In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium.

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20 degrees C and 40 degrees C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A TnphoA insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.     

Influence of genes encoding proton-translocating enzymes on suppression of Salmonella typhimurium growth and colonization.

Twenty-four-hour-old, aerobically grown, Luria-Bertani broth cultures of Salmonella typhimurium F98 suppressed the growth of a spectinomycin-resistant (Spcr) derivative of the same strain inoculated at 10(3) CFU ml(-1). This growth suppression is genus specific and RpoS independent, and it is not solely a result of nutrient depletion (P. A. Barrow, M. A. Lovell, and L. Zhang-Barber, J. Bacteriol. 178:3072-3076, 1996). Mutations in three genes are shown here to significantly reduce growth suppression under these conditions. The mutations were located in the nuo, cyd, and unc operons, which code for the NADH dehydrogenase I, cytochrome d oxidase, and F0F1 proton-translocating ATPase complexes, respectively. When cultures were grown under strictly anaerobic conditions, only the unc mutant did not suppress growth. Prior colonization of the alimentary tract of newly hatched chickens with the S. typhimurium F98 wild type or nuo or cyd mutants suppressed colonization by an S. typhimurium F98 Spcr derivative inoculated 24 h later. In contrast, the S. typhimurium unc mutant did not suppress colonization. The nuo and unc mutants showed poorer growth on certain carbon sources. The data support the hypothesis that growth suppression operates because of the absence of a utilizable carbon source or electron acceptor.     

Antagonistic effects of intestinal Lactobacillus isolates on pathogens of chicken

L.Z. JIN, Y.W. HO, N. ABDULLAH, M.A. ALI AND S. JALALUDIN. 1996. Twelve Lactobacillus strains isolated from chicken intestine, which demonstrated a strong and moderate capacity to adhere to the ileal epithelial cells in vitro , were used to investigate their inhibitory ability against five strains of salmonella, i.e. Salmonella enteritidis 935/79, Salm. pullorum, Salm. typhimurium, Salm. blockley and Salm. enteritidis 94/448, and three serotypes of Escherichia coli , viz. E. coli O1 : K1, O2 : K1 and O78 : K80. The results showed that all the 12 Lactobacillus isolates were able to inhibit the growth of the five strains of salmonella, and the three strains of E. coli in varying degrees. Generally, they were more effective in inhibiting the growth of salmonella than E. coli . Inhibition of the pathogenic bacteria was probably due to the production of organic acids by the Lactobacillus isolates.     

Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.     

Loss of Lactose Metabolism in Lactic Streptococci

Lactose-negative mutants occurred spontaneously in broth cultures of Streptococcus lactis C(2)F. Instability of lactose metabolism was noted in other strains of S. lactis, in strains of S. cremoris, and in S. diacetilactis. Colonies of S. lactis C(2)F grown with lactose as the carbohydrate source also possessed lac(-) cells. Treatment of lactic streptococci with the mutagen acriflavine (AF) increased the number of non-lactose-fermenting variants. The effect of AF on growth and on loss of lactose-fermenting ability in S. lactis C(2)F was consequently further examined. The presence of AF appears to favor competitively the growth of spontaneously occurring lactose-negative cells and appears to act in the conversion of lactose-positive to non-lactose-fermenting cells. The lactose-negative mutants partially revert to lactose-positive variants which remain defective in lactose metabolism and remain unable to coagulate milk. The lactose-negative cells become dominant in continuous culture growth and provide evidence that alterations in the characteristics of starter strains can be produced by continuous culture, in this case, the complete loss in ability to ferment lactose.     

Epithelial Attachment and Other Factors Controlling the Colonization of the Intestine of the Gnotobiotic Chicken by Lactobacilli

Four strains of lactobacilli with different abilities to adhere to chicken crop epithelial cells in vitro were administered to germfree chickens and their colonization of the intestine compared. Ability to colonize the gut effectively was related to adhesion index and, in the absence of adhesive ability, to growth rate. Tolerance of low pH was also a factor in determining good colonization of the small intestine. With only one strain did diet appear to affect colonization.     

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.     

Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165.

Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea. Putative factors important in the pathogenicity of E. coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin. Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E. coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant. In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence. Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165. Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.     

The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits

The antibacterial potential of four strains of Bacillus subtilis, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, previously selected on the basis of their antifungal activity and efficacy against cucurbit powdery mildew, was examined. Among these strains, UMAF6614 and UMAF6639 showed the highest antibacterial activity in vitro, especially against Xanthomonas campestris pv. cucurbitae and Pectobacterium carotovorum subsp. carotovorum. These strains produced the three families of lipopeptide antibiotics known in Bacillus spp.: surfactins, iturins, and fengycins. Using thin-layer chromatography analysis and direct bioautography, the antibacterial activity could be associated with iturin lipopeptides. This result was confirmed by mutagenesis analysis using lipopeptide-defective mutants. The antibacterial activity was practically abolished in iturin-deficient mutants, whereas the fengycin mutants retained certain inhibitory capabilities. Analyses by fluorescence and transmission electron microscopy revealed the cytotoxic effect of these compounds at the bacterial plasma membrane level. Finally, biological control assays on detached melon leaves demonstrated the ability of UMAF6614 and UMAF6639 to suppress bacterial leaf spot and soft rot; accordingly, the biocontrol activity was practically abolished in mutants deficient in iturin biosynthesis. Taken together, our results highlight the potential of these B. subtilis strains as biocontrol agents against fungal and bacterial diseases of cucurbits and the versatility of iturins as antifungal and antibacterial compounds.     

Culture of Ethiopian Strains of Borrelia recurrentis

A number of standard bacteriological media with supplements were tested for their ability to support in vitro growth of Ethiopian strains of Borrelia recurrentis. Propagation of 18 out of 21 strains occurred in Trypticase soy yeast broth to which bovine albumin (fraction V), N-acetyl glucosamine, and sodium pyruvate had been added. This medium supported a population of 10(7) organisms per ml and yielded a harvest of four to five times the original inoculum during the logarithmic phase of growth. Maximal yield varied from 1.4 x 10(7) to 3.4 x 10(7) organisms per ml. Generation time in optimal media was 11.3 h. Lesser multiplication of organisms occurred in other media tested. Strains from primary cultures were infective for the green monkey. Recovery of viable organisms from subculture has not been successful.     

Induction of melanogenesis suppression: cellular pharmacology and mode of differential action

Recent elucidation of regulatory mechanisms of eu- and pheomelanogenesis has led us towards an exciting new era of melanogenesis control. I will chiefly address our progress on inhibitory control of melanogenesis from the macromolecular level to human skin colour. In the past, the exploration and search for skin depigmenting agents has been focussed on and initiated from substances which can suppress isolated tyrosinase in vitro. Now, as I have classified below, many new melanogenic inhibitors have been discovered which, in spite of their non-suppressive effect on isolated naked tyrosinase, suppress melanin formation in the living pigment cell in vitro as well as in the natural world. I will also discuss a recently found unique disorder: unilateral suppression of mixed melanogenesis.     

Survival of lactococci during passage through mouse digestive tract

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.     

Phenotypic suppression by streptomycin of amber mutants in the ribonucleic Acid bacteriophage coat protein cistron

After mutagenesis with nitrosoguanidine or ultraviolet light, 298 streptomycin high-resistant and 98 streptomycin high-dependent mutants were isolated from HfrC Su⁻. They were tested for their ability to phenotypically suppress five different amber ribonucleic acid (RNA) bacteriophage mutants in the presence of streptomycin. The phage mutants are all in the coat protein, which is 129 amino acids long; the uracil-adenine-guanine codons were at the following positions: sus3 and amB2, 6; amB11, 50; amB21, 54; sus11, 70. Only sus3 and amB2 could be phenotypically suppressed by streptomycin; this was clearly demonstrated in nine mutant strains, seven str-HR and two str-HD. The suppression was always dependent upon added streptomycin and was dose-dependent in all cases. None of the mutants showed measurable suppression in absence of the drug. Among revertants to streptomycin independence from streptomycin-dependent strains that could show phenotypic suppression, most of those that were still resistant to streptomycin (10 μg or more) retained the capacity to show phenotypic suppression; whereas among those revertants sensitive to 10 μg of streptomycin or less, none retained the capacity. Eight different amber polar mutants (strong and weak) in gene 34 of phage T4 were also tested for pleiotypic suppression by streptomycin in all the streptomycin-resistant and -dependent strains isolated. No suppression was found in any of the 396 strains tested.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究

【目的】从牡丹(Paeonia suffruticosa Andr.)根部组织中分离鉴定内生细菌,测定拮抗菌株脂肽类活性物质的体外抑菌活性。【方法】采用平板对峙法筛选出对牡丹灰霉病菌(Botrytis paeoniae Oadem)、牡丹炭疽病菌(Gloeosporium sp.)、牡丹黑斑病菌(Altenaria sp.)、牡丹黄斑病菌(Phyllosticta commonsii)有拮抗作用的内生细菌。基于形态特征、生理生化特性和16S rRNA基因序列同源性鉴定拮抗菌株。根据脂肽类抗菌物质合成相关基因序列对拮抗菌株进行基因扩增检测,采用酸沉淀法提取拮抗菌株的脂肽类物质,平板对峙法测定脂肽类物质的体外抑菌活性。【结果】从牡丹根部组织中共分离获得62株内生细菌,其中菌株Md31和Md33对4种病原菌均有较明显的抑制作用。Md31和Md33被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过对菌株Md31和Md33进行5个脂肽类合成功能基因bmyB、fenD、ituC、srfAA和srfAB的检测,序列同源性分析,表明两个菌株具有合成脂肽类物质的能力。菌株Md31和Md33的脂肽类粗提物对所测试的牡丹病原真菌均具有不同程度的抑制作用。【结论】获得了2株对牡丹病原菌有良好抑制效果的解淀粉芽孢杆菌Md31和Md33,两个菌株的脂肽类粗提物也具有较强的体外抑菌活性,该研究为牡丹内生细菌的进一步开发应用奠定了基础。     

Nonsense suppression in thymine-requiring strains of Escherichia coli is a consequence of altered folate metabolism

Thymine-requiring strains of Escherichia coli suppress nonsense and frameshift mutants of T4 phage. We proposed that these mutants make errors during translation because of an imbalance in folate metabolism. A thymine-requiring strain grown under suppressing conditions has elevated levels of reduced folates. We tested the effect of either mutational blocks or the inhibition of various steps in folate biosynthesis on suppression. Conditions which prevent the accumulation of 5-methyl tetrahydrofolate inhibit suppression, suggesting that elevated levels of this folate are required for suppression. Furthermore, conditions that result in an accumulation in dihydrofolate inhibit suppression.     

Nonsense suppression in thymine-requiring strains of Escherichia coli is a consequence of altered folate metabolism

Thymine-requiring strains of Escherichia coli suppress nonsense and frameshift mutants of T4 phage. We proposed that these mutants make errors during translation because of an imbalance in folate metabolism. A thymine-requiring strain grown under suppressing conditions has elevated levels of reduced folates. We tested the effect of either mutational blocks or the inhibition of various steps in folate biosynthesis on suppression. Conditions which prevent the accumulation of 5-methyl tetrahydrofolate inhibit suppression, suggesting that elevated levels of this folate are required for suppression. Furthermore, conditions that result in an accumulation in dihydrofolate inhibit suppression.     

Pseudomonas fluorescens adhesion and transport through porous media are affected by lipopolysaccharide composition.

The objectives of this work were (i) to use transposon mutagenesis to produce mutants of Pseudomonas fluorescens that were altered in adhesion ability and transport through porous media and (ii) to identify the alterations in surface characteristics that were responsible for the changes in attachment. Mutants of P. fluorescens were generated with TnphoA, which enabled identification of mutants that were altered in surface proteins. Transposon mutants were screened for alterations in adhesion ability by attachment assays on hydrophobic polystyrene and water-wettable polystyrene. Four TnphoA mutants with increased adhesion to the hydrophobic surface and decreased adhesion to the water-wettable surface were obtained. Transport of the strains through porous media was evaluated by passing suspensions of each mutant and the parent through columns containing quartz sand and determining the number of cells retained in the columns. The mutants all demonstrated increased adhesion and retention in the columns. Southern analysis demonstrated two types of mutants with separate transposon insertion sites. Polyacrylamide gel electrophoresis of the strains demonstrated that the O antigen on the lipopolysaccharide was either attenuated or absent. Lack of this polysaccharide, and the consequent increased exposure of the lipid moiety of the lipopolysaccharide, is probably responsible for the increase in adhesion to the hydrophobic substrata and retention in the sand column. This work combined with previous studies of attachment of P. fluorescens demonstrates that more than one type of polymer can mediate the adhesion of this organism to nonbiological surfaces.     

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.