Search results for: E. faecium

Authors: Amin Abbasi, Mahdi Asghari Ozma

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Background and Objective:Enterococcus faecium is a normal flora of the human gastrointestinal tract that causes infection in the host body under conditions such as biofilm formation, in which the use of antibiotics causes changes in these pathogenic mechanisms. In this study, we aimed to evaluate comprehensively the changes in E.faecium when exposed to sub-MIC of the gentamicin,especiallythe biofilm formation rate. Materials and Methods: For this study, the keywords "Enterococcus faecium ", "Biofilm", and "Gentamicin" in the databases PubMed, Google Scholar, Sid, and MagIran between 2015 and 2021 were searched, and 14 articles were chosen, studied, and analyzed. Results: Gentamicin significantly had increased biofilm formation in most of the isolates in the studies. Increased expression of the genes (efaA and esp) and proteins involved in biofilm formation and decreased expression of the genes (gelE and cylA) involved in spreading and proteins involved in metabolism and cell division in E.faecium were the most significant cause of the biofilm formation, which were increased in sub-MIC gentamicin-treated situation. Conclusion: Inadequate use of gentamicin intensify biofilm formation of E.faecium, which can make the treatment of infections caused by this bacterium difficult.

Keywords: biofilm, enterococcus faecium, gentamicin, proteome

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Authors: C. Miranda, R. Soares, S. Cunha, L. Ferreira, G. Igrejas, P. Poeta

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Different animal species, including ornamental animals, are reported as potential reservoirs of antibiotic resistance genes. Consequently, these resistances can be disseminated in the environment and transferred to humans. Moreover, multidrug-resistant bacteria reduce the efficacy of antibiotics, as the case of vancomycin-resistant enterococci. Enterococcus faecalis and E. faecium are described as the main nosocomial pathogens. In this line, the aim of this study was to characterize resistance and virulence genes of enterococci species isolated from samples of food supplied to ornamental animals during 2020. The 29 enterococci isolates (10 E. faecalis and 19 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB and ermC), tetracycline (tetL, tetM, tetK and tetO), quinupristin/dalfopristin (vatD and vatE), gentamicin (aac(6’)-aph(2’’)-Ia), chloramphenicol (catA), streptomycin (ant(6)-Ia) and vancomycin (vanA and vanB). The same isolates were also tested for 10 virulence factors genes (esp, ace, gelE, agg, fsr, cpd, cylA, cylB, cylM and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. The most prevalent resistance genes detected in both enterococci species were ermB (n=15, 52%), ermC (n=7, 24%), tetK (n=8, 28%) and vatE (n=4, 14%). Resistance genes for vancomycin were found in ten (34%) E. faecalis and ten (34%) E. faecium isolates. Only E. faecium isolates showed the presence of ermA (n=2, 7%), tetL (n=13, 45%) and ant(6)-Ia gene (n=4, 14%). A total of nine (31%) enterococci isolates were classified as multidrug-resistant bacteria (3 E. faecalis and 6 E. faecium). In three E. faecalis and one E. faecium were not detected resistance genes. The virulence genes detected in both species were agg (n=6, 21%) and cylLL (n=11, 38%). In general, each isolate showed only one of these virulence genes. Five E. faecalis and eleven E. faecium isolates were negative for all analyzed virulence genes. These preliminary results showed the presence of multidrug-resistant enterococci in food supplied to ornamental animals, in particular vancomycin-resistant enterococci. This genotypic characterization reinforces the relevance to public health in the control of antibiotic-resistant bacteria.

Keywords: antibiotic resistance, enterococci, feed, ornamental animals

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Authors: Vandana Bali, Manab B. Bera, Parmjit S. Panesar

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Microbial production of antimicrobials as biopreservatives is the major area of focus nowadays due to increased interest of consumers towards natural and safe preservation of ready to eat food products. The agro-industrial byproduct based medium and optimized process conditions can contribute in economical production of bacteriocins. Keeping this in view, the present investigation was carried out on agro-industrial byproducts utilization for the production of bacteriocin using Enterococcus faecium BS13 isolated from local fermented food. Different agro-industrial byproduct based carbon sources (whey, potato starch liquor, kinnow peel, deoiledrice bran and molasses), nitrogen sources (soya okra, pea pod and corn steep liquor), metal ions and surfactants were tested for optimal bacteriocin production. The effect of various process parameters such as pH, temperature, inoculum level, agitation and time were also tested on bacteriocin production. The optimized medium containing whey, supplemented with 4%corn steep liquor and polysorbate-80 displayed maximum bacteriocin activity with 2% inoculum, at pH 6.5, temperature 40oC under shaking conditions (100 rpm).

Keywords: Bacteriocin, biopreservation, corn steep liquor, Enterococcus faecium, waste utilization, whey

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Authors: Salma H. Abu Hafsa, A. Mendonca, B. Brehm-Stecher, A. A. Hassan, S. A. Ibrahim

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Enterococci are important inhabitants of the animal intestine and are widely used in probiotic products. A probiotic strain is expected to possess several desirable properties in order to exert beneficial effects. Therefore, the objective of this study was to isolate and characterize strains of Enterococcus sp. from chicken cecal and fecal samples to determine potential probiotic properties. Enterococci were isolated from thirty one chicken cecal and fecal samples collected from a local farm. In vitro studies were performed to assess antibacterial activity (using agar well diffusion and cell free supernatant broth technique against Salmonella enterica serotype Enteritidis), susceptibility to antibiotics (amoxycillin, cotrimoxazole, chloramphenicol, cefuroxime, ceftriaxone, ciprofloxacin, and nalidixic acid), survival in acidic conditions, resistance to bile salts, and their survival during simulated gastric juice conditions at pH 2.5. Isolates were identified using biochemical and molecular assays (API 50 CHL, and API ZYM kits followed by 16S rDNA gene sequence analysis). Two strains were identified, of which, Enteroccocus faecium was capable of inhibiting the growth of S. enteritidis and was susceptible to a wide range of antibiotics. In addition, the isolated strain exhibited significant resistance under highly acidic conditions (pH=2.5) for 8 hours and survived well in bile salt at 0.2% for 24 hours and showing ability to survive in the presence of simulated gastric juice at pH 2.5. Based on these results, the E. faecium isolate fulfills some of the criteria to be considered as a probiotic strain and therefore, could be used as a feed additive with good potential for controlling S. enteritidis in chickens. However, in vivo studies are needed to determine the safety of the strain.

Keywords: acid tolerance, antimicrobial activity, Enterococcus faecium, probiotic

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Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

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Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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Authors: Piña-Ronces Laura Gabriela, Reyes-Escogido María de Lourdes

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Exist a large variability in Exopolysaccharides (EPS) produced by LAB depending on carbon source, they have multiple applications in food industry mainly, but they have become important for the health. In this study, we identified EPS-producing strains belonging to the BAL group; they were previously isolated from humans. After that, we extracted and evaluated the antioxidant activity of EPS produced by all strains. Antioxidant activity was determined by DPPH method using ascorbic acid as standard for both comparison and quantification. 31 strains (51.66 %) produced EPS at concentrations between 451 and 1.561 mg/l, 16 of EPS extracted showed antioxidant effect superior to ascorbic acid at the same concentrations. EPS-producing strains were L. plantarum, L. sp and L. fermentum corresponding to Lactobacillus genus and, E. faecium, E. durans, and E. hirae of Enterococcus genus. Antioxidant activity showed by EPS from 3 strains of L. plantarum and 3 strains of E. faecium was different into specie, while the antioxidant activity determined for EPS obtained from the other strains did not show difference at specie level, but was superior to ascorbic acid. EPS produced by L. plantarum and E. hirae had the best activity, it could be considerate for selection them as a possible new alternative for therapy or treatment of diseases related whit oxidative stress. Further studies about biological functions of EPS have to be conducted for new applications in health.

Keywords: oxidative stress, lactic acid bacteria, exopolysaccharides, antioxidant activity

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Authors: Asma Bashir, Neha Farid, Kashif Ali, Kiran Fatima

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Background: Bacteriocins are a subclass of antimicrobial peptides that are becoming extremely important in treatments. It is possible to utilise bacteriocins in place of or in addition to traditional antibiotics. It is possible to treat a variety of infections, including Vancomycin-Resistant Staphylococcus aureus (VRSA) and Methicillin-Resistant Staphylococcus aureus (MRSA), using the targeted spectrum of activity of these microorganisms. Method: This study aimed to examine the efficiency of antibiotics and bacteriocin against VRSA and MRSA. The effects of bacteriocins, such as enterocin KAE01, enterocin KAE03, enterocin KAE05, and enterocin KAE06 isolated from Enterococcus faecium strains, alone and in combination with vancomycin and methicillin antibiotics were examined. The selection technique utilized the minimum inhibitory concentrations (MICs) against Gram-positive indicator strain ATCC 6538 Methicillin-Resistant Staphylococcus aureus (MRSA) and indicator strain KSA 02 Vancomycin-Resistant Staphylococcus aureus (VRSA). Results: We report the isolation and identification of enterocins KAE01, KAE03, KAE05, and KAE06 from food isolates of Enterococcus faecium (KAE01, KAE03, KAE05, and KAE06). After isolating the protein, it was partially purified with ammonium sulphate precipitation and purified with fast protein liquid chromatography (FPLC) procedures. Combinations of enterocin KAE01, 1 citric acid, 1 lactic acid, and microcin J25, 1 reuterin, 1 citric acid, and microcin J25, 1 reuterin, 1 lactic acid shown synergistic benefits (FIC index = 0.5) against Vancomycin-Resistant Staphylococcus aureus (VRSA). In addition, a moderately synergistic (FIC index = 0.75) interaction was seen between pediocin PA-1, 1 citric acid, 1 lactic acid, and reuterin 1 citric acid, 1 lactic acid against L. ivanovii HPB28. In the presence of acids, nisin Z exhibited a modestly synergistic effect (FIC index = 0.625-0.75); however, it exhibited additive effects (FIC index = 1) when combined with reuterin or pediocin PA-1 against L. ivanovii HPB28. The efficacy of synergistic consortiums against Gram-positive bacteria was examined. Conclusion: Combining antimicrobials with various modes of action boosted efficacy and expanded the spectrum of inhibition, particularly against multidrug-resistant pathogens, according to our research.

Keywords: Enterococcus faecium, bacteriocin, antimicrobial resistance, antagonistic activity, vancomycin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus

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Authors: Preeyarach Whisetkhan, Malai Taweechotipatr, Ulisa Pachekrepapol

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Elevated level of blood cholesterol or hypercholesterolemia may lead to atherosclerosis and poses a major risk for cardiovascular diseases. Probiotics play a crucial role in human health, and probiotic bacteria that possesses bile salt hydrolase (BSH) activity can be used to lower cholesterol level of the host. The aim of this study was to investigate whether lactic acid bacteria (LAB) isolated from traditional Thai fermented foods were able to exhibit bile salt hydrolase activity and their use in fermented milk. A total of 28 isolates were tested for BSH activity by plate method on MRS agar supplemented with 0.5% sodium salt of taurodeoxycholic acid and incubated at 37°C for 48 h under anaerobic condition. The results showed that FN1-1 and FN23-3 isolates possessed strong BSH activity. FN1-1 and FN23-3 isolates were then identified for phenotype, biochemical characteristics, and genotype (16S rRNA sequencing). FN1-1 isolate showed 99.92% similarity to Lactobacillus pentosus DSM 20314(T), while FN23-3 isolate showed 99.94% similarity to Enterococcus faecium CGMCC1.2136 (T). Lactobacillus pentosus FN1-1 and Enterococcus faecium FN23-3 were tolerant of pH 3-4 and 0.3 and 0.8% bile. Bacterial count and pH of milk fermented with Lactobacillus pentosus FN1-1 at 37°C and 43°C were investigated. The results revealed that Lactobacillus pentosus FN1-1 was able to grow in milk, which led to decrease in pH level. Fermentation at 37°C resulted in faster growth rate than at 43 °C. Lactobacillus pentosus FN1-1 was a candidate probiotic to be used in fermented milk products to reduce the risk of high-cholesterol diseases.

Keywords: probiotics, lactic acid bacteria, bile salt hydrolase, cholesterol

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Authors: A. Cheriguene, F. Chougrani

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A total of 153 wild lactic acid bacteria were isolated from goat’s milk collected from different areas in Western Algeria. The strains were identified using phenotypical, biochemical and physiological properties. API system and SDS-PAGE technique was also used in identification of the strains. Six genera were found Enterococcus (41.83%), Lactobacillus (29.40%), Lactococcus (19.60%), Leuconostoc (4.57%), Streptococcus thermophilus (3.26%) and Pediococcus (1.30%). The most abundant species were Enterococcus faecium (24 isolates), Enterococcus durans (22 isolates), Lactococcus lactis subsp. lactis (25 isolates), Lactobacillus rhamnosus (09 isolates) and Lactobacillus delbrueckii subsp. bulgaricus (07 isolates). The strains were screened for production and technological properties such as acid production, aminopeptidase activity, autolytic properties, antimicrobial activity and exopolysaccharide production. In general most tested isolates showed a good biomass separation when collected by centrifugation; as for the production of the lactic acid, results revealed that our strains are weakly acidifying; nevertheless, lactococci showed a best acidifying activity compared to lactobacilli. Aminopeptidase activity was also weak in most strains; but, it was generally higher for lactobacilli compared to lactococci, where we recorded 30 units for Lactobacillus delbrueckii subsp. bulgaricus M14. Autolytic activity was generally higher for most strains, more particularly lactobacilli where we recorded values of 71.13% and 70% of autolysis rate respectively in Lactobacillus rhamnosus strains 9S10 and 9S7. Antimicrobial activity was detected in 50% of the isolates, particularly in lactobacilli where 80% of strains tested were able to inhibit the growth of other strains. Two strains could produce exopolysaccharides, E. faecium 8M6 and E. durans 7S8. Some strains were able to maintain two or three technological characteristics together.

Keywords: lactic acid bacteria, technological properties, acidification, aminopeptidase acivity (AP), autolysis

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Authors: C. Miranda, S. Cunha, R. Soares, M. Maia, G. Igrejas, F. Silva, P. Poeta

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The worldwide inappropriate use of antibiotics has increased the emergence of antimicrobial-resistant microorganisms isolated from animals, humans, food, and the environment. To combat this complex and multifaceted problem is essential to know the prevalence in livestock animals and possible ways of transmission among animals and between these and humans. Enterococci species, in particular E. faecalis and E. faecium, are the most common nosocomial bacteria, causing infections in animals and humans. Thus, the aim of this study was to characterize resistance and virulence factors genes among two enterococci species isolated from calf colostrums in Portuguese dairy farms. The 55 enterococci isolates (44 E. faecalis and 11 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB, and ermC), tetracycline (tetL, tetM, tetK, and tetO), quinupristin/dalfopristin (vatD and vatE) and vancomycin (vanB). Of which, 25 isolates (15 E. faecalis and 10 E. faecium) were tested until now for 8 virulence factors genes (esp, ace, gelE, agg, cpd, cylA, cylB, and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. All enterococci isolates showed resistance to erythromicyn and tetracycline through the presence of the genes: ermB (n=29, 53%), ermC (n=10, 18%), tetL (n=49, 89%), tetM (n=39, 71%) and tetK (n=33, 60%). Only two (4%) E. faecalis isolates showed the presence of tetO gene. No resistance genes for vancomycin were found. The virulence genes detected in both species were cpd (n=17, 68%), agg (n=16, 64%), ace (n=15, 60%), esp (n=13, 52%), gelE (n=13, 52%) and cylLL (n=8, 32%). In general, each isolate showed at least three virulence genes. In three E. faecalis isolates was not found virulence genes and only E. faecalis isolates showed virulence genes for cylA (n=4, 16%) and cylB (n=6, 24%). In conclusion, these colostrum samples that were consumed by calves demonstrated the presence of antibiotic-resistant enterococci harbored virulence genes. This genotypic characterization is crucial to control the antibiotic-resistant bacteria through the implementation of restricts measures safeguarding public health. Acknowledgements: This work was funded by the R&D Project CAREBIO2 (Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics - towards innovative theragnostic biomarkers), with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

Keywords: antimicrobial resistance, calf, colostrums, enterococci

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Authors: Jihane Saad, Thomas Lendormi, Caroline Le Marechal, Anne-marie Pourcher, Céline Druilhe, Jean-louis Lanoiselle

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Agricultural anaerobic digestion consists in the conversion of animal slurry and manure into biogas and digestate. They need, however, to be treated at 70 ºC during 60 min before anaerobic digestion according to the European regulation (EC n°1069/2009 & EU n°142/2011). The impact of such heat treatment on the outcome of bacteria has been poorly studied up to now. Moreover, a recent study¹ has shown that enterococci and clostridia are still detected despite the application of such thermal treatment, questioning the relevance of this approach for the hygienisation of digestate. The aim of this study is to establish the heat inactivation kinetics of two species of enterococci (Enterococcus faecalis and Enterococcus faecium) and two species of clostridia (Clostridioides difficile and Clostridium novyi as a non-toxic model for Clostridium botulinum of group III). A pure culture of each strain was prepared in a specific sterile medium at concentration of 10⁴ – 10⁷ MPN / mL (Most Probable number), depending on the bacterial species. Bacterial suspensions were then filled in sterilized capillary tubes and placed in a water or oil bath at desired temperature for a specific period of time. Each bacterial suspension was enumerated using a MPN approach, and tests were repeated three times for each temperature/time couple. The inactivation kinetics of the four indicator bacteria is described using the Weibull model and the classical Bigelow model of first-order kinetics. The Weibull model takes biological variation, with respect to thermal inactivation, into account and is basically a statistical model of distribution of inactivation times as the classical first-order approach is a special case of the Weibull model. The heat treatment at 70 ºC / 60 min contributes to a reduction greater than 5 log10 for E. faecium and E. faecalis. However, it results only in a reduction of about 0.7 log10 for C. difficile and an increase of 0.5 log10 for C. novyi. Application of treatments at higher temperatures is required to reach a reduction greater or equal to 3 log10 for C. novyi (such as 30 min / 100 ºC, 13 min / 105 ºC, 3 min / 110 ºC, and 1 min / 115 ºC), raising the question of the relevance of the application of heat treatment at 70 ºC / 60 min for these spore-forming bacteria. To conclude, the heat treatment (70 ºC / 60 min) defined by the European regulation is sufficient to inactivate non-sporulating bacteria. Higher temperatures (> 100 ºC) are required as far as spore-forming bacteria concerns to reach a 3 log10 reduction (sporicidal activity).

Keywords: heat treatment, enterococci, clostridia, inactivation kinetics

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Authors: M. S. Raza, A. Kapil, Sonu Tyagi, H. Gautam, S. Mohapatra, R. Chaudhry, S. Sood, V. Goyal, R. Lodha, V. Sreenivas, B. K. Das

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Background: Acute bacterial meningitis remains the major cause of mortality and morbidity. More than half of the survivors develop the significant lifelong neurological abnormalities. Diagnosis of the hospital acquired acute bacterial meningitis (HAABM) is challenging as it appears either in the post operative patients or patients acquire the organisms from the hospital environment. In both the situations, pathogens are exposed to high dose of antibiotics. Chances of getting multidrug resistance organism are very high. We have performed this experiment to find out the etiological agents of HAABM and its antibiotics susceptibility pattern. Methodology: A perspective study was conducted at the Department of Microbiology, All India Institute of Medical Sciences, New Delhi. From March 2015 to April 2018 total 400 Cerebro spinal fluid samples were collected aseptically. Samples were processed for cell count, Gram staining, and culture. Culture plates were incubated at 37°C for 18-24 hours. Organism grown on blood and MacConkey agar were identified by MALDI-TOF Vitek MS (BioMerieux, France) and antibiotic susceptibility tests were performed by Kirby Bauer disc diffusion method as per CLSI 2015 guideline. Results: Of the 400 CSF samples processed, 43 (10.75%) were culture positive for different bacteria. Out of 43 isolates, the most prevalent Gram-positive organisms were S. aureus 4 (9.30%) followed by E. faecium 3 (6.97%) & CONS 2 (4.65%). Similarly, E. coli 13 (30.23%) was the commonest Gram-negative isolates followed by A. baumannii 12 (27.90%), K. pneumonia 5 (11.62%) and P. aeruginosa 4(9.30%). Most of the antibiotics tested against the Gram-negative isolates were resistance to them. Colistin was most effective followed by Meropenem and Imepenim for all Gram-negative HAABM isolates. Similarly, most of antibiotics tested were susceptible to S. aureus and CONS. However, E. faecium (100%) were only susceptible to vancomycin and teicoplanin. Conclusion: Hospital acquired acute bacterial meningitis (HAABM) is becoming the emerging challenge as most of isolates are showing resistance to commonly used antibiotics. Gram-negative organisms are emerging as the major player of HAABM. Great care needs to be taken especially in tertiary care hospitals. Similarly, antibiotic stewardship should be followed and antibiotic susceptibility test (AST) should be performed regularly to update the antibiotic patter and to prevent from the emergence of resistance. Updated information of the AST will be helpful for the better management of the meningitis patient.

Keywords: CSF, MALDI-TOF, hospital acquired acute bacterial meningitis, AST

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Authors: Neha Farid

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Due to the abuse of antimicrobial medications in animal feed, the occurrence of multi-drug resistant (MDR) pathogens in foods is currently a growing public health concern on a global scale. MDR infections have the potential to penetrate the food chain by posing a serious risk to both consumers and animals. Food pathogens are those biological agents that have the tendency to cause pathogenicity in the host body upon ingestion. The major reservoirs of foodborne pathogens include food-producing fauna like cows, pigs, goats, sheep, deer, etc. The intestines of these animals are highly condensed with several different types of food pathogens. Bacterial food pathogens are the main cause of foodborne disease in humans; almost 66% of the reported cases of food illness in a year are caused by the infestation of bacterial food pathogens. When ingested, these pathogens reproduce and survive or form different kinds of toxins inside host cells causing severe infections. The genus Listeria consists of gram-positive, rod-shaped, non-spore-forming bacteria. The disease caused by Listeria monocytogenes is listeriosis or gastroenteritis, which induces fever, vomiting, and severe diarrhea in the affected body. Campylobacter jejuni is a gram-negative, curved-rod-shaped bacteria causing foodborne illness. The major source of Campylobacter jejuni is livestock and poultry; particularly, chicken is highly colonized with Campylobacter jejuni. Serious public health concerns include the widespread growth of bacteria that are resistant to antibiotics and the slowing in the discovery of new classes of medicines. The objective of this study is to provide some potential antibacterial activities with certain broad-range antibiotics and our desired bacteriocins, i.e., Enterococcus faecium from specific strains preventing microbial contamination pathways in order to safeguard the food by lowering food deterioration, contamination, and foodborne illnesses. The food pathogens were isolated from various sources of dairy products and meat samples. The isolates were tested for the presence of Listeria and Campylobacter by gram staining and biochemical testing. They were further sub-cultured on selective media enriched with the growth supplements for Listeria and Campylobacter. All six strains of Listeria and Campylobacter were tested against ten antibiotics. Campylobacter strains showed resistance against all the antibiotics, whereas Listeria was found to be resistant only against Nalidixic Acid and Erythromycin. Further, the strains were tested against the two bacteriocins isolated from Enterococcus faecium. It was found that bacteriocins showed better antimicrobial activity against food pathogens. They can be used as a potential antimicrobial for food preservation. Thus, the study concluded that natural antimicrobials could be used as alternatives to synthetic antimicrobials to overcome the problem of food spoilage and severe food diseases.

Keywords: food pathogens, listeria, campylobacter, antibiotics, bacteriocins

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Authors: K. Karapetyan, F. Tkhruni, A. Aghajanyan, T. S. Balabekyan, L. Arstamyan

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Introduction: Globalization of the food supply has created the conditions favorable for the emergence and spread of food-borne and especially dangerous pathogens (EDP) in developing countries. The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the processed foods product. Antimicrobial compounds of lactic acid bacteria (LAB) possess bactericidal or bacteriostatic activity against intestinal pathogens, spoilage organisms and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella. Endemic strains of LAB were isolated. The strains, showing broad spectrum of antimicrobial activity against food spoiling microorganisms, were selected. The genotyping by 16S rRNA sequencing, GS-PCR, RAPD PCR methods showed that they were presented by Lactobacillus rhamnosus109, L.plantarum 65, L.plantarum 66 and Enterococcus faecium 64 species. LAB are deposited in "Microbial Depository Center" (MDC) SPC "Armbiotechnology". Methods: LAB strains were isolated from different dairy products from rural households from the highland regions of Armenia. Serially diluted samples were spread on MRS (Merck, Germany) and hydrolyzed milk agar (1,2 % w/v). Single colonies from each LAB were individually inoculated in liquid MRS medium and incubated at 37oC for 24 hours. Culture broth with biomass was centrifuged at 10,000 g during 20 min for obtaining of cell free culture broth (CFC). The antimicrobial substances from CFC broth were purified by the combination of adsorption-desorption and ion-exchange chromatography methods. Separation of bacteriocins was performed using a HPLC method on "Avex ODS" C18 column. Mass analysis of peptides recorded on the device API 4000 in the electron ionization mode. The spot-on-lawn method on the test culture plated in the solid medium was applied. The antimicrobial activity is expressed in arbitrary units (AU/ml). Results. Purification of CFC broth of LAB allowed to obtain partially purified antimicrobial preparations which contains bacteriocins with broad spectrum of antimicrobial activity. Investigation of their main biochemical properties shown, that inhibitory activity of preparations is partially reduced after treatment with proteinase K, trypsin, pepsin, suggesting a proteinaceous nature of bacteriocin-like substances containing in CFC broth. Preparations preserved their activity after heat treatment (50-121 oC, 20 min) and were stable in the pH range 3–8. The results of SDS PAAG electrophoresis show that L.plantarum 66 and Ent.faecium 64 strains have one bacteriocin (BCN) with maximal antimicrobial activity with approximate molecular weight 2.0-3.0 kDa. From L.rhamnosus 109 two BCNs were obtained. Mass spectral analysis indicates that these bacteriocins have peptide bonds and molecular weight of BCN 1 and BCN 2 are approximately 1.5 kDa and 700 Da. Discussion: Thus, our experimental data shown, that isolated endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms of different taxonomic group, such as Salmonella sp., Esherichia coli, Bacillus sp., L.monocytogenes, Proteus mirabilis, Staph. aureus, Ps. aeruginosa. Obtained results proved the perspectives for use of endemic strains in the preservation of foodstuffs. Acknowledgments: This work was realized with financial support of the Project Global Initiatives for Preliferation Prevention (GIPP) T2- 298, ISTC A-1866.

Keywords: antimicrobial activity, bacteriocins, endemic strains, food safety

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Authors: A. Lardjam, R. Mazid, S. Y. Boudghene, A. Izarouken, Y. Dali, N. Djebli, H. Toumi

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Origanum glandulosum is an aromatic plant, common in Algeria and widely used by local people for its medicinal properties. The essential oil from this plant, which grows in the west of Algeria, was studied to evaluate and determine its antibacterial activity. The extraction of the essential oil was performed by water steam distillation; the yield obtained from the aerial parts (1.78 %) is interesting, its chromatographic profile revealed by TLC showed the presence of phenolic compounds thymol and carvacrol. The evaluation of the activity of the essential oil of Origanum glandulosum on bacterial strains of hospital origin, ATCC, MRB, and HRB, most implicated in nosocomial infections (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus resistant to meticillin, Enterococcus faecium, VA R and R TEC, Acinetobacter baumanii, IMP R and R CAZ, Klebsiella pneumonia carbapenemase-producing) by the method of aromatogramme and micro atmosphere, shows that the antibacterial potency of this oil is very high, expressed by significant inhibition diameters on all strains except Pseudomonas aeruginosa, and low MICs and is characterized by a bactericidal action.

Keywords: antibacterial activity, essential oil, HRB, MBR, nosocomial infections, origanum glandulosum

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Authors: Margita Čanigová, Jana Račková, Miroslav Kročko, Viera Ducková, Vladimíra Kňazovická

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The aim of the study was to test the milk samples in terms of enterococci presence and their counts. Tested samples were as follows: raw cow milk, raw cow milk stored at 10°C for 16 hours and milk pasteurised at 72°C for 15 seconds. The typical colonies were isolated randomly and identified by classical biochemical test - EN-COCCUS test (Lachema, CR) and by PCR. Isolated strains were tested in terms of antibiotic resistance by well diffusion method. Examined antibiotics were: vancomycin (30 μg/disc), gentamicin (120 μg/disc), erythromycin (15 μg/disc), teicoplanine (30 μg/disc), ampicillin (10 μg/disc) and tetracycline (30 μg/disc). Average value of enterococci counts in raw milk cistern samples (n=30) was 8.25 ± 1.37 ×103 CFU/cm3. Storage tank milk samples (n=30) showed an increase (P > 0.05) and average value was 9.16 ± 1.49 × 103 CFU/cm3. Occurrence of enterococci in pasteurized milk (n=30) was sporadic and their counts were mostly below 10 CFU/cm3. Overall, 96 enterococci strains were isolated. In samples of raw cow milk and stored raw cow milk, Enterococcus faecalis was a dominant species (58.1% and 71.7%, respectively), followed by E. faecium (16.3% and 0%, respectively). Enterococcus mundtii, E. casseliflavus, E. durans and E. gallinarum were isolated, too. Resistances to ampicillin, erythromycin, gentamicin, tetracycline and vancomycin were found in 7.29%, 3.13%, 4.00%, 13.54% and 10.42% of isolated enterococci strains, respectively. Resistance to teicoplanine was not found in any isolated strain. All Vancomycin-Resistant Enterococci (VRE) belonged to E. faecalis. Obtained results confirmed that raw milk is a potential risk of enterococci resistant to antibiotics transmission into the food chain.

Keywords: antibiotic resistance, enterococci, milk, biosystems engineering

Procedia PDF Downloads 342

Authors: Abdul Qadir Qadis, Satoru Goya, Minoru Yatsu, Yu-uki Yoshida, Toshihiro Ichijo, Shigeru Sato

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Bacterial probiotics are known to modulate the gut-associated lymphoid and epithelial tissue response to enhance the activities of intestinal and systemic immune system in human and animals. In cattle, the immune-stimulatory effects of probiotics have been evaluated during intestinal disorders. To investigate the effects of probiotic on the function of peripheral blood mononuclear cells, eight healthy Holstein calves (10 ± 3 weeks) were assigned to a 4 × 2 experimental design. The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given no probiotic served as the control. In the treatment group, increases in numbers of CD282+ monocytes, CD3+ T-cells and CD4+, CD8+ and WC1+ γδ T- cell subsets were noted on day 7 post-placement compared to pre-dose day and the control group. Expression of interleukin-6, interferon-gamma and tumor necrosis factor-alpha was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment. The 5-day repeated administration of a bacterial probiotic may enhance cellular immune function in weaned calves.

Keywords: bacterial-probiotic, calf, interleukin, leukocyte

Procedia PDF Downloads 631

Authors: N. Varsamis, M. Gerasimou, P. Christodoulou, S. Mantzoukis, G. Kolliopoulou, N. Zotos

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Purpose: Body fluids (pleural, peritoneal, synovial, pericardial, cerebrospinal) are an important element in the detection of microorganisms. For this reason, it is important to examine them in the Intensive Care Unit (ICU) patients. Material and Method: Body fluids are transported through sterile containers and enriched as soon as possible with Tryptic Soy Broth (TSB). After one day of incubation, the broth is poured into selective media: Blood, Mac Conkey No. 2, Chocolate, Mueller Hinton, Chapman and Saboureaud agar. The above selective media are incubated directly for 2 days. After this period, if any number of microbial colonies are detected, gram staining is performed. After that, the isolated organisms are identified by biochemical techniques in the automated Microscan system (Siemens) and followed by a sensitivity test on the same system using the minimum inhibitory concentration MIC technique. The sensitivity test is verified by Kirby Bauer-based plate test. Results: In 2017 the Laboratory of Microbiology received 60 samples of body fluids from the ICU. More specifically the Microbiology Department received 6 peritoneal fluid specimens, 18 pleural fluid specimens and 36 cerebrospinal fluid specimens. 36 positive cultures were tested. S. epidermidis was identified in 18 specimens, S. haemolyticus in 6, and E. faecium in 12. Conclusions: The results show low detection of microorganisms in body fluid cultures.

Keywords: body fluids, culture, intensive care unit, microorganisms

Procedia PDF Downloads 176

Authors: Theresa Campbell, Camille Bowen-Forbes, William Aalbersberg

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Differences in the sensory properties of two subtly distinct varieties of Rubus rosifolius lead to the examination of their anthocyanin, essential oil and polyphenol profiles. In both cases, notable differences were identified. Pelargonidin-3-rhutinoside (17.2 mg/100 g FW) and Cyanidin-3-glucoside (66.2 mg/100g FW) proved to be the dominant anthocyanins in the red and wine red varieties respectively. Linalool and terpineol were the major constituents of the essential oil from the red variety; however, those of the wine red variety are unidentified. In regard to phenolic compounds, caffeic acid and quercetin were in a higher concentration in the red variety (1.85 and 0.73 mg/100g FW respectively, compared to 1.22 and 0.34 mg/100g FW respectively in the wine red fruits); while ellagic acid and ferulic acid were of a higher concentration in the wine red variety (0.92 and 0.84mg/100g FW respectively, compared to 0.15 and 0.48 mg/100g FW respectively in the red variety). The methanol extract of both fruit varieties showed great antioxidant activity. Analysis of the antimicrobial activity of the fruit extracts against the growth of drug resistant pathogens revealed that they are active against methicillin resistant S. aureus (MRSA), rifampicin resistant S. aureus (RRSA), wild-type S. aureus (WTSA) and vancomycin-resistant Enterococcus faecium (VREF). Activity was also reported against several food-borne pathogens including two strains of E. coli, L. monocytogenes and Enterobacter aerogenes. The cytotoxicity of the various extracts was assessed and the essential oil extracts exhibited superior activity. The phenolic composition and biological activity of the fruits indicate that their consumption is beneficial to health and also that their incorporation into functional foods and nutraceuticals should be considered.

Keywords: phytochemicals, antimicrobial, cytotoxic, Rubus rosifolius

Procedia PDF Downloads 354

Authors: M. Gerasimou, S. Mantzoukis, P. Christodoulou, N. Varsamis, G. Kolliopoulou, N. Zotos

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Purpose: Microbial infection of the blood is a serious condition where bacteria invade the bloodstream and cause systemic disease. In such cases, blood cultures are performed. Blood cultures are a key diagnostic test for intensive care unit (ICU) patients. Material and method: The BacT/Alert system, which measures the production of carbon dioxide with metabolic organisms, is used. The positive result in the BacT/Alert system is followed by culture in the following selective media: Blood, Mac Conkey No 2, Chocolate, Mueller Hinton, Chapman and Sabaureaud agar. Gram staining method was used to differentiate bacterial species. The microorganisms were identified by biochemical techniques in the automated Microscan (Siemens) system and followed by a sensitivity test on the same system using the minimum inhibitory concentration MIC technique. The sensitivity test is verified by a Kirby Bauer-based test. Results: In 2017 the Laboratory of Microbiology received 3347 blood cultures. Of these, 170 came from the ICU. 116 found positive. Of these S. epidermidis was identified in 42, A. baumannii in 27, K. pneumoniae in 12 (4 of these KPC ‘Klebsiella pneumoniae carbapenemase’), S. hominis in 8, E. faecium in 7, E. faecalis in 5, P. aeruginosa in 3, C. albicans in 3, S. capitis in 2, K. oxytoca in 2, P. mirabilis in 2, E. coli in 1, S. intermidius in 1 and S. lugdunensis in 1. Conclusions: The study of epidemiological data and microbial resistance phenotypes is essential for the choice of therapeutic regimen for the early treatment and limitation of multivalent strains, while it is a crucial factor to solve diagnostic problems.

Keywords: blood culture, bloodstream, infection, intensive care unit

Procedia PDF Downloads 123

Authors: Rashad R. Al-Hindi

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The aim of this study was to isolate and identify lactic acid bacteria and evaluate their therapeutic and food preservation importance. Ninety-three suspected lactic acid bacteria (LAB) were isolated from thirteen different raw and fermented milk of indigenous sources in the Kingdom of Saudi Arabia. The identification of forty-six selected LAB strains and genetic relatedness were performed based on 16S rDNA gene sequence comparison. The LAB counts in certain samples were higher under microaerobic than anaerobic conditions. The identified LAB belonged to genera Enterococcus (16 strains), Lactobacillus (9 strains), Weissella (10 strains), Streptococcus (8 strains) and Lactococcus (3 strains). Phylogenetic tree generated from the full-length (~1.6 kb) sequences confirmed previous findings. Utilization of shorter 16S rDNA sequences (~1.0 kb) also discriminated among strains of which V2 region was the most effective. None of the strains exhibited resistance to clinically relevant antibiotics or undesirable hemolytic activity, while they differed in other probiotic characteristics, e.g., tolerance to acidic pH, resistance to bile salt, and antibacterial activity. In conclusion, the isolates Lactobacillus casei MSJ1, Lactobacillus casei Dwan5, Lactobacillus plantarum EyLan2 and Enterococcus faecium Gail-BawZir8 are likely the best probiotic LAB and we speculate that studying the synergistic effects of bacterial combinations might result in the occurrence of more effective probiotic potential. We argue that the raw and fermented milk of animals hosted in Saudi Arabia, especially stirred yogurt (Laban) made from camel milk, are rich in LAB with promising probiotics potential.

Keywords: fermented foods, lactic acid bacteria, probiotics, Saudi Arabia

Procedia PDF Downloads 170

Authors: Naila Safdar, Faria Riasat, Azra Yasmin

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Lychee is an emerging fruit crop in Pakistan. Two famous cultivars of lychee, Gola and Surakhi, were collected from Khanpur Orchard, Pakistan and their whole fruit (including peel, pulp and seed) was investigated for pomological features and therapeutic activities. Both cultivars differ in shape and size with Gola having large size (3.27cm length, 2.36cm width) and more flesh to seed ratio (8.65g). FTIR spectroscopy and phytochemical tests confirmed presence of different bioactive compounds like phenol, flavonoids, quinones, anthraquinones, tannins, glycosides, and alkaloids, in both lychee fruits. Atomic absorption spectroscopy indicated an increased amount of potassium, magnesium, sodium, iron, and calcium in Gola and Surakhi fruits. Small amount of trace metals, zinc and copper, were also detected in lychee fruit, while heavy metals lead, mercury, and nickel were absent. These two lychee cultivars were also screened for antitumor activity by Potato disc assay with maximum antitumor activity shown by aqueous extract of Surakhi seed (77%) followed by aqueous extract of Gola pulp (74%). Antimicrobial activity of fruit parts was checked by agar well diffusion method against six bacterial strains Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis, Bacillus sp. MB083, and Bacillus sp. MB141. Highest antimicrobial activity was shown by methanolic extract of Gola pulp (27mm ± 0.70) and seed (19.5mm ± 0.712) against Enterococcus faecalis. DPPH scavenging assay revealed highest antioxidant activity by aqueous extract of Gola peel (98.10%) followed by n-hexane extract of Surakhi peel (97.73%). Results obtained by reducing power assay also corroborated with the results of DPPH scavenging activity.

Keywords: antimicrobial evaluation, antitumor assay, gola, phytoconstituents, reactive oxygen species, Surakhi

Procedia PDF Downloads 370

Authors: O. Davies, K. Veravalli, P. Panwalkar, M. Tofighi, P. Butterick, B. Healy, A. Mofidi

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Vancomycin resistant enterococcus infection is a condition that usually impacts ICUs, transplant, dialysis, and cancer units, often as a nosocomial infection. After an outbreak in the acute trauma and orthopaedic unit in Morriston hospital, we aimed to access the conditions that predispose VRE infections in our unit. Thirteen cases of VRE infection and five cases of VRE colonisations were identified in patients who were treated for orthopaedic care between 1/1/2020 and 1/11/2021. Cases were reviewed to identify predisposing factors, specifically looking at age, presenting condition and treatment, presence of infection and antibiotic care, active haemo-oncological condition, long term renal dialysis, previous hospitalisation, VRE predisposition, and clearance (PREVENT) scores, and outcome of care. The presenting condition, treatment, presence of postoperative infection, VRE scores, age was compared between colonised and the infected cohort. VRE type in both colonised and infection group was Enterococcus Faecium in all but one patient. The colonised group had the same age (T=0.6 P>0.05) and sex (2=0.115, p=0.74), presenting condition and treatment which consisted of peri-femoral fixation or arthroplasty in all patients. The infected group had one case of myelodysplasia and four cases of chronic renal failure requiring dialysis. All of the infected patient had sustained an infected complication of their fracture fixation or arthroplasty requiring reoperation and antibiotics. The infected group had an average VRE predisposition score of 8.5 versus the score of 3 in the colonised group (F=36, p<0.001). PREVENT score was 7 in the infected group and 2 in the colonised group(F=153, p<0.001). Six patients(55%) succumbed to their infection, and one VRE infection resulted in limb loss. In the orthopaedic cohort, VRE infection is a nosocomial condition that has peri-femoral predilection and is seen in association with immunosuppression or renal failure. The VRE infection cohort has been treated for infective complication of original surgery weeks prior to VRE infection. Based on our findings, we advise avoidance of infective complications, change of practice in use of antibiotics and use radical surgery and surveillance for VRE infections beyond infective precautions. PREVENT score shows that the infected group are unlikely to clear their VRE in the future but not the colonised group.

Keywords: surgical site infection, enterococcus, orthopaedic surgery, vancomycin resistance

Procedia PDF Downloads 111

Authors: Latifat Afolake Ogunfolabo, Hakeem Babafemi Ogunfolabo

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The gastrointestinal tract is densely populated with micro-organism which closely and intensively interacts with the host and ingested feed. Food borne infections are some of the major international challenges that lead to high mortality and also, antimicrobial resistance, which has been classified as a serious threat by World Health Organization. Samples of slaughtered cattle and goats intestines were collected and standard culture methods were used for bacteria isolation and identification. Minimum inhibitory concentration of commonly used antibiotic using modification of the disk diffusion method was carried out on isolates. The samples cultured were all positive to Pseudomonas aeruginosa (95% and 90%), Escherichia coli (85%), Salmonella typhi (70% and 60%), Staphylococcus aureus (75%and 100%), Micrococcus luteus (55% and35%), Bacillus macerans (60% and 5%), Bacillus cereus (25% and 20%), Clostridium perfringens (20% and 5%), Micrococcus varians (20% and 5%), Bacillus subtilis (25% and 5%), Streptococcus faecalis (40% and 25%) and Streptococcus faecium (15% and 10%) in goat and cattle respectively. Also, Proteus mirabilis (40%), Micrococcus luteus (35%), Proteus vulgaris (30%), Klebsiella aerogenes(15%) were isolated from cattle. The total coliform (13.55 x10⁵cfu/gm ± 1.77) and (20.30 x10⁵cfu/gm ± 1.27) counts were significantly higher than the total bacteria count (8.3 x10⁵cfu/gm ± 1.41) and (16.60 x10⁵cfu/gm ±0.49) for goat and cattle respectively. Selected Bacteria count of isolates showed that Staphylococcus aureus had the highest significant value (6.9 x10⁵cfu/gm ± 0.57) and (16.80 x10⁵cfu/gm ± 0.57) Escherichia coli (4.60 x10⁵cfu/gm ± 0.42) and (7.05 x10⁵cfu/gm ± 0.64) while the lowest significant value was obtained in Salmonella/Shigella (1.7 x10⁵cfu/gm ± 0.00) and (1.5 x10⁵cfu/gm ± 0.00) for goat and cattle respectively. Susceptibility of bacteria isolated from slaughtered goat and cattle intestine to commonly used antibiotics showed that the highest statistical significant value for zone of inhibition for goat was obtained for Ciprofloxacin (30.00 ± 2.25, 23.75 ± 2.49, 17.17 ± 1.40) followed by Augmentin (28.33 ± 1.22, 21. 83 ± 2.44, 16.67 ± 1.49), Erythromycin (27.75 ±1.48, 20.25 ± 1.29, 16.67 ± 1.26) while the lowest values were obtained for Ofloxacin (27.17 ± 1.89, 21.42 ± 2.19, 16.83 ± 1.26) respectively and values obtained for cattle are Ciprofloxacin (30.64 ± 1.6, 25.79 ± 1.76, 8.07 ± 11.49) followed by Augmentin (28.29 ± 1.33, 22.64 ± 1.82, 17.43 ± 1.55) Ofloxacin (26.57 ± 2.02, 20.79 ± 2.75, 16.21 ± 1.19) while the lowest values were obtained for Erythromycin (26.64 ± 1.49, 20.29 ± 1.49, 16.29 ± 1.33) at different dilution factor (10⁻¹, 10⁻², 10⁻³) respectively. The isolates from goat and cattle were all susceptible to Augmentin at the three different dilution factors. Some goat isolates are intermediate to Ciprofloxacin and Erythromycin at 10⁻² and 10⁻³, while resistance to Ciprofloxacin at 10⁻³ dilution factor. Ciprofloxacin and Ofloxacin at the dilution factors of 10⁻³ and 10⁻¹ for some cattle isolate and resistance were observed for Ofloxacin and Erythromycin at dilution of 10⁻³. These results indicate the susceptibilities and the antimicrobial resistance to commonly used antibiotic.

Keywords: antibiotic susceptibility, bacteria, cattle, goat, identification

Procedia PDF Downloads 90

Authors: A. M. Isakhanyan, F. N. Tkhruni, N. N. Yakimovich, Z. I. Kuvaeva, T. V. Khachatryan

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Introduction: At present, the simbiotics based on different genera and species of lactic acid bacteria (LAB) and yeasts are used. One of the basic properties of probiotics is presence of antimicrobial activity and therefore selection of LAB and yeast strains for their co-cultivation with the aim of increasing of the activity is topical. Since probiotic yeast and bacteria have different mechanisms of action, natural synergies between species, higher viability and increasing of antimicrobial activity might be expected from mixing both types of probiotics. Endemic strains of LAB Enterococcus faecium БТK-64, Lactobaccilus plantarum БТK-66, Pediococcus pentosus БТK-28, Lactobacillus rhamnosus БТK-109 and Kluyveromyces lactis БТX-412, Saccharomycopsis sp. БТX- 151 strains of yeast, with probiotic properties and hight antimicrobial activity, were selected. Strains are deposited in "Microbial Depository Center" (MDC) SPC "Armbiotechnology". Methods: LAB and yeast strains were isolated from different dairy products from rural households of Armenia. The genotyping by 16S rRNA sequencing for LAB and 26S RNA sequencing for yeast were used. Combined cultivation of LAB and yeast strains was carried out in the nutrient media on the basis of milk whey, in anaerobic conditions (without shaker, in a thermostat at 37oC, 48 hours). The complex preparations were obtained by purification of cell free culture broth (CFC) broth by the combination of ion-exchange chromatography and gel filtration methods. The spot-on-lawn method was applied for determination of antimicrobial activity and expressed in arbitrary units (AU/ml). Results. The obtained data showed that at the combined growth of bacteria and yeasts, the cultivation conditions (medium composition, time of growth, genera of LAB and yeasts) affected the display of antimicrobial activity. Purification of CFC broth allowed obtaining partially purified antimicrobial complex preparation which contains metabiotics from both bacteria and yeast. The complex preparation inhibited the growth of pathogenic and conditionally pathogenic bacteria, isolated from various internal organs from diseased animals and poultry with greater efficiency than the preparations derived individually alone from yeast and LAB strains. Discussion. Thus, our data shown perspectives of creation of a new class of antimicrobial preparations on the basis of combined cultivation of endemic strains of LAB and yeast. Obtained results suggest the prospect of use of the partially purified complex preparations instead antibiotics in the agriculture and for food safety. Acknowledgments: This work was supported by the RA MES State Committee of Science and Belarus National Foundation for Basic Research in the frames of the joint Armenian - Belarusian joint research project 13РБ-064.

Keywords: co-cultivation, antimicrobial activity, biosafety, metabiotics, lactic acid bacteria, yeast

Procedia PDF Downloads 306

Authors: Hela Ghali, Sihem Ben Fredj, Mohamed Ben Rejeb, Sawssen Layouni, Salwa Khefacha, Lamine Dhidah, Houyem Said Laatiri

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Background: Antimicrobial resistance is a growing threat to public health and motivates to improve prevention and control programs both at international (WHO) and national levels. Despite their low pathogenicity, vancomycin-resistant enterococci (VRE) are common nosocomial pathogens in several countries. The high potential for transmission of VRE between patients and the threat to send its resistance genes to other bacteria such as staphylococcus aureus already resistant to meticilin, justify strict control measures. Indeed, in Europe, the proportion of Enterococcus faecium responsible for invasive infections, varies from 1% to 35% in 2011 and less than 5% were resistant to vancomycin. In addition, it represents the second cause of urinary tract and wound infections and the third cause of nosocomial bacteremia in the United States. The nosocomial outbreaks of VRE have been mainly described in intensive care services, hematology-oncology and haemodialysis. An epidemic of VRE has affected our hospital and the objective of this work is to describe the measures put in place. Materials/Methods: Following the alert given by the service of plastic surgery concerning a patient carrier of VRE, a team of the prevention and healthcare security service (doctor + technician) made an investigation. A review of files was conducted to draw the synoptic table and the table of cases. Results: By contacting the microbiology laboratory, we have identified four other cases of VRE and who were hospitalized in Medical resuscitation department (2 cases, one of them was transferred to the Physical rehabilitation department), and Nephrology department (2 cases). The visit has allowed to detect several malfunctions in professional practice. A crisis cell has allowed to validate, coordinate and implement control measures following the recommendations of the Technical Center of nosocomial infections. In fact, the process was to technically isolate cases in their sector of hospitalization, to restrict the use of antibiotics, to strength measures of basic hygiene, and to make a screening by rectal swab for both cases and contacts (other patients and health staff). These measures have helped to control the situation and no other case has been reported for a month. 2 new cases have been detected in the intensive care unit after a month. However, these are short-term strategies, and other measures in the medium and long term should be taken into account in order to face similar outbreaks. Conclusion: The efforts to control the outbreak were not efficient since 2 new cases have been reported after a month. Therefore, a continuous monitoring in order to detect new cases earlier is crucial to minimize the dissemination of VRE.

Keywords: hospitals, nosocomial infection, outbreak, vancomycin-resistant enterococci

Procedia PDF Downloads 272

Authors: S. H. Gasa, L. Singh, B. Pillay, A. O. Olaniran

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Wastewater treatment plants (WWTPs) have been implicated as the leading reservoir for antibiotic resistant bacteria (ARB), including Enterococci spp. and antibiotic resistance genes (ARGs), worldwide. Enterococci are a group of clinically significant bacteria that have gained much attention as a result of their antibiotic resistance. They play a significant role as the principal cause of nosocomial infections and dissemination of antimicrobial resistance genes in the environment. The main objective of this study was to ascertain the role of WWTPs in Durban, South Africa as potential reservoirs for antibiotic resistant Enterococci (ARE) and their related ARGs. Furthermore, the antibiogram and resistance gene profile of Enterococci species recovered from treated wastewater effluent and receiving surface water in Durban were also investigated. Using membrane filtration technique, Enterococcus selective agar and selected antibiotics, ARE were enumerated in samples (influent, activated sludge, before chlorination and final effluent) collected from two WWTPs, as well as from upstream and downstream of the receiving surface water. Two hundred Enterococcus isolates recovered from the treated effluent and receiving surface water were identified by biochemical and PCR-based methods, and their antibiotic resistance profiles determined by the Kirby-Bauer disc diffusion assay, while PCR-based assays were used to detect the presence of resistance and virulence genes. High prevalence of ARE was obtained at both WWTPs, with values reaching a maximum of 40%. The influent and activated sludge samples contained the greatest prevalence of ARE with lower values observed in the before and after chlorination samples. Of the 44 vancomycin and high-level gentamicin-resistant isolates, 11 were identified as E. faecium, 18 as E. faecalis, 4 as E. hirae while 11 are classified as “other” Enterococci species. High-level aminoglycoside resistance for gentamicin (39%) and vancomycin (61%) was recorded in species tested. The most commonly detected virulence gene was the gelE (44%), followed by asa1 (40%), while cylA and esp were detected in only 2% of the isolates. The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(3')-IIIa, and ant(6')-Ia detected in 43%, 45% and 41% of the isolates, respectively. Positive correlation was observed between resistant phenotypes to high levels of aminoglycosides and presence of all aminoglycoside resistance genes. Resistance genes for glycopeptide: vanB (37%) and vanC-1 (25%), and macrolide: ermB (11%) and ermC (54%) were detected in the isolates. These results show the need for more efficient wastewater treatment and disposal in order to prevent the release of virulent and antibiotic resistant Enterococci species and safeguard public health.

Keywords: antibiogram, enterococci, gentamicin, vancomycin, virulence signatures

Procedia PDF Downloads 188

Authors: António Inês, Ana Guimarães, José Maria, Vânia Laranjo, Armando Venâncio, Luís Abrunhosa

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Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP-PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 μg/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 μm pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Keywords: bio-detoxification, lactic acid bacteria, mycotoxins, food and feed

Procedia PDF Downloads 534

Authors: Z. Yousefniayejahr, S. Bolognini, A. Bonini, C. Campobasso, N. Poma, F. Vivaldi, M. Di Luca, A. Tavanti, F. Di Francesco

Abstract:

Pathogenic bacteria represent a threat to healthcare systems and the food industry, because their rapid detection remains challenging. Electrochemical biosensors are gaining prominence as a novel technology for the detection of pathogens due to intrinsic features such as low cost, rapid response time, and portability, which make them a valuable alternative to traditional methodologies. These sensors use biorecognition elements that are crucial for the identification of specific bacteria. In this context, bacteriophages are promising tools for their inherent high selectivity towards bacterial hosts, which is of fundamental importance when detecting bacterial pathogens in complex biological samples. In this study, we present the development of a low-cost and portable sensor based on the Zeno phage for the rapid detection of Staphylococcus aureus. Screen-printed gold electrodes functionalized with the Zeno phage were used, and electrochemical impedance spectroscopy was applied to evaluate the change of the charge transfer resistance (Rct) as a result of the interaction with S. aureus MRSA ATCC 43300. The phage-based biosensor showed a linear range from 101 to 104 CFU/mL with a 20-minute response time and a limit of detection (LOD) of 6 CFU/mL under physiological conditions. The biosensor’s ability to recognize various strains of staphylococci was also successfully demonstrated in the presence of clinical isolates collected from different geographic areas. Assays using S. epidermidis were also carried out to verify the species-specificity of the phage sensor. We only observed a remarkable change of the Rct in the presence of the target S. aureus bacteria, while no substantial binding to S. epidermidis occurred. This confirmed that the Zeno phage sensor only targets S. aureus species within the genus Staphylococcus. In addition, the biosensor's specificity with respect to other bacterial species, including gram-positive bacteria like Enterococcus faecium and the gram-negative bacterium Pseudomonas aeruginosa was evaluated, and a non-significant impedimetric signal was observed. Notably, the biosensor successfully identified S. aureus bacterial cells in a complex matrix such as a nasal swab, opening the possibility of its use in a real-case scenario. We diluted different concentrations of S. aureus from 108 to 100 CFU/mL with a ratio of 1:10 in the nasal swap matrices collected from healthy donors. Three different sensors were applied to measure various concentrations of bacteria. Our sensor indicated high selectivity to detect S. aureus in biological matrices compared to time-consuming traditional methods, such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and radioimmunoassay (RIA), etc. With the aim to study the possibility of using this biosensor to address the challenge associated with pathogen detection, ongoing research is focused on the assessment of the biosensor’s analytical performances in different biological samples and the discovery of new phage bioreceptors.

Keywords: electrochemical impedance spectroscopy, bacteriophage, biosensor, Staphylococcus aureus

Procedia PDF Downloads 25

Authors: Sonia A. Stoica, Lexi Frankel, Amalia Ardeljan, Selena Rashid, Ali Yasback, Omar Rashid

Abstract:

Introduction Enterococcus is a vast genus of lactic acid bacteria, gram-positivecocci species. They are common commensal organisms in the intestines of humans: E. faecalis (90–95%) and E. faecium (5–10%). Rare groups of infections can occur with other species, including E. casseliflavus, E. gallinarum, and E. raffinosus. The most common infections caused by Enterococcus include urinary tract infections, biliary tract infections, subacute endocarditis, diverticulitis, meningitis, septicemia, and spontaneous bacterial peritonitis. The treatment for sensitive strains of these bacteria includes ampicillin, penicillin, cephalosporins, or vancomycin, while the treatment for resistant strains includes daptomycin, linezolid, tygecycline, or streptogramine. Enterococcus faecalis CECT7121 is an encouraging nominee for being considered as a probiotic strain. E. faecalis CECT7121 enhances and skews the profile of cytokines to the Th1 phenotype in situations such as vaccination, anti-tumoral immunity, and allergic reactions. It also enhances the secretion of high levels of IL-12, IL-6, TNF alpha, and IL-10. Cytokines have been previously associated with the development of cancer. The intention of this study was to therefore evaluate the correlation between Enterococcus infections and incidence of bone carcinoma. Methods A retrospective cohort study (2010-2019) was conducted through a Health Insurance Portability and Accountability Act (HIPAA) compliant national database and conducted using International Classification of Disease (ICD) 9th and 10th codes for bone carcinoma diagnosis in a previously Enterococcus infected population. Patients were matched for age range and Charlson Comorbidity Index (CCI). Access to the database was granted by Holy Cross Health for academic research. Chi-squared test was used to assess statistical significance. Results A total number of 17,056 patients was obtained in Enterococcus infected group as well as in the control population (matched by Age range and CCI score). Subsequent bone carcinoma development was seen at a rate of 1.07% (184) in the Enterococcal infectious group and 3.42% (584) in the control group, respectively. The difference was statistically significant by p= 2.2x10-¹⁶, Odds Ratio = 0.355 (95% CI 0.311 - 0.404) Treatment for enterococcus infection was analyzed and controlled for in both enterococcus infected and noninfected populations. 78 out of 6,624 (1.17%) patients with a prior enterococcus infection and treated with antibiotics were compared to 202 out of 6,624 (3.04%) patients with no history of enterococcus infection (control) and received antibiotic treatment. Both populations subsequently developed bone carcinoma. Results remained statistically significant (p<2.2x10-), Odds Ratio=0.456 (95% CI 0.396-0.525). Conclusion This study shows a statistically significant correlation between Enterococcus infection and a decreased incidence of bone carcinoma. The immunologic response of the organism to Enterococcus infection may exert a protecting mechanism from developing bone carcinoma. Further exploration is needed to identify the potential mechanism of Enterococcus in reducing bone carcinoma incidence.

Keywords: anti-tumoral immunity, bone carcinoma, enterococcus, immunologic response

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