Cell-Free Culture Supernatant of Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 Reduces the Pathogenicity of NetB-Positive Clostridium perfringens in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77-85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity.

Development of the Ileal Microbiota in Three Broiler Breeds.

The development and succession of the microbiota in ileal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 3 and 42 days post hatch (d.p.h). Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 d.p.h, chicks were kept in three circular brooder pens ensuring a mixture of breeds in each brooder. From 22 d.p.h, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of ileal mucus and luminal contents taken from five birds of each breed at 3, 7, 14, 21, 28, and 42 d.p.h. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The initial microbiota in the ileum varied between breeds. The common features were a low diversity and general dominance by one or two taxa such as Enterococcus or Escherichia with relatively low numbers of Lactobacillus. Escherichia became the most abundant genus in samples where Enterococcus was previously the dominant taxa. The next phase of development was marked by an increase in the abundance of Candidatus Arthromitus in the mucus and Lactobacillus in the lumen. The high abundance of Candidatus Arthromitus persisted between 7 and 14 d.p.h after which Lactobacillus became the most abundant genus in both the mucus and lumen. Dominance of the ileal microbiota by Lactobacillus was a transient feature. By 42 d.p.h, the relative abundance of Lactobacillus had fallen while a range of other taxa including Escherichia, Turicibacter, and members of Clostridiales increased. This general pattern was followed by all breeds, however, the rate at which succession occurred differed as Ross matured quicker than Cobb with Hubbard as an intermediate.

Topical Application of Adult Cecal Contents to Eggs Transplants Spore-Forming Microbiota but Not Other Members of the Microbiota to Chicks.

The intestinal microbiota plays an essential role in the metabolism and immune competence of chickens from the first day after hatching. In modern production systems, chicks are isolated from adult chickens, instead hatching in a clean environment. As a result, chicks are colonized by environmental bacteria, including potential pathogens. There is a need to investigate methods by which chicks can be exposed to a more appropriate microbial community at hatching. Such methods must be easy to apply in a hatchery and produce consistent results. The development of the intestinal microbiota of chicks hatched from eggs sprayed with dilute adult cecal content during incubation was observed at 0, 3, 7, and 14 days posthatching (dph) across two experiments. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. A topical treatment of dilute adult cecal content was sufficient to transplant spore-forming bacteria such as Lachnospiraceae and Ruminococcaceae However, this treatment was not able to transplant other taxa that are considered to be core elements of the chicken cecal microbiota, such as Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae, and Burkholderiaceae The topical treatment significantly altered the microbiota of chicks immediately posthatching and accelerated the normal development of the microbiota with earlier colonization by Ruminococcaceae in the cecum and "Candidatus Arthromitus" in the ileum. The effect of the treatment on the cecal microbiota was maximal at 3 dph but diminished over time.IMPORTANCE Over the last 60 years poultry production has intensified in response to increased demand for meat. In modern systems, chicks hatch without contacting chickens and their gut bacteria. Consequently, they are colonized by environmental bacteria that may cause disease. The normal bacteria that live in the gut, or intestinal microbiota, play an important role in the development of the immune system. Therefore, it is essential to find easy ways to expose chicks to the more appropriate bacteria at hatching. This experiment investigated whether spraying eggs with adult cecal contents was sufficient to transfer an adult microbiota to chicks. Our findings show that spore-forming bacteria were transplanted, but other members of the microbiota were not. In this respect, the spray application was partially successful, but the timing of the spray needs to be modified to ensure that more bacteria are transferred.

Development of the Caecal Microbiota in Three Broiler Breeds.

The development of the caecal microbiota plays a role in the metabolism and immune competence of chickens. A detailed understanding of normal succession in the caecal microbiota can inform the use of probiotics and other interventions to optimize the caecal microbiota. The development of the microbiota in caecal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 0 and 42 days post hatch. Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 days post hatch, chicks were kept in brooder pens ensuring a mixture of breeds in each brooder. From 22 days post hatch, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of caecal mucus and luminal contents from five birds of each breed at 0, 3, 7, 14, 21, 28, and 42 days post hatch. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The early caecal microbiota was characterized by poor diversity and dominance by one or two bacterial species. Early colonizers of the caecum included Bifidobacteriaceae, Lachnospiraceae, Bacteroidaceae and Burkholderiaceae with some amplicon sequence variants (ASVs) assigned to Ruminococcaceae. Later colonizers of the caecal microbiota were most apparent from 14 d.p.h and included Ruminococcaceae, Clostridiales vadin BB60 group, Christensenellaceae and Bacillaceae. The caecal microbiota continued to change until 42 d.p.h when the microbiota was characterized by a high abundance of Bacteroidaceae, Lachnospiraceae and Ruminococcaceae. The lumen microbiota was significantly different to the mucus with some ASVs assigned to Lachnospiraceae, Ruminococcaceae, Christensenellaceae and Bacillaceae showing increased abundance in the mucus. ASVs assigned to Bacteroidaceae, Lactobacillaceae and Burkholderiaceae showed a preference for the lumen. Analysis of five caecal mucus samples from each breed at 42 days post hatch showed differences in microbiota composition between Ross and Cobb as well as between Ross and Hubbard. Since performance data was not collected no functional inferences as to the significance of this finding can be made.

In vitro inhibition of avian pathogenic Enterococcus cecorum isolates by probiotic Bacillus strains.

Enterococcus cecorum is a commensal bacteria and opportunistic pathogen that can cause outbreaks of Enterococcal spondylitis ("kinky back") in poultry, with a growing concern worldwide. Numerous Bacillus-based probiotic strains are commercially available with proven effects in supporting gut health and growth performance, but efficacy against pathogenic E. cecorum is unknown. This study compared the in vitro inhibitory potential of cell-free supernatants (CFSs) of 18 Bacillus strains (14 commercial probiotic strains, 1 internal negative control and 3 type strains) on the growth of 9 clinical E. cecorum isolates. Standardized biomass cultures of live Bacillus were harvested and filtered to obtain CFSs. Inhibitory potential against E. cecorum isolates was assessed via a microdilution assay in which the final pathogen concentration was ∼ 104 CFU/mL. Absorbance (OD) was measured every 15 min for 15 h and used to calculate percentage growth inhibition at an OD equivalent to 0.4 in the positive control (PC) (pathogen but no CFS), and growth delay vs. PC. Growth kinetic responses of pathogen isolate-Bacillus strain combinations ranged from total pathogen inhibition to partial inhibition, lag in growth, no effect, or increased growth vs. PC. Percentage inhibition of individual isolates varied markedly among Bacillus strains, from 100% to -100% (growth promotion as recorded for the type strain) (B. amyloliquefaciens DSM7T). Five B. amyloliquefaciens CFSs produced higher average inhibition rates (>75%) than 2 out of 3 Bacillus licheniformis CFSs (-2.5, and -8.39% vs. PC, respectively) and 1 out of 2 Bacillus subtilis CFSs (7.3% vs. PC) (P < 0.05). Commercial strain 3AP4 exhibited the highest average percentage inhibition vs. PC (85.0% ± 7.9) and the most consistent inhibitory effect across pathogen isolates. The findings indicate that some commercially available poultry probiotic Bacillus strains are effective at inhibiting pathogenic E. cecorum in vitro, but effects are highly strain and pathogen isolate-dependent. Further work is required to confirm effects in vivo and isolate the inhibitory substances.

Rough and smooth morphotypes isolated from Lactobacillus farciminis CNCM I-3699 are two closely-related variants.

This study focused on a pleomorphic strain Lactobacillus farciminis CNCM I-3699 known as probiotic for animal applications. On plating, this strain was characterized by the presence of rough and smooth morphotypes depending on experimental conditions. Dominant smooth (S) form, bright white, having smooth edges with moist, ropy, and creamy along with rough (R) form, pale white, having irregular edges and a dry and granular aspect were always obtained from the parent strain under aerobic culture conditions. In anaerobic conditions, only S form growth was observed. Biochemical dosage of capsular exopolysaccharides showed a significant difference between S and R forms (p<0.01), in agreement with a ropy or non ropy phenotype for the S or R form, respectively. These differences were confirmed by transmission electronic microscopy. The auto-aggregation profile revealed major differences in cultural behaviors. The R morphotype presented a highly auto-aggregative ability contrary to the S morphotype. However, biochemical and molecular analyses revealed that R and S morphotypes: 1) shared the same sugar fermentation pattern; 2) belonged to L. farciminis species using 16S rDNA sequencing; 3) had identical PFGE patterns using NotI and ApaI endonucleases; and 4) had identical CRISPR loci but different from those of other L. farciminis strains. Furthermore, the novelty and uniqueness of CRISPR spacer sequences in CNCM I-3699 provides a genetic support for the development of a molecular tracking tool for CNCM I-3699 and its variants. In conclusion, L. farciminis CNCM I-3699 is a pleomorphic strain giving reproducibly rise to two phenotypically distinct morphotypes R and S. This phenomenon may explain survival and growth abilities in in vitro fluctuating aerobic-anaerobic conditions along with modulation of exopolysaccharide synthesis and autoaggregation profile.

Genome Sequence of Lactobacillus plantarum Strain UCMA 3037.

Nucleic acid of the strain Lactobacillus plantarum UCMA 3037, isolated from raw milk camembert cheese in our laboratory, was sequenced. We present its draft genome sequence with the aim of studying its functional properties and relationship to the cheese ecosystem.

In vitro characterization of aggregation and adhesion properties of viable and heat-killed forms of two probiotic Lactobacillus strains and interaction with foodborne zoonotic bacteria, especially Campylobacter jejuni.

Bacterial aggregation and/or adhesion are key factors for colonization of the digestive ecosystem and the ability of probiotic strains to exclude pathogens. In the present study, two probiotic strains, Lactobacillus rhamnosus CNCM-I-3698 and Lactobacillus farciminis CNCM-I-3699, were evaluated as viable or heat-killed forms and compared with probiotic reference Lactobacillus strains (Lb. rhamnosus GG and Lb. farciminis CIP 103136). The autoaggregation potential of both forms was higher than that of reference strains and twice that of pathogenic strains. The coaggregation potential of these two beneficial micro-organisms was evaluated against several pathogenic agents that threaten the global safety of the feed/food chain: Escherichia coli, Salmonella spp., Campylobacter spp. and Listeria monocytogenes. The strongest coaggregative interactions were demonstrated with Campylobacter spp. by a coaggregation test, confirmed by electron microscopic examination for the two forms. Viable forms were investigated for the nature of the bacterial cell-surface molecules involved, by sugar reversal tests and chemical and enzymic pretreatments. The results suggest that the coaggregation between both probiotic strains and C. jejuni CIP 70.2(T) is mediated by a carbohydrate-lectin interaction. The autoaggregation potential of the two probiotics decreased upon exposure to proteinase, SDS or LiCl, showing that proteinaceous components on the surface of the two lactobacilli play an important role in this interaction. Adhesion abilities of both Lactobacillus strains were also demonstrated at significant levels on Caco-2 cells, mucin and extracellular matrix material. Both viable and heat-killed forms of the two probiotic lactobacilli inhibited the attachment of C. jejuni CIP 70.2(T) to mucin. In conclusion, in vitro assays showed that Lb. rhamnosus CNCM-I-3698 and Lb. farciminis CNCM-I-3699, as viable or heat-killed forms, are adherent to different intestinal matrix models and are highly aggregative in vitro with pathogens, especially Campylobacter spp., the most commonly reported zoonotic agent in the European Union. This study supports the need for further in vivo investigations to demonstrate the potential food safety benefits of Lb. rhamnosus CNCM-I-3698 and Lb. farciminis CNCM-I-3699, live or heat-killed, in the global feed/food chain.

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli.

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.

Safety assessment of dairy microorganisms: the Lactobacillus genus.

Lactobacilli are Gram positive rods belonging to the Lactic Acid Bacteria (LAB) group. Their phenotypic traits, such as each species' obligate/facultative, homo/heterofermentation abilities play a crucial role in souring raw milk and in the production of fermented dairy products such as cheese, yoghurt and fermented milk (including probiotics). An up to date safety analysis of these lactobacilli is needed to ensure consumer safety. Lactobacillus genus is a heterogeneous microbial group containing some 135 species and 27 subspecies, whose classification is constantly being reshuffled. With the recent use of advanced molecular methods it has been suggested that the extreme diversity of the Lactobacillus genomes would justify recognition of new subgeneric divisions. A combination of genotypic and phenotypic tests, for example DNA-based techniques and conventional carbohydrate tests, is required to determine species. Pulsed-Field gel Electrophoresis (PFGE) has been successfully applied to strains of dairy origin and is the most discriminatory and reproducible method for differentiating Lactobacillus strains. The bibliographical data support the hypothesis that the ingestion of Lactobacillus is not at all hazardous since lactobacillemia induced by food, particularly fermented dairy products, is extremely rare and only occurs in predisposed patients. Some metabolic features such as the possible production of biogenic amines in fermented products could generate undesirable adverse effects. A minority of starter and adjunct cultures and probiotic Lactobacillus strains may exceptionally show transferable antibiotic resistance. However, this may be underestimated as transferability studies are not systematic. We consider that transferable antibiotic resistance is the only relevant cause for caution and justifies performing antibiotic-susceptibility assays as these strains have the potential to serve as hosts of antibiotic-resistance genes, with the risk of transferring these genes to other bacteria. However, as a general rule, lactobacilli have a high natural resistance to many antibiotics, especially vancomycin, that is not transferable. Safety assessment requirements for Lactobacillus strains of technological interest should be limited to an antibiotic profile and a study to determine whether any antibiotic resistance(s) of medical interest detected is (or are) transferable. This agrees with the recent EFSA proposal suggesting attribution of a QPS status for 32 selected species of lactobacilli.

Beneficial lactobacilli in food and feed: long-term use, biodiversity and proposals for specific and realistic safety assessments.

Lactobacilli have played a crucial role in the production of fermented products for millennia. Their probiotic effects have recently been studied and used in new products. Isolated cases of lactobacillemia have been reported in at-risk populations, but lactobacilli present an essentially negligible biological risk. We analyzed the current European guidelines for safety assessment in food/feed and conclude that they are not relevant for the Lactobacillus genus. We propose new specific guidelines, beginning by granting a 'long-standing presumption of safety' status to Lactobacillus genus based on its long history of safe use. Then, based on the available body of knowledge and intended use, only such tests as are useful will be necessary before attributing 'qualified presumption of safety' status.

Safety and efficacy of probiotic lactobacilli in promoting growth in post-weaning Swiss mice.

Lactobacillus rhamnosus MA27/6B and L. acidophilus MA27/6R are strains used in feed as probiotics. Their safety profiles and growth-stimulating properties were investigated via in vivo studies on young Swiss mice. After repeated administrations of different probiotic preparations in drinking water, safety parameters determined from liver, spleen and total weight remained unchanged. The growth-stimulating properties of viable or dead lactobacilli were studied after supplementation in drinking water. The feed intake (FI), water intake and body weight gain (WG) of the animals were compared to those of control mice. The lactobacilli supplementation of a sub-optimal diet made of barley allow recording of measurable growth performance of mice. It significantly increased WG compared to control groups (P < 0.01), by +28.9% and +31.7% for L. rhamnosus MA27/6B and L. acidophilus MA27/6R, respectively. This WG was correlated with a decrease in the consumption index. The effect of the dose ingested was also investigated: 10(8) lactobacilli CFU/mouse/day produced greater WG than 10(2), 10(4) or 10(6) CFU/mouse/day. No significant differences in growth performance parameters were observed between mice fed with 10(8) cells of viable or nonviable preparations. The mouse assay described could be used as a preliminary criterion when screening candidate probiotics for growth performance properties.