甘草提取物对Staphylococcus aureus的抑菌活性及作用机理

食源性病菌为食品安全带来了巨大挑战,甘草提取物作为食品添加剂对金黄色葡萄球菌(Staphylococcus aureus)具有良好的抑菌作用,可作为天然食品防腐剂的候选原料,但目前对其抑菌机理的研究还不深入,影响了其应用。为探究甘草提取物对S.aureus的抑菌机理,该研究通过生长曲线、氧化损伤实验、细胞膜壁分析、蛋白质分析和DNA分析,评价了甘草提取物对S.aureus的抑菌作用机制。结果表明,甘草提取物导致S.aureus核酸渗漏,说明其膜完整性被破坏;同时,甘草提取物降低了几种能量代谢酶:琥珀酸脱氢酶(succinate dehydrogenase,SDH)和总ATP酶的活力;此外,光谱和竞争分析表明,甘草提取物与DNA发生了静电结合和凹槽结合。总之,甘草提取物主要是通过对S.aureus细胞壁膜、蛋白质合成、细菌代谢活力和遗传物质发挥作用,从而抑制其生长。该研究为甘草提取物在食品防腐方面的应用提供了理论基础。

The Effect of Culture Condition on Type 5 Capsular Polysaccharide Production of Staphylococcus aureus from Diary Cattle

[Objective] The effect of different culture conditions on type 5 capsular polysaccharide production of Staphylococcus aureus from diary cattle was studied to provide simple way for CP production and preparation and laid foundation for carrying out new polysaccharide vaccine research. [Method] Staph-ylococcus aureus was isolated from milk sample of sick dairy cattle and capsular polysaccharide serotypes were identified. Type 5 capsular polysaccharide was cultured on BHI,solid columbia and mod110 culture media. Glucose and lactose were taken as carbon sources for every culture media in solid and liquid state. Therefore 9 different culture conditions were taken to study the effect of culture conditions on capsular polysaccharide production. [Result] Different culture conditions indicated that compared with columbia culture media, BHI culture media could decline capsular polysaccharide production and mod110 culture media could increase capsular polysaccharide production. While for same culture media, solid culture media was better for capsular polysaccharide production,meanwhile,taken lactose as carbon source could increase capsular polysaccharide production.

金黄色葡萄球菌(Staphylococcus aureus)液体增菌培养基的优化

为研发一种金黄色葡萄球菌(Staphylococcus aureus)选择性增菌液,选用针对革兰氏阴性菌及非葡萄球菌属菌株具有选择抑制作用的7.5%氯化钠肉汤作为基础培养基,探索氨基酸、丙酮酸钠、甘露醇对金黄色葡萄球菌的促生长能力,通过正交试验确定培养基的最优配方,并使用优化后的培养基与原始培养基作用于来源不同的四株金黄色葡萄球菌标准菌株,对其增菌效果和抑制大肠杆菌(Escherichia coli)的效果进行对比。结果表明,最佳培养基配方为在基础培养基上添加L-丙氨酸1.0 g/L、甘露醇6.0 g/L、丙酮酸钠1.2 g/L。菌株在优化后培养基上增菌浊度及转接至Baird-Parker的菌落数上均有促进效果,最终菌株的OD550 nm值有0.28~0.34的增长,转接Baird-Parker后的菌落数也有10~100倍的增长,且在促进目标菌生长的同时对大肠杆菌的抑制性也没有受到影响,表明该增菌培养基选择性良好。

Staphylococcus aureus and biofilms:transmission, threats, and promising strategies in animal husbandry

Staphylococcus aureus(S. aureus) is a common pathogenic bacterium in animal husbandry that can cause diseases such as mastitis, skin infections, arthritis, and other ailments. The formation of biofilms threatens and exacerbates S. aureus infection by allowing the bacteria to adhere to pathological areas and livestock product surfaces, thus triggering animal health crises and safety issues with livestock products. To solve this problem, in this review, we provide a brief overview of the harm caused by S. aureus and its biofilms on livestock and animal byproducts(meat and dairy products). We also describe the ways in which S. aureus spreads in animals and the threats it poses to the livestock industry. The processes and molecular mechanisms involved in biofilm formation are then explained. Finally, we discuss strategies for the removal and eradication of S. aureus and biofilms in animal husbandry, including the use of antimicrobial peptides, plant extracts, nanoparticles, phages, and antibodies. These strategies to reduce the spread of S. aureus in animal husbandry help maintain livestock health and improve productivity to ensure the ecologically sustainable development of animal husbandry and the safety of livestock products.

Elimination of methicillin‑resistant Staphylococcus aureus biofilms on titanium implants via photothermally‑triggered nitric oxide and immunotherapy for enhanced osseointegration

Background:Treatment of methicillin-resistant Staphylococcus aureus(MRSA)biofilm infections in implant placement surgery is limited by the lack of antimicrobial activity of titanium(Ti)implants.There is a need to explore more effective approaches for the treatment of MRSA biofilm infections.Methods:Herein,an interfacial functionalization strategy is proposed by the integration of mesoporous polydopamine nanoparticles(PDA),nitric oxide(NO)release donor sodium nitroprusside(SNP)and osteogenic growth peptide(OGP)onto Ti implants,denoted as Ti-PDA@SNP-OGP.The physical and chemical properties of Ti-PDA@SNP-OGP were assessed by scanning electron microscopy,X-ray photoelectron spectroscope,water contact angle,photothermal property and NO release behavior.The synergistic antibacterial effect and elimination of the MRSA biofilms were evaluated by 2′,7′-dichlorofluorescein diacetate probe,1-N-phenylnaphthylamine assay,adenosine triphosphate intensity,O-nitrophenyl-β-D-galactopyranoside hydrolysis activity,bicinchoninic acid leakage.Fluorescence staining,assays for alkaline phosphatase activity,collagen secretion and extracellular matrix mineralization,quantitative real‑time reverse transcription‑polymerase chain reaction,and enzyme-linked immunosorbent assay(ELISA)were used to evaluate the inflammatory response and osteogenic ability in bone marrow stromal cells(MSCs),RAW264.7 cells and their co-culture system.Giemsa staining,ELISA,micro-CT,hematoxylin and eosin,Masson's trichrome and immunohistochemistry staining were used to evaluate the eradication of MRSA biofilms,inhibition of inflammatory response,and promotion of osseointegration of Ti-PDA@SNP-OGP in vivo.Results:Ti-PDA@SNP-OGP displayed a synergistic photothermal and NO-dependent antibacterial effect against MRSA following near-infrared light(NIR)irradiation,and effectively eliminated the formed MRSA biofilms by inducing reactive oxygen species(ROS)-mediated oxidative stress,destroying bacterial membrane integrity and causing leakage of intracellular components(P<0.01).In vitro experiments revealed that Ti-PDA@SNP-OGP not only facilitated osteogenic differentiation of MSCs,but also promoted the polarization of pro-inflammatory M1 macrophages to the anti-inflammatory M2-phenotype(P<0.05 or P<0.01).The favorable osteo-immune microenvironment further facilitated osteogenesis of MSCs and the anti-inflammation of RAW264.7 cells via multiple paracrine signaling pathways(P<0.01).In vivo evaluation confirmed the aforementioned results and revealed that Ti-PDA@SNP-OGP induced ameliorative osseointegration in an MRSA-infected femoral defect implantation model(P<0.01).Conclusions:Ti-PDA@SNP-OGP is a promising multi-functional material for the high-efficient treatment of MRSA infections in implant replacement surgeries.

Structural insights on anti-biofilm mechanism of heated slightly acidic electrolyzed water technology against multi-resistant Staphylococcus aureus biofilm on food contact surface

Slightly acidic electrolyzed water(SAEW)has proven to be an efficient and novel sanitizer in food and agriculture field.This study assessed the efficacy of SAEW(30 mg/L)at 40℃on the inactivation of foodbome pathogens and detachment of multi-resistant Staphylococcus aureus(MRSA)biofilm.Furthermore.the underlying mechanism of MRS A biofilm under heated SAEW at 40℃treatment on metabolic profiles was investigated.The results showed that the heated SAEW at 40℃significantly effectively against foodbome pathogens of 1.96-7.56(lg(CFU/g))reduction in pork,chicken,spinach,and lettuce.The heated SAEW at 40℃treatment significantly reduced MRS A biofilm cells by 2.41(lg(CFU/cm^(2))).The synergistic effect of SAEW treatment showed intense anti-biofilm activity in decreasing cell density and impairing biofilm cell membranes.Global metabolic response of MRSA biofilms,treated by SAEW at 40℃,revealed the alterations of intracellular metabolites,including amino acids,organic acid,fatty acid,and lipid.Moreover,signaling pathways involved in amino acid metabolism,energy metabolism,nucleotide synthesis,carbohydrate metabolites,and lipid biosynthesis were functionally disrupted by the SAEW at 40℃treatment.As per our knowledge,this is the first research to uncover the potential mechanism of heated SAEW treatment against MRSA biofilm on food contact surface.

Eugenol targeting CrtM inhibits the biosynthesis of staphyloxanthin in Staphylococcus aureus

Staphylococcus aureus is a serious foodborne pathogen threatening food safety and public health.Especially the emergence of methicillin-resistant Staphylococcus aureus(MRSA)increased the difficulty of S.aureus treatment.Staphyloxanthin is a crucial virulence factor of S.aureus.Blocking staphyloxanthin production could help the host immune system counteract the invading S.aureus cells.In this study,we first screened for staphyloxanthin inhibitors using a virtual screening method.The outcome of the virtual screening method resulted in the identification of eugenol(300μg/mL),which significantly inhibits the staphyloxanthin production in S.aureus ATCC 29213,S.aureus Newman,MRSA ATCC 43300 and MRSA ATCC BAA1717by 84.2%,63.5%,68.1%,and 79.5%,respectively.The outcome of the growth curve assay,field-emission scanning electron,and confocal laser scanning microscopy analyses confirmed that eugenol at the test concentration did not affect the morphology and growth of S.aureus.Moreover,the survival rate of S.aureus ATCC 29213 and MRSA ATCC 43300 under H_(2)O_(2) pressure decreased to 51.9%and 45.5%in the presence of eugenol,respectively.The quantitative RT-PCR and molecular simulation studies revealed that eugenol targets staphyloxanthin biosynthesis by downregulating the transcription of the crtM gene and inhibiting the activity of the CrtM enzyme.Taken together,we first determined that eugenol was a prominent compound for staphyloxanthin inhibitor to combat S.aureus especially MRSA infections.

The ever-changing microenvironment of Staphylococcus aureus in cutaneous infections

Background:Staphylococcus aureus is responsible for the majority of skin and soft tissue infections,which are often diagnosed at a late stage,thereby impacting treatment efficacy.Our study was designed to reveal the physiological changes at different stages of infection by S.aureus through the combined analysis of variations in the skin microenvironment,providing insights for the diagnosis and treatment of S.aureus infections.Methods:We established a murine model of skin and soft tissue infection with S.aureus as the infectious agent to investigate the differences in the microenvironment at different stages of infection.By combining analysis of the host immune status and histological observations,we elucidate the progression of S.aureus infection in mice.Results:The results indicate that the infection process in mice can be divided into at least two stages:early infection(1–3 days post-i nfection)and late infection(5–7 days post-i nfection).During the early stage of infection,notable symptoms such as erythema and abundant exudate at the infection site were observed.Histological examination revealed infiltration of numerous neutrophils and bacterial clusters,accompanied by elevated levels of cytokines(IL-6,IL-10).There was a decrease in microbial alpha diversity within the microenvironment(Shannon,Faith's PD,Chao1,Observed species,Simpson,Pielou's E).In contrast,during the late stage of infection,a reduction or even absence of exudate was observed at the infected site,accompanied by the formation of scabs.Additionally,there was evidence of fibroblast proliferation and neovascularization.The levels of cytokines and microbial composition gradually returned to a healthy state.Conclusion:This study reveals synchrony between microbial composition and histological/immunological changes during S.aureus-i nduced SSTIs.

Electrochemical and colorimetric dual-signal detection of Staphylococcus aureus enterotoxin B based on AuPt bimetallic nanoparticles loaded Fe-N-C single atom nanocomposite

Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.

Antimicrobial and synergistic effects of lemongrass and geranium essential oils against Streptococcus mutans,Staphylococcus aureus,and Candida spp.

BACKGROUND The oral cavity harbors more than 700 species of bacteria,which play crucial roles in the development of various oral diseases including caries,endodontic infection,periodontal infection,and diverse oral diseases.AIM To investigate the antimicrobial action of Cymbopogon Schoenanthus and Pelargonium graveolens essential oils against Streptococcus mutans,Staphylococcus aureus,Candida albicans,Ca.dubliniensis,and Ca.krusei.METHODS Minimum microbicidal concentration was determined following Clinical and Laboratory Standards Institute documents.The synergistic antimicrobial activity was evaluated using the Broth microdilution checkerboard method,and the antibiofilm activity was evaluated with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay.Data were analyzed by one-way analysis of variance followed by the Tukey post-hoc test(P≤0.05).RESULTS C.schoenanthus and P.graveolens essential oils were as effective as 0.12%chlorhexidine against S.mutans and St.aureus monotypic biofilms after 24 h.After 24 h P.graveolens essential oil at 0.25%was more effective than the nystatin group,and C.schoenanthus essential oil at 0.25%was as effective as the nystatin group.CONCLUSION C.schoenanthus and P.graveolens essential oils are effective against S.mutans,St.aureus,Ca.albicans,Ca.dubliniensis,and Ca.krusei at different concentrations after 5 min and 24 h.

Prevalence and antibiogram study of Salmonella and Staphylococcus aureus in poultry meat

Objective:To evaluate the presence and antibiogram pattern of Salmonella and Staphylococcus aureus(S.aureus)in retail poultry meat products.Methods:Foodborne pathogens(Salmonella and S.aureus)were isolated from poultry meat and confirmed with the help of biochemical and immunological test.Antibiogram of the isolates were examined by following CLS1 methods.Results:A total number of 209 poultry meat samples were collected and studied in this study.Out of which,5.26%were found contaminated with Salmonella while 18.18%were found contaminated with S.aureus.All the Salmonella and S.aureus isolates were found resistant to at least one antibiotic.About 72.72%of the Salmonella isolates showed resistance to tetracycline,while S.aureus isolates were also found highly resistant to tetracycline equal to 44.73%.One of the Salmonella isolates showed multi-drug resistance to almost six antibiotics out of nine antibiotics used in the study.Multidrug resistant S.aureus isolates were also found in the study.Conclusions:The study confirmed the presence of Salmonella and S.aureus in retail poultry meat.It is a potential threat to consumer health.To reduce the risk of contamination,good hygiene practices are necessary from processing to storage.

Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

A polymerase chain reaction （PCR） assay was employed for direct detection of Staphylococcus aureus without enrichment in dairy products. A solvent extraction procedure was successfully modified for the extraction of Staphylococcus aureus DNA from artificially contaminated whole milk, skim milk, and cheese. A primer targeting the thermostable nuclease gene （nuc） was used in the PCR. A DNA fragment of 279 bp was amplified. The PCR product was confirmed by DNA sequencing. In this study, the PCR, GB-4789.10-94, Perifilm RSA.Count Plate, and Baird-Parker ＋ RPF Agar were compared. The sensitivity of the PCR was 10 CFU mL^-1 of whole milk, skim milk, and 55 CFU g^-1 of cheese. The developed methodology allowed for detection of Staphylococcus aureus in dairy products in less than 6 h. The time taken for the development of this PCR assay was 12-24 h, less than the time taken by the general PCR assay using the enrichment method, and the coincidence rate of this developed PCR was 94.3%, the sensitivity was 100%. It was a rapid, sensitive, and effective method for PCR to detect Staphylococcus aureus in milk and milk products.

Antimicrobial activity and synergism of Sami-Hyanglyun-Hwan with ciprofloxacin against methicillin-resistant Staphylococcus aureus

Objective: To investigate the antibacterial activity of SHHextracted with either water or ethanol against methicillin-resistant Staphylococcus aureus(MRSA) and combinatory antimicrobial effect with ciprofloxacin(CIP) by time kill assay and checkerboard dilution test. Methods: The antibacterial activity determined by broth dilution method indicated that the antibacterial activity of Sami-Hyanglyun-Hwan(SHH) water extract(SHHW) and SHH ethanol extract(SHHE) ranged from 250 to 2000 μg/m L and 125 to 1000 μg/m L against MRSA, respectively. Results: In the checkerboard method, the combinations of SHHE with CIP had a partial synergistic or synergistic effect against MRSA. The time-kill curves showed that a combined SHHE and CIP treatment reduced the bacterial counts dramatically after 24 h. Conclusions: The present study demonstrates the therapeutic ability of SHHE against MRSA infections.

Surface-engineered liposomes for dual-drug delivery targeting strategy against methicillin-resistant Staphylococcus aureus(MRSA)

This study focused on the encapsulation of vancomycin(VAN) into liposomes coated with a red blood cell membrane with a targeting ligand, daptomycin–polyethylene glycol–1,2-distearoyl-sn-glycero-3-phosphoethanolamine, formed by conjugation of DAPT and Nhydroxysuccinimidyl-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.This formulation is capable of providing controlled and targeted drug delivery to the bacterial cytoplasm. We performed MALDI-TOF, NMR and FTIR analyses to confirm the conjugation of the targeting ligand via the formation of amide bonds. Approximately 45% of VAN could be loaded into the aqueous cores, whereas 90% DAPT was detected using UV–vis spectrophotometry. In comparison to free drugs, the formulations controlled the release of drugs for > 72 h. Additionally, as demonstrated using CLSM and flow cytometry, the resulting formulation was capable of evading detection by macrophage cells. In comparison to free drugs, red blood cell membrane–DAPT–VAN liposomes, DAPT liposomes, and VAN liposomes reduced the MIC and significantly increased bacterial permeability, resulting in > 80% bacterial death within 4 h. Cytotoxicity tests were performed in vitro and in vivo on mammalian cells,in addition to hemolytic activity tests in human erythrocytes, wherein drugs loaded into the liposomes and RBCDVL exhibited low toxicity. Thus, the findings of this study provide insight about a dual antibiotic targeting strategy that utilizes liposomes and red blood cell membranes to deliver targeted drugs against MRSA.

Detection of Pathogenic Bacteria <i>Staphylococcus aureus</i>and <i>Salmonella</i>sp. from Raw Milk Samples of Different Cities of Pakistan

Food-borne diseases are the main public health problem throughout the world. Milk is important component of human diet including fats, proteins, vitamins and minerals. It is a best source of calcium and phosphorus. Different types of pathogenic bacteria like S. aureus and Salmonella enter in milk and then multiply, after multiplication they become active in causing diseases. These bacteria create serious problems for human health. This study aimed to isolate and identify pathogenic bacteria Staphylococcus aureus and Salmonella from raw milk samples of different cities of Pakistan. Primary screening of raw milk samples was done on the basis of morphological, cultural and biochemical techniques. The final identification was made using 16SrRNA sequence analysis. A total of 200 raw milk samples were collected from different cities of Pakistan. Selective medium xylose lysine deoxycholate agar (XLD) and Mannitol salt agar were used for the identification of Salmonella sp. and S. aureus. Staphylococcus aureus produced yellow colonies with yellow zones on Mannitol salt agar. Staphylococcus aureus exhibited gram-positive character with purple coloration and it was detected as cocci-shaped. Biochemically 91 (45%) samples enhibited Catalase, Coagulase, DNase, Urease, Citrate, fermentation tests positive and indole, oxidase and H2S tests negative with nonmotile character, indicating the presence of Staphylococcus aureus. Salmonella sp. was detected as gram negative rods with pink coloration on gram staining. Biochemically 87 (43%) samples revealed catalase, citrate, H2S and fermentation tests positive while oxidase, DNase, Indole and urease tests negative, indicating the presence of Salmonella sp. in these samples. Of the 200 samples tested, 43% were positive for Salmonella, while 45% samples were contaminated with S. aureus. The 16SrRNA sequence analysis confirmed the results of biochemical and cultural characterization by depicting 99% identity of samples with S. aureus and 98% identity with Salmonella spp. The occurrence of high percentage of these pathogenic bacteria in raw milk may be linked to its contamination at the time of collection, processing, strorage and distribution. This quantitative data could be utilized to better establish the appropriate levels of protection for raw milk, dairy products and processing technologies.

Anti-viral activity of Staphylococcus aureus lysates against herpes simplex virus type-Ⅰinfection:an in vitro and in vivo study

AIM:To investigate the effect of Staphylococcus aureus(S.aures)lysates(SALs)on herpes simplex virus type-Ⅰ(HSV1)infection in human corneal epithelial(HCE)cells and in a mouse model of HSV1 keratitis.METHODS:HCE,Vero,HeLa,and BV2 cells were infected with HSV1[HSV1f strain,HSV1f;HSV-1-H129 with green fluorescent protein(GFP)knock-in,HSV1g].Pre-or post-infection,SAL at various concentrations was added to the culture medium for 24 h.GFP fluorescence in HSV1g or plaque formation by HSV1f were examined.The effects of heat-treated SAL,precooled acetone-precipitated SAL,and SAL subjected to ultrafiltration(100 kDa)were evaluated.The effects of other bacterial components and lysates on HSV1 infection were also tested,including lipoteichoic acid(LTA),peptidoglycan(PGN),staphylococcal protein A(SPA),andα-hemolysin from S.aureus(α-toxin)as well as lysates from a wild-type S.aureus strain,S.epidermidis,and Escherichia coli(W-SAL,SEL,and ECL,respectively).In addition,SAL eye drops were applied topically to BALB/c mice with HSV1 keratitis,followed by in vivo observations.RESULTS:The cytopathic effect,plaque formation(HSV1f),and GFP expression(HSV1g)in infected cells were inhibited by SAL in a dose-dependent manner.The active component of SAL(≥100 kDa)was heat-sensitive and retained activity after acetone precipitation.In HSV1ginfected cells,treatment with LTA-sa,α-toxin,PGN-sa,or SPA did not inhibit GFP expression.SAL,W-SAL,and SEL(but not ECL)decreased GFP expression.In mice with HSV1 keratitis,SAL reduced corneal lesions by 71%.CONCLUSION:The results of this study demonstrate that SAL can be used to inhibit HSV1 infection,particularly keratitis.Further studies are needed to determine the active components and mechanism underlying the effects of SAL.

Assessment of the inhibitory effects of sodium nitrite, nisin, potassium sorbate, and sodium lactate on Staphylococcus aureus growth and staphylococcal enterotoxin A production in cooked pork sausage using a predictive growth model

This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork sausage by inoculating sausage samples containing preservative with an S.aureus strain producing staphylococcal enterotoxin A(SEA)and then storing them at 37℃ for 36 h.Samples were analyzed every 3 h to count the S.aureus colonies and to detect SEA.The modified Gompertz model was used to describe S.aureus growth in the samples under various conditions,and the preservatives with a significant antimicrobial effect were selected.In addition,the antimicrobial effects of the selected preservatives under various concentrations were tested.Results showed that sodium nitrite,nisin,and potassium sorbate had a weak effect against S.aureus growth and had no effect against SEA production,whereas sodium lactate could significantly inhibit S.aureus growth and SEA production.Moreover,the antimicrobial effect of sodium lactate was concentration-dependent,wherein sodium lactate concentration<12 g/kg showed no inhibitory effect,but when the concentration was increased to 24 g/kg,sodium lactate could effectively inhibit S.aureus growth and SEA production,and at 48 g/kg,sodium lactate had a significant inhibitory effect.

Infectious Spondylitis-Associated Staphylococcus aureus with Virulence Gene pvl or tst Causes More Necrosis than Apoptosis in Human Alveolar Basal Epithelial Cell Line A549

Methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA, respectively) can cause non-tuberculosis infectious spondylitis. In 43 cases of bacterial infectious spondylitis, Mycobacterium tuberculosis and S. aureus were the two major causative pathogens. MRSA caused more anterior operations and thoracic infections, while MSSA caused more posterior infections and lumbar infections. Differences between six S. aureus isolates from infectious spondylitis were characterized. MLST and staphylococcal cassette chromosome mec (SCCmec) analysis identified MSSA ST959 and ST30 isolates, MRSA ST239/SCCmec IIIA isolates 2 and 3, ST59/SCCmec IIIA-like isolate 6, and ST30/SCCmec IV isolate 5. While all of the isolates were resistant to penicillin and ampicillin, the MRSA isolates were more resistant than the MSSA isolates. Carbapenem-resistant MRSA ST239/SCCmec IIIA and ST59/SCCmec IIIA-like isolates of the agr1 type were also resistant to clindamycin and erythromycin. Leukocidin genes (pvl or lukED) and hemolysin genes (hla, hld and hlg) were present in all of the isolates. All six isolates caused more necrosis than apoptosis in the human alveolar basal epithelial cell line A549;however, ST59/SCCmec IIIA-like isolate 6, ST30/ SCCmec IV isolate 5 with pvl genes, and MSSA ST30 isolates with tst caused greater than 40% cell death after the 4-h incubation. Regardless of the MRSA isolate and its SCCmec type or the MSSA isolate, the infectious spondylitis-associated S. aureus isolates differed genetically, and the pvl and tst genes may be important genes for cell necrosis.

Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.

Prevalence of Methicillin-Resistant <i>Staphylococcus aureus</i>in Raw Hamburgers and Ready-to-Eat Sandwiches Commercialized in Supermarkets and Fast Food Outlets in Brazil

Although well known, Staphylococcus aureus is a bacterium that remains widely studied because of its high pathogenic potential and its ability to develop resistance to antibiotics routinely used in clinical practice. The present study investigated the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in hamburgers and sandwiches sold in supermarkets and fast food outlets in Salvador, BA, Brazil. Fifty samples of frozen raw hamburgers (25: beef and 25: chicken) and 50 samples of ready-to-eat sandwiches (25: beef and 25: chicken) were collected and investigated for the presence of MRSA. MRSA was present in 32% of the hamburgers and 8% of the sandwiches. The frequency of MRSA was higher in the samples containing chicken meat. However, the statistical analysis showed no association between MRSA presence and the type of meat investigated (P > 0.05). The high prevalence of MRSA in hamburgers and the presence of the microorganism in ready-to-eat sandwiches are worrying and indicate the need for better control during food preparation to prevent the spread of bacteria.