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Drug resistance in Streptococcus faecalis, Streptococcus faecium and Staphylococcus epidermidis isolated from laboratory animals.

A total of 1,278 strains of Streptococcus faecalis, 688 of Str. faecium and 796 of Staphylococcus epidermidis were collected from laboratory animals of 20 colonies in 1980, and tested for their resistance to 20 drugs. Thirteen percent of Str. faecalis, 12% of Str. faecium and 25% of Staph. epidermidis were drug resistant. Multiple drug resistant organisms were frequently detected in laboratory animal colonies where antibiotic treatment had been undertaken. TC-resistant streptococci and EM-resistant Staph. epidermidis were distributed in about half of the colonies regardless of the history of antibiotic usage.

Animals↗

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis.

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 degrees C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 degrees C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.

Anti-Bacterial Agents↗

Susceptibility of enterococci. II. Inhibitory and bactericidal activity of drugs in combination against Streptococcus faecalis and Streptococcus faecium.

The microbiological utility of antibiotic combinations against Streptococcus faecalis and Streptococcus faecium strains was studied. The drugs used were netilmicin + amoxicillin (20 strains); netilmicin + piperacillin (20 strains); netilmicin- + vancomycin (20 strains); netilmicin + rifampicin (20 strains). Netilmicin used in combination with the penicillins was advantageous against Streptococcus faecalis, but not uniformly against Streptococcus faecium. The combinations of netilmicin with vancomycin or rifampicin were no more effective than the single drugs in most cases, although the response varied for the different strains of the two species.

Anti-Bacterial Agents↗

Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis.

Streptococcus faecalis transposon Tn917 was cloned in Escherichia coli on plasmid vector pBR325. The erythromycin resistance determinant of Tn917 was not expressed in the E. coli background. The nucleotide sequence of Tn917 was determined and found to be 5,257 base pairs in length. Six open reading frames (ORFs) were identified and designated 1 through 6 (5' to 3'); all were on the same DNA strand. A region exhibiting strong homology with known promoters was identified upstream from ORF1. ORFs 1 to 3 were virtually identical to the previously sequenced erythromycin resistance determinant on Streptococcus sanguis plasmid pAM77. At the 3' point, where the homology between Tn917 and pAM77 ends, was a 20-base-pair region about 80% homologous with a component of the res site of Tn3. The amino acid sequence of ORF4 showed homology with other site-specific recombination enzymes, including approximately 30% homology with the resolvase of Tn3. Contained within Tn917 was a directly oriented 73-base-pair duplication of the left terminus. The Tn917 sequence revealed that antibiotic-enhanced transposition might be due to extension of transcription from the resistance-related genes (in ORFs 1 to 3) into transposition genes (in ORFs 4 to 6). Transcription analyses resulted in data consistent with this interpretation.

Amino Acid Sequence↗

Expression of the hemolysin trait in a proteolytic transconjugant of Streptococcus faecalis.

Streptococcus faecalis 31H-1 is a proteolytic transconjugant of strain 31B, that harbors the 38.5 Mdal plasmid which codes for hemolysin in strain X14. It fails to express the hemolytic character in broth due to its proteolytic character. Hemolytic activity can be demonstrated when the proteolytic activity is inhibited by 10(-4) M EDTA. The effect of erythromycin (on the donor cells) and of proteinase K during conjugation suggests that the conjugation system involving the 38.5 Mdal plasmid of S. faecalis strain X14 is sex-pherormone mediated.

Conjugation, Genetic↗

Interaction of ciprofloxacin with ampicillin and vancomycin for Streptococcus faecalis.

Streptococcus faecalis is known to be resistant to killing by many of the commonly used antimicrobial agents. Ciprofloxacin, a member of the quinolone class of compounds, was studied for its ability to inhibit and to kill S. faecalis. The rate of killing by ciprofloxacin was slow and was comparable to that of ampicillin or vancomycin. The antimicrobial effect of combining ciprofloxacin with ampicillin or with vancomycin was also studied using checkerboard double-dilution and killing kinetics. There was no synergy or antagonism between ciprofloxacin and either cell wall active agent. Unlike the aminoglycosides, the addition of ciprofloxacin did not produce additional killing over that of ampicillin or of vancomycin alone.

Ampicillin↗

Aminoglycoside-modifying enzyme content of a multiply resistant strain of Streptococcus faecalis.

Streptococcus faecalis strain BM6217 was resistant to high levels of all the clinically useful aminoglycoside antibiotics. This broad aminoglycoside resistance was mediated by constitutively synthesized phosphotransferase, acetyltransferase, and adenylyltransferase activities. It was inferred that phosphorylation occurred at the 3', 5", and 2"-hydroxyl groups, acetylation at the 6'-amino group, and adenylylation, probably, at the 6-hydroxyl group of the aminoglycosides. Strain BM6217 remained susceptible to the aminocyclitol spectinomycin but the combination of penicillin G with that antibiotic appeared antagonistic to this strain.

Aminoglycosides↗

Induction and regulation of a nicotinamide adenine dinucleotide-specific 6-phosphogluconate dehydrogenase in Streptococcus faecalis.

Streptococcus faecalis grown with glucose as the primary energy source contains a single, nicotinamide adenine dinucleotide phosphate (NADP)-specific 6-phosphogluconate dehydrogenase. Extracts of gluconate-adapted cells, however, exhibited 6-phosphogluconate dehydrogenase activity with either NADP or nicotinamide adenine dinucleotide (NAD). This was shown to be due to the presence of separate enzymes in gluconate-adapted cells. Although both enzymes catalyzed the oxidative decarboxylation of 6-phosphogluconate, they differed from one another with respect to their coenzyme specificity, molecular weight, pH optimum, K(m) values for substrate and coenzyme, and electrophoretic mobility in starch gels. The two enzymes also differed in their response to certain effector ligands. The NADP-linked enzyme was specifically inhibited by fructose-1,6-diphosphate, but was insensitive to adenosine triphosphate (ATP) and certain other nucleotides. The NAD-specific enzyme, in contrast, was insensitive to fructose-1,6-diphosphate, but was inhibited by ATP. The available data suggest that the NAD enzyme is involved primarily in the catabolism of gluconate, whereas the NADP enzyme appears to function in the production of reducing equivalents (NADPH) for use in various reductive biosynthetic reactions.

Adenosine Triphosphate↗

Lactic acid translocation: terminal step in glycolysis by Streptococcus faecalis.

Streptococcus faecalis obtains metabolic energy chiefly from the conversion of glucose to lactic acid; the present experiments deal with the mechanism of lactic acid translocation across the cytoplasmic membrane. Efflux of [(14)C]lactate from preloaded cells was accelerated by raising the external pH, and also by the ionophores nigericin and valinomycin. These results suggest that lactate leaves the cell by an electroneutral process, presumably as lactic acid. Further evidence was obtained by studying the entry of [(14)C]lactate into nonmetabolizing cells. It appears that the membrane is essentially impermeable to the lactate anion, but allows passage of lactic acid. The most persuasive evidence is that, upon establishment of a pH gradient such that the cytoplasm was alkaline, l-[(14)C]lactate accumulated in the cells against the concentration gradient. Accumulation was transient, and dissipated in parallel with the collapse of the pH gradient. The concentration gradient attained at the peak was a function of the pH difference. Ionophores which are known to collapse a pH gradient, such as nigericin and valinomycin, abolished accumulation of l-lactate. We infer that lactic acid translocation, whether into the cells or outward, is an electroneutral process and for that reason the distribution of lactic acid across the membrane is a function of the pH of cytoplasm and medium. The specificity of translocation and its kinetic parameters suggest that it is mediated by a carrier of low specificity.

Carbon Radioisotopes↗

Identification and characterization of a small sequence located at two sites on the amplifiable tetracycline resistance plasmid pAMalpha1 in Streptococcus faecalis.

Streptococcus faecalis DT-11 harbors the 6.0-megadalton plasmid pAMalpha1, which determines resistance to tetracycline (TC). When this strain is grown in the presence of TC for a number of generations, a reversible gene amplification occurs, generating tandem repeats of a 2.65-megadalton segment of the plasmid. On the basis of heteroduplex studies between various forms of pAMalpha1 and fragments generated by the Escherichia coli restriction endonuclease EcoRI, we have obtained direct evidence for the presence of a small sequence designated RS1 (for recombination sequence) located on both sides of the TC resistance determinant. Corresponding points on the two sequences are separated by 2.65 megadaltons of deoxyribonucleic acid. The two RS1 sequences have the same polarity and are of a size corresponding to about 380 nucleotide base pairs. The data presented serve as strong support for amplification models that are based on recombinational events involving the RS1 sequences.

Base Sequence↗

Properties of erythromycin-inducible transposon Tn917 in Streptococcus faecalis.

Streptococcus faecalis strain DS16 harbors two plasmids, a conjugative plasmid, pAD1, which encodes hemolysin and bacteriocin activities, and a nonconjugative plasmid, pAD2, encoding resistance to streptomycin, kanamycin, and erythromycin, the latter of which is inducible. The erythromycin resistance determinant is located on a 3.3-megadalton transposable element designated Tn917, which could be transposed to pAD1 as well as to two other plasmids, pAm gamma 1 and pAM alpha 1. When strain DS16 was exposed to low (inducing) concentrations of erythromycin for a few hours, the frequency of Tn917 transposition from pAD2 to pAD1 increased by an order of magnitude. This induction paralleled induction of erythromycin resistance and was prevented by exposing the cells to inhibitors of deoxyribonucleic acid, ribonucleic acid or protein synthesis. The exposure of strain DS16 to inducing concentrations of erythromycin also enhanced the frequency of erythromycin-resistant transconjugants appearing during mating. Initially, cointegrate molecules, whose molecular weights were approximately the sum of pAD1 and pAD2, accounted for these transconjugants; however, as the induction time increased, pAD1::Tn917 became increasingly prominent.

Chloramphenicol↗

Enzymes of agmatine degradation and the control of their synthesis in Streptococcus faecalis.

Streptococcus faecalis ATCC 11700 uses agmatine as its sole energy source for growth. Agmatine deiminase and putrescine carbamoyltransferase are coinduced by growth on agmatine. Glucose and arginine were found to exert catabolite repression on the agmatine deiminase pathway. Four mutants unable to utilize agmatine as an energy source, isolated from the wild-type strain, exhibited three distinct phenotypes. Two of these strains showed essentially no agmatine deiminase, one mutant showed negligible activity of putrescine carbamoyltransferase, and one mutant was defective in both activities. Two carbamate kinases are present in S. faecalis, one belonging to the arginine deiminase pathway, the other being induced by growth on agmatine. These two enzymes have the same molecular weight, 82,000, and seem quite different in size from the kinases isolated from other streptococci.

Agmatine↗

Sodium/proton antiporter in Streptococcus faecalis.

Streptococcus faecalis, like other bacteria, accumulates potassium ions and expels sodium ions. This paper is concerned with the pathway of sodium extrusion. Earlier studies (D.L. Heefner and F.M. Harold, Proc. Natl. Acad. Sci. USA 79:2798-2802, 1982) showed that sodium extrusion is effected by a primary, ATP-linked sodium pump. I report here that cells grown under conditions in which sodium ATPase is not induced can still expel sodium ions. This finding suggested the existence of an alternate pathway. Sodium extrusion by the alternate pathway requires the cells to generate a proton motive force. This conclusion rests on the following observations. (i) Sodium extrusion required glucose. (ii) Sodium extrusion was observed at neutral pH, which allows the cells to generate a proton motive force, but not at alkaline pH, which reduces the proton motive force to zero. (iii) Sodium extrusion was inhibited by the addition of dicyclohexylcarbodiimide and of proton-conducting ionophores. (iv) In response to an artificial pH gradient (with the exterior acid), energy-depleted cells exhibited a transient sodium extrusion which was unaffected by treatments that dissipated the membrane potential and which was blocked by proton conductors. I propose that streptococci have two independent systems for sodium extrusion: an inducible sodium ATPase and a constitutive sodium/proton antiporter.

Carrier Proteins↗

Comparative killing activity and postantibiotic effect of streptomycin combined with ampicillin, ciprofloxacin, imipenem, piperacillin or vancomycin against strains of Streptococcus faecalis and Streptococcus faecium.

Nine strains of Streptococcus faecalis and Streptococcus faecium were studied with respect to ampicillin, ciprofloxacin, imipenem, piperacillin, vancomycin and streptomycin. Two strains were highly resistant (MIC greater than or equal to 2,000 micrograms/ml) to streptomycin. Evaluation and comparison of the killing activity with killing curves, and duration of the postantibiotic effect (PAE) after exposure for 1 h with regrowth curves was done with combinations of antibiotics or alone. The overall killing effect of ciprofloxacin with streptomycin was antagonistic, whereas synergism (greater than or equal to one log10 decrease in viable counts) was observed in streptomycin-susceptible strains with combinations of streptomycin and ampicillin, imipenem, piperacillin or vancomycin. In addition, prolongation of PAE (greater than or equal to 0.5 h) was seen only in streptomycin-susceptible strains. Thus, seven (100%) strains showed a synergistic increase in PAE to combinations with ampicillin and vancomycin, three (43%) to imipenem, four (57%) to piperacillin, but none to the combination of streptomycin and ciprofloxacin. A significant correlation was observed between the magnitude of increased killing and the extent of increase in recovery period with combinations of streptomycin with either ampicillin or vancomycin.

Ampicillin↗

Interaction of anti-kojibiose antibody with the lipoteichoic acids from Streptococcus faecalis and Streptococcus faecium.

Antisera prepared in rabbits by immunization with p-aminophenyl beta-kojibioside conjugated to bovine serum albumin (antikojibiose sera), readily agglutinated whole cells of Streptococcus faecalis or Streptococcus faecium, and showed specific reactions with the lipoteichoic acids (LTAs) of these streptococci by passive hemagglutination, microscale enzyme-linked immunosorbent assay, and crossed immunoelectrophoresis. The interaction of the antikojibiose sera with the LTAs was inhibited best by kojibiose [alpha-D-glucopyranosyl-(1----2)-D-glucose], somewhat less by the dextran from which the kojibiose was prepared, and not measurably by maltose [alpha-D-glucopyranosyl-(1----4)-D-glucose]. The sera reacted only minimally in only the most sensitive assay (microscale enzyme-linked immunosorbent assay) with LTA from group A streptococci (this LTA contains a single kojibiosyl residue as part of the glycolipid moiety of the molecule and failed to react with the Lactobacillus fermentum LTA which is substituted with alpha-D-galactopyranosyl-(1----2)-D -glucosyl units.

Antibody Specificity↗

Identification of Streptococcus faecalis and Streptococcus faecium and susceptibility studies with newly developed antimicrobial agents.

Identification and susceptibility studies were performed on 301 blood and urine Streptococcus faecalis and Streptococcus faecium isolates. Strep Trio-Tubes S4, S5, and S3 (Carr-Scarborough Microbiologicals, Inc.) were compared with conventional methods for accuracy and rapidity. Of 282 isolates identified as S. faecalis, 98% were identified by species in 4 h with Trio-Tubes; the same percentage of isolates analyzed by conventional methods were identified in 24 h. All 14 S. faecium isolates (approximately 5% of the total number of isolates) were identified by Trio-Tubes in 24 h. In vitro MIC susceptibility testing of the isolates was performed by the Dynatech 2000 microdilution technique (Dynatech Laboratories, Inc.). Several newly developed antimicrobial agents, including imipenem (a carbapenem) and some of the quinolone drugs, i.e., CI-934, ciprofloxacin, A-56619, A-56620, amifloxacin, norfloxacin, and enoxacin, were tested, as were ampicillin, erythromycin, and vancomycin. Both ampicillin and vancomycin showed good activity against S. faecalis, with MICs for 90% of isolates tested (MIC90S) of 1 and 2 micrograms/ml, respectively; with S. faecium, ampicillin exhibited an MIC90 of 16 micrograms/ml and vancomycin exhibited an MIC90 of 2 micrograms/ml. Of the newer antimicrobial agents, imipenem and CI-934 exhibited the greatest activity against S. faecalis strains, with MIC90S of 2 and 0.5 micrograms/ml, respectively. MBCs against the isolates were determined with CI-934, with 90% of S. faecalis strains showing MBCs of 1 microgram/ml or less.

Ampicillin↗

Susceptibility of enterococci. I. Inhibitory and bactericidal activity of several chemoantibiotics against Streptococcus faecalis and Streptococcus faecium.

The authors present a microbiological study of 100 strains of Enterococcus (70 strains of Streptococcus faecalis and 30 strains of Streptococcus faecium) tested for susceptibility to the following antibiotics, amoxicillin, ampicillin + flucloxacillin, piperacillin, rifampicin, vancomycin, netilmicin, ofloxacin, and norfloxacin. The assessment of minimum inhibitory and minimum bactericidal concentrations of these substances indicates that all have good inhibitory activity except netilmicin, which is active at higher concentrations; with rifampicin and vancomycin showing very poor bactericidal activity. The bactericidal activity of penicillins was hard to assess because of tolerance and paradoxical effect phenomena. The quinolones showed good inhibitory and bactericidal activity.

Anti-Bacterial Agents↗