trait of GBS in the neonatal context and stands as a promising target for the development of novel diagnostic and antibacterial strategies.

In vitro readthrough of termination codons by gentamycin in the Stüve-Wiedemann Syndrome.

The Stüve-Wiedemann Syndrome (SWS) is a frequently lethal chondrodysplasia caused by null mutations in the leukemia inhibitory factor receptor gene (LIFR) responsible for an impaired activation of the JAK-STAT pathway after LIF stimulation. Most LIFR mutations are nonsense mutations, thus prompting us to investigate the impact of aminoglycosides on the readthrough of premature termination codons (PTCs). Culturing skin fibroblasts from three SWS patients and controls for 48 h in the presence of gentamycin (200-500 microg/ml) partially restored the JAK-STAT3 pathway when stimulated by LIF. Consistently, quantitative RT-PCR analysis showed that gentamycin stabilized LIFR mRNAs carrying UGA premature termination codons. We conclude that high gentamycin concentrations can partially restore functional LIFR protein synthesis in vitro, prompting us to investigate PTC readthrough using less toxic and more efficient drugs in this presently untreatable lethal condition.

Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome).

Studying consanguineous families with Ghosal hematodiaphyseal dysplasia syndrome (GHDD), a disorder of increased bone density, we identified mutations in TBXAS1, which encodes thromboxane synthase (TXAS). TXAS, an enzyme of the arachidonic acid cascade, produces thromboxane A(2) (TXA(2)). Platelets from subjects with GHDD showed a specific deficit in arachidonic acid-produced aggregation. We also found that TXAS and TXA(2) modulated expression of TNFSF11 and TNFRSF11B (encoding RANKL and osteoprotegerin (OPG), respectively) in primary cultured osteoblasts.

Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan.

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.

Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium.

D-aspartate ligase has remained the last unidentified peptide bond-forming enzyme in the peptidoglycan assembly pathway of Gram-positive bacteria. Here we show that a two-gene cluster of Enterococcus faecium encodes aspartate racemase (Racfm) and ligase (Aslfm) for incorporation of D-Asp into the side chain of the peptidoglycan precursor. Aslfm was identified as a new member of the ATP-grasp protein superfamily, which includes a diverse set of enzymes catalyzing ATP-dependent carboxylate-amine ligation reactions. Aslfm specifically ligated the beta-carboxylate of D-Asp to the epsilon-amino group of L-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide. D-iso-asparagine was not a substrate of Aslfm, indicating that the presence of this amino acid in the peptidoglycan of E. faecium results from amidation of the alpha-carboxyl of D-Asp after its addition to the precursor. Heterospecific expression of the genes encoding Racfm and Aslfm in Enterococcus faecalis led to production of stem peptides substituted by D-Asp instead of L-Ala2, providing evidence for the in vivo specificity and function of these enzymes. Strikingly, sequencing of the cross-bridges revealed that substitution of L-Ala2 by D-Asp is tolerated by the d,d-transpeptidase activity of the penicillin-binding proteins both in the acceptor and in the donor substrates. The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium.

Impact of specific pbp5 mutations on expression of beta-lactam resistance in Enterococcus faecium.

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.

Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.

Flavobacterium johnsoniae CIP100931 is resistant to most beta-lactam antibiotics and has a decreased susceptibility to carbapenems. A beta-lactamase gene was cloned and expressed in Escherichia coli DH10B. The purified beta-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of approximately 27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams. Sequence analysis revealed that JOHN-1 is a molecular class B beta-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively). JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes. Although F. johnsoniae and Chryseobacterium spp. are phylogenetically related bacteria, this report further underlines the heterogeneity of class B beta-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these beta-lactamase genes.

Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes.

Myroides odoratus and Myroides odoratimimus (formerly designated in a single species as Flavobacterium odoratum) are gram-negative aerobes and sources of nosocomial infections in humans. They have variable susceptibility to beta-lactams and a decreased susceptibility to carbapenems. Using genomic DNAs of M. odoratus CIP 103105 and M. odoratimimus CIP 103073 reference strains, shotgun cloning of beta-lactamase genes was performed, followed by protein expression in Escherichia coli. The deduced amino acid sequences of these beta-lactamase genes revealed that TUS-1 and MUS-1 from M. odoratus CIP 103105 and M. odoratimimus CIP 103073, respectively, shared 73% amino acid identity. Mature proteins TUS-1 and MUS-1, with pI values of 7.8 and 5.2, respectively, had relative molecular masses of ca. 26 kDa. These beta-lactamases are members of the subclass B1 of metallo-beta-lactamases and are distantly related to other metalloenzymes, being most closely related to IND-1 from Chryseobacterium indologenes (42% amino acid identity). However, phylogenic analysis showed that TUS-1 and MUS-1 belong to the same phylogenic lineage of subclass B1 enzymes that groups the subclass B1 beta-lactamases of Flavobacterium species. Kinetic parameters of purified beta-lactamases TUS-1 and MUS-1 detailed their hydrolysis spectra, which encompass most beta-lactams except aztreonam. beta-Lactamases TUS-1 and MUS-1 were classified in functional subgroup 3a of metalloenzymes. This work further characterizes chromosome-encoded metalloenzymes from Flavobacteriaceae species that explain at least part of their intrinsic resistance to beta-lactams.