Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

Present state of lactic acid bacteria phage taxonomy

Recent developments in molecular taxonomy of bacteriophages of lactobacilli and lactic streptococci are presented. DNA homology appears to be the most valid criterion in studying phage taxonomy. For each bacterial species, phages can be classified in a few families according to this criterion. A fair correspondence is observed between the groups differentiated by DNA-DNA hybridization and those differenciated by protein composition. In 2 bacterial species, a close relatedness between some virulent and temperate phages has been demonstrated. The presence of homologous sequences in the genome of various phages may play an important role in their evolution. For future taxonomic studies, some phages representative of DNA homology groups are proposed as reference phages.     

Enumeration and identification of pediococci in powder-based products using selective media and rapid PFGE

In this study, pediococci selective medium (PSM) was evaluated for the enumeration of Pediococcus acidilactici and Pediococcus pentosaceus from probiotic animal feed and silage inoculants. PSM is based on the complex basal medium MRS supplemented with cysteine hydrochloride, novobiocin, vancomycin, and nystatin. No significant change in electivity was observed when pediococci where recovered from culture or powder-based products following incubation at 37 degrees C under anaerobic conditions for 24 h. The medium was suitable for the enumeration of pediococci in samples also containing bacilli, bifidobacteria, enterococci, lactobacilli, lactococci, propionibacteria, streptococci, and yeast components. However, to inhibit Lactobacillus plantarum and Lactobacillus casei, ampicillin was added and the revised medium, termed PSM+A, was also considered to be suitably elective for pediococci recovered from powder. In addition, a rapid PFGE protocol is presented, which allows Pediococcus species and strain verification from colonies in less than 3 days.     

Selective enumeration of Lactobacillus casei in yoghurt-type fermented milks based on a 15°C incubation temperature

A procedure was developed to enumerate selectively Lactobacillus casei populations in yoghurt-type fermented milks that can also contain strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium infantis. Commercial LBS agar was acidified to pH 5.4, and the plates were incubated at 15°C for 14 days under anaerobic conditions. Acidification prevented the development of streptococci, and incubation at 15°C limited the development of the lactobacilli and the bifidobacteria. L. casei formed colonies on HHD medium which were different from those obtained with L. bulgaricus. Counts of L. casei on HHD confirmed results obtained on LBS - pH 5.4 medium and incubated at 15°C. L. casei did not form colonies on M17, nor did L. acidophilus or L. bulgaricus.     

Resistance to antibiotics and histamine production at the bacteria, isolated from the stomatopharynx of the children with bronchial asthma]

1549 strains of bacteria (gram-positive and gram-negative bacteria and Candida) were isolated from the 117 children (65 with bronchial asthma and 52 healthy one) aged from 3 to 14 years old. Susceptibility of 1213 strains to 20 antibiotics was determined by disk-diffusion method. It was shown that 336 strains produce histidine decarboxylase and for 40 strains the quantity of the produced histamine was measured on the Moeller medium. Histamine-modifying activity was investigated at 32 strains in vitro by fluorometric method. Reduced colonization resistance of the pharynx of the children with bronchial asthma was shown. Spectrum of bacteria with histidine decarboxylase activity was more wide at the children with asthma. Histamine production was about 1 mcg/mL at streptococci, staphylococci and hemophilic bacteria, from 1 to 3 mcg/mL - at bacilli, corynebacteria and Candida, from 1 to 10 mcg/mL - at lactobacilli, enterobacteria, pseudomonades, acinetobacteria. Ability for partial histamine destruction in virowas demonstrated for some strains of lactobacilli, enterobacteria and pseudomonades (initial histamine concentration reduced on 19.4-35.8 per cent after 48-hour incubation).     

THE USE OF A SELECTIVE MEDIUM FOR THE ENUMERATION OF LACTOBACILLI IN CHEDDAR CHEESE

SUMMARY: The use of a selective solid medium for counting lactobacilli in Cheddar cheese is described. It has proved useful for this purpose, especially in the early period of ripening when large numbers of streptococci render other methods impracticable.
The medium, in a semisolid form, is more sensitive for the detection of heterofermentation in lactobacilli than media previously available. The possibility of using it to count heterofermentative lactobacilli and leuconostocs is discussed.     

Avoparcin and vancomycin: useful antibiotics for the isolation of brewery lactic acid bacteria

Vancomycin (30 μg) and avoparcin (20 μg) have no effect on growth of hetero-fermentative lactobacilli, mesophilic homofermentative lactobacilli, Lactobacillus salivarius and bacteria of the genera Leuconostoc and Pediococcus. The growth of thermophilic homofermentative lactobacilli (with the exception of Lacto salivarius ), enteric and lactic streptococci and all other Gram-positive bacteria encountered in breweries (and likely to be encountered in foods) is inhibited by these antibiotics. The recovery of sub-lethally stressed cells is slightly reduced by the presence of either antibiotic. Vancomycin and avoparcin are useful selective agents for the detection of lactic acid bacteria in the brewing industry and may also be of use, for quality control purposes, in the distilling, wine, cider, dairy and meat industries. These antibiotics may also find applications in differentiation of pediococci from streptococci, and in rapid identification of lactobacilli.     

Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey

AIMS: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups. METHODS AND RESULTS: High resolution gas chromatography-mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs. Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties. Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position. CONCLUSION: The study evidenced strains producing some volatile compounds responsible for food flavouring. Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.     

The adherence of lactic acid bacteria to the columnar epithelial cells of pigs and calves

The adhesion of various lactobacilli and streptococci to columnar epithelial cells of pigs and calves were studied, by in vitro methods. The porcine strains isolated most frequently were Lactobacillus delbrueckii, Lact. acidophilus and Lact. fermentum. Thirteen of the 22 lactobacilli were adhesive. All the streptococci isolated belonged to Lancefield's D-group; none of them adhered to pig epithelial cells. The adhesive strains (9 of 22) of calves were identified as Lact. fermentum. Adherence was variable even between strains of the same species. Isolates from plant material, cultured milk and cheese did not adhere to the columnar epithelial cells in vitro. The adhesive porcine strains tolerated low pH and bile acids, which is important for their survival under conditions in the stomach and intestine.     

THE SELECTIVE ACTION OF THALLOUS ACETATE FOR LACTOBACILLI

SUMMARY: The effect of thallous acetate has been examined on the growth of 23 strains of lactobacilli representing 13 different species. All strains grew satisfactorily in both broth and agar media containing 0.1% of thallous acetate. Of the 18 other organisms also examined the growth of the streptococci was unsuppressed, staphylococci, a micrococcus, B. licheniformis and Pr. mirabilis were partially inhibited and strains of Bact. coli, B. cereus, Chr. prodigiosum and Ps. fluorescens were almost completely inhibited by that concentration. By its use a valuable selective medium for isolating lactobacilli from natural habitats of mixed flora was obtained, other organisms, with the exception of streptococci, being almost completely eliminated.     

THE NORMAL INTESTINAL FLORA OF THE PIG. III. QUALITATIVE STUDIES OF LACTOBACILLI AND STREPTOCOCCI

SUMMARY: Streptococci and lactobacilli were isolated from the faeces and caecal contents of experimental pigs receiving various diets. Fifty-seven strains of streptococci and forty-one (representative of 220) lactobacilli were examined physiologically and serologically. Lactobacillus acidophilus and L. fermenti predominated among the lactobacilli, while the largest group of streptococci resembled the 'unclassified' strains of Barnes & Ingram (1955) and Barnes, Ingram & Ingram (1956) from bacon factory premises, pig faeces and canned hams.     

Optimization of the method of isolation of microamounts of plasmid DNA from lactobacilli

Modification of the alkaline lysis at elevated temperature technique is proposed isolation of plasmid DNA from lactobacilli. Modification consists of colorimetric control of culture phase during the biomass growth, pH control at the probes treatment with lysozyme and alkaline solution of natrium dodecylsulfate by adding the indicator bromcrezolpurple into the medium for biomass growth. The high concentration of lysozyme is used (10 mkg.ml-1). Lactobacilli are lysed at 2 min incubations of the probes with the lytic solution in the boiling water bath. The treatment of the probes by proteinase K, by the mixture of chloroform:phenol:isoamyl spirit (25:24:1 vol/vol/vol) and by diethylpirocarbonate increased considerably the quality of the obtained DNA preparations. The modified technique is suitable for isolation of the plasmid DNA from lactobacilli of different species, enterococci, streptococci and other lactic bacteria. The connection of antibiotic resistance marker and the plasmid profile of lactobacilli under different conditions with the presence of the plasmid DNA- protein complex is discussed.     

THE ENUMERATION OF LACTOBACILLI ON GRASS AND IN SILAGE

SUMMARY: For the enumeration of lactobacilli on grass and in silage the following medium has shown promise: peptone, meat extract and glucose, 10 g. each; tomato extract, 200 ml.; yeast autolysate, 50 ml.; Tween 80, 0.5 ml.; agar, 15 g., in a final volume of 1 1. and containing acetic acid/sodium acetate buffer in 0.2M concentration; pH 5–4. The medium was adjusted to pH 5–4 before sterilization and the requisite amount of concentrated pH 5.4 acetate buffer added just before plating. Double laver plates were used.
The only other silage organisms which in this medium formed colonies comparable in size with those of lactobacilli were heterofermentative streptococci and a micrococcus.     

Comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples.

The interest in functional foods, probiotics and prebiotics requires a proper method to determine specific bacterial groups in the intestinal flora, especially bifidobacteria and lactobacilli. Three media for lactobacilli (MRS, Rogosa, LAMVAB), three media for bifidobacteria (RB, NPNL, Beerens medium) and nine media for total anaerobes have been tested for selectivity and recovery. For total anaerobes Faecal Reinforced Clostridial Agar (FRCA) showed the highest cfu/g, followed by Columbia Blood Agar and BHI Blood agar. There were no significant differences between the media tested. Reduced physiological salt solution was found to be the best dilution medium. For bifidobacteria and lactobacilli samples of human faeces, cat faeces and pig ileal contents were used. Bifidobacteria could reliably be determined on all three media tested in human faeces, but not on pig ileal contents or cat faeces. Absolute counts were highest in human samples. No lactobacilli could be isolated on MRS in either sample, none of the colonies in the countable plates were lactobacilli. For Rogosa over 90% of the colony types observed in human samples were not lactobacilli. For cat faeces this was 58%, but no false positives were observed in the pig ileal samples. For LAMVAB the percentages of false positive colony types were 9, 14 and 0% for human, cat and pig samples. It can be concluded that for bifidobacteria RB and Beerens medium show comparable results, and can be used to quantify bifidobacteria in human faeces, but none of the media tested is suitable for reliably counting bifidobacteria from pig and cat samples. For lactobacilli LAMVAB shows the highest sensitivity.     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the Adhesion and Biofilm Formation of Streptococcus mutans

Probiotics have decreased the counts of salivary mutans streptococci (MS) in clinical studies. The aim of this study was to compare the effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the adhesion of a reference strain and a clinical isolate of Streptococcus mutans and on the counts of MS in a biofilm. The adhesion of S. mutans Ingbritt and the clinical isolate S. mutans 2366 to a smooth glass surface and saliva-coated hydroxyapatite (SHA) were studied in the presence of and without the lactobacilli. A three-species biofilm formed on saliva-coated hydroxyapatite discs was used in the biofilm experiments. The lactobacilli did not affect adhesion to the glass surface but interfered with binding to SHA. No effects of the lactobacilli were detected on the MS levels in the three-species biofilms. The results of the SHA binding experiments best reflected the results of the existing clinical studies.     

Normal vaginal flora in chimpanzees (Pan troglodytes): qualitative and quantitative study

Lactobacilli are the predominant microorganisms in the vaginal flora of human beings, and are known to play an important role in protecting them from genital infections. On the other hand, the composition of the vaginal flora differs among laboratory animal species, and lactobacilli are not the predominant vaginal microorganism in many laboratory animals. We speculated that the vaginal flora of chimpanzees would be more similar to those of human beings than to those of other animal species, because chimpanzees are phylogenetically close to human beings, and their reproductive physiology is similar to that of human beings. To clarify our speculation, we examined the development of the vaginal flora in chimpanzees (Pan troglodytes). Streptococci, lactobacilli, and members of the family Bacteroidaceae were the most predominant bacteria in the vagina of mature chimpanzees (9 to 22 years old). During development of the vaginal flora of chimpanzees, the total number of bacteria increased with age and reached a plateau just before sexual maturity (5 to 7 years of age; juvenile period). Lactobacilli were already one of the predominant bacteria before sexual maturity. In mature chimpanzees, the total number of bacteria (aerobes and anaerobes) in the vagina was highest during the swelling phase of the menstrual cycle. During the swelling phase in mature chimpanzees, streptococci, lactobacilli, and Bacteroidaceae were the most frequently isolated (100%) organisms, and the total number of organisms recovered from vaginal specimens from these three groups was the highest. In mature chimpanzees in which the number of bacteria was the highest, lactobacilli were the predominant bacteria. Taken together, these results suggest that these three bacterial groups (streptococci, lactobacilli, and Bacteroidaceae) are indigenous to the vagina of chimpanzees, and chimpanzees would be the most suitable laboratory animals for studying the role of lactobacilli in the vagina of human beings.     

Definition of bacteriophage groups according to their lytic action on mesophilic lactic streptococci

The lytic activity of 132 phages isolated during slow acid production in cheese factories situated in all the dairying regions of France during the past 16 years has been determined on 291 strains of mesophilic lactic streptococci. The results have been treated according to a method of analysis of data so as to establish a classification. Six groups of phages have thus been formed. Sixty-six percent of the phages studied, which are very similar and for the most part nonspecific to one species, have been gathered together in one group. On the other hand, a classification of the bacterial strains has been made on their sensitivity to the phages. Six groups, each corresponding to one of these groups of phages, have thus been defined. One of them accounts for 40% of the strains studied, of which certain ones are sensitive to a large number of phages.     

Displacement of Enterococcus faecalis from hydrophobic and hydrophilic substrata by Lactobacillus and Streptococcus spp. as studied in a parallel plate flow chamber.

The displacement of Enterococcus faecalis 1131 from hydrophobic and hydrophilic substrata by isolates of Lactobacillus casei 36 and Streptococcus hyointestinalis KM1 was studied in a parallel plate flow chamber. The experiments were conducted with either 10 mM potassium phosphate buffer or human urine as the suspending fluid, and adhesion and displacement were measured by real-time in situ image analysis. The results showed that E. faecalis 1131 was displaced by lactobacilli (31%) and streptococci (74%) from fluorinated ethylene propylene in buffer and that displacement by lactobacilli was even more effective on a glass substratum in urine (54%). The passage of an air-liquid interface significantly impacted on adhesion, especially when the surface had been challenged with lactobacilli (up to 100% displacement) or streptococci (up to 94% displacement). These results showed that the parallel plate flow system with real-time in situ image analysis was effective for studying bacterial adhesion and that uropathogenic enterococci can be displaced by indigenous bacteria.     

小单孢菌40027菌株噬菌体的分离及其生物学特性的研究

以福堤霉素A产生菌──小单孢菌40027菌株为指示菌,从土壤中分离得到三株噬菌体:ΦHAU7、ΦHAU9和ΦHAU11。三株噬菌体的寄主专一性较强,在测试的15株放线菌菌株中,三株噬菌体能感染小单孢菌40027菌株和A-M-01菌株,ΦHAU9和ΦHAU11还能感染蔷薇小单孢菌(Micromonospora purprea)。三株噬菌体都是由多面体的头部和尾部组成;形成噬菌斑时培养基中适宜的Ca2 、Mg2 浓度分别为32mM和30mM;ΦHAU7在储存液中适宜的pH范围为6~12,而其它两株噬菌体的适宜的pH范围为6~10;在储存过程中三株噬菌体适宜的温度范围为28~37℃,经60℃保温30min后,除ΦHAU7仍有53%活力之外,其它两株噬菌体全部失活。限制性内切酶酶切结果表明:三株噬菌体基因组DNA均为双链DNA;基因组大小分别约为60kb、58kb和55kb。高压脉冲电泳结果揭示:三株噬菌体基因组DNA均具有粘性末端。