An approach to integrated data assessment in a proficiency test on the enumeration of Escherichia coli.

AIMS: To develop appropriate statistical approaches to plan and evaluate proficiency tests for the enumeration of Escherichia coli, addressing, in particular, a possible but frequently unavoidable lack of test sample homogeneity. METHODS AND RESULTS: Each of 50 laboratories analysed two samples of a stabilized suspension of E. coli in duplicate, using various media, inoculation methods, and incubation times and conditions. In parallel, the E. coli suspension was tested by the organiser for homogeneity and stability. Escherichia coli counts followed a log-normal distribution. After eliminating, by Youden analysis, two data sets that were considered outliers and eight data sets for underperformance of the laboratories (substantial lack of repeatability), the standard deviation of the mean was about 0·06 log(10) units. There was no evidence of bimodality of the data. Lack of homogeneity of distribution of bacteria had a strong effect on measurement uncertainty, in addition to laboratory bias and method repeatability. The homogeneity decreases during storage of the individual test vials; this effect could be modelled by the known kinetics of inactivation of micro-organisms. The results were confirmed by Monte Carlo simulations. CONCLUSIONS: By a tailored analysis of proficiency testing data, it is possible to distinguish the effect of lack of homogeneity, laboratory bias and method repeatability, on the measurement uncertainty. SIGNIFICANCE AND IMPACT OF THE STUDY: A statistic tool is provided to solve problems related to lack of stability of microbiological test material and to separate the effects of sample inhomogeneity from the performance of the individual laboratory.

Effect of redox potential on Bacillus subtilis and Bacillus licheniformis in broth and in pasteurized sausage mixtures.

Bacillus licheniformis (a facultatively anaerobe) and B. subtilis (aerobic) may cause spoilage in certain meat products pasteurized in hermetically sealed containers if these are stored with inadequate refrigeration. After having optimized a method for determination of the redox potential in sausage mixtures, we investigated the contribution of low redox potential (Eh) and anaerobiosis to the inhibition of these bacilli. With the Eh values encountered in pasteurized meat products there was no effect of the redox potential on growth of B. licheniformis, either in model broths or in mildly heated bologna-type sausage mixtures. When cultured in a model broth with head space, B. subtilis grew to about 10(7)/ml and was little affected by the initial Eh value (range between +250 and -150 mV) and the initial dissolved oxygen concentration. During exponential growth of this bacterium in the absence of oxygen, the Eh value increased by about 180 mV. In sausage mixture with air inclusions, B. subtilis attained slightly higher cell densities than in mixtures protected against air uptake but it was subsequently overgrown by facultative anaerobic bacilli. We conclude that knowledge of the Eh value of a meat product does not permit a prediction of the growth potential of bacilli in pasteurized meat products, and that a sufficient heat treatment and proper refrigeration are essential to control these bacteria.

Utilization of microbes to process and preserve meat.

This paper discusses how, and to what extent, the addition of microorganisms to meats helps to meet the needs of consumers and industry. Lactic acid bacteria adapted to meats improve the safety of fermented sausages by means of acid formation. Using selected strains, the safety of certain non-fermented, perishable meat products may be improved without affecting their shelf life. Certain bacteriocin-forming cultures may reduce the levels of Listeria monocytogenes in some meat products significantly, but their effect on the overall safety of meats is limited by the resistance of Gram-negative bacteria. Data on the effect of microorganisms on the sensory properties of fermented meats are summarized. For bacteria to have a probiotic effect, they need to attain high numbers during fermentation and/or storage of meats. Genetic engineering of cultures may improve certain properties of the strains but benefits to consumers and industry are too small to make them acceptable by consumers and regulatory bodies in the near future.

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.

Coupling factor adenosine-5'-triphosphatase from Rhodospirillum rubrum: a simple and rapid procedure for its purification.

When photosynthetic membranes from Rhodospirillum rubrum, devoid of loosely bound small molecules and proteins, were passed through a French-pressure cell, the enzyme adenosine-5'-triphosphatase (EC 3.6.1.3.) (ATPase) was released into the soluble fraction. The solubilized ATPase was purified to homogeneity. In many respects it behaved like the enzyme purified by other workers, but it also hydrolyzed Mg-ATP with a small, but significant rate. Furthermore, it was much more stable. Maximal restoration of photophosphorylation in ATPase-depleted membranes was achieved by addition of about 1 mg purified ATPase per mg bacteriochlorophyll. For reconstitution of NAD+-photoreduction, about half of this amount was saturating.

Behaviour of Listeria monocytogenes in meat and its control by a bacteriocin-producing strain of Lactobacillus sake.

Lactobacillus sake Lb 706 can release a bacteriocin inhibitory to Listeria monocytogenes. In MRS broth, viable counts decreased rapidly when Lact. sake Lb 706 was added, whereas growth of the listerias was not affected by a bacteriocin-negative variant of the same Lactobacillus strain. Inhibition of L. monocytogenes was also observed in pasteurized minced meat inoculated with Lact. sake Lb 706. The bacteriocin produced is apparently effective in meat. However, the effect of the bacteriocin producer was less evident in minced meat than in broth. In comminuted cured raw pork filled into casings (German-type 'fresh Mettwurst'), L. monocytogenes was able to grow at a pH of 6.3, but addition of Lact. sake Lb 706 prevented the growth of listerias during the first few days after manufacture. At normal pH (5.7) L. monocytogenes did not multiply and addition of Lact. sake Lb 706 reduced viable counts of listerias by about one log cycle. Lactobacillus sake Lb 706 therefore may have some potential as a protective culture in meat products.

[Microbiological public health aspects in the use of rain water as water reservoirs for toilet flushing, garden irrigation and laundry]. / Mikrobiologisch-hygienische Aspekte bei der Nutzung von Regenwasser als Betriebswasser für Toilettenspülung, Gartenbewässerung und Wäschewaschen.

From a total of 102 rain water cisterns in use for toilet flushing, garden irrigation and laundering washing about 1,600 water samples were collected and subjected to microbiological analysis. The assays included aerobic heterotrophic microorganisms growing at 20 and 37 degrees C, respectively, as well as the identification of Escherichia coli, coliform organisms, faecal streptococci, Pseudomonas aeruginosa, staphylococci, yersiniae, salmonellae, shigellae, legionellae and yeasts. The median of the total number of cells per ml was 1,200 at 20 degrees C and 230 at 37 degrees C, respectively. Approximately 26 E. coli cells and 198 coliform organisms (median values) were found per 100 ml. In the case of cisterns manufactured of plastic the total number of cells was generally found to be lower than in samples collected from concrete or brick-made storage tanks. With the exception of the ubiquitously distributed organism Pseudomonas aeruginosa (found in 11.8% of the samples) and salmonella in only one sample, no other pathogens were detected. More than 95% of all analysed samples met the quality standards for bathing waters as set by the European Community. Provided certain precautions are taken, such as strict separation of mains for drinking water and rain water, as well as correct labelling of pipelines and collection sites, the use of rain water for toilet flushing, garden irrigation and laundry washing presents no unacceptable risk to public health.