Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

Antimicrobial edible packaging based on cellulosic ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus

Edible cellulosic films made with hydroxypropylmethylcellulose (HPMC) have proven to be inadequate moisture barriers. To improve its water vapor barrier properties, different hydrophobic compounds were incorporated into the HPMC matrix. Some fatty acids and derivatives were included into the film-forming solution prior to film formation. Stearic acid was chosen because of its high capacity to reduce significantly the water vapor transmission rate. Antimicrobial activity of edible HPMC film was obtained by the incorporation of nisin into the film-forming solution. Nisin is an antimicrobial peptide effective against gram-positive bacteria. The inhibitory activity of this bacteriocin was tested for inhibition of Listeria innocua and Staphylococcus aureus. The use of stearic acid was observed to reduce the inhibitory activity of active HPMC film against both selected strains. This phenomenon may be explained by electrostatic interactions between the cationic nisin and the anionic stearic acid. Further studies showed that antimicrobial activity of film varied with the nature of the hydrophobic compound incorporated, in decreasing order: film without lipid, methylstearate film, and stearic acid film. This corroborated the idea of electrostatic interactions.     

Low molecular weight chitosan prepared with the aid of cellulase,lysozyme and chitinase: Characterisation and antibacterial activity

A continuous set of low molecular weight chitosan (LMWC) products was successfully made for this study by coordinating three enzymes (chitinase, lysozyme and cellulase) and two different deacetylated chitosan substrates (80% and 92%). It was observed that the intrinsic viscosity molecular weight (M_V) and SEC-HPLC-determined M_W distribution of LMWC were directed by both the used enzyme and the degree of chitosan substrate acetylation. LMWC prepared using lysozyme and 92%-deacetylated chitosan had larger M_W and, therefore, possessed higher antibacterial activity, as compared to other combinations. LMWC enzyme-directed properties suggest chitinase is more predictable and flexible to produce the specified M_V of LMWC. LMWC’s solubility and antibacterial activity, determined as minimum inhibitory concentration (MIC), against Escherichia coli exhibited a negative linear relationship with log M_V. E. coli strains showed much higher susceptibility to LMWCs than Staphylococcus aureus strains did. Both species also showed intra-species sensitivity diversity toward LMWC.     

高分子质量阳离子聚丙烯酰胺的合成及其对漂白麦草浆的助留助滤性能

研究采用无机氧化还原引发体系,以阳离子单体DMC和丙烯酰胺(州)为共聚单体合成出一系列不同分子量和不同单体配此的阳离子聚丙烯酰胺(CPAM).与有机引发体系相比,无机引发体系具有反应平缓,可操作性强等优点.将所得产品应用于造纸湿部,结果表明,自制阳离子聚丙烯酰胺具有良好的助留助滤性能.     

Antibacterial effects of chitosan solution against Legionella pneumophila, Escherichia coli, and Staphylococcus aureus

Chitosan has been shown to have antibacterial activities on the growth of a wide variety of bacteria. Chitosan solution has been sold commercially for use as an antibacterial agent. Chitosan solution contains not only chitosan but also organic acids as solvents and desalted Japan Sea Proper Water (dJSPW). We aimed to clarify whether chitosan solution has antibacterial activity against bacteria invading bath water, and then to explore the causative factor among these ingredients. The antibacterial activity of full-strength chitosan solution and of 10(2)- and 10(4)-fold chitosan solution diluted with purified water was studied against Legionella pneumophila serogroups 1 (L. pneumophila SG1) and 6 (L. pneumophila SG6), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) for 7 days at 37 degrees C. To clarify the causative factor in the antibacterial activity against E. coli, the antibacterial activities of the full-strength and diluted chitosan solutions for 24 h were examined. L. pneumophila SG1 and SG6, and E. coli could not survive in the chitosan solution or in the 10(2)-fold dilute solution for over a day at 37 degrees C. The cells of S. aureus were found to have decreased more than 2.46 log cfu/ml after 1 day of incubation, not only in the chitosan solutions, but also in phosphate buffer solution as a control. No inhibitory effect of dJSPW on the growth of the bacteria was observed. The antibacterial activity of the chitosan solution was lower compared with those of the organic acids solutions, and it increased with decreasing pH value. We observed the antibacterial activity of chitosan solution against L. pneumophila SG1 and SG6, and E. coli, suggesting it may be due to the decreased pH value derived from organic acids rather than from chitosan itself or dJSPW.     

低分子量透明质酸的制备及其抗氧化活性的研究

首先研究了Fe2+-S2O2-8氧化体系硫酸根自由基(SO-4·)对透明质酸(HA)的氧化降解作用,初步确定了SO-4·氧化降解HA制备低分子量透明质酸(LMWHA)的实验条件。接着HA降解产物经过乙醇沉淀以及凝胶柱层析纯化,采用高效凝胶过滤色谱法(HPGFC)测得纯化后LMWHA分子量为1.45×105Da。最后采用不同的体外抗氧化实验,并以HA为对照,研究了LMWHA的抗氧化活性,发现SO-4·降解后生成的LMWHA的抗氧化活性高于降解前的。本实验说明用SO-4·降解HA制备具有抗氧化活性LMWHA是可行的,为LMWHA抗氧化药物的开发提供了理论依据。     

Tolerance of Salmonella Enteritidis and Staphylococcus aureus to surface cleaning and household bleach

Effective cleaning and sanitizing of food preparation sites is important because pathogens are readily spread to food contact surfaces after preparation of contaminated raw products. Tolerance of Salmonella Enteritidis and Staphylococcus aureus to surface cleaning by wiping with regular, microfiber, and antibacterial-treated cloths was investigated. Wiping with cleaning cloths resulted in a considerable reduction of microorganisms from surfaces, despite the greater difficulty in removing S. aureus than Salmonella Enteritidis. Depending on the cloth type, S. aureus were reduced on surfaces from initial numbers of approximately 10(5) CFU/100 cm2 to numbers from less than 4 CFU/100 cm2 (below the detection limit) to 100 CFU/100 cm2. Directly after the cloths were used to clean the contaminated surfaces, they contained high numbers of bacteria (10(4) to 10(5) CFU/100 cm2), except for the disposable antibacterial-treated cloths, in which no bacteria could be detected. The tolerance of these pathogens to sodium hypochlorite was studied in the suspension test and in cloths. S. aureus showed a better tolerance for sodium hypochlorite than Salmonella Enteritidis. Inactivation of microorganisms in cloths required a higher concentration of sodium hypochlorite than was needed in the suspension test. Repeated exposure to sodium hypochlorite, however, resulted in an increase in susceptibility to this compound. This study provides essential information about the transfer of bacteria when wiping surfaces and highlights the need for a hygiene procedure with cleaning cloths that sufficiently avoids cross-contamination in the household environment.     

Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.     

Extraction of chitin from prawn shells and conversion to low molecular mass chitosan

Extraction and depolymerisation of chitin and chitosan from prawn shells was carried out using various chemical procedures. Sodium hydroxide and hydrochloric acid solutions were used for deproteination and demineralisation, respectively, while acetone was used for decolourisation. The amount of chitin and subsequently chitosan obtained was ∼35% and 25% respectively of the dry weight of the shells. The chitin was deacetylated using sodium hydroxide at 100 °C and the influence of the concentration of the reagent and duration of the reaction was investigated. The degree of deacetylation (DD) of the chitosan was evaluated by FTIR and NMR spectroscopy and the molecular mass distribution was determined by Gel Permeation Chromatography. It was found that the final DD was significantly higher using 50% sodium hydroxide solution (73% ± 9%) compared to 25% sodium hydroxide solution (40% ± 5%). It was noted also that the deacetylation reaction was more than 80% completed after 2 h but the chitosan produced had higher molecular mass while chitosan produced after 10 h had lower molecular mass and higher degree of deacetylation. The molecular mass distribution was bimodal for all the samples and consisted of a broad high molecular mass peak (peak 1) and a sharp low molecular mass peak (peak 2). The Mw of peak 1 decreased from ∼1.3 × 10⁶ after 2 h reaction with sodium hydroxide to 3.1 × 10⁵ after 10 h reaction indicating that depolymerisation and deacetylation occurred simultaneously. Peak 2 had a Mw of ∼2.4–9.9 × 10³.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

Fate of Staphylococcus aureus, Salmonella enterica serovar typhimurium, and vibrio vulnificus in raw oysters treated with chitosan

The fate of Staphylococcus aureus, Salmonella enterica serovar Typhimurium, and Vibrio vulnificus in oysters treated with chitosan was investigated. Three concentrations (0.5, 1.0, and 2.0%) of chitosan in 0.5% hydrochloric acid were prepared and coated onto raw oysters, which were then stored at 4 degrees C for 12 days. Untreated oysters and oysters coated with 0.5% hydrochloric acid without chitosan were used as controls. S. aureus cells were most sensitive to 2.0% chitosan followed by 0.5 and 1.0%. In general, chitosan treatment of oysters produced a decline in the population of S. aureus by 1 to 4 log CFU/ml compared with the untreated control. Chitosan treatment had no influence on the reduction of Salmonella Typhimurium over the 12-day storage period; inhibition of Salmonella Typhimurium growth was similar in both the control samples and the chitosan-treated samples. However, time of storage had a major effect on the survival of Salmonella Typhimurium on oysters. Neither time nor chitosan concentration had a significant effect on the growth of V. vulnificus during storage. All treatments were similar in inhibiting V. vulnificus growth.     

Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and Staphylococcus aureus in cuajada, a semisolid dairy product manufactured in Spain

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in “cuajada” (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph. aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10°C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph. aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph. aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph. aureus in cuajada over 12 d at 10°C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products.     

低分子量南瓜多糖的提取、纯化及结构初步研究

采用水浸提法提取低分子量南瓜粗多糖.通过DEAE Sepharose Fast Flow离子交换层析和sephacryl s-100 HR凝胶过滤层析得到南瓜低分子量多糖(LwPP-Ia),通过高效液相色谱测定了LWPP-Ia的纯度,显示为单一峰,分子量为6267Da.运用高效液相离子色谱测定了单糖组成,为葡萄糖(27.75%)、果糖(7.70%)、半乳糖(9.12%).紫外扫描显示无蛋白、核酸杂质.红外光谱鉴定表明含有多糖的特征吸收峰,并含有吡喃糖环.     

Short communication: Activity of nisin,lipid bilayer fragments and cationic nisin-lipid nanoparticles against multidrug-resistant Staphylococcus spp. isolated from bovine mastitis

Staphylococci are the main etiological agents of bovine mastitis. Bacteriocins and nanoparticles have emerged as promising alternatives for the future development of antimicrobial agents. This study evaluated the activity of the bacteriocin nisin and bicelles of the synthetic cationic lipid dioctadecyldimethylammonium bromide, alone and in combination, against multidrug-resistant Staphylococcus spp. strains isolated from bovine mastitis. In summary, cationic nisin/dioctadecyldimethylammonium bromide nanoparticles are shown to be a promising alternative for the control of mastitis caused by multidrug-resistant Staphylococcus spp.     

Isolation and partial characterisation of two low molecular weight durum wheat (Triticum durum) glutenins

Two low molecular weight durum wheat (Triticum durum Desf) glutenins, DSG-1 and DSG-2 (durum-wheat sulphur-rich glutenin fractions) were isolated from two cultivars, Mondur of good technological quality and Kidur of poor technological quality. The glutenin fraction, composed mostly of DSG protein, was extracted using a low concentration of NA-tetradecanoate and then fractionted by by using molecular sieve chromatography (Bio-Gel P 30). The amino acid compositions and the N-terminal sequences of the pure DSG proteins were determined, and their hydrophobic characteristics, calculated on the basis of these data, showed that DSG-2 is more hydrophobic than DSG-1. The amino acid compositions of DSG-1 and DSG-2 were different. The N-terminal amino acids of DSG-1 and DSG-2 were also different but were identical for each of the two cultivars. In the case of DSG-1, in addition to the main chain a minor chain was found in which the first three amino acids of the main chain were missing. The minor chain represented about 30% int he DSG-1 of mondur and almost 50% in Kidur.     

高浓低黏高稳定性阳离子表面施胶淀粉的研制

以木薯淀粉为原料,采用低黏度化与阳离子化同时进行,然后用复合氧化相结合的方法研制高浓低黏高稳定性阳离子表面施胶淀粉.探讨了活化降黏剂用量、氧化剂A用量、氧化剂B用量、醚化降黏反应温度、醚化降黏反应时间等对阳离子表面施胶淀粉黏度及电位的影响.结果显示:在水介质中,阳离子醚化剂用量2.5%,活化降黏剂用量0.8%,醚化反应温度48℃,醚化反应时间5h,氧化剂A用量0.6%-0.8%,一次氧化反应温度42℃,一次氧化反应时间2h,氧化剂B用量1.0%～1.5%,二次氧化反应温度36℃,二次氧化反应时间2h,得到的阳离子淀粉糊液黏度低、稳定性高,能满足高浓度表面施胶的需要.     

Effects of chitosan molecular weight and degree of deacetylation on the properties of gelatine-based films

The effect of chitosan molecular weight (MW) and degree of deacetylation (DD) on the physicochemical properties of gelatine-based films was studied with the goal of improving the films. Determination of the dynamic viscoelastic properties (elastic modulus G′ and viscous modulus G″) of the film-forming solutions revealed that the interactions between gelatine and chitosan were stronger in the blends made with chitosan of higher molecular weights or higher degrees of deacetylation than the blends made with lower molecular weights or degrees of deacetylation. Fish gelatine films modified with chitosan of higher molecular weights or higher degrees of deacetylation had higher tensile strengths (TS, 72.4 MPa for 550 kDa vs. 62.2 MPa for 200 kDa; 63.2 MPa for 93.6% DD vs. 38.0 MPa for 76.5% DD) and higher glass transition temperatures (T_g, 130 °C for 550 kDa vs. 108 °C for 200 kDa; 108 °C for 93.6% DD vs. 103 °C for 76.5% DD). Fourier transform infrared spectra (FTIR) and X-ray diffraction (XRD) studies indicated that gelatine and chitosan interactions determined the properties of the film. Thus, combining gelatine and chitosan may be a method for improving the physicochemical properties of gelatine films, especially when using chitosan of higher molecular weights and higher degrees of deacetylation.     

Effect of low molecular weight dextrins on gelatinization and retrogradation of starch

!-Amylases, usually added to bread recipes as anti-firming agents, are known to produce low molecular weight dextrins by starch hydrolysis. The influence of these compounds on the gelatinization and retrogradation of starch was studied by differential scanning calorimetry. Adding oligosaccharides to starch caused a delay in gelatinization, although its extent was not quantified. However, oligosaccharides of degrees of polymerization (DP) 3-5 reduced the enthalpy of the retrogradation endotherm, shown as the staling endotherm. The addition of gluten to starch and starch/oligosaccharide mixtures had no effect on the gelatinization and retrogradation of starch. The retrogradation of starch in dough samples was also analysed, after 'baking' in the calorimeter, to obtain additional information about starch retrogradation during storage. Oligosaccharides of DP 3-5 also reduced the enthalpy of the retrogradation endotherm. This work provides evidence that oligosaccharides influence starch changes during the baking and storage of bread. These effects could be considered as the mechanism by which the bacterial !-amylase reduces starch retrogradation and acts as an anti-firming agent.