The electron microscopic study of cell-to-cell interactions between antagonistic microorganisms

The electron microscopic study of thin sections and positively stained specimens of cells taken from particular cocultures of Lactobacillus acidophilus D75, Lactobacillus casei YIT 9018, Shigella flexnery 2a, Bacillus subtilis ATCC 6633, and Staphylococcus aureus ATCC 25923 (some of these bacteria are antagonistic to others) showed the presence of specific ultrastructural elements indicating cell specialization and cooperation. The responses of antagonistic bacteria manifested themselves at the cellular and population levels.     

The range of antagonistic effects of Lactobacillus bacterial strains on etiologic agents of bacterial vaginosis

Bacterial vaginosis is caused by uncontrolled sequential overgrowth of some anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia, Bacteroides spp., Peptostreptococcus spp., Mobiluncus sp. usually occurring in stable numbers in the bacterial flora of healthy women. On the other hand, different species of bacteria belonging to the genus Lactobacillus, most frequently L. plantarum, L. rhamnosus and L. acidophilus, form a group of aerobic bacteria dominating in the same environment. The diversity and density of their populations depend on the age and health conditions. Thanks to their antagonistic and adherence properties bacteria of the genus Lactobacillus can maintain a positive balance role in this ecosystem. The aim of this study was to assess the antagonistic properties of Lactobacillus strains isolated from the vagina of healthy women against most common agents of bacterial vaginosis. It was found that nearly all of the tested Lactobacillus strains exerted distinct antagonistic activity against anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia and Peptostreptococcus anaerobius and quite a number also against Gram-negative rods, while only some of them were able to inhibit Gram-positive aerobic cocci as Enterococcus faecalis or Staphylococcus aureus.     

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine

Aims:  To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma.
Methods and Results:  Probiotic strains ( Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H₂S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs ( P  < 0·05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses.
Conclusions:  Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species.
Significance and Importance of the Study:  This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.     

双歧杆菌QJ405、乳杆菌QJ405在试管内对肠道致病菌抑杀效果的初步报告

目的 观察双歧杆菌ＱＪ４０５和乳杆菌ＱＪ４０５在试管内对肠道致病的抑菌和杀菌效果。方法 抑菌实验：实验组,双歧杆菌ＱＪ４０５和乳杆菌ＱＪ４０５分别与各致病菌共同培养;对照组,实验组中的细菌各自单独培养。然后观察一定间隔时间（每２ｈ或４ｈ）细菌的数量变化,并绘制生长曲线。杀菌实验：将２种菌的培养上清液定量滴在已均匀涂布定量致病菌的平板表面,然后观察滴过上清液的区域内是否有细菌生长。结果：４种肠道致病菌和２株标准菌株（ＡＴＣＣ２５２９２２,ＡＴＣＣ２５９２３）的生长明显受到抑制。乳杆菌ＱＪ４０５的代谢产物有杀菌作用。结论 双歧杆菌ＱＪ４０５和乳杆菌ＱＪ４０５具有益生菌的性质。     

阴道中益生特性乳杆菌的分离与功能评价

目的 分离健康女性阴道中的乳杆菌并鉴定其益生特性,为开发治疗妇科疾病的复方益生菌制剂提供新型菌株。方法 采集健康女性阴道分泌物并分离筛选乳杆菌,通过16S rDNA序列分析鉴定乳杆菌分离株,并对其产酸性能、产H2O2能力、抑菌能力、产生物膜能力进行检测。结果 从50名健康女性阴道内共分离出179株乳杆菌,其中卷曲乳杆菌101株、詹氏乳杆菌42株、格氏乳杆菌26株、植物乳杆菌5株、唾液乳杆菌3株以及干酪乳杆菌2株。179株乳杆菌中有146株具有产酸能力,发酵液pH值的最低的5株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、詹氏乳杆菌J87,植物乳杆菌J75以及格氏乳杆菌J35,其pH分别为4.20、4.23、4.24、4.26及4.36;产H2O2弱阳性菌株有87株、阳性有37株、强阳性有9株,这9株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、卷曲乳杆菌J20、詹氏乳杆菌J87,詹氏乳杆菌J90、詹氏乳杆菌J15、格氏乳杆菌J11、植物乳杆菌J75、植物乳杆菌J69以及植物乳杆菌J40;能拮抗大肠埃希菌的菌株有115株、拮抗金黄色葡萄球菌的有84株、拮抗白假丝酵母的有52株;经统计,对三者同时有拮抗作用且作用最强的只有6株,分别为卷曲乳杆菌J3、卷曲乳杆菌J50、卷曲乳杆菌J62、詹氏乳杆菌J87、詹氏乳杆菌J16和格氏乳杆菌J66;不同乳杆菌产生物膜能力数值范围在1.0~5.4,卷曲乳杆菌、詹氏乳杆菌、干酪乳杆菌的生物被膜形成能力显著高于其他三种菌（P<0.05）。在全部179株菌中,卷曲乳杆菌J3和詹氏乳杆菌J87既具有强的产酸能力和产过氧化氢能力,又有较强抑菌活性,同时产生物膜能力也最强。结论 卷曲乳杆菌J3和詹氏乳杆菌J87具有优良的生物学特性,有望成为用于治疗妇科疾病微生态制剂的备选菌株。     

Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species

The goal of this study was to develop a method allowing rapid identification of the lactic acid bacteria strains in use in the laboratory (Lactobacillus plantarum NCIMB8826; L. fermentum KLD; L. reuteri 100-23; L. salivarius UCC43321; L. paracasei LbTGS1.4; L. casei ATCC393), based on PCR amplification of 16S RNA coding sequences. First, specific forward oligonucleotides were designed in the variable regions of 16S RNA coding sequences of six Lactobacillus strains. The reverse oligonucleotide was designed in the region where the sequences were homologous for the six strains. The expected size of the amplification product was +/-1000 bp. The specificity of the method was tested on total chromosomal DNA. For five out of the six strains, the amplification of the fragment was strain-specific and the method was directly applicable to colonies. For the strain L. casei ATCC393, an additional argument to the classification of this bacteria in the paracasei group could be proposed. Validation of the developed method was performed by applying it to six Lactobacillus reference strains and to various species of bacteria.     

两种微生物法测定食品中叶酸含量的比较

比较酪乳酸杆菌(Lactobacillus casei,L.casei ATCC 7469)、粪链球菌(Streptococcus faecalis,S.aecalisATCC 8043)测定强化食品中叶酸的含量,结果表明,S.faecalis比L.casei检测时限短1d,实验操作容易把握,分别对两种菌检测结果重现性进行比较,P＞0.05,差异无统计学意义,两种方法实验结果比较,P＞0.05,差异无统计学意义.     

Screening of lactic starter from Tunisian fermented vegetables and application for the improvement of caper (Capparis spinosa) fermentation through an experimental factorial design

This study aims at designing a lactic starter for caper fermentation isolated from Tunisian fermented vegetables to improve the process and produce consistent and high-quality product. In this study, the lactic starter was isolated by exploring the lactic acid bacteria (LAB) of Tunisian artisanal fermented vegetables. Identification was carried out by partial 16S rRNA gene sequencing. Screening was based on salt tolerance and antagonistic activities against Escherichia coli ATCC 10536 and Enterococcus faecalis ATCC 10541. Caper fermentation was optimized through a full factorial experimental design (23), by exploring three factors: starter inoculum size, NaCl concentration, and acetate content. Differences in pH values, Total aerobic mesophilic bacteria and LAB counts between the beginning and end of fermentation are selected as responses and corresponding regression coefficients were calculated. The lactic microbiota is mainly represented by Lactobacillus plantarum group. Based on salt tolerance and antimicrobial activity, the strain Lactobacillus plantarum F3 was selected as starter for caper fermentation. The effect of NaCl concentration, acetate content, and inoculum size on acidity, total aerobic mesophilic bacteria count, and LAB count after 1 week and 1 month of caper fermentation was studied. Depending on the fermentation time, either 1 week or 1 month, the initial conditions should comprise 0% acetate, 108 CFU/mL inoculum, and 5% NaCl for 1 week against 5% acetate, 107 CFU/mL inoculum, and 10% NaCl for 1 month lasting caper fermentation. A protocol for caper fermentation was set up ensuring hygienic quality and LAB viability. Lb. plantarum F3 was selected as lactic starter for caper fermentation, and initial fermentation conditions were optimized through a full factorial design. This work has shown loss in LAB viability after 1 week of fermentation. Based on results obtained, an optimized fermentation protocol was set up. This protocol ensures LAB survival and high hygienic quality of the product.     

Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi

We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general, Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (Kodiak). Further incubation studies and scanning electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.     

Antagonistic activity of lactic acid bacteria as probiotics against selected bacteria of the Enterobaceriacae family in the presence of polyols and their galactosyl derivatives

Probiotic lactic acid bacteria were grown on erythritol, xylitol, sorbitol or lactitol and produced various derivatives: gal-erythritol, gal-xylitol, and gal-sorbitol as prebiotics. Galactosyl derivatives of erythritol, xylitol and sorbitol were metabolised by Lactobacillus spp. This resulted in their antagonistic activity against the test microflora. No activity was observed in the presence of xylitol and erythritol. Gal-sorbitol obtained by enzymatic transglycosylation from lactose had the same abilities of inducing the antagonistic activity of lactic acid bacteria that lactitol had.     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

Construction and use of a computerized DNA fingerprint database for lactic acid bacteria from silage

Efficient selection of new silage inoculant strains from a collection of over 10,000 isolates of lactic acid bacteria (LAB) requires excellent strain discrimination. Toward that end, we constructed a GelCompar II database of DNA fingerprint patterns of ethidium bromide-stained EcoRI fragments of total LAB DNA separated by conventional agarose gel electrophoresis. We found that the total DNA patterns were strain-specific; 56/60 American Type Culture Collection strains of 33 species of LAB could be distinguished. Enterococcus faecium strains ATCC19434 and ATCC35667 had identical total DNA patterns and RiboPrints. Lactobacillus rhamnosus strains ATCC7469 and ATCC27773 also had identical total DNA patterns, but different RiboPrints. EcoRI RiboPrint patterns could distinguish only about 9/23 Lactobacillus plantarum strains and about 6/10 Lactobacillus buchneri strains, whereas all 33 strains could be distinguished by EcoRI total DNA patterns. Despite gel-to-gel variation, new DNA patterns can be readily grouped with existing patterns using GelCompar II. The database contains large homogenous clusters of L. plantarum, E. faecium, L. buchneri, Lactobacillus brevis and Pediococcus species that can be used for tentative taxonomic assignment. We routinely use the DNA fingerprint database to identify and characterize new strains, eliminate duplicate isolates and for quality control of inoculant product strains. The GelCompar II database has been in continuous use for 7 years and contains more than 3600 patterns representing approximately 700 unique patterns from over 300 gels and is the largest computerized DNA fingerprint database for LAB yet reported.     

Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium

Pure strains of Lactobacillus ssp. isolated from a commercial probiotic consortium were checked in a double layer solid medium for their inhibition activities against selected pathogenic bacteria including serotypes of Listeria monocytogenes, Escherichia coli and Salmonella. The antagonistic properties of the Lactobacillus strains may be related to the production of bacteriocin-like compounds. All the pathogens tested were inhibited by one or a few strains of Lactobacillus, the best inhibition was observed against L. monocytogenes but the inhibition was also satisfactory against E. coli, Salm. typhimurium and Salm. enteritidis.     

Lipoquinones of some spore-forming rods, lactic-acid bacteria and actinomycetes.

The respiratory quinones of 73 strains of Gram-positive bacteria including spore-forming rods, lactic-acid bacteria and actinomyctes were examined. Menaquinones with seven isoprenoid units (MK-7) were the main quinone type found in representatives of the genus Bacillus and in Sporolactobacillus inulinus. However, a strain of B. thuringiensis produced MK-8 in addition to MK-7, and strains of B. lentus and B. pantothenticus appeared to produce MK-9 and MK-8, respectively, with no MK-7. In the clostridia and lactic-acid bacteria, no quinones were found, except in Pediococcus cerevisiae NCTC 8066 and Lactobacillus casei subsp. rhamnosus ATCC 7469, which contained menaquinones, and Streptococcus faecalis NCTC 775 and HIM 478-1, which contained demethylmenaquinones, in relatively low concentrations. Menaquinones were also found in the actinomycetes (except Actinomyces odontolyticus and Bifidobacterium bifidum which did not produce any quinones) and in Protaminobacter alboflavus ATCC 8458, the so-called Actinobacillus actinoides ATCC 15900 and Noguchia granulosis NCTC 10559.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Bacteriocin production by spray-dried lactic acid bacteria

AIMS: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. METHODS AND RESULTS: Bacterial cell concentrates were spray-dried and stored at 4 degrees C and 18 degrees C and 0.3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. CONCLUSIONS: A higher survival rate was obtained when survival was performed at 4 degrees C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.     

体外拮抗幽门螺杆菌的人嗜酸乳杆菌菌株的选育

目的 探讨人嗜酸乳杆菌对幽门螺杆菌（Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ,ＨＰ）毒力株的体外拮抗作用,筛选出对ＨＰ毒力株有明显拮抗作用的嗜酸乳杆菌菌株。方法 从健康人胃肠道中分离出５２株嗜酸乳杆菌可疑株,通过其培养特性,生理特性,生化反应及代谢产物测定等进行鉴定,获得２６株嗜酸乳杆菌。同时,从临床患者胃活检标本中分离出２３株ＨＰ菌株,用ＰＣＲ方法筛选出ｃａｇＡ阳性ＨＰ毒力株,然后,采用打孔法进行嗜酸乳杆菌培养上清拮抗ＨＰ毒力株的实验,以１％的乳酸作对照。结果 筛选出４株对ＨＰ毒力株有明显拮抗作用的嗜酸乳杆菌,这种拮抗作用不依赖嗜酸乳杆菌分泌的乳酸。结论 人嗜酸乳杆菌在体外对ＨＰ毒力株具有明显拮抗作用。该研究为应用微生态疗法治疗ＨＰ感染提供了理论基础。     

Effect of LGG yoghurt on Streptococcus mutans and Lactobacillus spp. salivary counts in children

The aim of this study was to establish effect of 14 day consumption of commercially available yoghurt containing Lactobacillus rhamnosus ATCC53103 - LGG (Bioaktiv LGG, Dukat, Croatia) on Streptococcus mutans and Lactobacillus spp. salivary counts in children. Twenty five patients, 6-10 yr old participated in the study. At the inclusion in the study caries risk for every patient was evaluated. The saliva samples were tested with chair side kits for saliva buffer capacity (CRT buffer, Vivadent, Schaan, Liechtenstein), S. Mutans and Lactobacillus counts (CRT bacteria test, Vivadent, Schaan, Liechtenstein). Seven, 14 and 30d after yoghurt consumption saliva samples were tested again with CRT buffer and CRT bacteria tests. Obtained data were analyzed using chi2 and Kruskal-Wallis tests. Results showed significant increase in saliva buffer capacity 30d after yoghurt consumption. S. Mutans salivary counts were significantly decreased after 30d. Significant differences in Lactobacillus counts were not observed. It could be concluded that daily consumption of yoghurt containing LGG have an inhibitory effect on oral pathogenic bacteria and may be beneficial in caries prevention.