The Probiotic Strain H. alvei HA4597® Improves Weight Loss in Overweight Subjects under Moderate Hypocaloric Diet: A Proof-of-Concept, Multicenter Randomized, Double-Blind Placebo-Controlled Study.

Background: Increasing evidence supports the role of the gut microbiota in the control of body weight and feeding behavior. Moreover, recent studies have reported that the probiotic strain Hafnia alvei HA4597® (HA), which produces the satietogenic peptide ClpB mimicking the effect of alpha-MSH, reduced weight gain and adiposity in rodent models of obesity. Methods: To investigate the clinical efficacy of HA, 236 overweight subjects were included, after written informed consent, in a 12-week prospective, double-blind, randomized study. All subjects received standardized counselling for a -20% hypocaloric diet and were asked to maintain their usual physical activity. Subjects of the HA group received two capsules per day providing 100 billion bacteria per day and subjects in the Placebo (P) group received two placebo capsules. The primary endpoint was the percentage of subjects achieving a weight loss of at least 3% after 12 weeks. Intention-to-treat statistical analysis was performed using exact-Fischer, Mann-Whitney and paired-Wilcoxon tests as appropriate. Results: In the HA group, significantly more subjects (+33%) met the primary endpoint than in the P group (54.9 vs. 41.4%, p = 0.048). In the HA group, an increased feeling of fullness (p = 0.009) and a greater loss of hip circumference (p < 0.001) at 12 weeks were also observed. Fasting glycemia at 12 weeks was significantly lower (p < 0.05) in the HA compared to P group. Clinical and biological tolerance was good in both groups. Conclusions: A 12-week treatment with the probiotic strain H. alvei HA4597® significantly improves weight loss, feeling of fullness and reduction of hip circumference in overweight subjects following moderate hypocaloric diet. These data support the use of H. alvei HA4597® in the global management of excess weight.

The effect of protective cultures on Staphylococcus aureus growth and enterotoxin production.

Staphylococcus aureus is the causative agent of staphylococcal food poisoning and is a common contaminant in milk. Despite efforts to control S. aureus, recalls and outbreaks continue to occur, highlighting the need for additional interventions. This study determined the potential for protective cultures (PC) that are commercially available to producers to control S. aureus growth in raw milk and attenuate virulence by impeding staphylococcal enterotoxin (SE) production in raw milk and laboratory medium. Cultures of Hafnia alvei and Lactococcus lactis effectively inhibited S. aureus growth in raw milk to counts ~5 log CFU/mL lower than control when cocultured following a cheesemaking time and temperature profile; two cultures of Lactobacillus plantarum inhibited growth to ~1.5 log CFU/mL less than control. Cocultures of S. aureus with Lc. lactis, H. alvei and Lb. plantarum in raw milk reduced SE levels by 24.9%, 62.4%, and 76%, respectively. Lc. lactis also decreased SE production in raw milk in the absence of PC-mediated growth inhibition. Significant reductions in SE production in the absence of pathogen growth inhibition were also achieved in laboratory medium. Together, these results demonstrate the potential for PCs to inhibit S. aureus growth and impede SE production in the absence of growth inhibition.

Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during digestion.

This study evaluated the ability of dairy matrices, different in composition (with and without fat) and structure (liquid and gel), to enhance microorganisms survival through digestion. The viability of three dairy microorganisms Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei was measured during in vitro and in vivo digestion. S. thermophilus was highly sensitive to gastric stress, and was not found in the duodenal compartment. B. auranticum was moderately sensitive to gastric stress but resistant to duodenal stress. H. alvei was highly resistant to both stresses. LIVE/DEAD confocal microscopy's images, probed the effect of low pH on microorganisms survival. However, in vivo analyses (16S rRNA gene metabarcoding) failed to confirm in vitro observations since tested microorganisms were not detected. Despite of the different evolutions during digestion on buffer capacity, lipolysis, and rheological characteristics, we did not observe any protective effect of the dairy matrices on microorganisms survival.

Quorum sensing activity of Hafnia alvei isolated from packed food.

Quorum sensing (QS) is a mechanism adopted by bacteria to regulate expression of genes according to population density. N-acylhomoserine lactones (AHLs) are a type of QS signalling molecules commonly found in Gram-negative bacteria which have been reported to play a role in microbial spoilage of foods and pathogenesis. In this study, we isolated an AHL-producing Hafnia alvei strain (FB1) from spherical fish pastes. Analysis via high resolution triple quadrupole liquid chromatography/mass spectrometry (LC/MS) on extracts from the spent supernatant of H. alvei FB1 revealed the existence of two short chain AHLs: N-(3-oxohexanoyl) homoserine lactone (3-oxo-C6-HSL) and N-(3-oxo- octanoyl) homoserine lactone (3-oxo-C8-HSL). To our knowledge, this is the first report of the production of AHLs, especially 3-oxo-C8-HSL, by H. alvei.

Recovery of Hafnia alvei from diseased brown trout, Salmo trutta L., and healthy noble crayfish, Astacus astacus (L.), in Bulgaria.

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.

Behavior of Escherichia coli O26:H11 in the presence of Hafnia alvei in a model cheese ecosystem.

This study was designed to evaluate the capacity of three Hafnia strains to inhibit the growth of an E. coli strain O26:H11 in an uncooked pressed model cheese, in the presence or absence of a microbial consortium added to mimic a cheese microbial community. Inoculated at 2 log CFU/ml into pasteurized milk without Hafnia, the E. coli O26:H11 strain reached 5 log CFU/g during cheese-making and survived at levels of 4 to 5 log CFU/g beyond 40 days. Inoculated into milk at 6 log CFU/ml, all three tested Hafnia strains (H. alvei B16 and HA, H. paralvei 920) reached values close to 8 log CFU/g and reduced E. coli O26:H11 counts in cheese on day 1 by 0.8 to 1.4 log CFU/g compared to cheeses inoculated with E. coli O26:H11 and the microbial consortium only. The Hafnia strains slightly reduced counts of Enterococcus faecalis (~-0.5 log from day 1) and promoted Lactobacillus plantarum growth (+0.2 to 0.5 log from day 8) in cheese. They produced small amounts of putrescine (~1.3 mmol/kg) and cadaverine (~0.9 mmol/kg) in cheese after 28 days, and did not affect levels of volatile aroma compounds. Further work on H. alvei strain B16 showed that E. coli O26:H11, inoculated at 2 log CFU/ml, was inhibited by H. alvei B16 inoculated at 6 log CFU/ml and not at 4.5 log CFU/ml. The inhibition was associated neither with lower pH values in cheese after 6 or 24h, nor with higher concentrations of lactic acid. Enhanced concentrations of acetic acid on day 1 in cheese inoculated with H. alvei B16 (4 to 11 mmol/kg) could not fully explain the reduction in E. coli O26:H11 growth. A synergistic interaction between H. alvei B16 and the microbial consortium, resulting in an additional 0.7-log reduction in E. coli O26:H11 counts, was observed from day 8 in model cheeses made from pasteurized milk. However, E. coli O26:H11 survived better during ripening in model cheeses made from raw milk than in those made from pasteurized milk, but this was not associated with an increase in pH values. In vitro approaches are required to investigate the mechanisms and causative agents of this interaction. H. alvei B16 appears to be a promising strain for reducing E. coli O26:H11 growth in cheese, as part of a multi-hurdle approach.

Inhibition of the early stage of Salmonella enterica serovar Enteritidis biofilm development on stainless steel by cell-free supernatant of a Hafnia alvei culture.

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.

Temperature influences the expression of fimbriae and flagella in Hafnia alvei strains: an immunofluorescence study.

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.

Escherichia albertii and Hafnia alvei are candidate enteric pathogens with divergent effects on intercellular tight junctions.

Attaching-effacing lesion-inducing Escherichia albertii and the related, but non-attaching-effacing organism, Hafnia alvei, are both implicated as enteric pathogens in humans. However, effects of these bacteria on epithelial cells are not well-characterized. Related enteropathogens, including enterohemorrhagic Escherichia coli O157:H7, decrease epithelial barrier function by disrupting intercellular tight junctions in polarized epithelia. Therefore, this study assessed epithelial barrier function and tight junction protein distribution in polarized epithelia following bacterial infections. Polarized epithelial (MDCK-I and T84) cells grown on filter supports were infected apically with E. coli O157:H7, E. albertii, and H. alvei for 16h at 37 degrees C. All strains decreased transepithelial electrical resistance and increased permeability to a dextran probe in a host cell-dependent manner. Immunofluorescence microscopy showed that both E. coli O157:H7 and E. albertii, but not H. alvei, caused a redistribution of the tight junction protein zona occludens-1. In contrast to E. coli O157:H7, E. albertii and H. alvei did not redistribute claudin-1. Western blotting of whole cell protein extracts demonstrated that each bacterium caused differential changes in tight junction protein expression, dependent on the host cell. These findings demonstrate that E. albertii and H. alvei are candidate enteric pathogens that have both strain-specific and host epithelial cell-dependent effects.

Impact on disinfection efficiency of cell load and of planktonic/adherent/detached state: case of Hafnia alvei inactivation by plasma activated water.

This paper describes the effects of initial microbial concentration and planktonic/adherent/detached states on the efficiency of plasma-activated water. This disinfecting solution was obtained by treating distilled water with an atmospheric pressure plasma produced by gliding electric discharges in humid air. The inactivation kinetics of planktonic cells of Hafnia alvei (selected as a bacterial model) were found to be of the first order. They were influenced by the initial microbial concentration. Efficiency decreased when the initial viable population N(0) increased, and the inactivation rate k(max) was linearly modified as a function of Log(10) (N(0)). This relation was used to compare planktonic, adherent, and detached cells independently from the level of population. Bacteria adhering to stainless steel and high-density polyethylene were also sensitive to treatment, but at a lower rate than their free-living counterparts. Moreover, cells detached from these solid substrates exhibited an inactivation rate lower than that of planktonic cells but similar to adherent bacteria. This strongly suggests the induction of a physiological modification to bacteria during the adhesion step, rendering adherent--and further detached--bacteria less susceptible to the treatment, when compared to planktonic bacteria.

Influence of environmental conditions on biofilm formation by Hafnia alvei strains.

Hafnia alvei is considered an opportunistic pathogen of animal and humans, affecting a wide range of homeothermic and poikilothermic hosts with different body temperatures. In this work, H. alvei strains isolated from different sources were studied with regard to their capacity to form biofilms under different environmental conditions. Strain, growth phase, temperature and culture media dependent changes of biofilm formation were semiquantitatively monitored using a microtiter plate method. Our study shows that all strains used could form biofilms in vitro, and that biofilm formation increases dramatically during growth at 25 degrees C but not at 37 degrees C, and decreases at both temperatures in presence of glucose. At 16 degrees C only one strain isolated from a lizard was able to form a dense biofilm showing that the ability to form biofilms in this species is regulated by environmental factors and is also strain specific.

Occurrence of proteolytic activity and N-acyl-homoserine lactone signals in the spoilage of aerobically chill-stored proteinaceous raw foods.

Proteolytic pseudomonads dominate the spoilage flora of aerobically chill-stored proteinaceous raw foods. Proteolysis during spoilage of these food systems affects both food quality and the dynamics of the bacterial community because it increases the availability of nutrients to the community as a whole. Quorum sensing, or cell-cell signaling, is associated closely with ecological interactions among bacteria in mixed communities. The potential role of quorum sensing in proteolytic food spoilage was examined, based on the evaluation of N-acyl-homoserine lactone (AHL) signal molecules. The occurrence of proteolytic activity and AHL signals was studied during spoilage of aerobically chill-stored ground beef, fish, chicken, and raw milk. Pseudomonads dominated the psychrotrophic flora, followed distantly by members of the Enterobacteriaceae. The growth of pseudomonads was correlated with the occurrence of proteolytic activity in all food systems. AHL concentration began increasing significantly only after the onset of proteolytic activity. Widely divergent AHL profiles were revealed by thin-layer chromatography analysis of the different food samples, and these profiles were likely determined by the undefined bacterial flora in these systems and by the characterized pseudomonads and Enterobacteriaceae. Although Hafnia alvei was a major component of the Enterobacteriaceae flora in all foods tested and a strong AHL producer, the signal molecules produced by H. alvei strain EB1 did not influence protease production by Pseudomonas fluorescens strain 395 in vitro. These results do not indicate any clear correlation between the overall detectable AHL signal molecules accumulated in the food samples and proteolytic activity.

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5.

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.