Antimicrobial activity of binary combinations of natural and synthetic phenolic antioxidants against Enterococcus faecalis.

This study investigated the antimicrobial activity of 3 natural (thymol, carvacrol, and gallic acid) and 2 synthetic [butylated hydroxyanisole (BHA) and octyl gallate] phenolic compounds, individually and in binary combinations, on 4 dairy isolates of Enterococcus faecalis with different virulence factors (ß-hemolytic, gelatinase, or trypsin activities; acquired resistance to erythromycin or tetracycline; and natural resistance to gentamicin). A checkerboard technique and a microdilution standardized method were used. All compounds individually tested exhibited antimicrobial activity against E. faecalis, with minimal inhibitory concentrations (MIC) ranging from 30 µg/mL (octyl gallate) to 3,150 µg/mL (gallic acid), although no significant differences were detected among strains to each phenolic compound. Carvacrol in combination with thymol or gallic acid, and gallic acid combined with octyl gallate showed partial synergistic inhibition of all E. faecalis strains. The most effective combinations were thymol+carvacrol and gallic acid+octyl gallate, as the MIC for each of these compounds was reduced by 67 to 75% compared with their respective individual MIC. These results highlight the possibility of using combinations of these phenolic compounds to inhibit the growth of potential virulent or spoilage E. faecalis strains by reducing the total amount of additives used in dairy foods.

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High ß-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.

Phenotypical characteristics of Shiga-like toxin Escherichia coli isolated from sheep dairy products.

AIMS: To analyse phenotypical characteristics of Shiga toxin-producing Escherichia coli (STEC) strains from ovine origin. METHODS AND RESULTS: A total of 13 STEC strains (eight O157 and five non-O157) isolated from sheep dairy products were used in this study. Biochemical traits, motility, haemolytic activity, resistance to tellurite-cefixime, maximum growth temperature and antibiotic resistance were determined. The STEC strains were grouped into nine biochemical and physiological biotypes (five for the O157 and four for the non-O157 strains). All STEC strains showed resistance to bacitracin, cloxacilin, penicillin and tylosin. CONCLUSIONS: Different biotypes and antibiotic resistance patterns of STEC isolated from sheep dairy products were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will be a contribution to the better characterization of STEC isolated from sheep dairy products, which have, to date, been scarcely studied, and to the better understanding of the risks associated with its consumption.

Evaluation of the spiral plating system for the routine assessment of indicator microorganisms in raw ewe's milk.

The numbers of members of different microbial groups in bulk raw ewe's milk used for cheesemaking (46 samples, taken on receipt at the dairy over 1 year) were assayed by the spiral plating system to determine the effectiveness of this method compared with that of widely accepted conventional methods in providing counts. The results indicated that for ewe's milk, the suitability of the spiral plating system depends to a great extent on the microbial group studied. Although "spiral" counts of mesophiles, psychrotrophs, coliforms, and Enterobacteriaceae could be considered equivalent to those obtained by conventional techniques (r > or = 0.90; variance between replicate platings approximately 0.005), the automated method was found not to be suitable for the assessment of other groups of indicator bacteria (thermodurics and enterococci). Counts of lactic acid bacteria and yeasts and molds were affected significantly (P < 0.05) by the plating method, although other statistical parameters were more favorable (r = 0.88 and r = 0.82, respectively; 95% confidence limits within 0.5 log units). Finally, counts of staphylococci, particularly on Baird-Parker medium, showed less variation and higher reproducibility with the spiral method. Nevertheless, for the routine microbiological analysis of ewe's milk, the spiral plating system, with its time-, effort-, and material-saving advantages, is preferred over the conventional method.

Temperature characterization of psychrotrophic and mesophilic Bacillus species from milk.

A total of 50 isolates of Bacillus spp. and one reference strain were investigated for their growth at 6.5 degrees C for 10 d, 30 degrees C for 3 d and 40 degrees C for 2 d. The results obtained differentiated three physiological groups: one clearly psychrotrophic (able to grow at 6.5 degrees C in 10 d, but not at 40 degrees C in 2 d), one intermediate in psychrotrophy (it grew at both 40 and 6.5 degrees C) and one mesophilic (capable of growth at 30 and 40 degrees C, but not at 6.5 degrees C). The proportion of strains in the second group was higher among isolates of B. cereus than for other Bacillus spp. However, the proportion of real mesophilic strains was lower for B. cereus isolates. Psychrotrophic B. cereus grew better at both 6.5 and 30 degrees C than other psychrotrophic Bacillus spp. Using eight strains, a correlation between differential growth at mesophilic temperatures (count at 30 degrees C minus count at 40 degrees C) and a standard psychrotrophic count at 6.5 degrees C for 10 d (r = 0.95) was obtained in mixed cultures when the psychrotrophic flora count was < or = 1 log (cfu/ml) lower than the mesophilic count.

Evaluation of different systems for the identification of Bacillus strains isolated from Spanish fermented sausages.

Sixty-nine isolates obtained during the manufacture and ripening of Spanish fermented sausages were identified to species level using the Vitek Bacillus biochemical card, the dichotomous key and table proposed by Berkeley et al. ((1984). In Methods in Microbiology, Vol. 16. Academic Press, London, p. 291), morphological and physiological tests and the API 20E miniaturized system. None of the tested systems was entirely satisfactory and the final identification was mainly done on the basis of cellular morphology and the table of test results. Our isolates belonged to the species: B. subtilis (37), B. megaterium (22), B. pumilus (5), B. circulans (3) and unidentified (2). Forty-five cultures (65.2%) were accurately identified with the dichotomous key. A similar figure for the Vitek Bacillus biochemical card was 36%. The results of the API 20E system were very reproducible, especially those of the Voges-Proskauer test. Most of the strains of B. megaterium were misidentified as B. subtilis with the dichotomous key. On the other hand, a high percentage of the cultures belonging to B. subtilis were misidentified as B. megaterium with the Vitek system.

Numerical characterization study of Micrococcaceae associated with lamb spoilage.

A computer-assisted characterization of 296 Micrococcaceae isolates obtained from aerobically chill-stored lamb carcasses was carried out using a probability matrix and Bayesian identification theorems, complemented with cluster analysis. Preliminary identification was done with an original probability matrix comprising 37 previously described taxa and 32 tests. Although its statistical quality was adequate, the percentage of identification of field strains to species level was only 70% (96.6% identified with genera). To achieve an improved characterization, cluster analysis was subsequently performed on this group and an additional 26% could be associated with defined species, with five more taxa defined. The combined use of both approaches was judged positive as new identifications and better discrimination could be achieved. The majority of our isolates belonged to the Staphylococcus species group. Many species and groups of staphylococci increased as the spoilage progressed.

Factors affecting spoilage microflora succession on lamb carcasses at refrigeration temperatures.

Three bacterial groups were detected during the shelf-life of lamb carcasses stored at refrigeration temperatures: coryneforms (mostly Brochothrix thermosphacta), Gram-positive cocci (staphylococci) and Gram-negative bacilli (Pseudomonas spp. and Moraxella spp.). Influence of categorical variables such as sampling day, sampling area and sampling method on their recovery was analysed by a chi-square test of independence and loglinear analysis. Hierarchical loglinear modelling proved to be very useful as several three-way interactions of factors were observed.

Modern microbiological methods for foods: colony count and direct count methods. A review.

Over the last years methods for enumeration of microorganisms in foods are changing rapidly. Techniques based on totally new concepts as well as instruments and miniaturized systems that allow the automation and simplification of existing microbiological procedures have been developed. These rapid methodologies should satisfy the increasing requirements for effective quality assurance of foods. In the present paper we review some of the more interesting methods based on colony count or direct bacterial count.

Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses.

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30 degrees C were Ps. fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7 degrees C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group (Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (> 74% SSM) and Ps. lundensis (> 80%). The recovery of pseudomonads seemed to be affected by the sampling day.

Numerical taxonomy of gram-negative, nonmotile, nonfermentative bacteria isolated during chilled storage of lamb carcasses.

A numerical taxonomic study using 75 characters was performed with 132 strains of gram-negative, nonmotile, nonfermentative bacteria selected on the basis of lack of motility and Gram reaction among 1,200 cultures isolated during aerobic storage of lamb carcasses. At the 80% similarity level (SSM), eight clusters were formed. Strains in clusters 1 to 6 could be identified as members of the family Moraxellaceae and, more specifically, as members of the Psychrobacter-[Moraxella] phenylpyruvica subgroup. Of these strains, clusters 1 and 2 (88 strains) were identified as [Moraxella] phenylpyruvica and cluster 3 (15 strains) was identified as Psychrobacter immobilis. Clusters 4, 5, and 6 were not identifiable with any species. Clusters 7 and 8 consisted of 14 strains considered nonmotile variants of Pseudomonas fragi. The highest separation indices corresponded to acid production from certain carbohydrates (melibiose, L-arabinose, and cellobiose). Although strains of Psychrobacter-Moraxella clusters were relatively frequently identified at the completion of slaughter, very few cultures were detected on spoiled carcasses. It appears, therefore, that this group of organisms has only low spoilage potential.

Biovars of coagulase-positive staphylococci isolated from bovine mastitic milk.

Fifty strains of coagulase-positive staphylococci associated with bovine mastitis were biotyped. Of them, 20 were identified as biovar C (cattle & sheep), 17 as biovar B (poultry & swine), 2 as biovar D (hares) and 1 as biovar F (pigeon & fox). Of the remaining strains, 2 were closely related to human biovar A, 2 failed one property to be classified as biovar B, 3 shared properties of biovars B and D and 3 could not be identified. Bovine strains belonging to biovar C formed an heterogeneous group showing differences in crystal violet growth type and production of alpha haemolysin. Only strains associated with biovar A were Tween 80 positive. The highest incidence of lytic reactions amongst all biovars was with phages 42 E (III) and 102 (IV). Antibiotic resistance was most frequently found in biovar C (65%). The 2 strains in biovar D, one classed as biovar C and one as intermediate, were enterotoxigenic (C or D toxins).