Microbial colonization of an in vitro model of a tissue engineered human skin equivalent – a novel approach

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to ' in vivo ' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis , Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10²–10⁶ CFU SE⁻¹ on the surface of replicate SEs. Growth of all species was supported for upto 72–120 h, with recovery densities of between 10⁴–10⁹ CFU SE⁻¹. A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10² to 10⁸ CFU SE⁻¹, as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.     

The Use of Sulphamezathine for Inhibiting Proteus Spp. on Baird-Parker's Isolation Medium for Staphylococcus aureus

S ummary : The addition of 50 μg of sulphamezathine/ml to egg-tellurite-glycine-pyruvate agar was effective in suppressing the growth and swarming of Proteus spp. Small numbers of Staphylococcus aureus (10³/g) could be recovered quantitatively on the modified medium in the presence of up to 10⁶/g of mixed Proteus vulgaris and Proteus mirabilis strains.     

RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 10³ 10⁵ cfu/mL, with R²= 0.99. The detection limit of this method was 1.09 × 10³ cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.     

Vitronectin and type-I collagen binding by Staphylococcus aureus and coagulase-negative staphylococci

Abstract Binding of ¹²⁵I-labelled type-I collagen and ¹²⁵I-labelled vitronectin (human serum spreading factor or S-protein) was studied using Staphylococcus aureus and coagulase-negative staphylococci of different species. Binding of collagen and vitronectin was time dependent for S. aureus ISP 546, and S. haemolyticus E 2498/86. Co-operative binding of vitronectin and collagen by staphylococcal cells was demonstrated. Binding to S. haemolyticus E 2498/86 was more rapid and was enhanced in vitronectin/collagen mixtures than for either protein separately. Furthermore, pre-incubation of staphylococcal cells with unlabelled collagen enhanced vitronectin binding. When cells of S. haemolyticus E 2498/86 were treated with pronase E, proteinase K, subtilopeptidase A or trypsin, vitronectin-binding was decreased by 50% or more, whereas collagen-binding was protease resistant. For the strains of S. aureus tested, both vitronectin and collagen binding were found to be protease sensitive. Type-I collagen peptides inhibited collagen-binding to S. haemolyticus E 2498/86, whereas vitronectin-binding was not affected perhaps indicating different receptors for these proteins. The binding of both collagen and vitronectin was shown to be reversible, since bound ¹²⁵I-collagen and ¹²⁵I-vitronectin were displaced after adding excess of the homologous protein.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

Occurrence and survival of indicator/pathogenic bacteria in kusaya gravy

Coliform group bacteria, Staphylococcus aureus , salmonella and proteus were not detected in four samples of kusaya gravy, a kind of brine used for the production of special salt-dried fish in Japan. Vibrio parahaemolyticus at a level of 10¹/ml was presumptively detected only in one sample. When inoculated into the gravy at a level of 10⁷-10⁸/ml, none of these bacteria grew. Vibrio parahaemolyticus, Escherichia coli and Proteus morganii rapidly decreased in number and were not detectable after 5 d. Staphylococcus aureus numbers reduced only 1 log cycle after incubation for 7 d in the gravy. These results indicate the safety of the gravy with respect to the organisms.     

The survival of Staphylococcus aureus during the fermentation and storage of yoghurt

The effect of yoghurt culture R _x on the survival of Staphylococcus aureus CCM 5984 added to milk in various concentrations was observed during the fermentation and storage of yoghurt. The end of the fermentation process (3.5 h) was only accompanied by a slight reduction. During the storage of yoghurt at 4°C a 1-2 log reduction was observed. No Staph. aureus was detected in yoghurt produced from milk contaminated by 10³ Staph. aureus cells 1^-1 after 48 h of cold storage. When a concentration of 10² Staph. aureus cells was used for milk contamination, the pathogen was not recovered from yoghurt during the fermentation and storage. The fermentation and storage of yoghurt was accompanied by increases in lactic acid and titrimetric acidity, as well as by a decrease in pH value.     

EVALUATION OF CULTURE PROTOCOLS AND OXYRASETM SUPPLEMENTATION FOR ARCOBACTER SPP.

Growth of Arcobacter butzleri was evaluated in Brain Heart Infusion broth incubated aerobically, microaerobically, and with Oxyrase^TM supplementation (anaerobically). At initial concentrations of10¹ to 10³ cells/mL, A. butzleri populations reached 7.5 to 8.0 log CFU/ml in 48h at 37C in Oxyrase^TM -supplemented broth. The organism quickly declined in the other two systems to undetectable levels during the initial 24h of incubation. Only moderate population levels (ca. 3 log CFU/ml) could be detected in aerobic and microaerobic systems after 56h incubation. Growth of five Arcobacter spp. strains was evaluated at 30C in Brucella-blood broth, modified Cary and Blair Transport Medium, and a biphase cultural system. Strains were inoculated at a level of ca. 10³ cells/ml and incubated with and without Oxyrase^TM supplementation to the system for 36h. Both the Brucella-blood broth and the biphase system supported good growth of the strains, with counts reaching ca. 8 to 9 log CFU/ml. Modified Cary and Blair Transport Medium was the least effective cultural system. Oxyrase^TM provided slight to moderate stimulation of growth for most strains.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Detection of the site of contamination by Staphylococcus aureus within the defeathering machinery of a poultry processing plant

Counts of Staphylococcus aureus from samples of neck skin taken from poultry carcasses at different stages of processing showed that the numbers decreased approximately 10-fold during scalding but increased by almost 1000-fold during plucking, reaching up to 10⁴/g. Swab samples taken from the defeathering machinery yielded counts of ca. 10³/swab at the entry to the first plucker but these increased to ca. 10⁷at the exit, with a subsequent decrease through the second and third machines. Counts of other organisms growing on the Baird-Parker isolation medium showed much lower levels at the exit to the first plucker, suggesting that this was the major site of contamination at which S. aureus grew preferentially. Data obtained from four visits to the processing plant over a period of 10 months suggested that the incidence of S. aureus on the birds is affected by the season (summer or winter) whereas levels in the plucking machines depended on the day of sampling.     

Enterotoxigenicity of Staphylococcus aureus strains isolated from poultry: raw poultry carcases as a potential food-poisoning hazard

Staphylococcus aureus strains were isolated from end-of-lay poultry carcases obtained from a plant at two different stages of processing before and after storage at different temperatures. These strains were supplemented with Staph. aureus strains isolated from poultry from a wide range of sources and biotyped, phage typed, and tested for production of enterotoxins A-E. The isolates were found to consist of poultry and human specific strains and each of these groups contained strains able to produce enterotoxin. Poultry strains produced only enterotoxin D whereas human strains produced enterotoxins A, C and D. The hen carcases used in storage experiments were found to be naturally contaminated with enterotoxin D producing staphylococci. No enterotoxin D could be detected on any of the carcases even after storage at temperatures which allowed multiplication of the organisms to occur (final Staph. aureus counts ranged from 10² to 10⁷/16 cm² of breast skin).     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

The proliferative response of rat liver parenchymal cells after partial hepatectomy A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine

Abstract. DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [³H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [³H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination.
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [³H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [³H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G¹-fractions was only 2 and 7%, respectively.     

Pressure-stress effects on the ultrastructure of cells of the dimorphic yeast Candida tropicalis

Abstract Starved cells of cadmium-sensitive Staphylococcus aureus 17810S accumulated ¹⁰⁹Cd via the Mn²⁺ porter energized by the membrane potential (ΔΨ) generated by l-lactate oxidation. However, Cd²⁺ accumulation did not result in inhibition of respiration and consequent generation of electrochemical proton gradient (Δμ_H+) via the respiratory chain. Thus, Δμ_H+-consuming processes, such as ATP synthesis and [¹⁴C]glutamate transport proceeded normally, despite the presence of Cd²⁺ in the cytoplasm. The mechanism of the intrinsic cadmium-insensitivity of the l-lactate oxidizing system is discussed.     

Characterization of carp thrombocytes with specific monoclonal antibodies

Monoclonal antibodies (WCL6) specific for carp Cyprinus carpio thrombocytes were produced by immunizing mice with membrane lysates of IgM-negative peripheral blood leucocytes (PBL) and selected on the negative reaction with B cells. WCL6 was reactive with a membrane molecule of approximately 90 kDa and to a lesser extent with molecules of approximately 95 and 110 kDa. In general, between 30 and 40% of PBL were WCL6⁺ and appeared to be round to spindle-shaped cells. Immunohistochemical analysis of cryo-sections showed much higher numbers of WCL6⁺ cells in the spleen than in the pronephros, intestine and thymus. Flow cytometric analysis of cell suspensions isolated from these organs only revealed WCL6⁺ cells (4–10%) in the spleen. Electron microscopy of immunogold-stained WCL6⁺ PBL showed round but also some spindle-shaped cells with canalicular and granular structures, and a more irregular and electron-dense nucleus than found in lymphocytes. WCL6⁺ cells with electrondense pyknotic nuclei (without a clear nuclear envelope) were found also and their frequency increased with the length of the isolation and staining procedure used. In the spleen, several differentiation steps of WCL6⁺ cells were found and hence the spleen seems to be the thrombopoietic organ in carp. Thrombocytes from blood could be activated with collagen; the collagen-activated cells showed a higher side (90°) scatter by flow cytometric analysis and finally considerable cell death.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas

D.CHOI, R.S.W. TSANG AND M.H. NG. 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10⁶/ml of a smooth wild strain of Salm. typhimurium , and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Transformation and expression of a staphylococcal plasmid in Escherichia coli

Abstract A multiple antibiotic-resistant Staphylococcus aureus , was found to possess three plasmid bands in agarose gel electrophoresis. A plasmid of approximately 4.3 kb (pMC790/2) was found to code for ampicillin and tetracycline resistance and to have one Eco RI site when transformed into S. aureus RN 4220. pMC790/2 in unmodified form was transformed into a recA⁻ E. coli at a frequency of 1.2×10⁴ transformants/μg of plasmid DNA. Plasmid (pMC790/2) replicated, maintained itself stably and expressed far better in the E. coli host than in S. aureus .