Lactic acid bacteria reduce bacterial diarrhea in rabbits via enhancing immune function and restoring intestinal microbiota homeostasis.

BACKGROUND: Numerous previous reports have demonstrated the efficacy of Lactic acid bacteria (LAB) in promoting growth and preventing disease in animals. In this study, Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 were isolated from the feces of healthy rabbits, and both strains showed good probiotic properties in vitro. Two strains (108CFU/ml/kg/day) were fed to weaned rabbits for 21 days, after which specific bacterial infection was induced to investigate the effects of the strains on bacterial diarrhea in the rabbits. RESULTS: Our data showed that Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 interventions reduced the incidence of diarrhea and systemic inflammatory response, alleviated intestinal damage and increased antibody levels in animals. In addition, Enterococcus faecium ZJUIDS-R1 restored the flora abundance of Ruminococcaceae1. Ligilactobaciiius animalis ZJUIDS-R2 up-regulated the flora abundance of Adlercreutzia and Candidatus Saccharimonas. Both down-regulated the flora abundance of Shuttleworthia and Barnesiella to restore intestinal flora balance, thereby increasing intestinal short-chain fatty acid content. CONCLUSIONS: These findings suggest that Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 were able to improve intestinal immunity, produce organic acids and regulate the balance of intestinal flora to enhance disease resistance and alleviate diarrhea-related diseases in weanling rabbits.

Control of Salmonella in low-moisture foods: Enterococcus faecium NRRL B-2354 as a surrogate for thermal and non-thermal validation.

Salmonella has been implicated in multiple foodborne outbreaks and recalls associated with low water activity foods (LawF). To verify the effectiveness of a process against Salmonella in LawF, validation using a nonpathogenic surrogate strain is essential. Enterococcus faecium NRRL B-2354 strain has been used as a potential surrogate of Salmonella in different processing of LawF. However, the survival of Salmonella and E. faecium in LawF during food processing is a dynamic function of aw, food composition and structure, processing techniques, and other factors. This review assessed pertinent literature on the thermal and non-thermal inactivation of Salmonella and its presumable surrogate E. faecium in various LawF and provided an overview of its suitibility in different LawF. Overall, based on the D-values, survival/reduction, temperature/time to obtain 4 or 5-log reductions, most studies concluded that E. faecium is a suitable surrogate of Salmonella during LawF processing as its magnitude of resistance was slightly greater or equal (i.e., statistical similar) as compared to Salmonella. Studies also showed its unsuitability which either does not provide a proper margin of safety or being overly resistant and may compromise the quality and organoleptic properties of food. This review provides useful information and guidance for future validation studies of LawF.

Enterococcus faecium PNC01 isolated from the intestinal mucosa of chicken as an alternative for antibiotics to reduce feed conversion rate in broiler chickens.

BACKGROUND: The development and utilization of probiotics had many environmental benefits for replacing antibiotics in animal production. Bacteria in the intestinal mucosa have better adhesion to the host intestinal epithelial cells compared to bacteria in the intestinal contents. In this study, lactic acid bacteria were isolated from the intestinal mucosa of broiler chickens and investigated as the substitution to antibiotic in broiler production. RESULTS: In addition to acid resistance, high temperature resistance, antimicrobial sensitivity tests, and intestinal epithelial cell adhesion, Enterococcus faecium PNC01 (E. faecium PNC01) was showed to be non-cytotoxic to epithelial cells. Draft genome sequence of E. faecium PNC01 predicted that it synthesized bacteriocin to perform probiotic functions and bacteriocin activity assay showed it inhibited Salmonella typhimurium from invading intestinal epithelial cells. Diet supplemented with E. faecium PNC01 increased the ileal villus height and crypt depth in broiler chickens, reduced the relative length of the cecum at day 21, and reduced the relative length of jejunum and ileum at day 42. Diet supplemented with E. faecium PNC01 increased the relative abundance of Firmicutes and Lactobacillus, decreased the relative abundance of Bacteroides in the cecal microbiota. CONCLUSION: E. faecium PNC01 replaced antibiotics to reduce the feed conversion rate. Furthermore, E. faecium PNC01 improved intestinal morphology and altered the composition of microbiota in the cecum to reduce feed conversion rate. Thus, it can be used as an alternative for antibiotics in broiler production to avoid the adverse impact of antibiotics by altering the gut microbiota.

Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms.

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.

Metabolome responses of Enterococcus faecium to acid shock and nitrite stress.

Enterococcus faecium is gaining increasing interest due to its virulence and tolerance to a range of stresses (e.g., acid shock and nitrite stress in human stomach). The chemical taxonomy and basic structural features of cellular metabolite can provide us a deeper understanding of bacterial tolerance at molecular level. Here, we used hierarchical classification and molecular composition analysis to investigate the metabolome responses of E. faecium to acid shock and nitrite stress. Our results showed that considerable high biodegradable compounds (e.g., dipeptides) were produced by E. faecium under acid shock, while nitrite stress induced the accumulations of some low biodegradable compounds (e.g., organoheterocyclic compounds and benzenoids). Complete genome analysis and metabolic pathway profiling suggested that E. faecium produced high biodegradable metabolites responsible for the proton-translocation and biofilm formation, which increase its tolerance to acid shock. Yet, the presence of low biodegradable metabolites due to the nitrite exposure could disturb the bacterial productions of surface proteins, and thus inhibiting biofilm formation. Our approach uncovered the hidden interactions between intracellular metabolites and exogenous stress, and will improve the understanding of host-microbe interactions.

Efficacy of probiotic Enterococcus faecium in combination with diclazuril against coccidiosis in experimentally infected broilers.

AIMS: The study was conducted to investigate the combination of a probiotic strain of Enterococcus faecium and diclazuril to control coccidiosis in broilers. METHODS AND RESULTS: A total of 240 one-day-old female broiler chicks were divided into eight groups (30 chicks per group): prophylactic groups (G1, G2 and G3) and therapeutic groups (G4, G5 and G6) and two control groups (untreated infected, G7 and untreated uninfected, G8 controls). In the prophylactic approach, diclazuril alone (G1), probiotic alone (G2) or a mixture of both probiotic and diclazuril (G3) was orally administered to the chicks via drinking water 10 days prior to the infection. However, in the therapeutic approach, G4, G5 and G6 birds were administered diclazuril alone, probiotic alone and diclazuril+probiotic mix, respectively, in drinking water for five consecutive days after the appearance of clinical signs of coccidiosis. Birds of both approaches and G7 were experimentally infected with 25 × 103 Eimeria-sporulated oocysts. Chicks in G3 showed the highest weight gain, the lowest lesion score, a low oocyst count and mortality rate among the challenged groups. Moderate lesion scores and oocyst counts were observed in chickens administered probiotics prophylactically. In the therapeutic approach, broilers in G6 but not G5 displayed a decreased mortality rate and lesion score in comparison to those in G7 and G8. However, the result of the probiotic-treated group was not significantly different from that in the untreated infected control group. CONCLUSION: The probiotic supplementation as a prophylactic approach can decrease the adverse effects of eimerian infection. In addition, the probiotic and diclazuril mix achieved a considerable improvement in the growth performance. Therefore, probiotic plus diclazuril combination achieved a synergistic effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation into the synergism/antagonism between a probiotic and diclazuril as anticoccidial agent and the difference in the timing of administration.

Administration of Enterococcus faecium HS-08 increases intestinal acetate and induces immunoglobulin A secretion in mice.

A probiotic is considered a live microbial feed supplement that has beneficial effects on the host. In this study, the probiotic property by which Enterococcus faecium HS-08 strengthens the immune system was investigated. Using a murine model, we evaluated the abilities of this strain to increase intestinal short-chain fatty acid contents and to induce the production of mucosal immunoglobulin A (IgA), which are crucial for mucosal immune systems. Various amounts (0%, 0.0038%, 0.038%, or 0.38%) of strain HS-08 cells were administered to BALB/cAJcl mice, which resulted in a dose-dependent increase of fecal IgA levels. A qRT-PCR analysis of Peyer's patch cells revealed that the gene expression of retinal-dehydrogenase, interleukin 6, B-cell-activating factor, and a proliferation-inducing ligand were increased, which leads to IgA secretion via a T-cell-independent mechanism. The administration of 0.038% and 0.38% of strain HS-08 cells also increased fecal acetate levels, which plays an important role for maintaining immune functions. This cecal floral analysis and the stability of strain HS-08 against gastrointestinal digestion suggest that this strain can inhabit the host intestine. In conclusion, the administration of E. faecium HS-08 increased intestinal acetate levels and enhanced IgA secretion, which may result in strengthening of the mucosal immune system.

Effect of microbial inoculation and particle size on fermentation profile, aerobic stability, and ruminal in situ starch degradation of high-moisture corn ensiled for a short period.

Dairy farmers are often challenged with the need to feed high-moisture corn (HMC) after less than 30 d of fermentation. The objective this study was to assess the effects of microbial inoculation and particle size on fermentation profile, aerobic stability, and ruminal in situ starch degradation of HMC ensiled for a short period. High-moisture corn was harvested, coarsely ground (3,798 ± 40 µm, on average) or finely ground (984 ± 42 µm, on average), then ensiled in quadruplicate vacuum pouches untreated (CON) or with the following treatments: Lactobacillus plantarum CH6072 at 5 × 104 cfu/g and Enterococcus faecium CH212 at 5 × 104 cfu/g of fresh forage (LPEF); or Lactobacillus buchneri LB1819 at 7.5 × 104 cfu/g and Lactococcus lactis O224 at 7.5 × 104 cfu/g (LBLL). Silos were allowed to ferment for 14 or 28 d. Ruminal in situ starch degradation increased when HMC was finely ground. In addition, in situ starch degradation was greater and aerobic stability increased approximately 5-fold with LBLL compared with CON and LPEF. An interaction between microbial inoculation and storage length occurred for lactic acid. At 14 d, concentrations of lactic acid were greatest in LPEF and lowest in LBLL. Lactic acid concentrations increased from 14 to 28 d with CON and LPEF, but decreased with LBLL. At 28 d, concentrations of lactic acid were lower in LBLL compared with CON and LPEF. An interaction between particle size, microbial inoculation, and storage length occurred for acetic acid and ammonia-N. At 14 and 28 d, acetic acid concentrations were greatest in finely ground LBLL followed by coarsely ground LBLL. Ammonia-N concentrations increased across all treatments from 0 to 28 d. At 14 and 28 d, concentrations of ammonia-N were greatest in finely ground LBLL and lowest in coarsely ground CON and coarsely ground LPEF. Results from this study suggest that L. buchneri LB1819 can produce acetic acid in as little as 14 d, and that by 28 d, it has the potential to improve the aerobic stability of HMC. Additionally, results indicate that L. buchneri LB1819 has the potential to improve ruminal degradation of starch by 28 d of storage. Finally, results confirm enhanced fermentation and improved ruminal starch degradation with finely ground HMC by 28 d of storage.

Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci.

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

Comparative genomic analysis revealed great plasticity and environmental adaptation of the genomes of Enterococcus faecium.

BACKGROUND: As an important nosocomial pathogen, Enterococcus faecium has received increasing attention in recent years. However, a large number of studies have focused on the hospital-associated isolates and ignored isolates originated from the natural environments. RESULTS: In this study, comparative genomic analysis was conducted on 161 isolates originated from human, animal, and naturally fermented dairy products. The results showed that the environment played an important role in shaping the genomes of Enterococcus faecium. The isolates from human had the largest average genome size, while the isolates from dairy products had the smallest average genome size and fewest antibiotic resistance genes. A phylogenetic tree was reconstructed based on the genomes of these isolates, which revealed new insights into the phylogenetic relationships among the dairy isolates and those from hospitals, communities, and animals. Furthermore, 202 environment-specific genes were identified, including 136 dairy-specific, 31 human blood-specific, and 35 human gastrointestinal-specific genes. Interestingly, five dairy-specific genes (namely lacF, lacA/B, lacD, lacG, and lacC) that constituted an integrated lactose metabolism pathway existed in almost all dairy isolates. The pathway conservation demonstrated an active role of the environment in shaping the genomes of Enterococcus faecium. CONCLUSIONS: This study shows that the Enterococcus faecium species has great genomic plasticity and high versatility to occupy broad ecological roles, dwelling as non-harmful dairy and animal gut commensals as well as significant nosocomial pathogens that disseminate antibiotic resistance genes.

2CS-CHXT Operon Signature of Chlorhexidine Tolerance among Enterococcus faecium Isolates.

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years that has recently acquired higher relevance in nosocomial infection control worldwide. Despite this, CHX tolerance among Enterococcus faecium bacteria, representing one of the leading agents causing nosocomial infections, has been poorly understood. This study provides new phenotypic and molecular data for better identification of CHX-tolerant E. faecium subpopulations in community and clinical contexts. The chlorhexidine MIC (MICCHX) distribution of 106 E. faecium isolates suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of the 2CS-CHXT operon, identified here. It encodes glucose and amino acid-polyamine-organocation family transporters, besides the DNA-binding response regulator ChtR, with a P102H mutation previously described only in CHX-tolerant clade A1 E. faecium, and the ChtS sensor. 2CS-CHXT seems to be associated with three regulons modulating diverse bacterial biological functions. Combined data from normal MIC distribution and 2CS-CHXT operon characterization support a tentative epidemiological cutoff (ECOFF) of 8 mg/liter to CHX, which is useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds calls for the prudent use of CHX in multiple contexts.IMPORTANCE Chlorhexidine is one of the substances included in the World Health Organization's list of essential medicines, which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic in health care (skin, hands, mouthwashes, eye drops) since the 1950s, its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here, we provide a comprehensive study on chlorhexidine tolerance among strains of Enterococcus faecium, one of the leading nosocomial agents worldwide, and identify a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Our data allowed for the proposal of a tentative epidemiological cutoff of 8 mg/liter, which is useful to detect tolerant E. faecium populations in surveillance studies in community and clinical contexts. The prediction of 2CS-CHXT regulons will also facilitate the design of future experimental studies to better uncover chlorhexidine tolerance among E. faecium bacteria.

Anti-adhesion of probiotic Enterococcus faecium WEFA23 against five pathogens and the beneficial effect of its S-layer proteins against Listeria monocytogenes.

Enterococcus faecium WEFA23 is a potential probiotic strain isolated from Chinese infant feces. In this study, the antagonistic activity of E. faecium WEFA23 on adhesion to pathogens was investigated. Enterococcus faecium WEFA23 was able to compete, exclude, and displace the adhesion of Escherichia coli O157:H7, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes CMCC54007, Staphylococcus aureus CMCC26003, and Shigella sonnei ATCC 25931 to Caco-2 cells. Among them, L. monocytogenes achieved the strongest inhibition rate in both competition and displacement assays. Those anti-adhesion capacities were related to the bacterial physicochemical properties (hydrophobicity, auto-aggregation, and co-aggregation) of the bacterial surface. For L. monocytogenes, the anti-adhesion capacity was affected by the heat treatment, cell density, and growth phase of E. faecium WEFA23; 108 colony-forming units of viable cells per millilitre at the stationary phase exhibited the strongest anti-adhesion activity. In addition, removal of S-layer proteins of E. faecium WEFA23 by treatment with 5 mol/L LiCl significantly decreased its adhesion capacity, and those S-layer proteins were able to compete, displace, and exclude L. monocytogenes at different levels. Both cells and S-layer proteins of E. faecium WEFA23 significantly reduced the apoptosis of Caco-2 cells induced by L. monocytogenes, which was mediated by caspase-3 activation. This study might be helpful in understanding the anti-adhesion mechanism of probiotics against pathogens.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing.

Salmonella is capable of surviving in a low moisture environment for long periods. Once adapted to the xeric conditions, the thermal resistance of Salmonella is enhanced. Cocoa powder is a low water activity (aw) food (LawF) that is an essential component in a wide variety of desserts and ready-to-eat (RTE) foods and drinks. The aim of this study was to evaluate the desiccation and thermal resistance of Salmonella in cocoa powder, as well as to examine the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing. Natural unsweetened cocoa powder was inoculated with a 3-strain Salmonella cocktail or E. faecium and was equilibrated to aw 0.30 and 0.45 at room temperature, then subjected to isothermal treatments at 70-80 °C or 12-month storage at RT (room temperature, 22.0 ±â€¯0.5 °C, aw 0.30). At 70 and 80 °C, D-values of both Salmonella and E. faecium increased with decreasing aw. D-values of Salmonella at aw 0.30 cocoa powder were 46.2 ±â€¯4.7, 20.5 ±â€¯1.7, and 11.5 ±â€¯0.9 min at 70, 75 and 80 °C, respectively. Higher heat resistance of E. faecium in aw 0.30 cocoa powder was observed with D-values of 59.9 ±â€¯5.0, 28.9 ±â€¯1.8, and 16.1 ±â€¯1.4 min at 70, 75, and 80 °C, respectively. However, E. faecium demonstrated less heat resistance than Salmonella when aw was increased to 0.45. D-values for Salmonella at aw 0.45 were 31.6-7.0 min at 70-80 °C compared to 25.8-4.7 min for E. faecium. During 12 months of storage at RT, surviving E. faecium population in aw 0.30 cocoa powder was higher than that of the Salmonella cocktail; the population decreased by 1.39 and 3.75 logs, respectively. These findings indicate that the suitability of E. faecium as a surrogate organism for Salmonella is influenced by aw of cocoa powder. The aw correlates with thermal inactivation rates in both Salmonella and E. faecium, and should be considered as a significant contributor to the thermal resistance of Salmonella in cocoa powder.

Radiofrequency pasteurization process for inactivation of Salmonella spp. and Enterococcus faecium NRRL B-2354 on ground black pepper.

Salmonella persistence in ground black pepper has caused several foodborne outbreaks and created public concern about the safety of low water activity (aw) foods. In this study, radiofrequency (RF) processing was evaluated for pasteurization of ground black pepper. Stability and homogeneity tests were done for both Salmonella spp. and E. faecium during moisture equilibration before RF heating to evaluate the inoculation method. Moisture content of samples were conditioned such that the final moisture content after RF heating reached the optimal storage moisture. RF heating was shown to provide more than 5.98 log CFU/g reduction for Salmonella spp. and the reduction of 3.89 log CFU/g for E. faecium with a 130 s of treatment time. The higher thermal resistance of E. faecium indicated its suitability as surrogate for Salmonella spp. during RF heating of ground black pepper. Piperine, total phenolics, volatile compounds, and antioxidant activity were assessed as quality parameters for ground black pepper. The results demonstrated that the RF processing provided effective inactivation of Salmonella spp. with insignificant (p > 0.05) quality deterioration.

Dry inoculation methods for nonfat milk powder.

Carriers with inoculated microorganisms are often used to validate low-moisture food safety interventions. In this study, we evaluated dry inoculation methods using silicon dioxide (SiO2) and a small portion of nonfat milk powder (NFMP) as dry carriers for NFMP. Silicon dioxide was characterized by vapor sorption analysis. One milliliter of inoculum of a 5-strain Salmonella cocktail (serovars Agona, Reading, Tennessee, Montevideo, and Mbandaka) or Enterococcus faecium NRRL B-2354 was inoculated onto 1 g of SiO2 or 10 g of NFMP as carriers. Both inoculated carriers were air-dried for 72 h [22°C, relative humidity (RH) ∼30%], equilibrated to water activity (aw) 0.25 ± 0.02 (24 h at 22°C, RH 25%), and mixed with preconditioned NFMP (aw = 0.25 ± 0.02) to reach an inoculation level of 8.2 ± 0.2 log cfu/g. Inoculated NFMP was stored at 22°C, RH 25%, and its bacterial populations were monitored for 30 d. Both sets in equilibrated NFMP were subjected to isothermal treatments in closed aluminum cells at 85, 90, and 95°C. Silicon dioxide maintained moisture content (0.29 ± 0.03%, dry basis) at different water activities. The NFMP inoculated with both carriers exhibited stable bacterial populations over 30 d at 22°C. Strains in NFMP inoculated with SiO2 showed equal or higher D-values but equal z-values compared with those inoculated with a small portion of NFMP. Enterococcus faecium exhibited comparable thermal resistance to Salmonella under all tested conditions. This study supports E. faecium as a Salmonella surrogate in thermal processing of NFMP and the use of SiO2 to inoculate NFMP.

Effect of oral supplementation of probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium on the composition of the faecal microbiota of foals.

Effects of probiotics on the intestinal microbiota of foals are yet insufficiently studied. The aim of this study was to investigate whether supplementation of Lactobacillus rhamnosus (DSM 7133) and Enterococcus faecium (DSM 7134) influences the bacterial composition of the faecal microbiota of foals. A total of 34 newborn foals were randomly assigned to the placebo group (PG, n = 16) and the treatment group (TG, n = 18). From day 1 to day 14 of life, foals orally received 3 ml of either a probiotic preparation (1.05 × 109 CFU E. faecium and 4.50 × 108 CFU L. rhamnosus) or placebo (carrier) once a day. Faeces were collected directly from the rectum immediately after birth (meconium) and at day 14 and day 56 of life. Samples of 12 foals per group were selected for microbiological analysis. DNA was extracted and used for polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR. No DNA or amplicons were obtained from meconium. There were no differences in richness of bands and Shannon index of diversity regarding the Clostridium cluster XIVa between groups. Cluster analysis and principal coordinate analysis of DGGE data showed a clear effect of age. Band-based similarity of bacterial clusters (Dice coefficient) decreased from day 14 to day 56 of life (p < 0.001) in PG foals only resulting in lower similarity in PG versus TG foals when 2 month old (p < 0.01). Five of thirty re-amplified bands were identified on species level. Others were assigned either to family (mainly Lachnospiraceae) or genus level (Akkermansia). The bands related to Akkermansia muciniphila or Akkermansia spp. appeared almost in all DGGE profiles. Two-week supplementation of the probiotic preparation to foals had no significant impact on the composition of the faecal microbiota but it appears to have prevented the reduction of bacterial similarity between 2 and 8 weeks of age observed in not treated foals.

Group IIA-Secreted Phospholipase A2 in Human Serum Kills Commensal but Not Clinical Enterococcus faecium Isolates.

Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to killing by serum, we uncover here that normal human serum effectively kills Enterococcus faecium Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in "noninflamed" serum was considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal concentration of hGIIA in serum effectively kills commensal E. faecium and that resistance of clinical E. faecium to hGIIA could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients.

Exponentially Increased Thermal Resistance of Salmonella spp. and Enterococcus faecium at Reduced Water Activity.

Salmonella spp. exhibit prolonged survivability and high tolerance to heat in low-moisture foods. The reported thermal resistance parameters of Salmonella spp. in low-moisture foods appear to be unpredictable due to various unknown factors. We report here that temperature-dependent water activity (aw, treatment temperature) plays an important role in the sharply increased thermal resistance of Salmonella enterica serovar Enteritidis PT 30 and its potential surrogate Enterococcus faecium NRRL B-2354. In our study, silicon dioxide granules, as carriers, were separately inoculated with these two microorganisms and were heated at 80°C with controlled relative humidity between 18 and 72% (resulting in corresponding aw,80°C values for bacteria between 0.18 and 0.72) in custom-designed test cells. The inactivation kinetics of both microorganisms fitted a log-linear model (R2, 0.83 to 0.97). Reductions in the aw,80°C values of bacterial cells exponentially increased the D80°C (the time needed to achieve a 1-log reduction in a bacterial population at 80°C) values for S Enteritidis and E. faecium on silicon dioxide. The log-linear relationship between the D80°C values for each strain in silicon dioxide and its aw,80°C values was also verified for organic wheat flour. E. faecium showed consistently higher D80°C values than S Enteritidis over the aw,80°C range tested. The estimated zaw (the change in aw,80°C needed to change D80°C by 1 log) values of S Enteritidis and E. faecium were 0.31 and 0.28, respectively. This study provides insight into the interpretation of Salmonella thermal resistance that could guide the development and validation of thermal processing of low-moisture foods.IMPORTANCE In this paper, we established that the thermal resistance of the pathogen S Enteritidis and its surrogate Enterococcus faecium, as reflected by D values at 80°C, increases sharply with decreasing relative humidity in the environment. The log-linear relationship between the D80°C values of each strain in silicon dioxide and its aw,80°C values was also verified for organic wheat flour. The results provide new quantitative insight into the way in which the thermal resistance of microorganisms changes in low-moisture systems, and they should aid in the development of effective thermal treatment strategies for pathogen control in low-moisture foods.

Microencapsulation increases survival of the probiotic Lactobacillus plantarum IS-10506, but not Enterococcus faecium IS-27526 in a dynamic, computer-controlled in vitro model of the upper gastrointestinal tract.

AIM: To test the effect of microencapsulation on the survival of two probiotic strains isolated from Dadih, Indonesian fermented buffalo milk, in a dynamic, computer-controlled in vitro model of the upper gastrointestinal (GI) tract (TIM-1), simulating human adults. METHODS AND RESULTS: Free or microencapsulated probiotics, Lactobacillus plantarum IS-10506 or Enterococcus faecium IS-27526, resuspended in milk were studied for survival in the complete TIM-1 system (stomach + small intestine) or in the gastric compartment of TIM-1 only. Hourly samples collected after the ileal-caecal valve or after the pylorus were plated on MRS agar (for Lactobacillus) or S&B agar (for Enterococcus). Survival of the free cells after transit through the complete TIM-1 system was on average for the E. faecium and L. plantarum 15·0 and 18·5% respectively. Survival of the microencapsulated E. faecium and L. plantarum was 15·7 and 84·5% respectively. The free cells were further assessed in only the gastric compartment of TIM-1. E. faecium and L. plantarum showed an average survival of 39 and 32%, respectively, after gastric passage. CONCLUSION: There is similar sensitivity to gastric acid as well as survival after complete upper GI tract transit of free cells, but microencapsulation only protected L. plantarum. SIGNIFICANCE AND IMPACT OF STUDY: Survival of microencapsulated L. plantarum IS-10506 is increased compared to free cells in a validated in vitro model of the upper GI tract. It increases its use as an ingredient of functional foods.

Norfloxacin salts of carboxylic acids curtail planktonic and biofilm mode of growth in ESKAPE pathogens.

AIMS: To enhance the antimicrobial and antibiofilm activity of norfloxacin against the planktonic and biofilm mode of growth in ESKAPE pathogens using chemically modified norfloxacin salts. METHODS AND RESULTS: Antimicrobial testing, synergy testing and time-kill curve analysis were performed to evaluate antibacterial effect of norfloxacin carboxylic acid salts against ESKAPE pathogens. In vivo efficacy to reduce bacterial bioburden was evaluated in zebrafish infection model. Crystal violet assay and live-dead staining were performed to discern antibiofilm effect. Membrane permeability, integrity and molecular docking studies were carried out to ascertain the mechanism of action. The carboxylic acid salts, relative to parent molecule norfloxacin, displayed two- to fourfold reduction in minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa, in addition to displaying potent bacteriostatic effect against certain members of ESKAPE pathogens. In vivo treatments revealed that norfloxacin tartrate (SRIN2) reduced MRSA bioburden by greater than 1 log fold relative to parent molecule in the muscle tissue. In silico docking with gyrA of S. aureus showed increased affinity of SRIN2 towards DNA gyrase. The enhanced antibacterial effect of norfloxacin salts could be partially accounted by altered membrane permeability in S. aureus and perturbed membrane integrity in P. aeruginosa. Antibiofilm studies revealed that SRIN2 (norfloxacin tartrate) and SRIN3 (norfloxacin benzoate) exerted potent antibiofilm effect particularly against Gram-negative ESKAPE pathogens. The impaired colonization of both S. aureus and P. aeruginosa due to improved norfloxacin salts was further supported by live-dead imaging. CONCLUSION: Norfloxacin carboxylic acid salts can act as potential alternatives in terms of drug resensitization and reuse. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study shows that carboxylic acid salts of norfloxacin could be effectively employed to treat both planktonic- and biofilm-based infections caused by select members of ESKAPE pathogens.