Protein supplementation changes gut microbial diversity and derived metabolites in subjects with type 2 diabetes.

High-protein diets are promoted for individuals with type 2 diabetes (T2D). However, effects of dietary protein interventions on (gut-derived) metabolites in T2D remains understudied. We therefore performed a multi-center, randomized-controlled, isocaloric protein intervention with 151 participants following either 12-week high-protein (HP; 30Energy %, N = 78) vs. low-protein (LP; 10 Energy%, N = 73) diet. Primary objectives were dietary effects on glycemic control which were determined via glycemic excursions, continuous glucose monitors and HbA1c. Secondary objectives were impact of diet on gut microbiota composition and -derived metabolites which were determined by shotgun-metagenomics and mass spectrometry. Analyses were performed using delta changes adjusting for center, baseline, and kidney function when appropriate. This study found that a short-term 12-week isocaloric protein modulation does not affect glycemic parameters or weight in metformin-treated T2D. However, the HP diet slightly worsened kidney function, increased alpha-diversity, and production of potentially harmful microbiota-dependent metabolites, which may affect host metabolism upon prolonged exposure.

The cnf1 gene is associated with an expanding Escherichia coli ST131 H30Rx/C2 subclade and confers a competitive advantage for gut colonization.

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2.

Specificities and Commonalities of Carbapenemase-Producing Escherichia coli Isolated in France from 2012 to 2015.

Carbapenemase-producing Escherichia coli (CP-Ec) represents a major public health threat with a risk of dissemination in the community as has occurred for lineages producing extended-spectrum ß-lactamases. To characterize the extent of CP-Ec spread in France, isolates from screening and infection samples received at the French National Reference Center (F-NRC) laboratory for carbapenemase-producing Enterobacterales were investigated. A total of 691 CP-Ec isolates collected between 2012 and 2015 and 22 isolates collected before 2012 were fully sequenced. Analysis of their genome sequences revealed some disseminating multidrug-resistant (MDR) lineages frequently acquiring diverse carbapenemase genes mainly belonging to clonal complex 23 (CC23) (sequence type 410 [ST410]) and CC10 (ST10 and ST167) and sporadic isolates, including rare ST131 isolates (n = 17). However, the most represented sequence type (ST) was ST38 (n = 92) with four disseminated lineages carrying blaOXA-48-like genes inserted in the chromosome. Globally, the most frequent carbapenemase gene (n = 457) was blaOXA-48. It was also less frequently associated with MDR isolates being the only resistance gene in 119 isolates. Thus, outside the ST38 clades, its acquisition was frequently sporadic with no sign of dissemination, reflecting the circulation of the IncL plasmid pOXA-48 in France and its high frequency of conjugation. In contrast, blaOXA-181 and blaNDM genes were often associated with the evolution of MDR E. coli lineages characterized by mutations in ftsI and ompC. IMPORTANCE Carbapenemase-producing Escherichia coli (CP-Ec) might be difficult to detect, as MICs can be very low. However, their absolute number and their proportion among carbapenem-resistant Enterobacterales have been increasing, as reported by WHO and national surveillance programs. This suggests a still largely uncharacterized community spread of these isolates. Here, we have characterized the diversity and evolution of CP-Ec isolated in France before 2016. We show that carbapenemase genes are associated with a wide variety of E. coli genomic backgrounds and a small number of dominant phylogenetic lineages. In a significant proportion of CP-Ec, the most frequent carbapenemase gene blaOXA-48, was detected in isolates lacking any other resistance gene, reflecting the dissemination of pOXA-48 plasmids, likely in the absence of any antibiotic pressure. In contrast, carbapenemase gene transfer may also occur in multidrug-resistant E. coli, ultimately giving rise to at-risk lineages encoding carbapenemases with a high potential of dissemination.

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection.

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.

Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producing Escherichia coli.

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 ß-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 ß-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131.

Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil.

The demand for sustainable and eco-friendly control methods of pests and insects is increasing worldwide. From this came the interest in Bacillus thuringiensis, an entomopathogenic bacterium capable of replacing chemical pesticides. However, the possibility of pests developing resistance to a particular strain may impair its use, and there is a need to identify novel strains of this species as potential commercial biopesticides. B. thuringiensis sv. israelensis is one of the most successful serovars, widely commercialized for its activity against black fly and mosquito larvae. In this study, we isolated, characterized, and sequenced a new Lebanese B. thuringiensis sv. israelensis isolate, strain AR23. Compared to the commercialized reference strain AM65-52 (Vectobac®, Sumitomo), AR23 showed an increased activity against several mosquito species. The genomic analysis revealed that this strain, compared to AM65-52, possesses a simplified plasmid content and an additional functional cry4Ba coding gene that most likely accounts for the increased effectiveness of this strain in mosquito larvae killing.

SME-4-producing Serratia marcescens from Argentina belonging to clade 2 of the S. marcescens phylogeny.

BACKGROUND: SME carbapenemases are increasingly reported, especially from North and South America. Here, we describe an SME-4-producing Serratia marcescens (SME-Sm) clinical isolate from Argentina and compare its genome with other SME-Sm and Sm isolates recovered from public databases. METHODS: Sm isolates were characterized by WGS using Illumina technology, susceptibility testing and MIC determination. Carbapenemase activity was revealed by biochemical tests based on imipenem hydrolysis. A whole-genome phylogeny was estimated for all the Sm isolates retrieved from public databases with kSNP3 and a whole-genome phylogenetic analysis based on non-recombinant core SNPs was inferred for Sm complete genomes and for those encoding any blaSME variants. RESULTS: Sm163 was resistant to amoxicillin, temocillin, aztreonam and carbapenems, remaining susceptible to extended-spectrum cephalosporins. WGS analysis of Sm163 revealed a genome of 5139329 bp and a chromosomally encoded blaSME-4 carbapenemase gene located on a genomic island closely related to SmarGI1-1 of Sm N11-02820. Comparison of the Sm genomes revealed that the 14 SME-Sm isolates possess this genomic island inserted at the same loci, that 13/14 belong to clade 1 and that 11/14 form a well-defined subcluster of cluster I of Sm clade 1, while Sm163 belongs to clade 2, suggesting that an SME-encoding genomic island may have been transferred between isolates from different clades. CONCLUSIONS: To the best of our knowledge this is the first report of an SME-4-encoding Sm from Argentina. The blaSME-4 gene is located on a SmarGI1-1-like genomic island. The genome of Sm163 belongs to clade 2, unlike all the other SME-Sm isolates, which belong to clade 1.

Evolutionary processes and environmental factors underlying the genetic diversity and lifestyles of Bacillus cereus group bacteria.

Bacteria of the Bacillus cereus group are environmental Gram-positive spore-forming bacteria ubiquitously distributed. Despite the high degree of genetic similarity among the different strains, they show strong phenotypic variability, from mammal or entomopathogen strains to soil-dwelling saprophytes, and from psychrophylic to thermotolerant strains. Most of the phenotypes are linked to the presence of large plasmids that encode for diverse toxins. However, other processes, like mutation or recombination, also participate in shaping the evolution and population structure of these bacteria. Here we review different aspects of the evolution of this group.

The cockroach Blattella germanica obtains nitrogen from uric acid through a metabolic pathway shared with its bacterial endosymbiont.

Uric acid stored in the fat body of cockroaches is a nitrogen reservoir mobilized in times of scarcity. The discovery of urease in Blattabacterium 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 the Blattabacterium genomes sequenced to date possess genes encoding for those enzymes. To test the host's contribution to the process, we have sequenced and analysed Blattella germanica transcriptomes 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.

Comparative genomics of Blattabacterium cuenoti: the frozen legacy of an ancient endosymbiont genome.

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.

Metabolic stasis in an ancient symbiosis: genome-scale metabolic networks from two Blattabacterium cuenoti strains, primary endosymbionts of cockroaches.

BACKGROUND: Cockroaches are terrestrial insects that strikingly eliminate waste nitrogen as ammonia instead of uric acid. Blattabacterium cuenoti (Mercier 1906) strains Bge and Pam are the obligate primary endosymbionts of the cockroaches Blattella germanica and Periplaneta americana, respectively. The genomes of both bacterial endosymbionts have recently been sequenced, making possible a genome-scale constraint-based reconstruction of their metabolic networks. The mathematical expression of a metabolic network and the subsequent quantitative studies of phenotypic features by Flux Balance Analysis (FBA) represent an efficient functional approach to these uncultivable bacteria. RESULTS: We report the metabolic models of Blattabacterium strains Bge (iCG238) and Pam (iCG230), comprising 296 and 289 biochemical reactions, associated with 238 and 230 genes, and 364 and 358 metabolites, respectively. Both models reflect both the striking similarities and the singularities of these microorganisms. FBA was used to analyze the properties, potential and limits of the models, assuming some environmental constraints such as aerobic conditions and the net production of ammonia from these bacterial systems, as has been experimentally observed. In addition, in silico simulations with the iCG238 model have enabled a set of carbon and nitrogen sources to be defined, which would also support a viable phenotype in terms of biomass production in the strain Pam, which lacks the first three steps of the tricarboxylic acid cycle. FBA reveals a metabolic condition that renders these enzymatic steps dispensable, thus offering a possible evolutionary explanation for their elimination. We also confirm, by computational simulations, the fragility of the metabolic networks and their host dependence. CONCLUSIONS: The minimized Blattabacterium metabolic networks are surprisingly similar in strains Bge and Pam, after 140 million years of evolution of these endosymbionts in separate cockroach lineages. FBA performed on the reconstructed networks from the two bacteria helps to refine the functional analysis of the genomes enabling us to postulate how slightly different host metabolic contexts drove their parallel evolution.

Evolutionary convergence and nitrogen metabolism in Blattabacterium strain Bge, primary endosymbiont of the cockroach Blattella germanica.

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.

Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers.

Blattabacteria are intracellular endosymbionts of cockroaches and primitive termites that belong to the class Flavobacteria and live only in specialized cells in the abdominal fat body of their hosts. In the present study we determined genome sizes as well as genome copy numbers for the endosymbionts of three cockroach species, Blattella germanica, Periplaneta americana and Blatta orientalis. The sole presence of blattabacteria in the fat body was demonstrated by rRNA-targeting techniques. The genome sizes of the three blattabacteria were determined by pulsed field gel electrophoresis. The resulting total genome sizes for the three symbionts were all approximately 650 +/- 15 kb. Comparison of the genome sizes with those of free-living Bacteroidetes shows extended reduction, as occurs in other obligatory insect endosymbionts. Genome copy numbers were determined based on cell counts and determination of DNA amounts via quantitative PCR. Values between 10.2 and 18.3 and between 323 and 353 were found for the symbionts of P. americana and B. orientalis respectively. Polyploidy in intracellular bacteria may play a significant role in the genome reduction process.