Production of enriched in B vitamins biomass of Yarrowia lipolytica grown in biofuel waste.

Yarrowia lipolytica as an oleaginous yeast is capable of growing in various non-conventional hydrophobic substrate types, especially industrial wastes. In this study, the content of thiamine (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), biotin (vitamin B7) and folic acid (vitamin B9) in the wet biomass of Y. lipolytica strains cultivated in biofuel waste (SK medium), compared to the standard laboratory YPD medium, was assessed. Additionally, the biomass of Y. lipolytica A-101 grown in biofuel waste (SK medium) was dried and examined for B vitamins concentration according to the recommended microbial methods by AOAC Official Methods. The mean values of these vitamins per 100 g of dry weight of Y. lipolytica grown in biofuel waste (SK medium) were as follows: thiamine 1.3 mg/100 g, riboflavin 5.3 mg/100 g, pyridoxine 4.9 mg/100 g, biotin 20.0 µg/100 g, and folic acid 249 µg/100 g. We have demonstrated that the dried biomass is a good source of B vitamins which can be used as nutraceuticals to supplement human diet, especially for people at risk of B vitamin deficiencies in developed countries. Moreover, the biodegradation of biofuel waste by Y. lipolytica is desired for environmental protection.

Bacterial aetiology of chronic otitis media with effusion in children - risk factors.

BACKGROUND: Otitis media with effusion (OME) may occur spontaneously because of poor Eustachian tube function or as an inflammatory response following AOM. Bacterial involvement in OME has been widely reported, with various available methods to identify pathogens from middle ear effusion, including traditional culture methods and polymerase chain reaction (PCR). OBJECTIVES: The primary goal of this study was to evaluate the bacteriological profile of middle ear effusion in OME. Risk factors of the bacterial OME aetiology were also identified. METHODS: Middle ear effusions (MEF) from 50 children, aged 2-8 years, diagnosed by ENT and undergoing routine tympanostomy tube placement were collected. MEF samples were streaked on standard microbiological media. Next, DNA was isolated from MEF samples and analysed with multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Alloiococcus otitidis. RESULTS: In multiplex PCR assay 37 (74%) of 50 children were positive for at least one of the four microorganisms. In 27.0% positive children multiple bacterial pathogens were identified. A. otitidis was the most frequently identified in positive MEF children (59.5%). By multiplex PCR, H. influenzae, S. pneumoniae and M. catarrhalis were detected in 24, 18 and 8% of OME patients, respectively. There was significant association between bilateral infection and H. influenzae aetiology of OME. CONCLUSIONS: Overall we found OME predominantly a single otopathogen infection caused mainly by A. otitidis, which is difficult in identification using standard culture method, ahead to S. pneumoniae and H. influenzae. However, one third of MEF samples had multiple bacterial pathogens.

Statistical evaluation of growth parameters in biofuel waste as a culture medium for improved production of single cell protein and amino acids by Yarrowia lipolytica.

Yarrowia lipolytica is an oleaginous yeast species with the ability to grow on a number of substrates types, especially industrial wastes. This paper concerns the statistical optimization of fermentation parameters and media to ensure consistent and improved Y. lipolytica protein production. A strain of Y. lipolytica A-101 was observed to be proficient in producing single cell protein, amino acids, and vitamin B12 while utilizing biofuel waste instead of a complete YPD medium for yeast growth. A fractional fractal design experiment was then applied, and the two fermentation parameters of temperature and pH were recognized to have a significant effect on the protein and amino acid production. Subsequently, the response surface methodology with a three-level complete factorial design was employed to optimize these influential parameters. Therefore, five different measuring systems were utilized to construct a quadratic model and a second-order polynomial equation. Optimal levels of parameters were then obtained by analysis of the model and the numerical optimization method. When the Y. lipolytica A-101 was cultivated at optimized pH (5.0) using biofuel waste as a medium, the protein concentration was increased to 8.28-a 44% enhancement as compared to the original (3.65). This study has thus demonstrated a beneficial way to cultivate Y. lipolytica A-101 on biofuel waste for enhanced production of single cell protein and amino acids for use in human diet and in animal feed.

New biological activity of the polysaccharide fraction from Cantharellus cibarius and its structural characterization.

One of the most commonly consumed mushrooms in Europe is Cantharellus cibarius, also known as chanterelles. Therefore, the investigation of the pro-health properties of crude polysaccharides from this species was performed. The obtained results indicate that the polysaccharide fraction from C. cibarius inhibits the activity of both COX-1 and COX-2. Moreover, the prebiotic potential was revealed in relation to Lactobacillus strains. Crude polysaccharides were found to inhibit the proliferation of colon cancer cells with the simultaneous absence of toxicity towards normal cells. The purification and structural characterization of the examined polysaccharide fraction from C. cibarius indicates that it consists of one monosaccharide in the repeating unit →6)-α-D-Manp-(1→. The presented activities indicate for the first time that this edible mushroom possesses interesting chemopreventive potential, especially against colon cancer.

The preliminary study of prebiotic potential of Polish wild mushroom polysaccharides: the stimulation effect on Lactobacillus strains growth.

PURPOSE: According to the vast body of evidence demonstrating that the intestinal microbiota is undoubtedly linked with overall health, including cancer risk, searching for functional foods and novel prebiotic influencing on beneficial bacteria is necessary. The present study aimed to investigate the potential of polysaccharides from 53 wild-growing mushrooms to stimulate the growth of Lactobacillus acidophilus and Lactobacillus rhamnosus and to determine the digestibility of polysaccharide fractions. METHODS: Mushroom polysaccharides were precipitated with ethanol from aqueous extracts. Determination of growth promoting activity of polysaccharides was performed in U-shaped 96-plates in an ELISA reader in relation to the reference strain of L. acidophilus and two clinical strains of L. rhamnosus. The digestibility of mushroom polysaccharides was investigated in vitro by exposing them to artificial human gastric juice. RESULTS: Obtained results revealed that fungal polysaccharides stimulate the growth of Lactobacillus strains stronger than commercially available prebiotics like inulin or fructooligosaccharides. Moreover, selected polysaccharides were subjected to artificial human gastric juice and remain undigested in more than 90%. CONCLUSION: Obtained results indicate that mushroom polysaccharides are able to pass through the stomach unchanged, reaching the colon and stimulating the growth of beneficial bacteria. Majority of 53 polysaccharide fractions were analysed for the first time in our study. Overall, our findings suggest that polysaccharide fractions from edible mushrooms might be useful in producing functional foods and nutraceuticals.

First report of a Staphylococcus caprae isolated from middle ear fluid of an infant with recurrent acute otitis media.

Staphylococcus caprae was originally isolated from goat milk. This uncommon coagulase-negative staphylococcus, usually associated with animals, has only infrequently been detected in human clinical specimens. Its association with acute otitis media has not been demonstrated so far. The study reports the first isolation of S. caprae from the middle ear fluid of a 12-month-old infant with recurrent, bilateral acute otitis media. Biochemical traits and susceptibility pattern of the isolated strain are also presented.

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis.

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus. The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance:ermA, ermB, ermC,msrA, mphC, and linA/A'. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistantS. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination wasermC, mphC, linA/A'. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus. The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance:ermA, ermB, ermC,msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistantS. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination wasermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

ANTI-ADHESIVE AND ANTI-BIOFILM ACTIVITIES IN VITRO OF LINEZOLID, VANCOMYCIN, TIGECYCLINE AND DAPTOMYCIN AGAINST STAPHYLOCOCCUS HAEMOLYTICUS.

Biofilm may be formed on wide variety of surfaces, including indwelling medical devices, leading to several infectious diseases, e.g., bacteremia and sepsis. The most,important pathogens related with infections associated with medical devices are coagulase-negative staphylococci, including Staphylococcus haeinolyticus - bacterial species which express quite often the multidrug resistance. The four clinical multiresistant and methicillin-resistant S. haenzolyticus were included in the present study. The evaluation of drug susceptibility was performed by using disc-diffusion method and broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The biofilm formation on the Nelaton catheter and the effect of linezolid, vancomycin, tigecycline and daptomycin on the biofilm formation and disruption of mature structure was based on the method with TTC (2,3,5-triphenyltetrazolium chloride). The adhesion process of S. haenzolyticus to the Nelaton catheter was inhibited by antibiotics, as follows: line-zolid at concentration 0.25-0.5 x MIC, vancomycin - concentration 0.5 x MIC, tigecycline - concentration 0.25-4 x MIC and daptomycin - concentration 0.06-1 x MIC, depending on the isolate. Linezolid inhibited the biofilm formation at concentration between 0.5-1 x MIC, vancomycin - 1-2 x MIC, tigecycline - 0.5-4 x MIC and daptomycin - 0.06-2 x MIC. The concentration of linezolid eradicating the mature biofilm was found to be 1-2 x MIC, vancomycin - 2-8 x MIC, tigecycline - 2-4 x MIC and daptomycin - 0.06-2 x MIC. The most active antibiotic against S. haentolyticus biofilm formation and disruption of mature structure seems to be daptomycin.

Empirical validation of Polish guidelines for the management of acute streptococcal pharyngitis in children.

OBJECTIVE: Group A Streptococcus (GAS) pharyngitis is currently the only commonly occurring form of acute pharyngitis for which antibiotic therapy is definitely indicated. Polish guidelines advocate the use of modified Centor score (MCS) to assess the probability of GAS pharyngitis. They advise performing throat culture or rapid antigen detection test (RADT) in children with score 2-3 in MCS and treating with antibiotic only those in whom GAS was detected. Negative RADT results should be confirmed by culture. In children with score 4, the guidelines allow to introduce empiric antibiotic therapy. Phenoxymethyl penicillin is recommended as a drug of choice to treat GAS pharyngitis. The aim of our study was to evaluate the accuracy of strategy recommended by Polish guidelines in identifying those children with acute pharyngitis who require antibiotic treatment. Hence, diagnostic values of score 4 in MCS and RADT were assessed using throat culture as a reference standard. Phenoxymethyl penicillin efficacy in GAS eradication and prevention of post-streptococcal complications were estimated as well. METHODS: Ninety children between 2 and 15 years of age with acute pharyngitis symptoms suggesting GAS etiology (MCS ≥ 2), participated in our study. At the initial visit MCS was evaluated and two throat swabs were collected to perform RADT and culture. In children with GAS pharyngitis treated with penicillin, microbiological cure was assessed by performing two control throat cultures. Next, children were under observation for 3 months. RESULTS: Positive predictive value of score 4 in MCS turned out to be 48.05% (95% CI: 36.5-59.7%). RADT sensitivity, specificity and diagnostic accuracy proved to be 100%, 96%, and 98%, respectively. GAS eradication rate in children treated with penicillin turned out to be 92.5%. No post-streptococcal sequelae occurred in any child in 3-month observation. CONCLUSIONS: Empiric antibiotic therapy in children with score 4 in MCS will result in significant overtreatment of those with nonstreptococcal pharyngitis. New generation RADT diagnostic value in GAS detection proved to be equivalent to that of culture, which obviates the need of backup culture in children with negative RADT results. Phenoxymethyl penicillin revealed high eradication efficacy and proved to prevent post-streptococcal sequelae in children with acute GAS pharyngitis.

A comparative analysis of phenotypic and genotypic methods for the determination of the biofilm-forming abilities of Staphylococcus epidermidis.

The collection of 146 Staphylococcus epidermidis strains isolated from the nasopharynx of lung cancer patients has been studied for the ability of slime secretion and biofilm formation using the Congo red agar (CRA) test and the microtiter plate (MtP) method, respectively. The prevalence of the icaAD and the aap genes was also analyzed. Some isolates (35.6%) were biofilm positive by the MtP method, while 58.9% of isolates exhibited a slime-positive phenotype by the CRA test. The sensitivities of the CRA test evaluated using the MtP method as a gold standard of biofilm production were 73.1%, 97.3% and 13.3% for all the strains screened, ica-positive and ica-negative strains, respectively. The genotype ica(+)aap(+) was correlated with a strong biofilm-producer phenotype. Interestingly, some of the ica(-)aap(-) isolates could also form a biofilm. The correlation between the presence of icaAD genes and the biofilm-positive phenotype by the MtP method as well as slime production by the CRA test was statistically significant (P<0.0001). However, some S. epidermidis strains possess the potential ability of ica-independent biofilm formation; thus, further studies are needed to determine reliable, short-time criteria for an in vitro assessment of biofilm production by staphylococci.

Bacteriostatic or bactericidal effect of linezolid against multiresistant Streptococcus pneumoniae.

The nasopharynx plays a critical role as the reservoir of Streptococcus pneumoniae, including drug-resistant strains particularly in children attending day care centers. A total 58 nasopharyngeal, multiresistant isolates of S. pneumoniae collected from healthy pre-school children were susceptible to linezolid (MIC = 0.25-1 mg/l), irrespective of serotype and drug resistance pattern. The majority of them (about 94%) were sensitive to the bactericidal effect of linezolid with MBCs = 0.5-4 mg/l. One isolate was killed at 8 mg/l of linezolid, while two at higher concentrations of this antibiotic with MBCs 16 or 32 mg/l, suggesting tolerance of linezolid. BOX-PCR fingerprinting data imply that two linezolid-tolerant strains belonged to distinct clones. Linezolid tolerance was confirmed by monitoring the viability of these isolates during exposure to 4 or 20 mg/l of this antibiotic. The linezolid-tolerant strains were sensitive to the bactericidal effect of vancomycin. Linezolid tolerance in clinical isolates of pneumococci may represent a potential therapeutic risk, especially in infections in which bactericidal activity of drug is critical for eradication of bacteria.

EDTA as a potential agent preventing formation of Staphylococcus epidermidis biofilm on polichloride vinyl biomaterials.

Polichloride vinyl (PCV) is a widely used thermoplastic polymer, also in the production of medical devices. In the present study we assess the influence of EDTA in vitro on the biofilm structure formed by Staphylococcus epidermidis isolates on PCV biomaterials (Nelaton and Thorax catheters). The 6 strains of S. epidermidis were isolated from nasopharynx of hospitalised patients. It was found that all isolates were able to form the biofilm on both PCV biomaterials, irrespective of adhesion properties (cell surface properties, ability to slime production, minimal time needed for adhesion). The EDTA showed bacteriostatic effect against planktonic cells of the isolates (MIC = 0.25-0.5 mmol/l; MBC = 10.0- >25.0 mmol/l; MBC/MIC = 20, 30, 40, >50). The adhesion process and also formation of the biofilm was inhibited by EDTA at concentrations 1.0-2.0 mmol/l (2-8 x MIC). The eradication of the mature biofilm was achieved at 2.0-4.0 mmol/l EDTA (4-8 x MIC ) for two strains, while for the other four isolates, concentration of EDTA needed for eradication effect was >32 mmol/l (> 128 x MIC ). Data obtained in this paper suggest that EDTA may be regarded as a useful agent preventing formation of the S. epidermidis biofilm on PCV biomaterials.

[Drug susceptibility of Haemophilus sp. and Staphylococcus aureus strains colonizing respiratory tract in children with mucoviscidosis]. / Lekowrazliwosc szczepów Haemophilus sp. i Staphylococcusa aureus kolonizujacych drogi oddechowe u dzieci z mukowiscydoza.

OBJECTIVE: The aim of our studies was to isolate haemophili rods and S. aureus from children with mucoviscidosis and to assess antimicrobial agent susceptibility of the isolates. METHODS: Sputa and nasopharynx swabs from 12 children with mucoviscidosis were screened for the prevalence ofHaemophilus sp. and S. aureus. Susceptibility to antimicrobial agents was performed by agar diffusion method using appropriate media. Beta-lactamase activity was determined with the nitrocefin discs, macrolide-lincosamide-streptogramin B resistance using D-test. MAIN OBSERVATIONS: Of 12 mucoviscidosis-positive patients, 7 (58.33%) were colonisated by haemophili rods (a total of 8 H. parainfluenzae, 2 H. influenzae or 2 Haemophilus sp. isolates) and by S. aureus (8 isolates), in these species were isolated alone or in mixed cultures. Two ampicillin-resistant Haemophilus sp. but without beta-lactamases production were obtained. Also two methicillin resistant S. aureus with constitutive phenotype of resistance to macrolides-lincosamides and streptogramins B isolates and two methicillin-sensitive (with constitutive and inducible resistance to macrolides-lincosamides and streptogramins B) were isolated.

Slime production and cell surface hydrophobicity of nasopharyngeal and skin staphylococci isolated from healthy people.

The collection of 314 staphylococcal strains including Staphylococcus aureus and coagulase-negative staphylococci (CNS) was isolated from skin or nasopharynx of healthy people. It was found that the majority of staphylococci possessed the ability to produce slime intensively or moderately, irrespective of ecological niche-nose, throat or skin. Most of them showed the hydrophilic cell surface. However, among S. aureus skin isolates or CNS throat isolates predominated strains with hydrophobic cell surface. There was a slight correlation between slime production and the nature of cell surface among CNS isolates but not among S. aureus strains. It was found that most of slime-producing CNS strains showed hydrophilic cell surface, while slime-negative isolates usually possessed hydrophobic cell surface. Our data suggest that slime production but not cell surface hydrophobicity can be regarded as an essential colonization factor responsible for staphylococci adherence to skin or mucous membranes of upper respiratory tract. These data also suggest that slime production seems to be a general feature of staphylococci isolated from various niches of healthy people.