Novel TLR4-Activating Vaccine Adjuvant Enhances the Production of Enterococcus faecium-binding Antibodies.

Vaccines are one of the greatest achievements of modern medicine. Due to their safer profile, the latest investigations usually focus on subunit vaccines. However, the active component often needs to be coupled with an adjuvant to be effective and properly trigger an immune response. We are developing a new synthetic monosaccharide-based TLR4 agonist, such as glucosamine-derived compounds FP18 and FP20, as a potential vaccine adjuvant. In this study, we present a new FP20 derivative, FP20Hmp, with a hydroxylated ester linked to the glucosamine core. We show that the modification introduced improves the activity of the adjuvant and its solubility. This study presents the synthesis of FP20Hmp, its in vitro characterization, and in vivo activity while coupled with the ovalbumin antigen or in formulation with an enterococcal antigen. We show that FP20Hmp enables increased production of antigen-specific antibodies that bind to the whole bacterium.

Exploring the feasibility of bacteriocins EntK1 and EntEJ97s in treatment of systemic vancomycin resistant enterococci infections in mice.

AIMS: Enterocins K1 and EJ97 have specific antimicrobial activity against Enterococcus faecium and Enterococcus faecalis, respectively. The aim of this study was to investigate the utility of these enterocins for in vivo treatment of systemic enterococcal infections. METHODS AND RESULTS: The antimicrobial effect in blood was analysed and compared against the effect in saline. Colony forming unit counts revealed that the enterocins killed all the bacteria within 1 hour. Additionally, the bactericidal effect against E. faecalis was more rapid in blood, indicating a possible synergy between EntEJ97 and blood. Importantly, no enterocin resistant mutants emerged in these experiments. Injecting the enterocins intraperitoneally in an in vivo mouse model and using fluorescence and minimum inhibitory concentration determination to estimate concentrations of the peptides in plasma, indicate that the enterocins exist in circulation in therapeutic concentrations. Alanine aminotransferase detection, and haemolysis analysis indicates that there is no detectable liver damage or haemolytic effect after injection. CONCLUSIONS: The study revealed that EntK1 and EntEJ97 are able to kill all bacteria ex vivo in the presence of blood. In vivo experiments determine that the enterocins exist in circulation in therapeutic concentrations without causing liver damage or haemolysis. Future experiments should test these peptides for treatment of infection in a relevant in vivo model.

Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections. IMPORTANCE: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Effects of Enterococcus faecium (R8a) on nonspecific immune gene expression, immunity and intestinal flora of giant tiger shrimp (Penaeus monodon).

In this study, Penaeus monodon were gave basic feed supplemented with three levels of Enterococcus faecium. Then, the expression of non-specific immunity-related genes, and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (AKP), phenol oxidase (PO) were evaluated. Meanwhile, the disease resistance test and intestinal flora determination were conducted. The results showed that the MDA levels of 2% and 5% E. faecium groups were significantly lower than that of the control group (P < 0.05). While the SOD and T-AOC and ACP and AKP of experimental groups were significantly higher (P < 0.05), the PO of experimental groups were significantly lower than that of the control group (P < 0.05). In addition, the expressions of immunity-related genes (tlr22, dorsal, lysozyme, crustin, imd, and relish) in the 2% and 5% E. faecalis groups were significantly greater than those in the control group (P < 0.05). After P. monodon was challenged with Vibrio parahaemolyticus for 7 days, the average cumulative mortality of P. monodon in the 2% and 5% groups were significantly lower than that in the 0% group (P < 0.05). With the increase of feeding time, the number of effective OTUs in each group showed a downward trend. At the 14th d, Proteobacteria, Bacteroidetes and Firmicutes, the dominant flora in the intestinal tract of P. monodon. In summary, supplied with E. faecium could increase the expression of non-specific immunity-related genes, enhance the immune capacity of P. monodon.

The therapeutic effects of fermented milk with lactic acid bacteria from traditional Daqu on hypertensive mice.

Lactic acid bacteria (LAB), a type of microorganism widely used in functional foods, has gained notable research attention in recent years. Certain strains possess the proteolytic ability to release potentially antihypertensive peptides from dairy proteins, which prompted us to explore the LAB strains from an understudied and unique ingredient, Daqu. We screened for 67 strains of LAB strains from traditional Daqu using the calcium dissolution ring method. Sixteen strains exhibiting angiotensin-converting enzyme inhibition (ACE-I) activity exceeding 50% were chosen for 16S rDNA sequencing and safety assessment. It is noteworthy that Enterococcus faecium CP640 and Lacticaseibacillus rhamnosus CP658 exhibited significant ACE-I activity, which was the result of strain fermentation in reconstituted skim milk. These 2 strains did not exhibit hemolytic activity or antibiotic resistance. They also did not produce biogenic amines and showed high survival rates in the gastrointestinal tract. Additionally, Enterococcus faecium CP640 and Lacticaseibacillus rhamnosus CP658 fermented milk exhibited a notable reduction in blood pressure levels in spontaneously hypertensive rats (SHR) compared with negative controls in SHR. Importantly, no adverse effect was observed in normal Wistar-Kyoto rats. Through the analysis of physiological, serum, and urine-related indicators, it was observed that Enterococcus faecium CP640 and Lacticaseibacillus rhamnosus CP658 have the potential to promote weight gain in SHR, alleviate excessive heart rate, improve renal function indicators, and effectively regulate blood sugar and uric acid levels in SHR. These 2 strains showed optimal properties in lowering blood pressure and have the potential to be used in functional dairy products in the future.

Emergence of vancomycin-resistant enterococci from vancomycin-susceptible enterococci in hospitalized patients under antimicrobial therapy.

OBJECTIVES: Enterococci are opportunistic pathogens with plastic genomes that evolve, acquire, and transmit antimicrobial-resistant determinants such as vancomycin resistance clusters. While vancomycin-resistant enterococci (VRE) have emerged as successful nosocomial pathogens, the mechanism by which vancomycin-susceptible enterococci (VSE) transform to VRE in hospitalized patients remains understudied. METHODS: Genomes of Enterococcus faecium from two critically ill hospitalized patients subjected to multiple antibiotic therapies, including broad-spectrum antibiotics, were investigated. To identify mechanisms of resistance evolution, genomes of vancomycin-susceptible and -resistant isolates were compared. RESULTS: While VSE isolates were initially identified, VRE strains emerged post-vancomycin therapy. Comparative genomics revealed horizontal transmission of mobile genetic elements containing the Tn1549 transposon, which harbours the vanB-type vancomycin resistance gene cluster. This suggests that broad-spectrum antibiotic stress promoted the transfer of resistance-conferring elements, presumably from another gut inhabitant. CONCLUSION: This is one of the first studies investigating VSE and VRE isolates from the same patient. The mechanism of transmission and the within-patient evolution of vancomycin resistance via mobile genetic elements under antibiotic stress is illustrated. Our findings serve as a foundation for future studies building on this knowledge which can further elucidate the dynamics of antibiotic stress, resistance determinant transmission, and interactions within the gut microbiota.

Performance responses of lactating Rahmani ewes fed diet supplemented with Enterococcus faecium NRC-3 or Lactobacillus rhamnosus.

Production of new types of probiotics for animal nutrition mainly depends on the appropriate bacterial strain and efficient substrate. Therefore, this study aimed to evaluate the impact of two probiotic strains containing 1.2 × 108 (CFU/g), produced on permeate media on performance responses of Rahmani ewes. Thirty early lactating ewes (about 2-3 years old and weighting on average 43.2 ± 0.3 kg) were randomly divided into three groups of 10 animals each using a completely randomized design. The 1st group was fed the basal diet (60% concentrate feed mixture (CFM) + 30% Egyptian clover + 10% bean straw). While the ewes in 2nd and 3rd groups were fed the basal diet + 2 g of Enterococcus faecium NRC-3(EF) and Lactobacillus rhamnosus (LR), respectively for 9 weeks. Ewes' diet supplementation with EF or LR increased (p < 0.05) dry matter, organic matter, crude protein, neutral detergent fiber, acid detergent fiber, and non-structural carbohydrates digestibility compared to ewes of the control group. Glucose, total protein, and albumin concentrations significantly increased in the blood of EF ewes than those of LR and control. Probiotics increased ewes' milk yield as well as milk protein, fat, and lactose yields, but no differences were observed between treatments when milk components were expressed as percentage. Milk fatty acids profile not changed due to EF or LR supplementation. Probiotics (E. faecium and L. rhamnosus) produced on cheese industry waste (permeate) have proven their ability to improve the productive performance of the lactating Rahmani ewes.

Transfer of Enterococcus faecium and Salmonella enterica during simulated postharvest handling of yellow onions (Allium cepa).

Bacterial transfer during postharvest handling of fresh produce provides a mechanism for spreading pathogens, but risk factors in dry environments are poorly understood. The aim of the study was to investigate factors influencing bacterial transfer between yellow onions (Allium cepa) and polyurethane (PU) or stainless steel (SS) under dry conditions. Rifampin-resistant Enterococcus faecium NRRL B-2354 or a five-strain cocktail of Salmonella was inoculated onto onion skin or PU surfaces at high or moderate levels using peptone, onion extract, or soil water as inoculum carriers. Transfer from inoculated to uninoculated surfaces was conducted using a texture analyzer to control force, time, and number of contacts. Transfer rates (ratio of recipient surface to donor surface populations) of E. faecium (4-5%) were significantly higher than those of Salmonella (0.5-0.6%) at the high (7 log CFU/cm2) but not moderate (5 log CFU/cm2) inoculum levels. Significantly higher populations of E. faecium transferred from onion to PU than from PU to onion. The transfer rates of E. faecium were impacted by inoculum carrier (61% [onion extract], 1.6% [peptone], and 0.31% [soil]) but not by inoculation level or recipient surface (PU versus SS). Bacterial transfer during dry onion handling is significantly dependent on bacterial species, inoculation levels, inoculum carrier, and transfer direction.

First Report of a Wastewater Treatment-Adapted Enterococcus faecalis ST21 Harboring vanA Gene in Brazil.

Enterococcus faecalis has emerged as an important opportunistic pathogen due to its increasing resistance to antimicrobials, mainly to vancomycin, which leads substantial cases of therapeutic failures. Wastewater treatment plants (WWTP), in turn, are considered hotpots in the spread of antimicrobial resistance according to One Health perspective. In this study, we present the first report of a vancomycin-resistant E. faecalis strain recovered from treated effluent in Brazil. For this purpose, the whole-genome sequencing (WGS) was carried out aiming to elucidate its molecular mechanisms of antimicrobial resistance and its phylogenetic relationships amongst strains from other sources and countries. According to Multilocus Sequence Typing (MLST) analysis this strain belongs to ST21. The WGS pointed the presence of vanA operon, multiple antibiotic resistance and virulence genes, and a significant pathogenic potential for humans. The phylogenomic analysis of E. faecalis 6805 was performed with ST21 representatives from the PubMLST database, including the E. faecalis IE81 strain from clinical sample in Brazil, which had its genome sequenced in this study. Our results demonstrated a strain showing resistance to vancomycin in treated effluent. To the best of our knowledge, this is an unprecedented report of vanA-carrying E. faecalis ST21. Furthermore, it is the first description of a vanA-harboring strain of this species from environmental sample in Brazil. Our data highlight the role of WWTP in the spread of AMR, since these environments are favorable for the selection of multidrug-resistant pathogens and the treated effluents, carrying antibiotic resistance genes, are directed to receiving water bodies.

Revisiting ESKAPE Pathogens: virulence, resistance, and combating strategies focusing on quorum sensing.

The human-bacterial association is long-known and well-established in terms of both augmentations of human health and attenuation. However, the growing incidents of nosocomial infections caused by the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) call for a much deeper understanding of these organisms. Adopting a holistic approach that includes the science of infection and the recent advancements in preventing and treating infections is imperative in designing novel intervention strategies against ESKAPE pathogens. In this regard, this review captures the ingenious strategies commissioned by these master players, which are teamed up against the defenses of the human team, that are equally, if not more, versatile and potent through an analogy. We have taken a basketball match as our analogy, dividing the human and bacterial species into two teams playing with the ball of health. Through this analogy, we make the concept of infectious biology more accessible.

Aminopenicillins vs non-aminopenicillins for treatment of enterococcal lower urinary tract infections.

Aminopenicillins (APs) achieve urinary concentrations that exceed typical minimum inhibitory concentrations for enterococcal lower urinary tract infection (UTI). The local clinical microbiology laboratory discontinued routine susceptibilities on enterococcal urine isolates, and reports that 'APs are predictably reliable for uncomplicated enterococcal UTI'. The objective of this study was to compare outcomes of APs with non-APs (NAPs) for enterococcal lower UTIs. This was an institutional-review-board-approved, retrospective cohort of adults hospitalized with symptomatic enterococcal lower UTIs from 2013 to 2021. The primary endpoint was composite clinical success at 14 days, defined as resolution of symptoms without new symptoms and no repeat culture growth of the index organism. A non-inferiority analysis was utilized with a 15% margin, and logistic regression evaluated characteristics associated with 14-day failure. In total, 178 subjects were included: 89 AP patients and 89 NAP patients. Vancomycin-resistant enterococci (VRE) were identified in 73 (82%) AP patients and 76 (85%) NAP patients (P=0.54); in total, 34 (38.2%) AP patients and 66 (74.2%) NAP patients had confirmed Enterococcus faecium (P<0.001). Amoxicillin (n=36, 40.5%) and ampicillin (n=36, 40.5%) were the most commonly used APs, and linezolid (n=41, 46%) and fosfomycin (n=30, 34%) were the most commonly used NAPs. Fourteen-day clinical success rates for APs and NAPs were 83.1% and 82.0%, respectively [1.1% difference, 97.5% confidence interval (CI) -0.117 to 0.139]. Among the E. faecium subgroup, 14-day clinical success was observed in 27/34 (79.4%) AP patients and 53/66 (80.3%) NAP patients (P=0.916). On logistic regression, APs were not associated with 14-day clinical failure (adjusted odds ratio 0.84, 95% CI 0.38-1.86). APs were non-inferior to NAPs for treating enterococcal lower UTIs, and may be considered irrespective of susceptibility results.

In-vitro evaluation of the probiotic potential and the fermentation profile of Pediococcus and Enterococcus strains isolated from Moroccan camel milk.

The promotion of human health through natural approaches like functional foods and probiotics is in high demand. The medicinal plants are the major feed of Moroccan dromedary, which improves the functional properties of their milk. A few studies have reported the probiotic and functional aptitudes of lactic acid bacteria (LAB) of this milk. In this context, our study aimed to identify LAB isolated from Moroccan raw camel milk and investigate their probiotic features and their fermentation profile. The molecular identification of twelve isolates indicated that they belong to Pediococcus pentosaceus, Enterococcus faecium, and Enterococcus durans. All LAB strains displayed high tolerance to gastrointestinal conditions (survival rate of 31.85-96.52% in pH 2.5, 35.23-99.05% in 0.3 bile salts, and 26.9-90.96% in pepsin), strong attachment abilities (auto-aggregation and hydrophobicity ranged from 28.75 to 95.9% and from 80.47 to 96.37%, respectively), and high co-aggregation ability with pathogenic bacteria. Importantly, they did not present antibiotic resistance or hemolytic activity. Our LAB strains demonstrated antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, and Salmonella enterica. Moreover, they could acidify cow milk (ΔpH of 2.55 after 24 h) and improve its antioxidant ability (inhibition of 36.77% of DPPH). Based on the multivariate analysis, Pediococcus pentosaceus Pd24, Pd29, Pd38, Enterococcus faecium Ef18, and Enterococcus durans Ed22 were selected as the most promising probiotics. Therefore, we propose that Pediococcus pentosaceus isolated from camel milk could be used as potential probiotic strains and/or starter cultures in functional milk fermentation.

Pangenome profiling of novel drug target against vancomycin-resistant Enterococcus faecium.

Enterococcus faecium is a frequent causative agent of nosocomial infection mainly acquired from outgoing hospital patients (Hospital Acquired Infection-HAIs). They are largely involved in the outbreaks of bacteremia, UTI, and endocarditis with a high transmissibility rate. The recent emergence of VRE strain (i.e. vancomycin resistant enterococcus) turned it into high priority pathogen for which new drug research is of dire need. Therefore, in current study, pangenome and resistome analyses were performed for available antibiotic-resistant genomes (n = 216) of E. faecium. It resulted in the prediction of around 5,059 genes as an accessory gene, 1,076 genes as core and 1,558 genes made up a unique genome fraction. Core genes common to all strains were further used for the identification of potent drug targets by applying subtractive genomics approach. Moreover, the COG functional analysis showed that these genomes are highly enriched in metabolic pathways such as in translational, ribosomal, proteins, carbohydrates and nucleotide transport metabolism. Through subtractive genomics it was observed that 431 proteins were non-homologous to the human proteome, 166 identified as essential for pathogen survival while 26 as potential and unique therapeutic targets. Finally, 3-dehydroquinate dehydrogenase was proposed as a potent drug target for further therapeutic candidate identification. Moreover, the molecular docking and dynamic simulation technique were applied to performed a virtual screening of natural product libraries (i.e., TCM and Ayurvedic compounds) along with 3-amino-4,5-dihydroxy-cyclohex-1-enecarboxylate (DHS) as a standard compound to validate the study. Consequently, Argeloside I, Apigenin-7-O-gentiobioside (from Ayurvedic library), ZINC85571062, and ZINC85570908 (TCM library) compounds were identified as potential inhibitors of 3-dehydroquinate dehydrogenase. The study proposed new compounds as novel therapeutics, however, further experimental validation is needed as a follow-up.Communicated by Ramaswamy H. Sarma.

Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei.

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

Effect of benzoic acid, Enterococcus faecium, and essential oil complex on intestinal microbiota of laying hens under coccidia and Clostridium perfringens challenge.

The objective of this study was to investigate whether dietary supplementation with benzoic acid, Enterococcus faecium, and essential oil complex (BEC) could help laying hens recover from coccidia and Clostridium perfringens type A challenge. A total of 60 (35-wk-old) Lohmann-laying hens were randomly assigned to 3 experimental groups (10 replicates with 2 hens per replicate): I) control group (CON), II) challenge group (CC), and III) BEC group (2,000 mg/kg BEC). The total experimental period was 8 wk. The results shown that the challenge layers had lower egg-laying rate and average daily feed intake (ADFI) (P < 0.05), and addition of BEC after challenge increased egg-laying rate (P < 0.05). The content of propionic acid (PA) and butyric acid (BA) in short-chain fatty acid (SCFA) was significantly decreased by challenge (P < 0.05). CC and BEC groups had lower villus height to crypt depth ratio (V/C) and higher pathological scores in duodenum (P < 0.05), whereas the BEC group had lower pathological scores in jejunum when compared with the CC group (P < 0.05). The challenge increased the concentration of proinflammatory cytokines (IL-1ß and IL-6) (P < 0.05). An increase in the abundance of Bacteroidoes (genus), Bacteroidaceae (family), Bacteroidoes sp. Marseille P3166 (species), Bacteroidoes caecicola (species) was observed in the CC group, whereas the BEC group had higher abundance of Bacteroides caecigallinarum (species). The genera Faecalibacterium and Asterolplasma were positively correlated with egg-laying rate (r = 0.57, 0.60; P < 0.01); and the genera Bacteroides and Romboutsia were negatively correlated with egg-laying rate (r = -0.58, -0.74; P < 0.01). The genera Bacteroides, Lactobacillus, and Rombutzia were positively correlated with jejunal mucosa proinflammatory factor IL-1ß level (r = 0.61, 0.60, 0.59; P < 0.01), which were negatively correlated with genera Rikenbacteriaceae RC9, Faecalibacterium, and Olsenlla (r = -0.56, -0.57, -0.61; P < 0.01). There genera UCG.005 was positively correlated with proinflammatory factor IL-6 level in jejunal mucosa (r = 0.58; P < 0.01), which was negatively correlated with Rikenbacteriaceae RC9 (r = -0.62; P < 0.01). The experiment results revealed that the addition of BEC to the diet restored the production performance of the laying hens. In addition, supplementation of 2,000 mg/kg BEC modulated gut health by reducing gut damage scores and modulating microbial composition, thereby promoting recovery of laying hens after coccidia and Clostridium perfringens challenge.

Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum.

Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5 kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96 kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6 h and moderate at 24 h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.

Enterococcus faecium supplementation prevents enteritis caused by Escherichia coli in goats.

The probiotic Enterococcus faecium is a gut microbe with immunomodulatory effects, which has been widely used to prevent diarrhoea in pigs and birds. Escherichia coli is a common pathogen that causes inflammatory bowel disease in animals. The aim of this study was to investigate the protective effects of E. faecium on enteritis in goats. Forty goats were randomly divided into 4 treatment groups: control, E. faecium, E. coli, and E. faecium + E. coli. The changes of physiological indicators and diarrhoea scoring were evaluated on days -4, -2, 0, 2, 4, 6, and 8. The pathological examination, inflammatory cytokines mRNA expression and bacterial counts in jejunum and caecum were detected on day 4 and 8. The results showed that body temperature, respiratory rate, heart rate and leukocyte counts all increased from the 2nd to the 6th day after feeding with E. coli, and the diarrhoea score was significantly increased. However, E. faecium-pretreated goats had lower body temperatures and fewer leukocytes than E. coli-treated goats on day 2, as well as decreased diarrhoea scores. E. coli treatment caused histopathological damage and morphological changes in the jejunum and caecum, while pretreatment with E. faecium significantly alleviated these injuries. E. faecium pretreatment can reduce the load of E. coli and increase the prevalence of Lactobacillus, thereby balancing the microbiota in the intestine. Furthermore, E. coli-infected goats pretreated with E. faecium showed obvious inhibition of Toll-like receptor 4, interleukin (IL)-1ß, IL-6, IL-8 and tumour necrosis factor-α mRNA expression in the jejunum and caecum compared to that in the E. coli treatment group. In conclusion, the addition of E. faecium to goat feed is beneficial for improving clinical symptoms, maintaining intestinal mucosa integrity, balancing the microbiota and decreasing inflammatory responses in E. coli-induced intestinal injury.

Novel Bio-Functional Aloe vera Beverages Fermented by Probiotic Enterococcus faecium and Lactobacillus lactis.

Aloe vera has been medicinally used for centuries. Its bioactive compounds have been shown to be very effective in the treatment of numerous diseases. In this work, a novel functional beverage was developed and characterized to combine the health benefits of probiotic bacteria with the Aloe vera plant itself. Two Aloe vera juices were obtained by fermentation either by a novel isolated Enterococcus faecium or a commercial Lactococcus lactis. The extraction of Aloe vera biocompounds for further fermentation was optimized. Extraction with water plus cellulase enhanced the carbohydrates and phenolic compounds in the obtained extracts. The biotransformation of the bioactive compounds from the extracts during fermentation was assessed. Both probiotic bacteria were able to grow on the Aloe vera extract. Lactic acid and short-chain fatty acids (SCFA) together with fourteen individual phenolic compounds were quantified in the produced Aloe vera juice, mainly epicatechin, aloin, ellagic acid, and hesperidin. The amount of total phenolic compounds was maintained through fermentation. The antioxidant activity was significantly increased in the produced juice by the ABTS method. The novel produced Aloe vera juice showed great potential as a functional beverage containing probiotics, prebiotics, SCFA, and phenolic compounds in its final composition.

Nigella sativa oil extract: A natural novel specific conjugal transfer inhibitor of vancomycin resistance from vanA/B-resistant Enterococcus faecium to Staphylococcus aureus.

AIM: The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) has been identified as one of the most challenging problems in healthcare settings worldwide. Specific conjugation inhibitors' development is critical in the fight against the spread of emerging VRSA. The impact of Nigella sativa oil on VR genes conjugal transfer from Enterococcus faecium (VREtfm) to vancomycin-sensitive S. aureus (VSSA) was investigated in this study. METHODS AND RESULTS: Enterococciwere isolated from retail broilers, fish, cows' milk, and human urine. VR E. faecalis and VREtfm VanA phenotypes were prevalent in retail broiler samples. The VREtfm isolates were dominant, exhibiting high levels of resistance to gentamycin and ciprofloxacin antibiotics, as well as the existence of both vanA and vanB genes and virulence traits (ESP+ , asa1+ ) as determined by PCR. Transconjugant VREtfm strains containing vanA/vabB and 20 kb plasmids (transfer frequency around 103 ) and carrying the Tn1546 transposon were identified. Tn1546 transposon transfer with its VR markers to VSSA was effectively inhibited in treated VREtfm donor strains with a sub-minimum inhibitory concentration of N. sativa oil. THE SIGNIFICANCE AND IMPACT OF THE STUDY: This work offers new insights for overcoming VR conjugal transfer utilizing natural N. sativa oil, as well as a suggestion for a novel specialized conjugation inhibitor that could effectively facilitate the difficulty of eliminating VR bacteria from healthcare settings.

Alleviating effect of dietary supplementation of benzoic acid, Enterococcus faecium and essential oil complex on coccidia and Clostridium perfringens challenge in laying hens.

The purpose of this experiment is to explore the effects of dietary supplementation of benzoic acid, Enterococcus faecium, and essential oil complex (BEC) on coccidia and Clostridium perfringens challenge in laying hens. A total of 80 Lohmann gray laying hens (35 wk old) were allocated to 4 treatments in a 2 × 2 factorial arrangement with the main effects of Clostridium perfringens type A (CP) and coccidia challenge (with or without challenge) and 2 BEC levels (0 and 1,000 mg/kg). The total experimental period was 6 wk. The results showed that: the challenge group significantly decreased the laying rate and average daily feed intake (ADFI) of laying hens (PChallenge < 0.01). The BEC + challenge group significantly increased the laying rate and decreased the feed conversion ratio (FCR) of laying hens (PBEC < 0.05). The challenge significantly decreased the thickness, strength, and relative weight of eggshell (PChallenge < 0.05). The BCE + challenge group significantly increased the relative weight and strength of the eggshell (PBEC < 0.05). The challenge significantly increased the crypt depth of the duodenum, jejunum and ileum, and decreased the villus-to-crypt ratio (V/C) (PChallenge < 0.01). The BEC + challenge group decreased the crypt depth of the duodenum and jejunum, and increased the V/C of the duodenum (PBEC < 0.01). The pathological scores of duodenum and jejunum of the challenge group were significantly higher than other groups (PChallenge < 0.01), while the BEC + challenge group had lower pathological scores of jejunum (PBEC < 0.01). The challenge significantly decreased the mRNA expression of Occludin, Mucin-2, Zonula occluden-1 (ZO-1) (Pchallenge < 0.05); whereas the BEC group significantly increased the expression of Occludin, Mucin-2, and Claudin-1 mRNA (PBEC < 0.05). The challenge significantly increased the level of interleukin 1ß (IL-1ß) in the jejunum (PChallenge < 0.05). Taken together, adding BEC to the diet can improved production performance and egg quality of layers, by protecting intestinal health against Clostridium perfringens type A (CP) and coccidia challenge.