Phages and cancer immunity: Gut bacteria are involved in the education of T cell immune responses, and the intestinal ecosystem influences anticancer immunity. Fluckiger et al. report microbial antigens that might cross-react with antigens associated with tumor cells. They found that a type of intestinal bacteria called enterococci harbor a bacteriophage that modulates immune responses. In mouse models, administration of enterococci containing the bacteriophage boosted T cell responses after treatment with chemotherapy or programmed cell death protein 1 (PD-1) blockade. In humans, the presence of the bacteriophage was associated with improved survival after PD-1 immunotherapy. A fraction of human T cells specific for naturally processed melanoma epitopes appeared to be able to recognize microbial peptides. This “molecular mimicry” may represent cross-reactivity between tumors and microbial antigens. Science , this issue p. 936

An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The “Staphylococcal Regulatory RNA Database” (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNA-seq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature.

Mycobacterium abscessus complex, the third most frequent mycobacterial complex responsible for community- and health care-associated infections in developed countries, comprises of M. abscessus subsp. abscessus and M. abscessus subsp. bolletii reviously referred as Mycobacterium bolletii and Mycobacterium massiliense. The diversity of this group of opportunistic pathogens is poorly described. In-depth analysis of 14 published M. abscessus complex genomes found a pan-genome of 6,153 proteins and core-genome of 3,947 (64.1%) proteins, indicating a non-conservative genome. Analysing the average percentage of amino-acid sequence identity (from 94.19% to 98.58%) discriminates three main clusters C1, C2 and C3: C1 comprises strains belonging to M. abscessus, C2 comprises strains belonging to M. massiliense and C3 comprises strains belonging to M. bolletii; and two sub-clusters in clusters C2 and C3. The phylogenomic network confirms these three clusters. The genome length (from 4.8 to 5.51-Mb) varies from 5.07-Mb in C1, 4.89-Mb in C2A, 5.01-Mb in C2B and 5.28-Mb in C3. The mean number of prophage regions (from 0 to 7) is 2 in C1; 1.33 in C2A; 3.5 in C2B and five in C3. A total of 36 genes are uniquely present in C1, 15 in C2 and 15 in C3. These genes could be used for the detection and identification of organisms in each cluster. Further, the mean number of host-interaction factors (including PE, PPE, LpqH, MCE, Yrbe and type VII secretion system ESX3 and ESX4) varies from 70 in cluster C1, 80 in cluster C2A, 74 in cluster C2B and 93 in clusters C3A and C3B. No significant differences in antibiotic resistance genes were observed between clusters, in contrast to previously reported in-vitro patterns of drug resistance. They encode both penicillin-binding proteins targeted by β-lactam antibiotics and an Ambler class A β-lactamase for which inhibitors exist. Our comparative analysis indicates that M. abscessus complex comprises three genomospecies, corresponding to M. abscessus, M. bolletii, and M. massiliense. The genomics data here reported indicate differences in virulence of medical interest; and suggest targets for the refined detection and identification of M. abscessus.

To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates. The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments. Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases. There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not).

The Mycobacterium abscessus complex is a group of rapidly growing, multiresistant mycobacteria previously divided into three species. Proposal for the union of Mycobacterium bolletii and Mycobacterium massiliense into one subspecies, so-called M. abscessus subsp. massiliense, created much confusion about the routine identification and reporting of M. abscessus clinical isolates for clinicians. Results derived from multigene sequencing unambiguously supported the reinstatement of M. massiliense and M. bolletii as species, culminating in the presence of erm(41)-encoded macrolide resistance in M. bolletii . Present genome-based analysis unambiguously supports the reinstatement of M. massiliense and M. bolletii as species after the average nucleotide identity values of 96.7 % for M. abscessus versus M. bolletii , and 96.4 % for M. abscessus versus M. massiliense , and the 96.6 % identity between M. bolletii and M. massiliense was put into the perspective of a larger, 28-species analysis. Accordingly, DNA–DNA hybridization values predicted by the complete rpo B gene sequencing analysis were between 68.7 and 72.3 % in this complex. These genomic data as well as the phenotypic characteristics prompted us to propose to reinstate the previously known M. massiliense and M. bolletii into two distinct species among the M. abscessus complex.

Objectives: To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France. Methods: The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A. Results: As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization. Conclusions: Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).