Phage typing of Staphylococcus aureus associated with subclinical bovine mastitis

Six hundred and seventeen isolates of Staphylococcus aureus from subclinical clinical mastitis cases in 63 dairy herds in Northern Ireland were typed using a set of 25 phages. Ninety-four per cent of the isolates were typable, with nine phages, predominantly from groups I and III, being responsible for almost all of the lysis. Although 68 phage patterns were found, six of them typed 47.2% of the isolates. One strain accounted for 14.7% of the isolates, but the largest number of strains (44) was restricted to individual farms. The epidemiological significance of these findings for on-farm mastitis control is discussed.     

1 株金黄色葡萄球菌烈性噬菌体的生物学特性及其裂解效果

以耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）为宿主菌从奶牛养殖场污水中分离到1 株烈性噬菌体P65。透射电镜表明,噬菌体P65头部呈多面体结构,属于有尾噬菌体目长尾噬菌体科。噬菌体P65最佳感染复数为0.01,一步生长曲线显示,噬菌体P65潜伏期为10 min,裂解周期为90 min,裂解量为35.46 PFU/cell。在pH 4～10和温度30～45 ℃条件下具有稳定的裂解活性。紫外线照射70 min后,噬菌体P65效价由1.91×108 PFU/mL降至2.65×104 PFU/mL。经噬菌体处理48 h后,菌株MRSA 2和MRSA 24的生物被膜清除率分别为91.3%和92.2%。在25 ℃条件下,噬菌体P65在牛奶和牛肉细菌污染模型中有一定的抑菌效果。本研究表明噬菌体P65具有较宽的宿主谱和稳定的裂解杀菌能力,在清除生物被膜和食品抗菌方面有一定的运用潜能。     

噬菌体vB_SauM_RS对乳源金黄色葡萄球菌生物被膜的清除作用

目的:以金黄色葡萄球菌为宿主菌,分离裂解性噬菌体vB_SauM_RS,测定其生物学特性,并探究其在两种牛奶中的抑菌效果及对生物被膜的清除作用.方法:将噬菌体与金黄色葡萄球菌以1∶100的比例(菌体数量比)分别接种于脱脂牛奶和全脂牛奶中,分别在4℃和25 ℃环境下处理,测定不同时间脱脂牛奶和全脂牛奶中细菌浓度和噬菌体效价...     

鲜湿米粉中金黄色葡萄球菌污染状况研究

监测市售鲜湿米粉中的金黄色葡萄球菌的污染情况,并进行耐甲氧西林金黄色葡萄球菌菌株(MRSA)鉴定和肠毒素分型,为预防湿米粉中金黄色葡萄球菌中毒及食品安全风险积累数据。监测结果为139份样品中有38个样品检出金黄色葡萄球菌。在分离出的38株分离株中,21株鉴定为耐甲氧西林金黄色葡萄球菌菌株(MRSA),32株携带肠毒素基因。结果表明:湿米粉中金黄色葡萄球菌检出率高,菌株产肠毒素能力较强,存在的食品安全隐患不容忽视。     

Prevalence of methicillin-resistant Staphylococcus aureus in a fresh meat pork production chain

The objective of this study was to investigate the prevalence of methicillin-resistant Staphylococcus aureus on different stages of a fresh pork production chain to reveal potential carryover from live animals to meat. Samples were collected at different stages of the production process in a large German abattoir with an integrated processing unit for fresh pork. Samples included nasal swabs from pigs at stunning, environmental samples from the slaughter line, surface samples from carcasses, environmental and meat samples from the processing unit, and samples from final products. Samples were analyzed with an established two-step selective enrichment method, and isolates were characterized with respect to their S. aureus protein A gene (spa) and staphylococcal cassette chromosome mec (SCCmec; which harbors the mecA gene) types. Contamination rate was highest (64.7%) in nasal swabs and lower (6.0%) on carcasses, meat at processing (4.2%), and final products (2.8%). Environmental samples were positive along the slaughter line (12%) but not in the processing unit. spa types t011 and t034 and SCCmec type V predominated the isolates. Heterogeneity of spa types was highest in nasal swabs. Results show that methicillin-resistant S. aureus can be identified at all stages of the production chain. Further studies are needed to identify potential control points to reduce the carryover from farm animals to the final products.     

Relationships between the gross,non-volatile and volatile compositions and the sensory attributes of eight hard-type cheeses

Relationships between individual flavour attributes of eight hard-type cheeses and their volatile compounds, free amino acids (FAA), free fatty acids (FFA) and gross compositional constituents were determined. Relationships were also determined between individual texture attributes and gross compositional constituents. A trained panel of 15 assessors described the sensory characteristics of the cheeses using eight odour, twenty flavour, four appearance and nine texture attributes. Volatile compounds were isolated using a model-mouth device. FAA, FFA and gross compositional constituents were determined using standard methods. Relationships were determined by using partial least squares regression coupled with a new jack-knife method for identification and elimination of non-contributing variables. Eight flavour attributes were found to be correlated with subsets of volatile compounds, FAA, FFA and gross compositional constituents. For instance, the “nutty” flavour of Emmental was found to be positively correlated with the concentrations of propionic acid, ethyl acetate and 2-pentanone. “Nutty” flavour was negatively correlated with the concentrations of salt in moisture and pH 4.6-soluble nitrogen (pH 4.6-SN). Four texture attributes were correlated with subsets of gross compositional constituents. For example, “firmness” was positively correlated with concentrations of protein, calcium and phosphorous and negatively correlated with pH value and level of pH 4.6-SN.     

Predicting heat inactivation of Staphylococcus aureus under nonisothermal treatments at different pH

The aim was to assess whether heat resistance data obtained from isothermal treatments allow the estimation of survivors of Staphylococcus aureus under nonisothermal conditions and to find a model that accurately predicts its heat inactivation at constantly rising heating rates (0.5-9 degrees C/min) in media of different pH (4.0-7.4). S. aureus showed a higher heat resistance under isothermal treatments at pH 4.0 than at pH 5.5-7.4. However, under nonisothermal treatments S. aureus increased its heat resistance at pH 5.5-7.4 and became more thermotolerant than at pH 4.0. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments did not adequately fit experimental values. Whereas the number of survivors was much higher than estimated at pH 5.5-7.4, that obtained at the slower heating rates at pH 4.0 was lower. An equation based on the Weibullian-like distribution (log10 S(t) = (t/delta)p) accurately described survival curves obtained under nonisothermal conditions. A nonlinear relationship was observed among the scale parameter (delta) and the heating rate which allowed the development of two equations capable of predicting the inactivation rate of S. aureus under nonisothermal treatments. This study might contribute to prevent public health risks in foods requiring long heating lag phases during their processing.     

Selection and identification of protective culture for controlling Staphylococcus aureus in fresh Domiati like cheese

Antibacterial activity of forty lactic acid bacteria (LAB) isolates toward Staphylococcus aureus was evaluated. The selected strains were then used as protective culture in artificial contaminated Domiati like cheese with S. aureus. The effect of using these strains on physicochemical properties and overall acceptability of fresh cheese was evaluated. Depending on its antibacterial activity, three strains of Lactobacillus rhamnosus 130RZFAAU, 131RZFAAU, and 190RZFAAU were selected for cheese making. No negative sensory properties were observed by the panelists when LAB strains were used as a single culture in the fresh cheese making. The application of these strains as protective culture in artificial contaminated cheesemaking process give a positive results. S. aureus was detected in cheese samples by culture method and propidium mono azide–quantitative polymerase chain reaction method. The results recommended that the strain L. rahmnosus 131RZFAUU that used in this study has antimicrobial activity against S. aureus and could be used as protective culture for improving the safety of Egyptian soft cheese.

Practical applications

Detection of pathogenic bacteria by classical tests can take several days. It would be useful to have a rapid detection protocol to screen for the presence of Staphylococcus aureus in milk and cheese. Application of real‐time PCR in cheese is sufficient in characterization the S. aureus communities in raw milk and follow the dynamics of the entire populations in cheese. Recently, some scientific publications have shown that the naturally cheese microflora can efficiently prevent the growth of pathogenic or spoilage microorganisms. The control of spoilage and pathogens bacteria has been traditionally done by chemical additives, but the application of promising protective cultures, especially for traditionally cheeses made from raw milk, is limited. This work present some protective culture selected for controlling S. aureus in soft cheese. This work confirm the PMA‐q PCR method for detection live cells of S. aureus in cheese rapidly.     

Modelling inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation at different temperatures

A detailed study of the inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation was performed at, respectively, 25 and 35 different combinations of process temperature and process pressure covering a range of 5-50 degrees C and 100-300 MPa. It appeared that in the entire studied pressure-temperature domain, S. aureus was more resistant to high-pressure homogenisation than Y. enterocolitica. Furthermore, the effect of the process pressure on the inactivation of S. aureus was considerably smaller than on the inactivation of Y. enterocolitica. Also, temperature between 5 and 40 degrees C did not affect inactivation of S. aureus by high-pressure homogenisation, while Y. enterocolitica inactivation was affected by temperature over a much wider range. Different mathematical models were compared to describe the inactivation of both bacteria under the experimental conditions applied. Such pressure-temperature inactivation models form the engineering basis for design, evaluation and optimisation of high-pressure homogenisation processes as a new preservation technique.     

冻生虾仁金黄色葡萄球菌预测模型的建立及应用

为得到生虾仁中金黄色葡萄球菌的生长模型,以期对加工中遭受金黄色葡萄球菌污染后的增长状况进行预测,通过把金黄色葡萄球菌接种到生虾仁,按生产工序拟定四个温度(4、10、13、20℃)并测定该菌在不同温度下菌落数的变化,采取3种常用方程进行拟合,比较后得出各温度的最适一级生长模型,并建立二级模型。结果表明,10、20℃适合采用Gompertz模型,13℃为Logistic模型,4℃采用Linear模型;预测表明金黄色葡萄球菌在解冻工序增长较明显,增长率9.7%;采用平方根方程拟合得到二级预测模型:umax=0.0129T-0.0220;模型验证表明预测值和实测值的残差在±0.01内,偏差因子和准确因子在可接受范围内,表明模型有效,用于生产预测,具有一定参考价值。     

Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin

High-intensity pulsed electric fields (HIPEF) can be used as a nonthermal preservation method that is believed to enhance the effect of nisin on microorganisms such as Staphylococcus aureus. The survival of S. aureus inoculated into skim milk and treated with nisin, with HIPEF, or with a combination of nisin-HIPEF was evaluated. Nisin dose, milk pH, and HIPEF treatment time were the controlled variables that were set up at 20 to 150 ppm, pH 5.0 to 6.8, and 240 to 2,400 micros, respectively. HIPEF strength and pulse width were kept constant at 35 kV/cm and 4 micros, respectively. No reduction in S. aureus concentration was observed in skim milk at its natural pH after treatment with nisin, but 1.1 log units were recovered after 90 min of treatment at pH 5.0 with 150 ppm nisin. A reduction in viable S. aureus counts of 0.3 and 1.0 log unit in skim milk treated with HIPEF at its natural pH was observed at 240 and 2,400 micros, respectively. The nisin-HIPEF treatment design was based on a response surface methodology. The combined effect of nisin and HIPEF was clearly synergistic. However, synergism depended on pH. A maximum microbial inactivation of 6.0 log units was observed at pH 6.8, 20 ppm nisin, and 2,400 micros of HIPEF treatment time, whereas a reduction of over 4.5 log units was achieved when pH, nisin concentration, and HIPEF treatment times were set at 5.0, 150 ppm, and 240 micros, respectively.     

金黄色葡萄球菌噬菌体的分离筛选

以金黄色葡萄球菌为宿主菌,采用富集法从污水、粪便样品中分离筛选噬菌体,纯化后观察噬菌斑特征,电镜形态,并测定其效价、RTD（RoutineTestDilution,常规实验稀释度）值,初步分析该噬菌体的宿主谱。结果获得一株对金黄色葡萄球菌敏感的噬菌体,该噬菌体在宿主菌平板上能形成直径1～2mm、边界清晰、圆形透明的噬菌斑,效价是3.2×1010PFU/mL,RTD值为10-3,电镜观察显示此噬菌体由直径约为30nm的多面体头部和约50nm的尾部组成,可以裂解宿主菌及其它2株金黄色葡萄球菌,不能裂解大肠杆菌,鼠伤寒沙门氏菌及痢疾志贺氏菌。本文为深入研究金黄色葡萄球菌噬菌体的生物学特性及其功能提供了依据,为进一步利用噬菌体检测食源性金黄色葡萄球菌、防治由金黄色葡萄球菌引起的疾病提供了理论基础。      

Efficacy of bovicin HC5 and nisin combination against Listeria monocytogenes and Staphylococcus aureus in fresh cheese

Fresh and soft cheeses provide a suitable medium for the growth of many microorganisms and have been frequently associated with foodborne diseases. This study aimed to evaluate the effects of the bacteriocins bovicin HC5 and nisin on Listeria monocytogenes and Staphylococcus aureus in Minas Frescal cheese, a traditional Brazilian fresh product. The cheese was inoculated with 10⁴ CFU g^?1 of both pathogens, and the bacteriocins were added at 600 AU g^?1 of each one. After 9 days of storage at 4 °C, L. monocytogenes Scott A were not detected in 25 g samples. Although the bacteriocins reduced the population of S. aureus ATCC 6538, increasing numbers were detected after 15 days of storage at 4 °C. Staphylococcal enterotoxin C was detected in cheeses added of bacteriocins and stored under abusive temperature of 15 °C. The results demonstrate the potential application of combination of bovicin HC5 and nisin in controlling the pathogens growth assessed in Minas Frescal cheese.     

Presence and growth prediction of Staphylococcus spp. and Staphylococcus aureus in Minas Frescal cheese,a soft fresh cheese produced in Brazil

Physical-chemical characteristics of Minas Frescal cheese (MFC) favor the growth of Staphylococcus spp. and allow the production of enterotoxins by specific strains. Here, we aimed to characterize the physical-chemical aspects (pH, storage temperature, and salt content) and the presence of Staphylococcus spp. in MFC samples (n = 50) to support a modeling study for the growth by this microorganism. Coagulase-positive staphylococci isolates were obtained and subjected to PCR assays to identify them as Staphylococcus aureus (nuc) and to detect staphylococcal enterotoxin-related genes (sea, seb, sec, sed, see). Staphylococcus aureus growth kinetics (maximum growth rate, Grmax, and lag time) were predicted based on ComBase model and MFC physical-chemical aspects. Mean counts of Staphylococcus spp. ranged from 3.3 to 6.7 log cfu/g, indicating poor hygiene practices during production. Selected isolates (n = 10) were identified as S. aureus, but none presented classical enterotoxin-related genes. pH, temperature, and salt content ranged from 5.80 to 6.62, 5°C to 12°C, and 0.85% to 1.70%, respectively. The Grmax values ranged from 0.012 to 0.419 log cfu/g per h. Independent of the storage temperature, the lowest Grmax values (0.012 to 0.372 log cfu/h) were obtained at pH 5.80 associated with salt content of 1.7%; independent of the pH and salt content, the best temperature to avoid staphylococcal growth was 7.5°C. Hygienic conditions during MFC production must be adopted to avoid staphylococcal contamination, and storage at temperatures lower than 7.5°C can prevent staphylococcal growth and the potential production of enterotoxins.     

A polyphasic approach to detect enterotoxigenic Staphylococcus aureus and diarrheagenic Escherichia coli in raw milk Italian cheeses by multiplex PCR

A polyphasic approach was evaluated for the detection of eight staphylococcal enterotoxin (SE)-encoding genes (sea, sec, sed, seg, seh, sei, sej, sel) and the Escherichia coli genes most commonly associated with virulence factors (eae, elt, ipaH, stx) in traditional soft cheeses, manufactured artisanally from whole raw milk in the Lombardy region (northern Italy). To determine the presence of the target genes, two multiplex PCRs were performed on DNA extracted both directly from cheese samples (culture-independent approach) and from whole cultivable cells, formed by harvesting colonies from the first serial dilution agar plates of selective media, as representative of cultivable community ("bulk"). Genes associated with enteroinvasive E. coli, ipaH, and Shiga toxin-producing E. coli, stx, were detected in two of the bulk samples analyzed; no virulence genes were found by amplifying DNA directly extracted from cheeses. SE-encoding genes were found in three cheeses (sea in all three samples, associated with sed and sej in two of these). More SE-encoding genes were detected by amplifying DNA obtained from bulk samples: sea, sed, sej, sec, seg, sel, and sei. No samples harbored the gene encoding for SE type H. The polyphasic approach followed has been useful in enhancing detection of target genes. Our results indicate that some of the artisanal cheeses examined may constitute a potential hazard for consumer health.     

脉冲强光对馒头表面金黄色葡萄球菌的杀菌效果

目的研究脉冲强光技术在馒头表面金黄色葡萄球菌杀菌中的效果及其机制。方法控制闪照距离为9~21 cm,单脉冲能量为100~500 J,闪照次数为0~20次,分析不同条件下金黄色葡萄球菌的失活规律,并将该优化条件应用于馒头表面金黄色葡萄球菌的杀菌试验,最后采用透射电子显微镜技术分析脉冲强光技术对金黄色葡萄球菌形态结构的影响,从而分析其杀菌机制。结果脉冲强光在闪照距离为9 cm,单脉冲能量为500 J的情况下,闪照16次能对金黄色葡萄球菌产生良好的杀菌效果。该脉冲强光处理技术能降低馒头表面的金黄色葡萄球菌1.72 log CFU/g。透射电镜观察结果说明了脉冲强光可破坏金黄色葡萄球菌的细胞膜和菌体胞质内容物,从而造成细胞的失活。结论脉冲强光可对金黄色葡萄球菌产生较强的杀菌效果,在馒头加工业中具有一定的应用前景。     

Synergetic effects of high-pressure carbon dioxide and nisin on the inactivation of Escherichia coli and Staphylococcus aureus

Synergetic effects of high-pressure carbon dioxide (HPCD) and nisin on Escherichia coli and Staphylococcus aureus were evaluated. Changes in morphology, interior structure, and membrane permeability were analyzed by scanning and transmission electron microscopy, and flow cytometry. Synergetic effects were found, especially in S. aureus. HPCD alone or with nisin led to morphological and intracellular alterations in both bacteria, but nisin alone led to these damages only in S. aureus. A positive correlation between membrane damage and inactivation was found, but ratios of inactivation were higher, probably because of viable but non-culturable state. Mechanisms were proposed for synergism: for E. coli, outer membrane was damaged first by HPCD, and then HPCD and nisin jointly acted on and destroyed the cytoplasmic membrane, leading to further intracellular damage by HPCD; for S. aureus, HPCD and nisin acted on the cytoplasmic membrane together leading to cell death.Industrial RelevanceEscherichia coli and Staphylococcus aureus are two common microorganisms, which exist widely in the environment and easily contaminate food such as vegetables and dairy products, respectively. Considering heat treatment may destroy some heat-sensitive quality of the products, this study evaluated synergetic effects of high-pressure carbon dioxide (HPCD) combined with the bacteriocin nisin. The investigations provided evidence for potentially combined application of HPCD and nisin to help keep food safe in the industry.     

Optimization of inactivation of Staphylococcus aureus by low concentration electrolyzed water using response surface methodology

The objective of this study was to investigate the combined effect of temperature (15–35°C), pH (3-9), and dipping time (1–5 min) on the inactivation of Staphylococcus aureus in broth treated with low concentration electrolyzed water (LcEW). Reductions of 1.44–7.12 log CFU/mL were observed in different combinations of the 3 factors. Also, a quadratic equation for S. aureus inactivation kinetic was developed by multiple regression analysis using response surface methodology. The predicted values were shown to be significantly in good agreement with experimental values as a result of the level of significance was p<0.0001. Besides, the developed model was validated by fitting with literature data and the results showed that the predictions had a good agreement with the observed data with a satisfied determination of coefficient (R₂=0.963).