Maria Cristina Ossiprandi

Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low μg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or non-clostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.

Insights into the biodiversity of the gut microbiota of broiler chickens

The gastrointestinal tract of poultry is densely populated with microorganisms, which are presumed to interact with the host and ingested feed. Comparison of the gut microbiota of chickens used for large-scale commercial production (Broiler Chicken, BC) and those grown in semi-wild conditions (Free-Range Chicken, FRC) revealed that at phylum level Firmicutes was the dominant phylum of the gut community in BC, while the gut microbiota of FRC contained higher levels of Bacteroidetes and Proteobacteria. Such differences may be due to the diet and/or the intensive use of antibiotics in BC. Indeed, analysis of the resistome of the cecal microbiomes showed a marked richness in BC datasets, with a modulation of the cecal microbiota toward antibiotic resistant bacteria. Functional characterization of the microbiome of FRC samples revealed an increase in gene pathways involved in degradation of complex carbohydrates. Furthermore, in silico analyses of the microbiomes of FRC and BC revealed a higher presence in genes involved in formate production in BC samples. Notably, compared to the BC microbiomes the FRC microbiomes were shown to contain a higher abundance of genes involved in the pathway for acetate production.

Prophages of the genus Bifidobacterium as modulating agents of the infant gut microbiota

Phage predation is one of the key forces that shape genetic diversity in bacterial genomes. Phages are also believed to act as modulators of the microbiota composition and, consequently, as agents that drive bacterial speciation in complex bacterial communities. Very little is known about the occurrence and genetic variability of (pro)phages within the Bifidobacterium genus, a dominant bacterial group of the human infant microbiota. Here, we performed cataloguing of the predicted prophage sequences from the genomes of all currently recognized bifidobacterial type strains. We analysed their genetic diversity and deduced their evolutionary development, thereby highlighting an intriguing origin. Furthermore, we assessed infant gut microbiomes for the presence of (pro)phage sequences and found compelling evidence that these viral elements influence the composition of bifidobacterial communities in the infant gut microbiota.

Unveiling bifidobacterial biogeography across the mammalian branch of the tree of life

Internally transcribed spacer (ITS) rRNA profiling is a novel tool for detailed analysis of microbial populations at low taxonomic ranks. Here we exploited this approach to explore species-level biogeography of the Bifidobacterium genus across 291 adult mammals. These include humans and 13 other primates, domesticated animals, such as dogs, cats, cows, sheep, goats, horses and pigs, and 46 additional species. The collected profiles revealed the presence of 89 putative novel bifidobacterial taxa in addition to 45 previously described species. Remarkably, in contrast to what is currently known for many gut commensals, we did not observe host-specialization among bifidobacterial species but rather their widespread distribution across mammals. Moreover, ITS rRNA profiling of wild relatives of domesticated dogs, rabbits and pigs clearly indicates that domestication and close contact with humans have impacted on the composition of the fecal bifidobacterial population. These data were complemented by analysis of bifidobacterial communities in milk of eight mammalian families, showing that bifidobacteria represent prototypical early gut microbiota members which are inherited by newborns from their lactating mother. Thus this study highlights the role of bifidobacteria as pioneering gut colonizers of a wide range of mammals.

Preliminary molecular analysis of Clostridium difficile isolates from healthy horses in northern Italy

Clostridium difficile, associated with a wide spectrum of diseases in humans, as well as in several animal species, is an important cause of colitis in adult horses and foals. The aim of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. difficile strains isolated from 42 faeces of healthy horses. Both toxin genes, tcdA and tcdB, were present in only 1 isolate (7.1%). Six isolates (42.9%) demonstrated tcdA-/tcdB+ genotype, and seven isolates (50.0%) were tcdA-/tcdB-. All strains were binary toxin genes negative (cdtA-/cdtB-). The PCR-positive strains, except for the tcdA+/tcdB+ isolate, tested negative for, in vitro, A and/or B toxins production by EIA. Eleven distinct ribotypes were observed. In conclusion, C. difficile can be present in the normal intestinal flora of healthy adult horses, in addition to foals. These animals could therefore play an important role as potential reservoirs of toxigenic strains.

Exploring Amino Acid Auxotrophy in Bifidobacterium bifidum PRL2010

The acquisition and assimilation strategies followed by members of the infant gut microbiota to retrieve nitrogen from the gut lumen are still largely unknown. In particular, no information on these metabolic processes is available regarding bifidobacteria, which are among the first microbial colonizers of the human intestine. Here, evaluation of amino acid auxotrophy and prototrophy of Bifidobacterium bifidum, with particular emphasis on B. bifidum strain PRL2010 (LMG S-28692), revealed a putative auxotrophy for cysteine. In addition, we hypothesized that cysteine plays a role in the oxidative stress response in B. bifidum. The use of glutathione as an alternative reduced sulfur compound did not alleviate cysteine auxotrophy of this strain, though it was shown to stimulate expression of the genes involved in cysteine biosynthesis, reminiscent of oxidative stress response. When PRL2010 was grown on a medium containing complex substrates, such as whey proteins or casein hydrolysate, we noticed a distinct growth-promoting effect of these compounds. Transcriptional analysis involving B. bifidum PRL2010 cultivated on whey proteins or casein hydrolysate revealed that the biosynthetic pathways for cysteine and methionine are modulated by the presence of casein hydrolysate. Such findings support the notion that certain complex substrates may act as potential prebiotics for bifidobacteria in their ecological niche.

Bifidobacterium vansinderenii sp. nov., isolated from faeces of emperor tamarin (Saguinus imperator)

A novel Bifidobacterium strain, Tam10BT, i.e. LMG 30126T, was isolated from emperor tamarin (Saguinus imperator). Cells were Gram-positive, non-motile, non-sporulating, non-haemolytic, facultative anaerobic and fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA genes as well as multilocus sequences (representing hsp60, rpoB, dnaJ, dnaG and clpC genes) and the core genome revealed that Bifidobacterium Tam10BT exhibited close phylogenetic relatedness to Bifidobacterium tissieri DSM 100201T. Comparative analysis of 16S rRNA gene sequences confirmed the phylogenetic results showing the highest gene sequence identity with strain B. tissieri DSM 100201T (96.5 %). Furthermore, genotyping based on the genome sequence of Tam 10B, in combination with phenotypic analyses, clearly showed that strain Tam10BT is distinct from each of the type strains of the so far recognized Bifidobacterium species. The type strain Tam10BT (=LMG 30126T=CCUG 70655T) represents a novel species, for which the name Bifidobacteriumvansinderenii sp. nov is proposed.

Next generation sequencing-based multigene panel for high throughput detection of food-borne pathogens

Contamination of food by chemicals or pathogenic bacteria may cause particular illnesses that are linked to food consumption, commonly referred to as foodborne diseases. Bacteria are present in/on various foods products, such as fruits, vegetables and ready-to-eat products. Bacteria that cause foodborne diseases are known as foodborne pathogens (FBPs). Accurate detection methods that are able to reveal the presence of FBPs in food matrices are in constant demand, in order to ensure safe foods with a minimal risk of causing foodborne diseases. Here, a multiplex PCR-based Illumina sequencing method for FBP detection in food matrices was developed. Starting from 25 bacterial targets and 49 selected PCR primer pairs, a primer collection called foodborne pathogen - panel (FPP) consisting of 12 oligonucleotide pairs was developed. The FPP allows a more rapid and reliable identification of FBPs compared to classical cultivation methods. Furthermore, FPP permits sensitive and specific FBP detection in about two days from food sample acquisition to bioinformatics-based identification. The FPP is able to simultaneously identify eight different bacterial pathogens, i.e. Listeria monocytogenes, Campylobacter jejuni, Campylobacter coli, Salmonella enterica subsp. enterica serovar enteritidis, Escherichia coli, Shigella sonnei, Staphylococcus aureus and Yersinia enterocolitica, in a given food matrix at a threshold contamination level of 101cell/g. Moreover, this novel detection method may represent an alternative and/or a complementary approach to PCR-based techniques, which are routinely used for FBP detection, and could be implemented in (parts of) the food chain as a quality check.

Phylogenetic classification of six novel species belonging to the genus Bifidobacterium comprising Bifidobacterium anseris sp. nov., Bifidobacterium criceti sp. nov., Bifidobacterium imperatoris sp. nov., Bifidobacterium italicum sp. nov., Bifidobacterium margollesii sp. nov. and Bifidobacterium parmae sp. nov.

Six Bifidobacterium strains, i.e., Goo31D, Ham19E, Rab10A, Tam1G, Uis4E and Uis1B, were isolated from domestic goose (Anser domesticus), European hamster (Cricetus cricetus), European rabbit (Oryctolagus cuniculus), emperor tamarin (Saguinus imperator) and pygmy marmoset (Callithrix pygmaea). Cells are Gram-positive, non-motile, non-sporulating, facultative anaerobic and fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA, ITS-, multilocus- sequences and the core genome revealed that bifidobacterial strains Goo31D, Ham19E, Rab10A, Tam1G, Uis4E and Uis1B exhibit close phylogenetic relatedness with Bifidobacterium choerinum LMG 10510, Bifidobacterium hapali DSM 100202, Bifidobacterium saguini DSM 23967 and Bifidobacterium stellenboschense DSM 23968. Genotyping based on the genome sequence of the isolated strains combined with phenotypic analyses, clearly show that these strains are distinct from each of the type strains of the so far recognized Bifidobacterium species. Thus, Bifidobacterium anseris sp. nov. (Goo31D=LMG 30189T=CCUG 70960T), Bifidobacterium criceti sp. nov. (Ham19E=LMG 30188T=CCUG 70962T), Bifidobacterium imperatoris sp. nov. (Tam1G=LMG 30297T=CCUG 70961T), Bifidobacterium italicum sp. nov. (Rab10A=LMG 30187T=CCUG 70963T), Bifidobacterium margollesii sp. nov. (Uis1B=LMG 30296T=CCUG 70959T) and Bifidobacterium parmae sp. nov. (Uis4E=LMG 30295T=CCUG 70964T) are proposed as novel Bifidobacterium species.

Natural zeolite (chabazite/phillipsite) dietary supplementation influences faecal microbiota and oxidant status of working dogs

Abstract We evaluated whether chabazite/phillipsite dietary supplementation might affect the faecal microbiota, oxidant and antioxidant status of working dogs at rest undergone to a trial test. Forty English Setter dogs were involved in two replicate trials. At each replicate, dogs were divided into two homogeneous groups (10 dogs/group). During a period of 28 days, diet was supplemented (Z group) or not supplemented (C group) with chabazite/phillipsite at the dose of 5 g/head/day. On day 29, dogs were subjected to a trial test. Faecal characteristics were assessed at 0 and 29 days (within two hours from the end of the trial test). Faecal consistency was not affected by dietary supplementation (p > .05). On day 29, Lactobacillus spp. and Enterococcus spp. counts were higher and Enterobacteriaceae were lower in Z than in C group (p < .05). At day 0 and at day 29, immediately before and within one hour from the end of the trial test, blood samples were recorded to evaluate the oxidant and antioxidant status. A reduction of 40% in thiobarbituric acid reactive substances (TBARS) levels was observed in Z compared to the C group (p < .05). Differences in nitric oxide and antioxidant enzymatic system levels were not significant (p > .05). Our results suggest that chabazite/phillipsite dietary supplementation, improves the intestinal microbiota ecosystem and may counteract the oxidative damage caused by physical stress in hunting dogs at the beginning of the working season.