Dominique Clermont

The Intestinal Microbiota Modulates the Anticancer Immune Effects of Cyclophosphamide

The Microbiota Makes for Good Therapy The gut microbiota has been implicated in the development of some cancers, such as colorectal cancer, but—given the important role our intestinal habitants play in metabolism—they may also modulate the efficacy of certain cancer therapeutics. Iida et al. (p. 967) evaluated the impact of the microbiota on the efficacy of an immunotherapy [CpG (the cytosine, guanosine, phosphodiester link) oligonucleotides] and oxaliplatin, a platinum compound used as a chemotherapeutic. Both therapies were reduced in efficacy in tumor-bearing mice that lacked microbiota, with the microbiota important for activating the innate immune response against the tumors. Viaud et al. (p. 971) found a similar effect of the microbiota on tumor-bearing mice treated with cyclophosphamide, but in this case it appeared that the microbiota promoted an adaptive immune response against the tumors. The gut microbiota promote the efficacy of several antineoplastic agents in mice. Cyclophosphamide is one of several clinically important cancer drugs whose therapeutic efficacy is due in part to their ability to stimulate antitumor immune responses. Studying mouse models, we demonstrate that cyclophosphamide alters the composition of microbiota in the small intestine and induces the translocation of selected species of Gram-positive bacteria into secondary lymphoid organs. There, these bacteria stimulate the generation of a specific subset of “pathogenic” T helper 17 (pTH17) cells and memory TH1 immune responses. Tumor-bearing mice that were germ-free or that had been treated with antibiotics to kill Gram-positive bacteria showed a reduction in pTH17 responses, and their tumors were resistant to cyclophosphamide. Adoptive transfer of pTH17 cells partially restored the antitumor efficacy of cyclophosphamide. These results suggest that the gut microbiota help shape the anticancer immune response.

Listeria rocourtiae sp. nov.

A Listeria-like strain isolated in Austria from pre-cut lettuce fitted the description of the genus Listeria although it could not be assigned to any of the known species. Comparison of the rrs gene (encoding 16S rRNA) sequence and gene content by DNA-array indicated affiliation to the genus Listeria. Phylogenetic distance from known species of the genus Listeria indicated that it represents a novel species. Since it can be differentiated from all other known species of the genus Listeria by using phenotypic tests, the name Listeria rocourtiae sp. nov. is proposed for the novel species. The type strain is CIP 109804(T) (=DSM 22097(T) =Allerberger 700284/02(T)). The type strain is avirulent as assessed by cell culture assays and inoculation of mice.

The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome.

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1‐C CRISPR2 is present in few strains but type 2‐A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre‐exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer‐associated motif‐shuffling demonstrated that the GG motif is sufficient to discriminate self and non‐self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II‐A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population.

The Genomic Diversification of the Whole Acinetobacter Genus: Origins, Mechanisms, and Consequences

Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

Novel class A beta-lactamase Sed-1 from Citrobacter sedlakii: genetic diversity of beta-lactamases within the Citrobacter genus.

ABSTRACT Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with β-lactam and quinolone antibiotics. The isolate produced an inducible class A β-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene,blaSed-1, was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded β-lactamases from Citrobacter koseri(formerly C. diversus) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), andProteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from blaSed-1. This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from C. koseriand 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae, respectively. By using DNA-DNA hybridization, a blaSed-1-like gene was identified in two reference strains, C. sedlakii(CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains ofC. sedlakii CIP-105037 and C. rodentiumCIP-104675 using two primers specific forblaSed-1. They shared 98 and 80% identity withblaSed-1, respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found inCitrobacter.

Identification of 50 Class D β-Lactamases and 65 Acinetobacter-Derived Cephalosporinases in Acinetobacter spp.

ABSTRACT Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D β-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D β-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D β-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of β-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of β-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

Natural occurrence of structures in oral streptococci and enterococci with DNA homology to Tn916.

Seventeen oral streptococci and 18 enterococci were tested for the presence of DNA sequences homologous to the conjugative transposon Tn916 encoding tetracycline resistance. All the strains were resistant to tetracyclines, including minocycline, and most of them were resistant to other antibiotics. Tn916-like structures, identified by hybridization of HincII-digested DNA, were found on the chromosomes of 11 oral streptococci and four enterococci and on two plasmids, pIP1549 and pIP1440, one harbored by an Enterococcus hirae strain and the other harbored by an Enterococcus faecalis strain. Sequences homologous to Tn916, only some of which corresponded to its internal HincII structure (Tn916-modified elements), were chromosomally located in three oral streptococci and two enterococci and were plasmid borne in pIP614 harbored by an E. faecalis strain. Nine enterococci and three oral streptococci carried either the Tet M or the Tet O determinant chromosomally, but they carried no other sequences homologous to Tn916. Images

Global phylogeography and evolutionary history of Shigella dysenteriae type 1.

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries1. A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission2. This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries1,3,4 and the first isolation of Sd1 in Japan in 18975. Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

Microbacterium binotii sp. nov., isolated from human blood

Two strains of non-spore-forming, rod-shaped, Gram-positive bacteria, CIP 101303(T) and CIP 102116, were isolated from human blood in 1976 and 1977, respectively. These strains had chemotaxonomic markers that were consistent with classification in the genus Microbacterium, i.e. MK-10, MK-11 and MK-12 as the major menaquinones, predominant iso- and anteiso-branched cellular fatty acids, galactose, mannose and rhamnose as the cell-wall sugars and ornithine as the diamino acid in the cell-wall peptidoglycan. The DNA G+C content was 70-72 mol%. Comparative 16S rRNA gene sequence studies revealed that strains CIP 101303(T) and CIP 102116 belonged to the genus Microbacterium and that they were related closely to Microbacterium halotolerans. The level of DNA-DNA relatedness showed that the two isolates represented a separate genomic species. Based on phenotypic and genotypic results, it is proposed that strains CIP 101303(T) and CIP 102116 be assigned to a novel species, Microbacterium binotii sp. nov. The type strain is CIP 101303(T) (=DSM 19164(T)).