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.     

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml⁻¹. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10³ cfu ml⁻¹ at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3·2 ng g⁻¹ of cheese made with an initial population of 10³–10⁶ cfu ml⁻¹. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

F eresu , S. & N yati , H. 1990. Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products. Journal of Applied Bacteriology 69 , 814–821.
The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20C for 24 h and then stored at either 20C or 5C for 96 h.
Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20C and 5C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10⁷-10⁹/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10³-10⁶ and 10²-10⁵ during storage of the fermented product at 20C and 5C respectively. Higher maximum numbers, 10⁹-10¹⁰, of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10⁵-10⁸ and 10⁴-10⁷ during storage of the fermented product at 20C and 5C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

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).     

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell-free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from10⁵ cfu ml⁻¹ to 10³ cfu ml⁻¹ after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.     

Interactive Growth of Staphylococcus aureus Strains with a Poultry Skin Microflora in a Diffusion Apparatus

The growth of different biotypes of Staphylococcus aureus strains isolated from poultry was studied using the NBS Ecologen in which the interacting mixed culture was derived from the microfiora of hen skin and separated from the Staph. aureus culture by a membrane of 0.4 μm pore size. Inhibition of growth of the Staph. aureus cultures occurred with strains from each biotype. Marked inhibition of growth was always accompanied by the production of large numbers (>10⁹/ml) of plaque forming units (phage). In the mixed culture chamber poultry phage group C strains became the predominant Staph. aureus type. The phages produced by the mixed culture showed a wide spectrum of lytic activity for the propagating strains of the human, bovine and poultry phage sets.     

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.     

Irish kefir-like grains: their structure, microbial composition and fermentation kinetics

The structure of six Irish kefir samples was studied in the electron microscope, and the microbial composition and fermentation kinetics during growth in 10% reconstituted skim milk at 21°C. The microbial composition of the six samples was similar; at the end of the fermentation the counts of lactococci, leuconostocs, lactobacilli, acetic acid bacteria and yeasts were 10⁹, 10⁸, 5 × 10⁶, 10⁵ and 10⁶ ml⁻¹ respectively; the levels of acetic acid bacteria and lactobacilli showed some intersample differences. Lactate was the major metabolite followed in order by ethanol, acetate and acetoin. The final concentrations of L-lactate produced (66–90 mmol kg⁻¹) were 10-fold higher than those of D-lactate. Acetate and ethanol concentrations varied from 4 to 14 and 2 to 40 mmol kg^−'1 respectively. The rates of citrate utilization and concentration of acetoin produced during growth differed between samples. Scanning electron microscopy showed not only variation between the interior and exterior of the sample but also large variation between different sections of the interiors and exteriors of the same sample. Long and short, and straight and curved rods and yeasts were seen in all samples, the curved rods observed in the interior, but lactococci were seen on the surface of only one sample. There were no gross differences in structure between samples.     

Interaction between the effects of bacteria and dry storage on the opening and water relations of cut rose flowers

W.G. VAN DOORN AND K. D'HONT. 1994. Flowering stems of four rose cultivars (Sonia, Madelon, Jacaranda and Frisco) were placed in aqueous suspensions of bacteria at 10⁴ and 10⁸ colony-forming units (cfu) ml^-1 for 24 h at 5C, then stored dry or held in water for 24 h at 8C and subsequently placed in vase-water at 20C. The effects of these treatments on vase-water uptake were similar to the effects on flower opening. In Sonia and Madelon roses flower opening was negatively affected both by 10⁸ cfu ml^-1 of bacteria and by dry storage. No effect was found at 10⁴ cfu ml^-1, but this concentration had a detrimental effect on flower opening when combined with dry storage. Although flower development in Jacaranda roses was not affected by the bacteria treatments it was inhibited by dry storage. This inhibition was progressively greater when the stems had previously been pulse-treated with a larger number of bacteria. Flower opening in Frisco roses was not affected by even the highest concentration of bacteria, nor by the period of dry storage. It is concluded that placing flowers in water containing bacteria (up to 10⁸ cfu ml^-1) may not always have a negative effect on flower development in cut rose flowers but, together with the effects of dry storage, the presence of even a low number of exogenous bacteria (10⁴ cfu ml^-1) inhibits the development in several cultivars. Such bacterial counts are nearly always found in samples of water used for standing roses during distribution to the consumers.     

Post-processing microflora and the shelf stability of gari marketed in Port Harcourt

Gari was examined for its post-processing microbial content. Aerobic mesophilic bacteria and fungi were isolated from all samples. The total viable bacterial counts ranged from 2.0 × 10² to 8.0 × 10⁴ cfu/g. Fungal counts ranged from 1.0 × 10² to 1.5 × 10⁴ cfu/g. The total viable counts of fresh samples were much lower than those of market and packaged samples. Bacillus, Micrococcus and Proteus spp. were the bacteria isolated, Aspergillus niger, Aspergillus flavus and Penicillium spp. the fungi. Food borne parasites and pathogens such as Staph. aureus and Clostridium perfringens were not found. The gari samples were quite stable, having a shelf life of 3–6 months. The water activities of the samples ranged from 0.52 to 0.68. Based on the microbial counts of the samples, the critical upper limit for the safety of gari was set at 10⁴ cfu/g dry sample.     

Ethanol Production from Glucose by Torulopsis glabrata Occurring Naturally in the Stomachs of Newborn Animals

S ummary : The elucidation of the mechanism of ethanol production in the stomachs of newborn animals is described. The condition was reproduced in lambs and piglets by feeding glucose in fat-free milk, and shown to arise from fermentation by the naturally occurring yeast Torulopsis glabrata , which can reach population densities of 10⁶ viable cells/ml of stomach contents. These yeasts ferment glucose, producing up to 500 mg of ethanol/100 ml of contents in the stomach, but do not ferment sucrose or lactose. High levels of ethanol in plasma of lambs are accompanied by obvious symptoms of intoxication. The characteristics of a constantly occurring coliform type bacterium, found at densities up to 10⁸ viable cells/ml in association with the Torulopsis yeasts were examined. Its precise role is not clear.     

Contamination of broiler chickens by Staphylococcus aureus during processing; incidence and origin

Staphylococcus aureus was present in only small numbers ( ca 10/g) on the skin of broiler chickens before processing. During processing, contamination of carcases with this organism increased to > 10³/g of skin. Based on the results of phage typing, it was shown that the increase in contamination was due to a strain of Staph. aureus which was indigenous to the processing plant. Plucking and evisceration appeared to be the main stages at which contamination of carcases with Staph. aureus occurred.     

A Simple Sensitive Method for Determining Staphylococcal Thermonuclease in Cheese

A simple quantitative method is described for the determination of staphylococcal thermostable nuclease in cheese. The method does not require concentration or purification of the nuclease prior to determination because of the increased sensitivity of the assay (0.5 ng/ml). Assay results of cheddar cheese made from pasteurized milk inoculated with enterotoxin-A producing Staphylococcus aureus strains demonstrated the sensitivity and reliability of the method for indicating the presence of Staph. aureus at concentrations of ≥ 1.4 × 10⁶/g of cheese.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Association of bacteria with the fungal fermentation of soybean tempe

Bacteria grew to viable populations of 10⁸–10⁹ cfu/g during the fermentation of soybeans into tempe with the fungus, Rhizopus oligosporus. Bacillus pumilus and B. brevis were the predominant bacterial species, reaching populations of approximately 10⁸ cfu/g during the 48 h fermentation. Species of Streptococcus faecium, Lactobacillus casei, Klebsiella pneumoniae and Enterobacter cloacae also contributed to the fermentation and achieved populations of 10⁶–10⁷ cfu/g. and accepted 25 May 1989     

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.     

Fungal growth during rice tapé fermentation

Fungal growth was quantified during Indonesian rice tapé fermentation using an agar-film technique following sample homogenization for 1 min at 25000 rev/min. After 72 h fermentation, mould hyphal length was 0·68 km/g, yeast hyphal length 2·1 km/g and the numbers of mould chlamydospores and single yeast cells were 14 times 10⁵/g and 5·1 times 10⁷/g respectively. The estimated fungal biomass in rice tapé after 72 h was 25 mg/g dry weight with 62% of this being mould hyphae, 24% mould chlamydospores, 13% yeast hyphae and 1% yeast cells.     

Yoghurt: an unlikely source of Campylobacter jejuni/coli

Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2–5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10⁵–10⁸ cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3–5 logs in 30 min. Strong inorganic acid HC1, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni , it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.