Damien Dubois

Identification of a variety of Staphylococcus species by MALDI-TOF mass spectrometry

ABSTRACT Whole-cell fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in combination with a dedicated bioinformatic software tool (MALDI Biotyper 2.0) was used to identify 152 staphylococcal strains corresponding to 22 staphylococcal species. Spectra of the 152 isolates, previously identified at the species level using a sodA gene-based oligonucleotide array, were analyzed against the main spectra of 3,030 microorganisms. A total of 151 strains out of 152 (99.3%) were correctly identified at the species level; only one strain was identified at the genus level. The MALDI-TOF MS method revealed different clonal lineages of Staphylococcus epidermidis that were of either human or environmental origin, which suggests that the MALDI-TOF MS method could be useful in the profiling of staphylococcal strains. The topology of the dendrogram generated by the MALDI Biotyper 2.0 software from the spectra of 120 Staphylococcus reference strains (representing 36 species) was in general agreement with that inferred from the 16S rRNA gene-based analysis. Our findings indicate that the MALDI-TOF MS technology, associated with a broad-spectrum reference database, is an effective tool for the swift and reliable identification of Staphylococci.

Colonization of the human gut by E. coli and colorectal cancer risk

Purpose: The intestinal microbiota is potentially involved in the development of colorectal carcinoma via various mechanisms. Escherichia coli are commensal bacteria of the human gut microbiota, but some pathogenic strains have acquired the ability to induce chronic inflammation and/or produce toxins, such as cyclomodulin, which could participate in the carcinogenesis process. Here, we analyzed the E. coli population associated with mucosa of patients with colon cancer in relation to clinicopathologic characteristics. We assessed carcinogenic properties of a colon cancer–associated E. coli strain in multiple intestinal neoplasia (Min) mice. Experimental design: Mucosa-associated or internalized E. coli were quantified and characterized from tumors and mucosa of patients with colon cancer and the healthy mucosa of diverticulosis controls. Min mice were inoculated with a colon cancer–associated E. coli strain (11G5). The number of colonic polyps was evaluated at 7 weeks after infection. Results: An increased level of mucosa-associated and internalized E. coli was observed in the tumors compared with normal tissue. A relationship between poor prognostic factors for colon cancer (tumor–node–metastasis stage) and colonization of mucosa by E. coli was observed. Pathogenic cyclomodulin-positive E. coli strains were more prevalent on mucosa of patients with stages III/IV than those with stage I colon cancer. Proliferative index and E. coli colonization level of the mucosa distant from the tumor significantly correlated. Min mice infected with the E. coli strain 11G5 displayed a marked increase in the number of visible colonic polyps compared with controls. Conclusion: These findings support that pathogenic E. coli could be a cofactor in pathogenesis of colorectal cancer. Clin Cancer Res; 20(4); 859–67. ©2013 AACR.

Performances of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Rapid Identification of Bacteria in Routine Clinical Microbiology

ABSTRACT Rapid and cost-effective matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based systems will replace conventional phenotypic methods for routine identification of bacteria. We report here the first evaluation of the new MALDI-TOF MS-based Vitek MS system in a large clinical microbiology laboratory. This system uses an original spectrum classifier algorithm and a specific database designed for the identification of clinically relevant species. We have tested 767 routine clinical isolates representative of 50 genera and 124 species. Vitek MS-based identifications were performed by means of a single deposit on a MALDI disposable target without any prior extraction step and compared with reference identifications obtained mainly with the VITEK2 phenotypic system; if the identifications were discordant, molecular techniques provided reference identifications. The Vitek MS system provided 96.2% correct identifications to the species level (86.7%), to the genus level (8.2%), or within a range of species belonging to different genera (1.3%). Conversely, 1.3% of isolates were misidentified and 2.5% were unidentified, partly because the species was not included in the database; a second deposit provided a successful identification for 0.8% of isolates unidentified with the first deposit. The Vitek MS system is a simple, convenient, and accurate method for routine bacterial identification with a single deposit, considering the high bacterial diversity studied and as evidenced by the low prevalence of species without correct identification. In addition to a second deposit in uncommon cases, expanding the spectral database is expected to further enhance performances.

ClbP Is a Prototype of a Peptidase Subgroup Involved in Biosynthesis of Nonribosomal Peptides

The pks genomic island of Escherichia coli encodes polyketide (PK) and nonribosomal peptide (NRP) synthases that allow assembly of a putative hybrid PK-NRP compound named colibactin that induces DNA double-strand breaks in eukaryotic cells. The pks-encoded machinery harbors an atypical essential protein, ClbP. ClbP crystal structure and mutagenesis experiments revealed a serine-active site and original structural features compatible with peptidase activity, which was detected by biochemical assays. Ten ClbP homologs were identified in silico in NRP genomic islands of closely and distantly related bacterial species. All tested ClbP homologs were able to complement a clbP-deficient E. coli mutant. ClbP is therefore a prototype of a new subfamily of extracytoplasmic peptidases probably involved in the maturation of NRP compounds. Such peptidases will be powerful tools for the manipulation of NRP biosynthetic pathways.

Structural insights into substrate recognition and product expulsion in CTX-M enzymes.

beta-Lactamase-mediated resistance to beta-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among beta-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the beta-lactamase family and probably for most carboxypeptidases.

Analysis of structure-function relationships in the colibactin-maturating enzyme ClbP.

pks genomic island of Escherichia coli is involved in the synthesis of the non-ribosomal peptide-type genotoxin colibactin, which has been suggesting as affecting the host immune response and having an impact on cancer development. The pks-encoded enzyme ClbP is an atypical peptidase that contributes to the synthesis of colibactin. In this work, we identified key features of ClbP. Bacterial fractionation and Western-blot analysis revealed the docking of ClbP to the bacterial inner membrane via a C-terminal domain harboring three predicted transmembrane helices. Whereas only one helix was necessary for the location in the inner membrane, the complete sequence of the C-terminal domain was necessary for ClbP bioactivity. In addition, the N-terminal sequence of ClbP allowed the SRP/Sec/YidC- and MreB-dependent translocation of the enzymatic domain in the periplasmic compartment, a feature also essential for ClbP bioactivity. Finally, the comparison of ClbP structure with that of the paralogs FmtA-like and AmpC revealed at an extremity of the catalytic groove a negative electrostatic potential surface characteristic of ClbP. Site-directed mutagenesis experiments identified in this zone two aspartic residues that were important for ClbP bioactivity. Overall, these results suggest a model for precolibactin activation by ClbP and pave a way for the design of inhibitors targeting colibactin production.

Cyclomodulins in Urosepsis Strains of Escherichia coli

ABSTRACT Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

Maternally acquired genotoxic Escherichia coli alters offspring's intestinal homeostasis.

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

CTX-M β-Lactamase Production and Virulence of Escherichia coli K1

We report a patient with neonatal meningitis caused by a CTX-M-1–producing Escherichia coli K1 strain. The influence of CTX-M production on virulence was investigated in cell culture and a newborn mouse model of meningitis. CTX-M production had no influence on virulence but was a major factor in clinical outcome.

Streptobacillus moniliformis as the Causative Agent in Spondylodiscitis and Psoas Abscess after Rooster Scratches

ABSTRACT We report a case of Streptobacillus moniliformis spondylodiscitis accompanied by a psoas abscess in an 80-year-old man scratched by a rooster. S. moniliformis was identified from abscess fluid by use of 16S rRNA gene sequencing. After 18 weeks of antimicrobial therapy, the clinical condition of the patient improved.