Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.     

Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR-dCas9

Bacteriolytic enzymes (cell lytic enzymes) are promising alternatives to antibiotics especially in killing drug-resistant bacteria. However, some bacteria slowly become resistant to various classes of peptidoglycan hydrolases, for reasons not well studied, in the presence of growth-supporting nutrients, which are prevalent at sites of infection. Here, we show that Staphylococcus aureus, a human and animal pathogen, while susceptible to the potent staphylolytic enzyme lysostaphin (Lst) in buffered saline, is highly resistant in the rich medium tryptic soy broth (TSB). Through a series of biochemical analysis, we identified that the resistance was due to prevention of Lst-cell binding mediated by the wall teichoic acids (WTAs) present on the cell surface. Inhibition or deletion of the gene tarO responsible for the first step of WTA biosynthesis greatly reduced S. aureus resistance to Lst in TSB. To overcome the resistance, we took advantage of the gene regulation potential of CRISPR-dCas9 and demonstrated that downregulation of tarO, tarH, and/or tarG gene expression, the latter two encoding enzymes that anchor WTAs in the outer layer of cell wall peptidoglycan, sensitized S. aureus to Lst and enabled eradication of the bacterium in TSB in 24 hr. As a result, we elucidate a key mechanism of Lst resistance in metabolically active S. aureus and provide a potential approach for treating life-threatening or hard-to-treat infections caused by Gram-positive pathogens.     

Adsorption of Staphylococcal Bacteriophage by Milk Proteins

Propagation of homologous bacteriophage in a culture of Staphylococcus aureus (1:1 ratio of phage to bacteria) in Trypticase Soy Broth (TSB) and in skim milk indicated more activity of phage in TSB. Early lysis of bacteria in skim milk followed by a pronounced rise in bacterial population suggested that staphylococcal phages were being inactivated by milk. Titration of phages from skim milk, whey, and TSB indicated about 90% adsorption of phages by acid- and heat-precipitable proteins of skim milk, whereas numbers recovered from whey were quite comparable to those recovered from TSB. Reducing the pH from 6.5 to 4.0 increased the percentage of phages recoverable from skim milk from 10 to 56%. Apparently, the changes in electrical charges on the casein micelles at this low pH were responsible for release of many phages from their complex with casein.     

Doxycycline-Dependent Inducible and Reversible RNA Interference Mediated by a Single Lentivirus Vector

Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species.     

Plume height and surface coverage analysis of methicillin-resistant Staphylococcus aureus isolates grown in a CDC biofilm reactor

Biofilm formation is a dynamic process that leads to mature communities over time. Despite a general knowledge of biofilm community formation and the resultant limitations of antibiotic therapy, there is a paucity of data describing specific plume heights, surface coverage and rates of maturation. Furthermore, little is published on the effect that the broth medium might have on the degree of biofilm maturation. In this study, three strains of methicillin-resistant Staphylococcus aureus (MRSA) (USA300, USA400 and a clinical isolate) were grown in brain heart infusion broth (BHI) or tryptic soy broth (TSB). Following growth, SEM images were captured for 3-D analysis to assess plume height. TSB produced significantly higher plume heights of USA300 and USA400 compared to BHI. Broth type was less influential on the clinical isolate. The data indicate that broth type and time may be important factors to consider when assessing maturation and plume height formation of MRSA biofilms.     

Effects of beta-lactam antibotics on thymidine incorporation of bacteria

Five strains each ofEscherichia coli, Proteus mirabilis, andStaphylococcus aureus were grown in Trypticase soy broth (TSB) with or without penicillin, oxacillin, ampicillin, amoxicillin, or cefamandole. [³H]Thymidine incorporation and the optical density of the cultures were determined at an hourly interval for the duration of incubation. All strains ofP. mirabilis showed after 2 and 3 h of incubation of 5-to 16-fold increase in the specific activity of [³H]thymidine incorporated in the presence of MIC of the antibiotics as compared to controls grown without the drugs.E. coli andS. aureus showed smaller increases in thymidine incorporation than didP. mirabilis. After 4–5 h the antibiotics produced an inhibition of [³H]thymidine incorporation. At the MAC, the responses were of a smaller magnitude. Regardless of whether these changes are the result of specific interference or just lack of [³H]thymidine incorporation, they are directly related to the antibiotic activity of agents known not to affect DNA synthesis. The monitoring of [³H]thymidine incorporation detects early antibiotic activity probably earlier than other current systems which are used for this purpose.     

Adherence ofStaphylococcus epidermidis to human pharyngeal epithelial cells: Evidence for lipase-sensitive adhesin and glycoprotein receptor

The adherence of eight strains ofStaphylococcus epidermidis to human pharyngeal epithelial cells (PEC) was investigated. All bacterial strains were nonencapsulated and non-slime producers. Binding was mannose resistant and was not related to surface hydrophobicity and surface charge of bacteria. Adherence to epithelial cells was reduced four- to tenfold (P<0.01) on pretreatment of bacteria with lipase while neuraminidase, phospholipase C, trypsin, and sodium periodate did not alter their binding. The surface carbohydrate profile of bacteria was studied by monitoring adherence to Lectin-Sepharoses. The bacteria did not conform to any pattern, and there was no relation to strain variation. The pretreatment of PEC with trypsin and sodium metaperiodate produced a marked reduction in bacterial binding (three- to 25-fold, P<0.01), but neuraminidase, phospholipase C, and lipase did not have any such effect. These findings provide evidence that the receptors on the surface of PEC are glycoprotein in nature, while the bacterial adhesin is a lipase-sensitive material.     

Adherence ofStaphylococcus epidermidis to pharyngeal epithelial cells mediated by lipoteichoic acid

Prior treatment of pharyngeal epithelial cells (PEC) with lipoteichoic acid (LTA) derived fromStaphylococcus epidermidis produced a marked inhibition of adherence of the homologous strain and two heterologous strains. The inhibition was dose dependent and saturable with 100 µg/ml of LTA. However, pretreatment of PEC with deacylated LTA did not block the adherence of the three strains tested. A similar but less marked blocking effect on the adherence ofS. epidermidis to PEC was also observed with LTAs derived fromS. aureus andStreptococcus pyogenes. On treatment of bacteria with substances capable of binding to LTA, such as polyclonal mouse anti-LTA antibodies or with human albumin, a marked inhibition of bacterial adherence was observed. Immunofluorescence studies showed that anti-LTA antiserum bound readily to the surface of bacterial cells. These findings provide clear evidence that the lipid component of LTA located on the bacterial surface is centrally involved in the adherence ofS. epidermidis to human mucosal cells.     

Comparison of Several Selective Media for Isolation and Differentiation of Coagulase-Positive Strains of Staphylococcus aureus

Six coagulase-positive strains of Staphylococcus aureus which had been cultivated in Brain Heart Infusion broth, milk, and brine were plated on seven isolation media. A significant difference in the growth patterns of the individual strains was found as well as a significant effect resulting from the previous cultivation history before plating. Brine and, to a lesser extent, milk were found to reduce maximal cell concentrations attained, but strains grown in brine and milk showed greater ability to withstand the selective action of the isolation media. Fibrinogen applied to the surface of five of the media allowed the formation of characteristic halos by coagulase-positive strains of S. aureus. Only half of the strains studied produced a zone of precipitation on SM110-Egg Yolk agar. The isolation medium containing cycloheximide and a high level of polymxin B was most inhibitory to the organisms.     

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia

Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co‐culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air–liquid interface to allow an in‐depth evaluation of a simulated colonization state. Exposure to wild‐type, S. aureus bacteria or conditioned media killed airway epithelial cells within 1 day, while mutant S. aureus strains lacking alpha‐toxin (hla) persisted on viable cells for at least 2 days. Recent S. aureus CC30 isolates are natural hla mutants, and we observed that these strains displayed reduced toxicity toward airway epithelial cells. Quantitative real‐time polymerase chain reaction of known virulence factors showed the expression profile of S. aureus grown in co‐culture correlates with results from previous human colonization studies. Microarray analysis indicated significant shifts in S. aureus physiology in the co‐culture model toward lipid and amino acid metabolism. The development of the in vitro colonization model will enable further study of specific S. aureus interactions with the host epithelia.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Structural investigation of human S. aureus-targeting antibodies that bind wall teichoic acid

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.     

Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus

Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. ¹³C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.Abbreviations PYK Peptode - Yeast extract K₂HPO₄     

Staphylococci Bind Heparin-Binding Host Growth Factors

Staphylococcus aureus, which mediated binding to heparan sulfate, and also strains of coagulase-negative staphylococci (CNS) adhered in high numbers to polymers with end-point attached heparin. A characteristic feature of several cell growth factors is strong affinity for heparin. In the present study, binding of the ¹²⁵I-labeled heparin-binding growth factors (HBGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and platelet-derived growth factor (PDGF) by S. aureus and CNS strains was examined. Staphylococcal strains used in this study bind bFGF and PDGF, but not aFGF. The binding of bFGF and PDGF was time dependent, influenced by pH and ionic strength for S. aureus Cowan 1. Preincubation of staphylococcal cells with unlabeled bFGF enhanced bFGF binding, but heparin, protamine sulfate, poly-L-lysine, and suramin were potent inhibitors of ¹²⁵I-bFGF binding to cells of S. aureus Cowan 1. Glycosaminoglycans of comparable size (chondroitin sulfate), other polysulfated polymers (λ-carrageenan, fucoidan), and some polysulfated polysaccharides (dextran sulfate, pentosan polysulfate) inhibited binding of both GFs to various extents. The partial inhibition of binding of both GFs after protease and periodate treatments indicates that both proteinaceous and other carbohydrate moieties participate in the binding. A lysozyme cell surface extract and bacterial lysates of S. aureus Cowan 1 competitively inhibited binding of ¹²⁵I-bFGF and ¹²⁵I-PDGF. These results suggest that staphylococci have the ability to bind two of the HBGFs, bFGF and PDGF, but not aFGF, via more than one cell structure. These binding structures seem to be exposed on the cell surface and deeply anchored in the cytoplasmic membrane as well.     

GWAS研究大肠杆菌和金黄色葡萄球菌种间互作进化机制

【目的】通过实验室培养模拟自然环境微生物相互作用,进而找到影响细菌基因型和表型的基因。【方法】将大肠杆菌和金黄色葡萄球菌在实验室条件下进行单独培养和两两混合培养并连续转接,通过得到的数量表型与最大生长速率表型做全基因组关联分析(GWAS),对得到的与表型相关的SNP进行注释与分析。【结果】162个SNP位点影响到大肠杆菌原始菌株与共培养菌株的生长,36个SNP位点影响大肠杆菌菌株在单独培养和共同培养的生长。总共有85个SNP位点影响金黄色葡萄球菌的原始菌株与单独培养。其中5个基因在之前文献中已有报道。对影响不同时间点细菌数量变化形状的SNP位点进行功能注释,大肠杆菌中有706个与生长性能相关。金黄色葡萄球菌中,129个和不同的生长性能相关。大肠杆菌SNP位点的13个基因在之前的研究中已有报道。【结论】混合培养和单独培养都检测到与生长相关的显著基因,本研究表明了GWAS在研究细菌互作进化机制方面的潜力。     

Effect of medium on growth and subsequent survival,after freeze-drying,ofLactobacillus delbrueckii subsp.bulgaricus

Summary Growth ofLactobacillus delbrueckii subsp.bulgaricus, grown at a constant pH (5.7) in papain-or pepsin-treated whey was superior to that in whey or whey permeate, but inferior to growth in milk; it was not significantly different from that found in a 1:1 whey: milk medium. When 2% sodium citrate was added to the fermented medium, the cell recovery levels (59–75%) upon centrifugation were not significantly different in papain-treated media compared to untreated substrates. Cultures obtained from papain-treated milk or whey showed similar mortality levels following freeze-drying (99%) to those grown on untreated milk or whey. Addition of Tween 80 to the growth medium improved survival rate by a factor of ten.     

Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important pathogen for both humans and animals, and it has been an ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. Elimination of S. aureus with classic antibiotics is difficult, and the current study aimed to evaluate the efficacy of ethanolic extracts of propolis (EEP) against S. aureus cultivated in complex media or milk. EEP (0–0.5 mg ml⁻¹) decreased growth of S. aureus in BHI media and 1 mg ml⁻¹ was bactericidal against washed cell suspensions (10⁷ CFU ml⁻¹). Propolis extracts also killed S. aureus cells resuspended in milk, but the bactericidal dose was at least 20-fold greater. Cultures that were transferred for at least 60 generations with sub-lethal doses of propolis did not change much their sensibility to EEP. Atomic force microscopy images revealed changes in morphology and cell size of S. aureus cells exposed to EEP (0.5 mg ml⁻¹). Our results indicate that propolis extracts might be effective against mastitis-causing S. aureus strains in vivo, but milk constituents affect the inhibitory activity of propolis. Considering that propolis-resistance appears to be a phenotype not easily selected, the use of EEP combined or not with other antimicrobial agents might be useful for mastitis control in vivo.     

Effect of milk on surface properties of Staphylococcus aureus from bovine mastitis

Abstract The surface hydrophobicity of cells of Staphylococcus aureus strains isolated from bovine mastitis grown on conventional agar and broth media was drastically reduced after incubation with bovine milk. Strains grown in high carbohydrate-high salt media yielded cells with reduced surface hydrophobicity compared to cells grown in conventional media, and adding bovine milk to minimal medium also yielded cells with reduced surface hydrophobicity, as determined by hydrophobic interaction chromatography and the salt aggregation test. Incubation of strains in milk and growth in a medium supplemented with bovine milk also significantly changed bacterial surface charge as determined by free-zone electrophoresis. Strains with high or with decreased adsorptive and aggregating properties did not produce surface capsule or slime. Heat treatment (60° C or 80° C) of the bacterial suspensions did not significantly change their adsorptive and aggregating properties.     

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.