Challenges for heat stress: Intestinal culturable bacteria of Lohmann Brown chickens.

This study aimed to assess how heat stress, specifically within the range of 35-38 °C, affects the populations of culturable intestinal lactobacilli, enterococci, and Escherichia coli, as well as the expression of Heat Shock Proteins (HSP70), in Lohmann Brown chickens. It also explored the influence of the chickens' blood transferrin and ceruloplasmin genotypes on these responses. Thirty chickens underwent eight hours of heat stress, maintained at an average temperature of 37 °C and a relative humidity of 75-80%, with continuous access to food and water. Behavioral monitoring was conducted throughout to prevent excessive heat-related mortality. The Lohmann Brown chickens from the Yerevan "Arax" poultry farm were initially classified based on their blood transferrin and ceruloplasmin genotypes to investigate potential correlations between intestinal bacterial composition and variations in these polymorphisms. A significant correlation was found between heat stress and the abundance of culturable enterococci within the intestinal microbiota, regardless of chicken TfAB, TfBC, CpAB, CpCC and TfAB, TfBC, CpAB, CpCD genotypes. Heat stress led to nearly double the HSP70 levels in chicken blood, along with a reduction in the culturable enterococci population by at least 10,000-fold in the intestinal microbiota. These findings are significant for targeted management strategies to mitigate heat stress in chicken populations.

Enterococcus mundtii A2 biofilm and its anti-adherence potential against pathogenic microorganisms on stainless steel 316L.

Pathogenic bacterial biofilms present significant challenges, particularly in food safety and material deterioration. Therefore, using Enterococcus mundtii A2, known for its antagonistic activity against pathogen adhesion, could serve as a novel strategy to reduce pathogenic colonization within the food sector. This study aimed to investigate the biofilm-forming ability of E. mundtii A2, isolated from camel milk, on two widely used stainless steels within the agri-food domain and to assess its anti-adhesive properties against various pathogens, especially on stainless steel 316L. Additionally, investigations into auto-aggregation and co-aggregation were also conducted. Plate count methodologies revealed increased biofilm formation by E. mundtii A2 on 316L, followed by 304L. Scanning electron microscopy (SEM) analysis revealed a dense yet thin biofilm layer, playing a critical role in reducing the adhesion of L. monocytogenes CECT 4032 and Staphylococcus aureus CECT 976, with a significant reduction of ≈ 2 Log CFU/cm2. However, Gram-negative strains, P. aeruginosa ATCC 27853 and E. coli ATCC 25922, exhibit modest adhesion reduction (~ 0.7 Log CFU/cm2). The findings demonstrate the potential of applying E. mundtii A2 biofilms as an effective strategy to reduce the adhesion and propagation of potentially pathogenic bacterial species on stainless steel 316L.

Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Novel pyrazoles as potent growth inhibitors of staphylococci, enterococci and Acinetobacter baumannii bacteria.

Background: Methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and Acinetobacter baumannii cause serious antibiotic-resistant infections. Finding new antibiotics to treat these infections is imperative for combating this worldwide menace. Methods & Results: In this study, the authors designed and synthesized potent antimicrobial agents using 4-trifluoromethylphenyl-substituted pyrazole derivatives. In addition to their potency against planktonic bacteria, potent compounds effectively eradicated S. aureus and Enterococcus faecalis biofilms. Human cells tolerated these compounds with good selectivity factors. Furthermore, the authors provide evidence for the mode of action of compounds based on time-kill kinetics, flow cytometry analysis of propidium iodide-treated bacteria and oxygen uptake studies. Conclusion: This study demonstrated 20 novel compounds with potent antibacterial activity that are tolerated by human cell lines.

In Vitro Activity of Rifampin, Rifabutin, and Rifapentine against Enterococci and Streptococci from Periprosthetic Joint Infection.

After staphylococci, streptococci and enterococci are the most frequent causes of periprosthetic joint infection (PJI). MICs and minimum biofilm bactericidal concentrations of rifampin, rifabutin, and rifapentine were determined for 67 enterococcal and 59 streptococcal PJI isolates. Eighty-eight isolates had rifampin MICs of ≤1 µg/ml, among which rifabutin and rifapentine MICs were ≤ 8 and ≤4 µg/ml, respectively. There was low rifamycin in vitro antibiofilm activity except for a subset of Streptococcus mitis group isolates. IMPORTANCE Rifampin is an antibiotic with antistaphylococcal biofilm activity used in the management of staphylococcal periprosthetic joint infection with irrigation and debridement with component retention; some patients are unable to receive rifampin due to drug interactions or intolerance. We recently showed rifabutin and rifapentine to have in vitro activity against planktonic and biofilm states of rifampin-susceptible periprosthetic joint infection-associated staphylococci. After staphylococci, streptococci and enterococci combined are the most common causes of periprosthetic joint infection. Here, we investigated the in vitro antibiofilm activity of rifampin, rifabutin, and rifapentine against 126 Streptococcus and Enterococcus periprosthetic joint infection isolates. In contrast to our prior findings with staphylococcal biofilms, there was low antibiofilm activity of rifampin, rifabutin, and rifapentine against PJI-associated streptococci and enterococci, apart from some Streptococcus mitis group isolates.

Transcriptomics Reveal the Survival Strategies of Enterococcus mundtii in the Gut of Spodoptera littoralis.

The complex interaction between a higher organism and its resident gut flora is a subject of immense interest in the field of symbiosis. Many insects harbor a complex community of microorganisms in their gut. Larvae of Spodoptera littoralis, a lepidopteran pest, house a bacterial community that varies both spatially (along the length of the gut) and temporally (during the insect's life cycle). To monitor the rapid adaptation of microbes to conditions in the gut, a GFP-tagged reporter strain of E. mundtii, a major player in the gut community, was constructed. After early-instar S. littoralis larvae were fed with the tagged microbes, these were recovered from the larval fore- and hindgut by flow cytometry. The fluorescent reporter confirmed the persistence of E. mundtii in the gut. RNA-sequencing of the sorted bacteria highlighted various strategies of the symbiont's survival, including upregulated pathways for tolerating alkaline stress, forming biofilms and two-component signaling systems for quorum sensing, and resisting oxidative stress. Although these symbionts depend on the host for amino acid and fatty acids, differential regulation among various metabolic pathways points to an enriched lysine synthesis pathway of E. mundtii in the hindgut of the larvae.

Tenacity of Enterococcus cecorum at different environmental conditions.

AIMS: Our aim was to analyse the survival of Enterococcus cecorum (EC) at various temperatures, relative air humidities and on different substrates commonly existing in broiler houses. METHODS AND RESULTS: A pathogenic EC isolate (EC14) was used to inoculate sterile litter, polyvinyl chloride (PVC) and dust samples. Incubation at 37, 25 or 15°C with either 32% relative humidity (RH) or 78% RH followed. At defined time points (0-4272 h post-inoculation), samples were examined in triplicate for the total viable count. Selected combinations were repeated for a non-pathogenic and two additional pathogenic EC strains. For EC14, the measured survival time ranged from 48 to 4272 h (178 days) depending on the substrate-humidity-temperature combination. The longevity was the highest on litter, followed by dust and then PVC. Lower temperatures facilitated its survival, lower relative air humidity favoured the survival only in combination with 25 or 15°C. All three pathogenic strains showed longer survival times (up to 432 h, 18 days) compared to the non-pathogenic EC strain (168 h, 7 days) under the same conditions. CONCLUSIONS: Enterococcus cecorum demonstrates a high persistence in the environment especially at 15°C and 32% RH. SIGNIFICANCE AND IMPACT OF THE STUDY: Hygiene management plans should consider the durability of EC and the risk of a carry-over to control consecutive EC outbreaks.

Anti-Proliferative and Anti-Biofilm Potentials of Bacteriocins Produced by Non-Pathogenic Enterococcus sp.

The incidence of cancer is increasing worldwide; likewise, the emergence of antibiotic-resistant biofilm-forming pathogens has led to a tremendous increase in morbidity and mortality. This study aimed to evaluate the probiotic properties of bacteriocin-producing Enterococcus sp. with a focus on their anti-biofilm and anticancer activities. Three of 79 Enterococcus isolates (FM43, FM65, FM50) were identified as producers of broad-spectrum bioactive molecules and were molecularly characterized as Enterococcus faecium by 16S rRNA sequencing. Phenotypic and genotypic screening for potential virulence factors revealed no factors known to promote pathogenicity. Treatment with proteinase K resulted in diminished antimicrobial activity; PCR-based screening for bacteriocin genes suggested the presence of both entA and entB genes that encode enterocins A and B, respectively. Maximum antimicrobial activity was detected during the early stationary phase, while activity disappeared after 24 h in culture. Bacteriocins from these isolates were stable at high temperatures and over a wide range of pH. Interestingly, crude supernatants of Ent. faecium FM43 and Ent. faecium FM50 resulted in significant destruction (80% and 48%, respectively; P < 0.05) of Streptococcus mutans ATCC 25175-associated preformed biofilms. Moreover, in vitro cytotoxicity assays revealed that extracts from Ent. faecium isolates FM43, FM65, and FM50 inhibited Caco-2 cell proliferation by 76.9%, 70%, and 85.3%, respectively. Taken together, the multifunctional capabilities of the microbial-derived proteins identified in our study suggest potentially important roles as alternative treatments for biofilm-associated infections and cancer.

Impact of diet and synbiotics on selected gut bacteria and intestinal permeability in individuals with excess body weight - A Prospective, Randomized Study.

Overweight and obese individuals may have leaky intestinal barrier and microbiome dysbiosis. The aim of this study was to determine whether body mass reduction with diet and synbiotics in an adult person with excess body mass has an influence on the gut microbiota and zonulin concentration. The study was a single blinded trial. 60 persons with excess body mass were examined. Based on randomization, patients were qualified either to the intervention group (Synbiotic group) or to the control group (Placebo group). Anthropometric measurements, microbiological assessment of faecal samples and zonulin concentration in the stool were performed before and after observation. After 3-months, an increase in the variety of intestinal bacteria (increase in the Shannon-Weaver index and the Simpson index) and a decrease in concentration of zonulin in faecal samples were observed in the Synbiotic group. Also, statistically significant correlation between zonulin and Bifidobacterium spp. (Spearman test, R=-0.51; p=0.0040) was noticed. There were no significant relationships between the body mass, BMI and changes in the intestinal microbiota or zonulin concentrations. The use of diet and synbiotics improved the condition of the microbiota and intestinal barrier in patients in the Synbiotic group.

Altered gut microbiota correlated with systemic inflammation in children with Kawasaki disease.

Kawasaki disease (KD) is a multi-systemic vasculitis of unknown etiology that occurs mainly in children, and the disturbance of gut microbiota is generally believed to cause a hyperimmune reaction triggering KD. The aim of the study was to investigate the alterations in the fecal microbiota and assess its relationship with systemic inflammation. Totally 30 KD children were enrolled and followed up for 6 months, with another group of 30 age- and sex-matched healthy children as controls. Phylotype profiles of fecal microbial communities were analyzed using 16S rRNA gene sequencing. Serum inflammatory markers were detected by flow cytometer. We showed that KD children exhibited a significant reduction in fecal microbial diversity in the acute phase compared with the healthy controls. Enterococcus, Acinetobacter, Helicobacter, Lactococcus, Staphylococcus and Butyricimonas in acute KD children were significantly higher than the healthy children. Levels of systemic inflammation biomarkers, including IL-2, IL-4, IL-6, IL-10, TNF-α, and INF-γ, were significantly elevated in the acute KD children. Altered microbiota genera Enterococcus and Helicobacter abundances were shown to be correlated positively with IL-6, which were never previously reported in KD. This study suggested that gut microbiota alteration is closely associated with systemic inflammation, which provides a new perspective on the etiology and pathogenesis of KD.

Molecular mechanisms of enterococcal-bacteriophage interactions and implications for human health.

Once overlooked as passive bystanders of the human intestinal microbiota, new evidence is shedding light on the importance of enterococci and their bacteriophages (phages) in shaping human health. Natural predators of enterococci, phages represent a narrow spectrum, precision targeting modality for the eradication of problematic enterococci within the microbiota or infected tissue. The identification of enterococcal phage receptors, absorption factors, and transcriptional responses following phage infection reveals a complex predator-prey relationship that modulates enterococcal cell surface architecture, susceptibility to antibiotics, and adaptation to host associated environments. Considering the dry up of contemporary antibiotic discovery pipelines in the pharmaceutical industry and a continued emergence of multidrug-resistant enterococci, enterococcal phages may serve as bonafide therapeutics. We highlight current advances in enterococcal phage biology with emphasis on recent breakthroughs in potential therapeutic applications that place enterococcal phages at the forefront of next-generation biologics.

Probiotic and technological features of Enterococcus and Weissella isolates from camel milk characterised by an Argane feeding regimen.

The objectives of this study were to isolate lactic acid bacteria (LAB) from a raw Moroccan camel milk collected after the incorporation of a specific Argane by-products diet, and to investigate their technological properties as well as their probiotic features. The molecular identification of the isolates indicated that they belong to Weissella confusa, Weissella cibaria or Enterococcus durans species. Our results revealed that the tested isolates have a fast acidifying ability (values ranging between 0.045 ± 0.01 to 0.93 ± 0.01 after only 4 h incubation), important proteolysis, autolysis, lipolytic activities and an important diacetyl and exopolysaccharides production. All these isolates demonstrated a high tolerance to gastrointestinal conditions, namely to gastric simulated juice (survival rate ranged between 75.05 ± 7.88 and 85.55 ± 1.77%) and to bile salts (survival rate between 42.79 ± 1.11 and 82.75 ± 1.01%). The autoaggregation, hydrophobicity and antioxidant activity mean values of the isolates were 13.26-41.16%, 13.23-54.47% and 47.57-63.31%, respectively. Importantly, LAB cultures exhibited antibacterial activity against Gram-negative and Gram-positive pathogenic bacteria and none of the tested isolates presented antibiotic resistance, haemolytic or DNase activities. This study revealed interesting properties for LAB isolated and supported their utilization as autochthone starters for camel milk fermentation that represent a challenge process. These results presented as well the probiotic potential for a possible human consumption.

The gut microbes, Enterococcus and Escherichia-Shigella, affect the responses of heart valve replacement patients to the anticoagulant warfarin.

Numerous algorithms based on patient genetic variants have been established with the aim of reducing the risk of GI bleeding and thromboembolism during warfarin administration. However, approximately 35 % of individual warfarin sensitivity still remains unexplained. Few of warfarin algorithms take into account gut microbiota profiles. The identification of certain microbiome will provide new targets and new strategies for reducing the risk of bleeding and thromboembolism during warfarin administration. In this study, we collected plasma and stool samples from 200 inpatients undergoing heart valve replacement (HVR), which were classified as low responder (LR), high responder (HR) and normal responder (NR). Significant differences were observed in the diversity and relative abundance of the gut microbiota among the three groups. The genus Escherichia-Shigella was enriched significantly in the LRs (P = 3.189e-11), while the genus Enterococcus was enriched significantly in the HRs (P = 1.249e-11). The amount of VK2 synthesized by gut microbiota in LR group was much higher than that in HR group (P = 0.005). Whole genome shotgun sequencing indicated that the relative abundance of enzymes and modules associated with VK biosynthesis was significantly higher in LRs than in HRs or NRs. The 12 microbial markers were identified through tenfold cross-validation with a random forest model. The results provided a new microbial diagnostic model that can be used to inform modulation of warfarin dosage on the basis of patient intestinal flora composition.

Parallel Genomics Uncover Novel Enterococcal-Bacteriophage Interactions.

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

Ability of Three Lactic Acid Bacteria to Grow in Sessile Mode and to Inhibit Biofilm Formation of Pathogenic Bacteria.

In this study, we explored the effect of three lactic acid bacteria (LAB), i.e. Enterococcus sp CM9, Enterococcus sp CM18 and Enterococcus faecium H3, and their supernatants, on seven biofilm-forming pathogenic strains isolated from human urinary tract or nose infections. By quantitative biofilm production assay, a strong adherence ability of Enterococcus sp CM9 and Enterococcus sp CM18 was revealed while E. faecium H3 resulted to be moderately adherent. Inhibition tests demonstrated an antimicrobial activity of LAB against pathogens.The presence of cell free supernatant (CFS) of CM9 and CM18 strains significantly decreased the adhesion of S. aureus 10,850, S. epidermidis 4,296 and E. coli FSL24. The CFS of H3 strain was effective against S. epidermidis 4,296 and P. aeruginosa PA1FSL biofilms only. Biofilm formation of K. pneumoniae Kp20FSL, A. baumannii AB8FSL and ESBL+ E. coli FS101570 have not been affected by any CSF while P. aeruginosa PA1FSL biofilm increase in presence of CM9 and CM18 CFS.Confocal Laser Scanning Microscopy revealed that K. pneumoniae Kp20FSL biofilm was inhibited by Enterococcus sp CM9, when grown together.Our results suggest that the LAB strains and/or their bacteriocins can be considered as potential tools to control biofilm formation of some bacterial pathogens.

What Is Wrong with Enterococcal Probiotics?

Enterococci, being the common part of indigenous human microbiota and present in numerous fermented food products and probiotics, are often positioned as health-threatening bacterial pathogens. This makes medical community, especially part of it which is not involved in microbiology research, in an uncertainty regarding the legitimacy of using enterococcal probiotics in medical practice or even consuming them in foods such as cheese or salami. However, the difference between the clinical strains and strains used as probiotics is more than significant. The present paper tries to shed light on enterococci based on the contemporary knowledge of molecular microbiology.

The effect of some environmental conditions on planktonic growth and biofilm formation by some lactic acid bacteria isolated from a local cheese in Turkey.

OBJECTIVE: The purpose of this study was to determine the effect of some environmental conditions (different temperature degrees and pH values, different salt, glucose and lactose concentrations) on the planktonic growth and biofilm formation ability of the lactic acid bacteria (LAB) isolated from a local cheese in Turkey. RESULTS: It was determined that Enterococcus lactis EC61 and Enterococcus faecalis EC41 are the most resistant bacteria to the changing environmental conditions and they can stably maintain their planktonic growth in the pH values of 6.5, 7.0, 7.5, and 8.0; in the salt concentrations of 4% and 6.5%; in the glucose concentration of 0.5%; and in the lactose concentrations of 0.5%, 1.5%, and 2.5%. It was found that all strains had the biofilm formation ability and especially the biofilm formation of Enterococcus lactis EC61 and Enterococcus faecalis EC41 strains significantly increased in the acidic pH values and in the increasing glucose and lactose concentrations, and significantly decreased in the increasing salt concentration. CONCLUSIONS: When considered in terms of LAB potential as a starter culture, specifying the effect of some environmental conditions on the planktonic growth and biofilm formation ability is important for the food industry. As a conclusion, it was determined that lactic acid bacteria, which were previously determined to have some starter culture characteristics, had additional properties on the way to being an starter culture.

Virulence of beta-hemolytic streptococci in infective endocarditis.

BACKGROUND: Streptococci involved in infective endocarditis (IE) primarily comprise alpha- or non-hemolytic streptococci (ANHS). Moreover, beta-hemolytic streptococci (BHS) can be involved, and guidelines recommend the addition of gentamicin for the first 2 weeks of treatment and the consideration of early surgery in such cases. This study compared the morbidity and mortality associated with IE depending on the microorganisms involved (BHS, ANHS, staphylococci, and enterococci). METHODS: We conducted a retrospective observational study between 2012 and 2017 in a single hospital in France. The endpoints were overall in-hospital mortality, 1-year mortality and the occurrence of complications. RESULTS: We analyzed 316 episodes of definite IE including 150 (38%), 96 (25%), 46 (12%), and 24 cases (6%) of staphylococcal, ANHS, enterococcal, and BHS IE, respectively. In-hospital mortality was significantly higher in the staphylococcal (n = 40; 26.7%) and BHS groups (n = 6; 25.0%) than in the ANHS (n = 9; 9.4%) and enterococcal groups (n = 5; 10.9%) (all p < 0.01). The rates of septic shock and cerebral emboli were also higher in the BHS group than in the ANHS group [n = 7 (29.2%) vs. n = 3 (3.1%), p < 0.001; n = 7 (29.2%) vs. n = 12 (12.5%); p = 0.05, respectively]. CONCLUSION: This study confirmed that BHS IE has a more severe prognosis than ANHS IE. The virulence of BHS may be similar to that of staphylococci, justifying increased monitoring of these patients and more 'aggressive' treatments such as early surgery.

Eutrophication and Related Antibiotic Resistance of Enterococci in the Minjiang River, China.

Antimicrobial resistance (AMR) in the aquatic environment has received increasing attention in recent years, and growing eutrophication problems may contribute to AMR in aquatic ecosystems. To evaluate whether and how eutrophication affects AMR, 40 surface water samples were collected from the Minjiang River, Fujian Province, China. Total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (CODMn) were measured as eutrophication factors. Additionally, enterococci species were isolated and their resistance to six common antibiotics was tested. Eutrophication generally showed a trend of increasing with the flow direction of the Minjiang River, with 25 sites (62.5%) having a TN/TP value over the Redfield value (16:1), which indicated that eutrophication in this region was of phosphorus limitation. High nutrition sites were in or near urban areas. Poor quality water was found in the middle and lower reaches of the Minjiang River system. The resistance frequency of 40 enterococci isolates to the six antibiotics tested was as follows: oxytetracycline > erythromycin > ciprofloxacin > chloramphenicol > ampicillin > vancomycin (70, 50, 17.5, 12.5, 2.5, 0%), and the multi-resistant rate reached 50% with eight resistance phenotypes. AMR also increased along the direction of water flow downstream, and most of the sites with the highest AMR were in or near urban areas, as was true for nutrition levels. Positive correlations between AMR and eutrophication factors (TN, TP, and CODMn) were identified using the Pearson's correlation coefficient, and TN/TP generally was negatively related to AMR. These results indicated that eutrophication may induce or selective for resistance of water-borne pathogens to antibiotics, with a high resistance level and a wide resistance spectrum.