Evaluation of Chromocult enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers

AIMS: To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult enterococci agar) recently developed by Merck. METHODS AND RESULTS: The study was carried out comparing Chromocult enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed. CONCLUSIONS: A good sensitivity of 98% was obtained for Chromocult agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P<0.01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult enterococci agar. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.     

Evaluation of three decarboxylating agar media to detect histamine and tyramine-producing bacteria in ripened sausages

Histidine- and tyrosine-decarboxylase activity of 175 strains of bacteria isolated from eight retail samples of Spanish ripened sausages was tested in three decarboxylating agars (Niven medium, Joosten and Northolt medium and modified decarboxylating agar of Maijala) and confirmed by an enzymic method (histamine) and thin-layer chromatography (tyramine). Enterobacteria and pseudomonads showed the highest percentage of positive responses to histamine and tyramine in the three decarboxylating agars, but only enterobacteria were subsequently confirmed as histamine-producing. Confirmed tyramine-producing strains were all identified as enterococci or lactic acid bacteria. The medium described by Joosten and Northolt was more sensitive and faster at detecting tyramine-producing micro-organisms. However, all three media failed to detect one histamine-positive strain of lactic acid bacteria used as a control.     

Use of a methylene blue azide medium for isolation of enterococci

A methylene blue azide medium (MBA), developed by Schaedler, Dubos, and Costello to isolate enterococci from the gastrointestinal tract of animals, was evaluated. This was done by comparing the isolation of enterococci from feces and saliva on the medium. Fifty-two catalase-negative, gram-positive cocci from human feces isolated from MBA were classified as enterococci. All strains grew in S F, 6.5% NaCl, and streptomycin broths, and all fermented mannitol. The isolates were provisionally subdivided into Streptococcus faecalis and S. faecium groups. S. faecalis-like strains fermented glycerol and pyruvate aerobically and produced acid in Snyder's medium (initial pH, 4.8). The S. faecium group fermented raffinose. Among all strains, several tests were variable. These included growth at 45 C, in 0.1% tellurite and in methylene blue milk. Three methods were employed to isolate and identify enterococci from the oral cavity. Direct streaking of MBA with saliva failed to produce any growth on the medium. Two other methods, with the use of various selective broths to promote the recovery of oral enterococci, failed to produce any bacteria capable of growing on MBA. The MBA-isolated fecal strains and oral viridans streptococci were generally indistinguishable on Mitis-Salivarius and K F agars. In experiments with fecal material, no gram-negative bacilli were found among the isolates selected. The MBA medium was judged as a high selectivity-low yield medium, and may provide a means of separating fecal and nonfecal enterococci.     

Characterization, Production, and Purification of Carnocin H, a Bacteriocin Produced by Carnobacterium 377

Carnocin H, a bacteriocin produced by a Carnobacterium sp., inhibited lactic acid bacteria, clostridia, enterococci, and some Staphylococcus aureus strains. Some strains of Listeria and Pediococcus were also sensitive to carnocin H. The bacteriocin was produced during the late stationary growth phase. Carnocin H was purified by cation exchange chromatography and reverse phase chromatography. Amino acid sequence and composition indicate that carnocin H is a novel bacteriocin belonging to the class II bacteriocins. The bacteriocin consists of approximately 75 amino acid residues with a highly cationic N-terminal containing six succeeding lysines. Activity, as measured by agar diffusion zones, was reduced at increased pH values, levels of indicator bacteria, NaCl, agar, and soy oil.     

Evaluation of the fermentability of oat fractions obtained by debranning using lactic acid bacteria

Aims: The overall kinetics of the fermentation of four oat fractions obtained by debranning using three potentially probiotic lactic acid bacteria were investigated. The main objective was to study the suitability of these fractions as fermentation media for the growth and the metabolic production of bacteria isolated from human intestine. Methods and Results: The cell growth, lactic acid production and substrate uptakes of the three lactobacilli was monitored for 30 h. An unstructured mathematical model was used to describe and fit the experimental data. In the medium from fraction B (1–3% pearlings or β-glucan-rich fraction) all strains reached the highest cell populations, maximum growth rates and maximum lactic acid productions. This could be because of the high levels of total fibre and β-glucan of this fraction. Limited growth and lactic acid formation was found in medium A (0–1% pearlings or bran-rich fraction). Conclusions: Medium B (1–3% pearling fraction) is the most suitable for fermentation and produces considerably higher probiotic cell concentrations. Significance and Impact of the Study: Debranning technology could be used to separate fractions from cereal grains for the production of functional formulations with higher probiotic levels than the ones that were obtained with the whole grain.     

Inhibitory effect of selected lactic acid bacteria on microflora associated with ready-to-use vegetables

The addition of selected lactic acid bacteria strains had a remarkable inhibitory effect on the growth dynamics of microflora associated with ready-to-use vegetables, during refrigerated storage. In particular, coliforms and enterococci were strongly reduced or eliminated from the products from the third day of storage. Lactobacillus casei strains proved more effective than pediococci. The use of lactic cultures able to produce bacteriocins and to grow at low temperatures could be a useful tool to preserve fresh vegetables and to ensure their microbiological safety.     

The lactic acid enterococci Enterococcus faecium and Enterococcus durans: nucleotide sequence diversity in 16S rRNA genes

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.     

Improvement of the membrane filter technique for enumeration of enterococci in water

The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2,3,5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC.MF technique), which was incubated at 37 C for 18 hr and further at 45 C for 24 hr. By this AC.MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC.MF technique was similar to that by the AC.MPN technique. The AC.MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.     

青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性及低温发酵菌株的筛选

【目的】探究青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性,筛选在低温条件下(10、15和25°C)生长性能较好的优良菌株。【方法】将垂穗披碱草青贮饲料中分离纯化的乳酸菌进行形态特征观察及16S r RNA基因测序鉴定;用MRS液体培养基在10、15和25°C条件下分离、初筛乳酸菌,选取高吸光度值的菌株作为优势菌株。用绿汁发酵液在10、15和25°C条件下培养测定其p H值,选取低p H值菌株作为优势菌株,并综合MRS培养基筛选结果确定优良菌株。【结果】从不同温度和发酵阶段的垂穗披碱草青贮饲料中共分离得到108个乳酸菌菌株,它们分属于6个属、18个种。其中,清酒乳杆菌LS-24在15°C条件下发酵液p H值显著降低(P0.05),戊糖片球菌PP-63在发酵初期生长速度较快,植物乳杆菌LP-21在15°C条件下发酵液p H值降至3.9且有最大活菌数。【结论】在青藏高原垂穗披碱草青贮饲料中发现的乳酸菌属基本涵盖了前人在常温青贮饲料中发现的所有属,但种数略少;在108株菌中,清酒乳杆菌LS-24、戊糖片球菌PP-63和植物乳杆菌LP-21在低温条件下均表现出较好的繁殖和发酵特性,可作为青贮饲料低温发酵的备选菌株。     

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

Elucidation of the taxonomic status of industrial strains of thermophilic lactic acid bacteria by sequencing of 16S rRNA genes

Phenotypic characteristics and results of PCR tests for the presence of species-specific genes indicate that a number of strains of thermophilic lactic acid bacteria previously considered as belonging to Streptococcus thermophilus are actually closely related to enterococci. In the present study, partial (over 500 nucleotides) sequencing of 16S rRNA genes from 12 strains of thermophilic lactic acid bacteria used as starters for manufacturing sour milk products on the territory of the Commonwealth of Independent States (CIS) has been performed. According to the results of the sequencing, seven of the strains have been classified with Enterococcus durans. The earlier classification (based on PCR tests) of two of the strains as S. thermophilus and three of the strains as E. faecium has been confirmed. The data obtained demonstrate that the enterococci E. durans and E. faecium are widely used as thermophilic starters for manufacturing sour milk products on the territory of the CIS.     

The Lactic Acid Enterococci Enterococcus faecium and Enterococcus durans: Nucleotide Sequence Diversity in 16S rRNA Genes

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.     

Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method

AIMS: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. METHOD: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). RESULTS: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

Development of detection medium for hard-to-culture beer-spoilage lactic acid bacteria

Aims:  To develop a detection medium for hard-to-culture beer-spoilage lactic acid bacteria (LAB). Methods and Results:  Four hard-to-culture beer-spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild-type strains in beer. To develop a countermeasure against these hard-to-culture beer-spoilage LAB, a beer-based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard-to-culture beer-spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer-based medium. Further comparative study was performed with five other media widely used for the detection of beer-spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer-spoiler detection (ABD) medium, possessed superior sensitivity for hard-to-culture beer-spoilage LAB and comparable sensitivity with easy-to-culture beer-spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro-organisms, and thereby allow the selective growth of beer-spoilage LAB. Conclusions:  Advanced beer-spoiler detection medium is considered as an effective tool for comprehensive detection of beer-spoilage LAB in breweries. Significance and Impact of the Study:  The detection by ABD medium can be used as an indicator for differentiating the beer-spoilage ability of LAB without further confirmatory tests in breweries.     

The adhesion of lactic acid bacteria to the intestinal epithelium of agricultural animals

The lactic acid bacteria isolated from different ecological niches are established to be able to adhere in vitro to the intestine epithelium of pigs and cattle. The studied strains of enterococci have a higher index of adhesiveness (5.7-2.24) as compared to lactic acid bacilli (3.34-1.08). Detection of the lactic acid bacteria ability to adhere to intestine enterocytes of agricultural animals served as a premise to use those bacteria when constructing preparations of the directed action on the basis of living cultures.     

Changes in microbial population during fermentation of tropical freshwater fish viscera

Freshwater fish viscera (FV) was homogenized, mixed with 10% (w/w of FV) molasses and 0, 2 or 4% salt and allowed to ferment at ambient temperature (26·2°C) under microaerophilic conditions. The results revealed a reduction in total viable count and the number of spores, coliforms, Escherichia coli , staphylococci and enterococci and an increase in yeasts and moulds and lactic acid bacteria during fermentation. Coliforms and E. coli were found to be absent after 6 d and enterococci on 8th day. The presence of salt resulted in a marginally lower number of all organisms except yeasts, moulds and lactic acid bacteria. Inclusion of either 0·5% propionic acid, 0·3% calcium propionate or 0·1% sorbic acid suppressed growth of yeasts and moulds with propionic acid being the most effective. The study indicated that a microbiologically stable product could be prepared by ensiling fish viscera with 10% molasses and 0·5% propionic acid.     

Study of intraspecific variations of the bacterium Lactococcus lactis in adaptation to high acidity of the medium

This paper reports on the study of acid tolerance of lactic acid bacteria as a property of cells, determining their ability to divide efficiently and retain viability under conditions of increased nutrient medium acidity during bacterial growth. The bacteria of the strain TV2, isolated from a self-soured curd, similar to the bacteria of the strain STE05 (Russian National Collection of Industrial Microorganisms), were assigned to the species Lactococcus lactis according to their G+C composition (36.7-36.5 mol %) and the high level of DNA-DNA hybridization (93%). However, these strains were essentially different in the number and size of the plasmids and the chromosomal DNA restriction fragments, as well as in the sensitivity to phages of lactic acid bacteria. It was found that bacteria of the strain TV2 were stable (i.e., they divided efficiently at a pH as low as 5.3) and tolerant to the lactic acid that they produced while growing (i.e., they retained viability at pH 4.4). Bacteria of the strain STE05 lacked acid tolerance (at pH below 6.5, growth was retarded, and pH 5.0 was the lowest value at which the cells remained viable). The acid tolerance and phage resistance of TV2 bacteria are likely to characterize their higher adaptive capacity in comparison with STE05 bacteria. Acid tolerance is inherited in a stable manner and retained by the segregants of TV2 strain obtained in the course of long-term storage of the bacteria. Specifically, the strains TV29, TV13, and TV 229, which displayed this property, had altered physiological and biochemical characteristics (accumulation of biomass and fermentation of lactose) in spite of their genetic identity to the original strain (pulsed-field gel electrophoresis of chromosomal DNA restriction fragments).     

Growth, maintenance and fermentation pattern of the salt-tolerant lactic acid bacterium Tetragenococcus halophila in anaerobic glucose limited retention cultures

The homofermentative lactic acid bacterium Tetragenococcus halophila showed mixed acid fermentation at low growth-rates under glucose limiting conditions and in the presence of 10% NaCl. Maximum growth yields in fermentors with cell retention were not affected by pH, but maintenance requirement was at pH 5.2 four times higher than at pH 7.0. Despite the high salt-concentration of the medium, maintenance requirements were low compared to other lactic acid bacteria. The possible causes of the observed differences in maintenance requirements are discussed.