Bacterial endosymbionts of insects play a central role in upgrading the diet of their hosts. In certain cases, such as aphids and tsetse flies, endosymbionts complement the metabolic capacity of hosts living on nutrient-deficient diets, while the bacteria harbored by omnivorous carpenter ants are involved in nitrogen recycling. In this study, we describe the genome sequence and inferred metabolism of Blattabacterium strain Bge, the primary Flavobacteria endosymbiont of the omnivorous German cockroach Blattella germanica. Through comparative genomics with other insect endosymbionts and free-living Flavobacteria we reveal that Blattabacterium strain Bge shares the same distribution of functional gene categories only with Blochmannia strains, the primary Gamma-Proteobacteria endosymbiont of carpenter ants. This is a remarkable example of evolutionary convergence during the symbiotic process, involving very distant phylogenetic bacterial taxa within hosts feeding on similar diets. Despite this similarity, different nitrogen economy strategies have emerged in each case. Both bacterial endosymbionts code for urease but display different metabolic functions: Blochmannia strains produce ammonia from dietary urea and then use it as a source of nitrogen, whereas Blattabacterium strain Bge codes for the complete urea cycle that, in combination with urease, produces ammonia as an end product. Not only does the cockroach endosymbiont play an essential role in nutrient supply to the host, but also in the catabolic use of amino acids and nitrogen excretion, as strongly suggested by the stoichiometric analysis of the inferred metabolic network. Here, we explain the metabolic reasons underlying the enigmatic return of cockroaches to the ancestral ammonotelic state. Bacterial endosymbionts from insects are subjected to a process of genome reduction from the moment they interact with their host, especially when the symbiosis is strict (the partners live together permanently) and the endosymbiont is maternally inherited. The type of genes that are retained correlates with specific metabolic host requirements. Here, we report the genome sequence of Blattabacterium strain Bge, the primary endosymbiont of the German cockroach B. germanica. Cockroaches are omnivorous insects and Blattabacterium cooperates with their metabolism, not only with essential nutrient metabolism but also through an efficient use of amino acids and the nitrogen excretion by the combination of a urea cycle and urease activity. The repertoires of functions that are maintained in Blattabacterium are similar to those already observed in Blochmannia spp., the primary endosymbiont of carpenter ants, also an omnivorous insect. This constitutes a nice example of evolutionary convergence of two endosymbionts belonging to very different bacterial phyla that have evolved a similar repertoire of functions according to the host. However, the current set of genes and, more importantly, those that were lost in the process of genome reduction in both endosymbiont lineages have also contributed to a different involvement of Blattabacterium and Blochmannia in nitrogen metabolism.

Background: Carbapenem-resistant Enterobacteriaceae are considered by WHO as “critical” priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes. Methods: We first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing. Results: In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination. Conclusions: Lineages of CP-Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by β-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131.

OXA-244 is a single-point-mutant derivative of OXA-48 displaying reduced carbapenemase activity. Here, we report the microbiological features of seven OXA-244-producing Escherichia coli isolates. Only one isolate grew on ChromID Carba Smart medium (bioMérieux), but six of the seven isolates grew on ChromID extended-spectrum-β-lactamase (ESBL) medium (bioMérieux), as they coproduced an ESBL and/or a plasmid-encoded cephalosporinase. The production of a carbapenemase was detected in 57.1%, 71.4%, 71.4%, and 100% of the E. coli isolates using the Carba NP test, the Rapidec Carba NP test (bioMérieux), a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) hydrolysis assay (Bruker), and the OXA-48 K-SeT assay (Coris BioConcept), respectively. Our results indicate that OXA-244-producing E. coli isolates are difficult to detect, which may lead to their silent spread.

Many insect species have established long-term symbiotic relationships with intracellular bacteria. Symbiosis with bacteria has provided insects with novel ecological capabilities, which have allowed them colonize previously unexplored niches. Despite its importance to the understanding of the emergence of biological complexity, the evolution of symbiotic relationships remains hitherto a mystery in evolutionary biology. In this study, we contribute to the investigation of the evolutionary leaps enabled by mutualistic symbioses by sequencing the genome of Blattabacterium cuenoti, primary endosymbiont of the omnivorous cockroach Blatta orientalis, and one of the most ancient symbiotic associations. We perform comparative analyses between the Blattabacterium cuenoti genome and that of previously sequenced endosymbionts, namely those from the omnivorous hosts the Blattella germanica (Blattelidae) and Periplaneta americana (Blattidae), and the endosymbionts harbored by two wood-feeding hosts, the subsocial cockroach Cryptocercus punctulatus (Cryptocercidae) and the termite Mastotermes darwiniensis (Termitidae). Our study shows a remarkable evolutionary stasis of this symbiotic system throughout the evolutionary history of cockroaches and the deepest branching termite M. darwiniensis, in terms of not only chromosome architecture but also gene content, as revealed by the striking conservation of the Blattabacterium core genome. Importantly, the architecture of central metabolic network inferred from the endosymbiont genomes was established very early in Blattabacterium evolutionary history and could be an outcome of the essential role played by this endosymbiont in the host’s nitrogen economy.

Uric acid stored in the fat body of cockroaches is a nitrogen reservoir mobilized in times of scarcity. The discovery of urease inBlattabacterium cuenoti, the primary endosymbiont of cockroaches, suggests that the endosymbiont may participate in cockroach nitrogen economy. However, bacterial urease may only be one piece in the entire nitrogen recycling process from insect uric acid. Thus, in addition to the uricolytic pathway to urea, there must be glutamine synthetase assimilating the released ammonia by the urease reaction to enable the stored nitrogen to be metabolically usable. None of theBlattabacteriumgenomes sequenced to date possess genes encoding for those enzymes. To test the host's contribution to the process, we have sequenced and analysedBlattella germanicatranscriptomes from the fat body. We identified transcripts corresponding to all genes necessary for the synthesis of uric acid and its catabolism to urea, as well as for the synthesis of glutamine, asparagine, proline and glycine, i.e. the amino acids required by the endosymbiont. We also explored the changes in gene expression with different dietary protein levels. It appears that the ability to use uric acid as a nitrogen reservoir emerged in cockroaches after its age-old symbiotic association with bacteria.

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish.