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PubMed1975

Phage typing scheme for group D streptococci isolated from human urogenital tract.

Streptococci of Lancefield group D were isolated from 52.2% of pharyngeal, 55.5% of urethral, 56.6% of endocervical, and 75.4% of ano-rectal specimens. Seventeen phages isolated from sewage and urogenital specimens were selected for phage typing. Four of these lysed only the strains of Streptococcus faecalis and its variety liquefaciens. Another six phages lysed only the strains of Streptococcus faecium and its variety durans. With the help of seven bacteriophages, 346 of 384 (90.1%) strains of S. faecalis and its variety liquefaciens could be classified into 27 types. Similarly, with the help of 10 other bacteriophages, 97 of 117 (82.9%) strains of S. faecium and its variety durans could be grouped into 22 types. In 72 of 87 (82.7%) cases, similar phage types were obtained at different culture sites of the same individual.

Bacteriophage Typing↗

PubMed2026

Novelty, diversity, and genetic dark matter in enterococci of invertebrates.

Enterococci appear to have originated in the guts of early terrestrializing arthropods and invertebrates over 425 million years ago-hosts that are now highly diverse and widespread in nature today. Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined Enterococcus association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and Enterococcus-adapted genes, 40% (eight of 20) of the Enterococcus species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of E. casseliflavus and E. flavescens in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci-diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains.IMPORTANCEEnterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years-the "antibiotic era"-E. faecalis and E. faecium gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of Enterococcus biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.

Animals↗

PubMed1979

Species-specific resistance to antimocrobial synergism in Streptococcus faecium and Streptococcus faecalis.

Combinations of penicillin with various aminoglycosidic aminocyclitols were tested against a collection of clinical isolates of Streptococcus faecium in vitro and were used to treat endocarditis caused by S. faecium in the rabbit model. S. faecium proved more resistant to penicillin than Streptococcus faecalis. Even more striking, however, was the resistance to in vitro synergism by combinations of penicillin and various aminoglycosides. At clinically achievable concentrations, penicillin-gentamicin was the only combination that was synergistic against all strains that were tested. Combinations of penicillin and streptomycin and penicillin and amikacin were synergistic only against those strains that were not highly resistant to streptomycin and kanamycin, respectively. Combinations of penicillin with kanamycin, tobramycin, sisomicin, or netilmicin failed to produce synergism against any of these strains. The possible clinical significance of these findings was verified by use of the rabbit model of endocarditis. Combinations of penicillin with gentamicin or streptomycin were synergistic in the therapy of endocarditis that was produced by a strain of S. faecium that did not have a high level of resistance to aminoglycosides. However, the combination of penicillin and netilmicin was no more effective than penicillin alone.

Aminoglycosides↗

PubMed1975

Synthesis of N-10-methyl-4-thiofolic acid and related compounds.

Compound 21 (N10-methyl-4-thiofolic acid) and related compounds were prepared as potential inhibitors of the cofactor forms of tetrahydrofolate. The preparation of 2-acetylamino-4-(benzylthio)-6-chloro-5-nitropyrimidine (4) provided an intermediate that was allowed to react with methyl p-[(3-aminoacetonyl)methylamino]benzoate oxime (16). The oxime function of the resulting 6-substituted aminopyrimidine 6 was hydrolyzed to give the corresponding acetonylaminopyrimidine 7, which on reductive cyclization gave methyl p-[[[2-amino-4-(benzylthio)-7,8-dihydro-6-pteridinyl]methyl]methylamino]benzoate (9). This dihydropteridine was oxidized with potassium permanganate, and the product was treated successively with sodium hydrosulfide to replace the benzylthio group and with aqueous sodium hydroxide to hydrolyze the ester function to give p-[[(2-amino-3,4-dihydro-4-thioxo-6-pteridinyl)methyl]methylamino]benzoic acid (N10-methyl-4-thiopteroic acid, 12). Another route to 12 involved the interaction of 2,5-diamino-4,6-dichloropyrimidine (15) with 16 to give methyl p-[[(2-amino-4-chloro-7,8-dihydro-6-pteridinyl)methyl]methylamino]benzoate (13). Displacement of the chloro group of 13 with sodium hydrosulfide followed by the simultaneous air oxidation of the dihydropteridine ring and saponification of the ester group gave 12. After protection of the 2-amino and 4-thioxo moieties of 12, the resulting intermediate benzoic acid was coupled with diethyl L-glutamate. The product of this reaction was deblocked to give 21. Methylation of 21 gave the corresponding 4-(methylthio) derivative 22, which on reaction with hydrazine gave the 4-hydrazino analog 23 of methotrexate. Reduction of 12 and 21 with sodium hydrosulfite gave the dihydropteridines 24 and 25, respectively. The title compound was an excellent inhibitor of the growth of Streptococcus faecium ATCC 8043. However, this and related compounds were ineffective inhibitors of dihydrofolic reductase and showed no significant activity in either the KB cell culture screen or against L1210 leukemia cells in mice.

Animals↗

PubMed1978

Carriage of group D streptococci in the human bowel.

Sixty faecal samples, 39 from adults and 21 from neonates, were investigated by means of a simple scheme to isolate and identify any group D streptococci present. A number of differences were found between the two groups. Group D streptococci were absent from 43% of the neonates compared with only 13% of the adults; Streptococcus bovis was commoner in the neonates (23.8%) than in the adults (5%), and Streptococcus faecium was not isolated from any of the infant samples although it was common in the adult samples (25%). The viable counts of Strep. faecium were found to be, on average, 100-fold lower than those of Streptococcus faecalis. The methods and results are discussed with reference to the clinical significance of group D streptococci, especially in bacterial endocarditis and carcinoma of the colon.

Adult↗

PubMed1976

Distribution of group D streptococci in rivers and streams.

A study was undertaken to determine sources of bacteriological contamination of rivers as well as the ecology of faecal streptococci in natural and contaminated waters. Media used to characterize and speciate group D streptococci were also evaluated using isolates from the water and sediment. Due to the low concentrations of faecal coliforms (FC) and faecal streptococci (FS) in river water samples, the FC/FS ratio was shown to be of little value in determining sources of contamination. High concentrations of FC and FS from sediment samples gave more credible FC/FS ratios. The actual distribution of group D streptococcal species in rivers and streams was also shown. High concentrations of all division II species (S. faecium, S. faecium var. casseliflavus, and S. faecium var. durans) were found in the water and sediment samples, with S. faecium var. durans comprising 50% of the total number of isolates. Low concentrations of division I group D streptococci (S. faecalis and varieties) were found, while only one isolation of division III group D streptococci (S. bovis and S. equinus) was made.

Enterococcus faecalis↗

PubMed1978

Use of pyruvate fermentation compared with tetrazolium reduction in the differentiation of group D streptococci.

The reduction of 2, 3, 5 triphenyltetrazolium chloride in the original medium of Barnes (Journal of General Microbiology, 14, 57, 1956), and in a modified medium, was compared with the ability to ferment pyruvate as a means of separating Streptococcus faecalis from all other group D streptococci. The tetrazolium reduction test gave an occasional negative reaction with Str. faecalis strains. In addition, a number of strains gave a weakly positive reaction in the test, as did some strains of Str. faecium and Str. bovis. With some batches of tetrazolium, these weak reactions with Str. faecalis were so frequent that interpretation of the results was difficult. On the other hand, all Str. faecalis strains, and no other group D streptococci, gave a positive pyruvate-fermentation reaction in 48 hours.

Bacteriological Techniques↗

PubMed1979

Effects of cerulenin on antibiotic-induced lysis of streptococcus faecalis (S. faecium).

Addition of the antibiotic cerulenin to cultures lowered the minimal effective concentration of penicillin G or methicillin required to produce bacterial lysis and killing. This effect was most pronounced at subinhibitory antibiotic concentrations. Cerulenin had no significant effects on lysis or killing induced in the presence of D-cycloserine, fosfomycin, bacitracin, or vancomycin.

Anti-Bacterial Agents↗

PubMed1975

Synthesis and biological activity of 5-fluoro-4'-thiouridine and some related nucleosides.

The synthesis of a series of 4'-thio-5-halogenopyrimidine nucleosides, including the 5-fluoro, chloro, bromo and iodo derivatives, has been carried out by condensation of the 2,4-bis-O-trimethylsilyl derivatives of the corresponding pyrimidine bases with the protected 4-thio-D-ribofuranosyl chloride. Among these, the alpha and beta anomers of 4'-thio-5-fluorouridine inhibited the growth of leukemia L1210 cells at concentrations of 4 x 10(-7) and 2 x 10(-7) M, respectively, and that of S. faecium at 4 x 10(-9) and 6 x 10(-10) M, respectively. These compounds retained marked activity against strains of S. faecium resistant to 10(-3) M 5-fluorouracil or 5-fluorouridine. As determined in S. faecium cultures, 4'-thio-5-fluorouridine decreased the total protein content of the cells more markedly than it did their RNA or DNA content. X-Ray crystallography showed that substitution of sulfur for the oxygen in the carbohydrate ring markedly changes the conformation of that moiety.

Animals↗

PubMed1975

Quinazolines as inhibitors of dihydrofolate reductase. 3. Analogs of pteroic and isopteroic acids.

A series of 19 quinazoline analogs of pteroic and isopteroic acid was prepared with particular emphasis being placed upon carboxylic acid esters. Each compound was evaluated as an inhibitor of the dihydrofolate reductases from rat liver as well as from Streptococcus faecium. Several of the more potent inhibitors were found to be inactive against L1210 leukemia in mice at low dose levels and were lethal to mice at 100 mg/kg. Six compounds were also evaluated for antimalarial activity against Plasmodium berghei in mice. Three of these were found to be curative at higher levels, while the remaining compounds were found to be toxic.

Animals↗

PubMed1978

Incidence and relationship of group D streptococci with other indicator organisms in meats.

Raw and processed meats were analyzed for presumptive group D streptococci using KF streptococcus agar. Counts were compared with coliform, presumptive Escherichia coli, and Enterobacteriaceae counts but no meaningful relationships were observed. Results indicated that group D streptococci and E. coli type I were principally contaminants from the packing plant, rather than at retail level. The predominating group D streptococcus in both beef and pork cuts was Streptococcus faecalis, while in processed meat (bologna), the predominating group D streptococci were Streptococcus faecium var. durans and Streptococcus faecium. Streptococcus bovis was not detected among the isolates from any meat samples. Marked differences were noted in numbers of group D streptococci in processed meat from different manufacturers. The results did not support the use of group D streptococci as alternative indicator organisms for meats. However, the association of group D streptococci with packing plant contamination may prove to be of value.

Animals↗

PubMed1978

[The etiology of foul brood].

Five hundred and thirty-six samples of honeycombs were examined in a laboratory in the years 1971-1974. In all the samples clinically determined as the foul brood, B. alvei was isolated as a pure culture, and enterococci, or both microorganisms were isolated in mixed form. Twenty-five strains of the isolated streptococci were analyzed microbiologically and biochemically; on the basis of their culture and biochemical characteristics five strains were designated as Streptococcus faecalis, 14 strains as Streptococcus faecalis var. liquefaciens, five strains as Streptococcus faecium and one strain as Streptococcus durans. After checking the used taxonomic key of the culture and biochemical classification of B. alvei it may be stated that the culture and biochemical characteristics are stable. The strains of B. alvei (very dried strains), which persisted in the dried slant meat-peptone agar, were viable under the laboratory conditions, which proves the high resistance of the spores to the environment.

Animals↗

PubMed1979

Enzymatic deacylation of lipoteichoic acid by protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790).

High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the former was incubated in the presence of growing protoplasts of Streptococcus faecium (S. faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid LTA(X) from this organism or from chemically deacylated LTA. Native LTA(X) was shown to contain less than one fatty acid equivalent per 18 LTA(X) molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA.

Enterococcus faecalis↗

PubMed1975

Purification and Characterization of two bacteriocins from Streptococcus faecium.

Two bacteriocins were found in the supernatant fluid and in an extract of Streptococcus faecium strain EI. The small soluble enterocin EIA represented more than 90% of the total activity in the supernatant fluid, and was purified 400-fold by ammonium sulphate fractionation, gel filtration on Sephadex G-75 and chromatography on DEAE-cellulose. Enterocin EIB, with a particle weight greater than 4 x 10(6), was the predominant type in the extract. It was released in appreciable quantities after breakage of the bacteria and was purified 100-fold by differential centrifugation, chromatography on Sepharose 4B and density gradient ultracentrifugation. Enterocin EIA, a basic substance with a molecular weight of about 10000, was resistant to heat and was attacked by trypsin, whereas enterocin EIB was less thermostable and insensitive to proteolytic enzymes. The activity of enterocin EIB was unchanged by treatment with DNAase. Sensitivity to enterocin action was confined to certain strains of various enterococcus species, Streptococcus salivarius and Listeria monocytogenes; all the other Gram-positive and Gram-negative bacteria tested for sensitivity were unaffected by either enterocin.

Ammonium Sulfate↗

PubMed1979

Precursor-product relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium.

Exponential biosynthesis and excretion of lipoteichoic acid (LTA) during the exponential phase of growth, and continued synthesis and excretion during valine starvation of Streptococcus faecium (S. faecalis ATCC 9790), were shown. During exponential growth, extracellular LTA (LTAx) accounted for approximately 13% of the total LTA in cultures, whereas during valine starvation, this percentage increased to approximately 60% within 4 h. LTAx was present in a low-molecular-weight, apparently deacylated form, whereas intracellular (LTAi) was present primarily in an apparently high-molecular-weight, acylated and micellar form. Experiments utilizing chases of either fully equilibrated or short pulses of [14C]- or [3H]glycerol were used to demonstrate that LTAx was derived directly from LTAi.

Acetates↗

PubMed1976

Effects of penicillin and lysozyme on the immunofluorescent and precipitin reactivity of group D streptococci.

Damage to the cell wall by growth in the presence of penicillin or by treatment with lysozyme enhanced the immunofluorescent (fluorescent antibody, FA) reactivity to group D streptococci. The optimum concentration and time of treatment with lysozyme varied inversely with the initial FA reactivity of the strain. Speciation of the organisms by a series of biochemical and physiologic tests suggested that the differences in initial FA reactivity were species-related. Thus, S. faecalis strains were the most FA-reactive and most sensitive to lysozyme. S. faecium strains were less FA-reactive and lysozyme-sensitive. S. bovis strains proved to be least FA-sensitive and were most resistant to lysozyme. Treatment with lysozyme was also effective in preparing extracts of group D antigen from all three species for Lancefield grouping by the precipitin test. The lysozyme extracts, moreover, produced much stronger reactions than those made from comparable volumes of cells by the methods of Lancefield or of Rantz and Randall.

Antibodies, Heterophile↗

PubMed1979

Effect of growth rate on lipid and lipoteichoic acid composition in Streptococcus faecium.

The lipid composition of Streptococcus faecium (S. faecalis ATCC 9790) was analyzed at various growth rates. Diphosphatidylglycerol and the non-ionic lipid fraction containing diacylglycerols and neutral glycolipids appeared to accumulate relative to cellular mass as the culture mass doubling time increased from 30 to 80 min. Within the same range of doubling times the non-ionic lipid fraction appeared to become substantially enriched with diacylglycerols. All lipid species and cellular lipoteichoic acid accumulated relative to the cellular mass at doubling times exceeding 80 min, although diacylglycerol accumulation exceeded that of all other compounds studied.

Enterococcus faecalis↗

PubMed1978

Generation rates of faecal streptococci in various vehicles.

Potassium tellurite media gave the highest detrimental effects on the generation number of all tested faecal streptococci species. This applies to all investigated vehicles. However, drastic tellurite effects became more conspicuous with certain vehicles than with others. This is particularly true with acid and alkaline foods and water, especially with strains like Streptococcus bovis, Str. faecium, and Str. durans. Again, tellurite becomes more influencial with regard to old cells rather than young cells in the logarithmic phase. On the other hand, the use of thallous acetate as a concentration agent secured better results than either the tellurite or azide almost with all tested vehicles. Yet, sodium azide may be regarded as holding an intermediate position between tellurite and thallous acetate in terms of vehicles, Streptococcus species, and age of pollution. A lower number of generation resulted with all tested faecal streptococci species, using liquid media bearing more than one concentrating agent. This applied to water and milk examination.

Azides↗