The Vitamin Requirements of Staphylococci Isolated from Human Skin

The vitamin requirements of 46 strains representing nine species of Staphylococcus isolated from human skin together with nine authentic reference strains of these species were determined using a chemically defined medium. Strains of Staphylococcus saprophyticus and Staphylococcus cohnii were isolated on selective media. All the strains investigated required nicotinic acid and thiamine for growth. Biotin was essential or stimulatory for all coagulase negative strains except one strain of Staphylococcus capitis. Oleic acid substituted for biotin in all cases except with one strain of Staphylococcus haemolyticus. No species pattern of biotin requirement or of the ability of oleic acid to substitute for biotin was apparent. Five out of six strains of Staph. cohnii required pantothenic acid.     

Human peripheral blood mononuclear cells: ; Inhibition of biotin transport by reversible competition with pantothenic acid is quantitatively minor

A transporter present in intestinal cells and in choriocarcinoma cells has been shown to transport both pantothenic acid and biotin at similar transporter affinities. However, the concentration of pantothenic acid in most foods and biological fluids is approximately 200 times the concentration of biotin; theoretically, pantothenic acid might substantially reduce biotin transport via competition. In the present study, we sought to determine whether pantothenic acid reduces biotin transport by the biotin transporter in peripheral blood mononuclear cells (PBMC). PBMC were isolated from human blood by gradient centrifugation. Incubations with [(3)H]biotin and pantothenic acid were conducted at physiologic concentrations. Intracellular [(3)H]biotin was quantified after washing by liquid scintillation counting. Pantothenic acid at 10 to 1,000 nmol/L reduced biotin (475 pmol/L) uptake by less than 12% (P < 0.05). Based on Lineweaver-Burk plots, the competition was reversible. Several structural analogs of pantothenic acid at 1,000 nmol/L reduced biotin transport by only 7 to 15% (P = 0.13). No pattern of molecular structure required for recognition by the transporter was apparent. Extracellular pantothenic acid did not affect biotin efflux from [(3)H]biotin-loaded PBMC (P > 0.05), suggesting that countertransport of extracellular pantothenic acid and intracellular biotin does not increase biotin efflux from PBMC. We conclude that the physiologic effects of pantothenic acid on the transport of biotin in PBMC are likely to be quantitatively minor.     

Synthesis of siderophores by strains of Staphylococcus cohnii isolated from various environments

Siderophore activity as the feature of microorganisms enabling colonization of human body and the survival in inanimate environment was investigated in 108 strains of Staphylococcus cohnii; S. cohnii ssp. cohnii (50 strains) and S. cohnii ssp. urealyticus (58 strains). Strains were isolated from people, hospital and non-hospital environment. Highest siderophore activity was noted in strains S. cohnii ssp. urealyticus particularly from the inanimate environments origin. In 86% analyzed strains siderophores of hydroxamate class were detected. Larger amounts of these compounds were synthesized in strains S. cohnii ssp. urealyticus. Strains belonging to both subspecies from human origin showed lower activity of siderophores (total pool) and did not produce hydroxamate class chelators or produced very small amounts of these compounds.     

Staphylococcus cohnii subspecies: Staphylococcus cohnii subsp. cohnii subsp. nov. and Staphylococcus cohnii subsp. urealyticum subsp. nov.

Two major subspecies of Staphylococcus cohnii, namely S. cohnii subsp. cohnii, from humans, and S. cohnii subsp. urealyticum, from humans and other primates, are described on the basis of a study of 14 to 25 strains and 18 to 33 strains, respectively. DNA-DNA hybridization studies conducted in our laboratory in 1983 (W. E. Kloos and J. F. Wolfshohl, Curr. Microbiol. 8:115-121, 1983) demonstrated that strains representing the different subspecies were significantly divergent. S. cohnii subsp. urealyticum can be distinguished from S. cohnii subsp. cohnii on the basis of its greater colony size; pigmentation; positive urease, beta-glucuronidase, and beta-galactosidase activities; delayed alkaline phosphatase activity; ability to produce acid aerobically from alpha-lactose; and fatty acid profile. The type strain of S. cohnii subsp. cohnii is ATCC 29974, the designated type strain of S. cohnii Schleifer and Kloos 1975b, 55. The type strain of S. cohnii subsp. urealyticum is ATCC 49330.     

Vitamin and Amino Acid Requirements of Pediococcus soyae and Pediococcus acidilactici Kitahara’s Strain

Nutritional requirements of three strains of Ped. soyae, one strain of Ped. acidilactici (Kitahara’s strain) were determined from a complete synthetic medium. Upon inspection by the single omission method, Ped. soyae required glycine-betaine, uracil, riboflavine, pyridoxine or pyridoxal, pantothenic acid, nicotinic acid, leucovorin, biotin, glutamic acid, arginine, histidine, isoleucine, leucine, phenyl alanine, valine, tryptophane, methionine, cystine and serine with exception of the P-factor. In addition to the above mentioned nutrients, a representative strain of the organism required xanthine, folic acid, thiamine, aspartic acid and threonine for the minimum synthetic medium. Ped. acidilactici Kitahara’s strain required riboflavine, pyridoxine, pantothenic acid, nicotinic acid, biotin and all components of the basal medium consisting of amino acids except methionine, but it did not require leucovorin, glycine-betaine and organic bases. Nutritional requirements of Ped. pentosaceus, Kitahara’s strain was proved to be quite identical with Ped. cerevisiae Pederson (= Ped. pentosaceus Mees).     

Growth requirements of san francisco sour dough yeasts and bakers' yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Galactosidase activity of lactose-positive Neisseria

The chromogenic substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG) was hydrolyzed by lactose-positive Neisseria. Eight strains of pharyngeal origin were examined. In culture reactions, seven strains resembled Neisseria meningitidis with the exception that they produced acid from 1% (w/v) lactose. An eighth strain (V8) differed in that it did not form acid from maltose or from 1% lactose. However, acid formation was observed in 10% lactose cultures of strain V8, suggesting that entry of lactose occurred by passive diffusion, rather than as a result of permease activity. The enzymes which hydrolyzed ONPG were produced constitutively by the cells of all eight strains. Thus, specific activity in these strains was not increased by prior exposure to lactose, or to two other possible inducers, isopropyl-beta-d-thiogalactoside or methyl-beta-d-thiogalactoside. Study of cell-free extracts of one strain showed that the enzyme was heat-labile, having a half-life of 10 min at 45 C. The enzyme was unstable at low protein concentrations, but it was protected completely or partially when albumin or manganous ions were added. The enzyme appeared to be a typical beta-galactosidase: alpha-galactosides (melibiose and p-nitrophenyl-alpha-d-galactopyranoside) were not hydrolyzed, activity against ONPG was not dependent upon inorganic phosphate, and galactose was released by cleavage of ONPG. ONPG hydrolysis provided a simple and rapid method for detecting lactose-positive Neisseria.     

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

A Synthetic Medium for Comparative Nutritional Studies of Lactobacilli

The composition of a synthetic medium supporting the growth of lactobacilli is given (Table 1). The medium, containing glucose, amino acids, vitamins, mineral salts, purines and pyrimidines, allows the study of nutritional requirements of different strains of lactobacilli under identical environmental conditions. It was found that all the strains tested needed L-glutamic acid, L-valine and L-leucine, and a group of them also required L-arginine, L-tyrosine and L-tryptophan. Some strains required vitamins, e.g. L. bulgaricus (pantothenic acid), L. fermenti (pantothenic acid and niacin). These results are compared with those found by others employing different media.     

Carbohydrate Metabolism by the Acetic Acid Bacteria

Vitamin requirements for the growth of the acetic acid bacteria were investigated extensively on a. taxonomical viewpoint and the following new findings were pointed out. Neither Acetobacter nor Intermediate strain required vitamin for the growth.

Gluconobacter required generally pantothenic acid. And some strains belonging to it did moreover somewhat of thiamine, nicotinic acid and p-aminobenzoic acid, although there was a difference of requirements between strains even in the same species. Riboflavin, pyridoxine, vitamin B₁₂, folic acid, biotin and inositol were unnecessary for the growth of the acetic acid bacteria. A taxonomical division of the acetic acid bacteria based on the vitamin requirements agreed well with that on basis of the oxidative activities for carbohydrates.     

Nutritional requirements and biochemical activities of pineapple pink disease bacterial strains from Hawaii

Bacteria which cause pink disease of pineapple, identified on the basis of their nutritional and biochemical activities, were found to belong to three genera. These bacteria include the following species: Gluconobacter oxydans, Acetobacter aceti, and Erwinia herbicola. Several pink disease strains required one to three vitamins for growth. Both G. oxydans strains 303D and 180 required biotin, nicotinic acid, and pantothenic acid for growth; E. herbicola 189 required only nicotinic acid; however, A. aceti 295 was able to grow without any added supplements in glucose mineral salts medium. Optimal vitamin concentrations for maximal growth and optimal pH for the maximal number of generations per hour was established for a few pink disease strains.Journal Series Paper No. 2373 of the Hawaii Agricultural Experiment Station, Honolulu.     

Biotin Transport Through the Blood-Brain Barrier

The unidirectional influx of biotin across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [3H]biotin. Biotin was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 100 microM. The permeability-surface area products were 10(-4) s-1 with a biotin concentration of 0.02 microM in the perfusate. Probenecid, pantothenic acid, and nonanoic acid but not biocytin or biotin methylester (all 250 microM) inhibited biotin transfer through the blood-brain barrier. The isolated rabbit choroid plexus was unable to concentrate [3H]biotin from medium containing 1 nM [3H]biotin. These observations provide evidence that: biotin is transported through the blood-brain barrier by a saturable transport system that depends on a free carboxylic acid group, and the choroid plexus is probably not involved in the transfer of biotin between blood and cerebrospinal fluid.     

Biosynthesis of biotin: synthesis of 7,8-diaminopelargonic acid in cell-free extracts of Escherichia coli

Cell-free extracts prepared from a biotin auxotroph of Escherichia coli were active in catalyzing the synthesis of 7,8-diaminopelargonic acid, an intermediate of the biotin pathway, from 7-oxo-8-aminopelargonic acid. The product was identified on the basis of its chromatographic characteristics and its biotin activities for biotin auxotrophs of E. coli. Enzyme activity was determined in a reaction coupled with the desthiobiotin synthetase system, which is required for the conversion of 7,8-diaminopelargonic acid to desthiobiotin, and by measuring the amount of desthiobiotin formed by microbiological assay. The reaction was stimulated by l-methionine and pyridoxal-5'-phosphate. l-Methionine could not be replaced by any other amino acids tested. Pyridoxamine and pyridoxamine-5'-phosphate were as active as pyridoxal phosphate. The enzyme, presumably an aminotransferase, was demonstrable in the parent strain of E. coli and all mutant strains tested with the exception of a strain which is able to grow on diaminopelargonic acid but not on 7-oxo-8-aminopelargonic acid. Furthermore, the enzyme was repressible by biotin. The results were consistent with the hypothesis that the biosynthesis of 7,8-diaminopelargonic acid from 7-oxo-8-aminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.     

Abnormal concentrations of B vitamins and amino acids in plasma and B vitamins in bile of rabbits with aflatoxicosis.

The dosages of aflatoxin B1 (AFB1) required to produce significant changes in concentrations of B vitamins in plasma and bile and of amino acids in plasma of rabbits were determined. Folate increased by 98% in plasma, whereas concentration of thiamine, vitamin B6, and biotin decreased by more than 50%. In bile, choline and biotin increased 14- and 18-fold, respectively, whereas folate and niacin decreased by more than 50%. All amino acids in plasma increased between 76 and 155%. The dosages of AFB1 required to induce these changes were usually between 12.5 and 37.5 microgram/kg of body weight per day. Except for changes in biliary concentrations of pantothenic acid, folic acid, and biotin, lower threshold dosages of aflatoxin were required to produce weight loss and anorexia (5.0 and 8.5 microgram of AFB1/kg per day, respectively) than for changes in vitamins and amino acids (approximately 25 to 50 microgram of AFB1/kg per day). The data indicated that AFB1 interfered with the metabolism of B vitamins and amino acids in rabbits.     

Enzyme activities of Lyme disease and relapsing fever borreliae

Activities of glycosidases ( n = 8), esterases ( n = 10), arylamidases ( n = 63), acid phosphatase, alkaline phosphatase and phosphoamidase were tested in 47 Borrelia strains. Forty-four were B. burgdorferi strains; 22 of which were isolated from human specimens (skin 13, cerebrospinal fluid six, and one each from blood, heart muscle and synovia), 13 were isolated from various organs of laboratory animals infected via tick bite or with human isolates, and nine from ticks. The remaining three were the relapsing fever strains B. coriaceae , B. hermsii , and B. turicatae. Strains were of low and high passage but the number of subcultures did not influence the enzyme patterns obtained by utilization of chromogenic substrates of constitutive enzymes. Glycosidase activity was absent in almost all strains tested. Esterase activity was high on molecules of chain length 9 carbons. 2-Naphthylamide derivatives were utilized by strains of human, rodent or tick origin in a range of 66.6 to 83.1%. Almost all strains utilized substrates for acid and alkaline phosphatase and phosphoamidase. Chymotrypsin activity was only found in three and two strains from specimens of human and rodent origin, respectively; and γ-glutamyltransferase activity only in three human skin isolates. No strain tested displayed trypsin activity. Overall, the specific activities of constitutive enzymes of the Borrelia strains tested are widely similar. Thus, the enzyme profiles did not discriminate between human, animal and tick isolates, or between human isolates of Borrelia whether cultivated from cerebrospinal fluid or from skin biopsy of Lyme borreliosis patients.     

Abnormal concentrations of B vitamins and amino acids in plasma and B vitamins in bile of rabbits with aflatoxicosis

The dosages of aflatoxin B1 (AFB1) required to produce significant changes in concentrations of B vitamins in plasma and bile and of amino acids in plasma of rabbits were determined. Folate increased by 98% in plasma, whereas concentration of thiamine, vitamin B6, and biotin decreased by more than 50%. In bile, choline and biotin increased 14- and 18-fold, respectively, whereas folate and niacin decreased by more than 50%. All amino acids in plasma increased between 76 and 155%. The dosages of AFB1 required to induce these changes were usually between 12.5 and 37.5 microgram/kg of body weight per day. Except for changes in biliary concentrations of pantothenic acid, folic acid, and biotin, lower threshold dosages of aflatoxin were required to produce weight loss and anorexia (5.0 and 8.5 microgram of AFB1/kg per day, respectively) than for changes in vitamins and amino acids (approximately 25 to 50 microgram of AFB1/kg per day). The data indicated that AFB1 interfered with the metabolism of B vitamins and amino acids in rabbits.     

Biotin vitamers content of lactose and beet molasses

The biotin activity of beet and lactose molasses against the test strain Saccharomyces cerevisiae 225 by auxanographic method was evaluated. The level of lactose molasses biotin activity is almost twice as high as that obtained in the case of beet molasses. The results of bioautography with test strains Saccharomyces cerevisiae 225 and Lactobacillus arabinosus 17-5 indicate the qualitative composition of biotin derivatives (vitamers) in both molasses. Depending on the various technological steps e.g. sterilization or clarification one may find differences in the content and qualitative composition of biotin vitamers.     

Nutritional properties ofCorynebacterium laevaniformans

The nutritional profiles ofCorynebacterium laevaniformans and the other levan synthesizing coryneform organism isolated by Henis and Aschner (1954) have been studied.C. laevaniformans required biotin, thiamine and pantothenic acid for growth while the Henis and Aschner strain required the former two vitamins only. Two of the six strains ofC. laevaniformans had, in addition, a requirement for glutamate.C. laevaniformans has been shown to be able to degrade levan in growing cultures. Some properties of a cell-free levansucrase are described.     

Some Aspects of the Enzyme Activities Involved in Coenzyme A Biosynthesis in Various Microorganisms

The distribution of the enzyme activities relating to CoA biosynthesis from pantothenic acid in various microorganisms and the effect of CoA on these activities are described.

High activities of partial reactions involved in CoA biosynthesis were surveyed in various type culture strains involving bacteria, actinomycetes, lactic acid bacteria, molds, and yeasts. Generally, higher activities were found in bacteria. CoA inhibited the phosphorylation of pantothenic acid, and resulted in a decrease of CoA production in all the CoA producing strains, while only a little inhibition by CoA was observed in the other reactions, and CoA production from pantothenic acid 4′-phosphate by Brevibacterium ammoniagenes IFO 12071 was not repressed even in the presence of 4mm of CoA. Extracellular excretion of the enzymes of CoA biosynthesis was observed when cells were in contact with sodium lauryl sulfate. Degrading activity against CoA and that against AMP were relatively lower in CoA producing strains when compared with those in other strains. It was confirmed that Brown’s route of CoA biosynthesis operates in Brevibacterium ammoniagenes IFO 12071.     

Staphylococcus cohnii--resident of hospital environment: cell-surface features and resistance to antibiotics

Staphylococcus cohnii strains dominated in the environment of investigated hospitals. We isolated 420 strains of the species mainly from hospitals environments, but also from infants--Intensive Care Units patients, its medical staff and non-hospital environments. S. cohnii subspecies cohnii was seen to dominate (361 strains). Seventy seven percent of these strains expressed cell-surface hydrofobicity, most of them were slime producers (61%) and this feature was correlated with their methicillin resistance. Among S. cohnii ssp. cohnii strains isolated from ICU environment 90% were resistant to methicillin, 43% expressed high-level resistance to mupirocin and high percentages were resistant to many other antibiotics. These strains may constitute a dangerous reservoir of resistance genes in a hospital.