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PubMed2006

7-O-malonyl macrolactin A, a new macrolactin antibiotic from Bacillus subtilis active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia.

We report here the discovery, isolation, and chemical and preliminary biological characterization of a new antibiotic compound, 7-O-malonyl macrolactin A (MMA), produced by a Bacillus subtilis soil isolate. MMA is a bacteriostatic antibiotic that inhibits a number of multidrug-resistant gram-positive bacterial pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia. MMA-treated staphylococci and enterococci were pseudomulticellular and exhibited multiple asymmetric initiation points of septum formation, indicating that MMA may inhibit a cell division function.

Animals↗
PubMed1975

Disk diffusion and serial dilution tests of susceptibility of some pathogenic gram-negative bacilli and enterococci to carbenicillin and ampicillin.

Tests for susceptibility to ampicillin and carbenicillin were performed with 35 strains each of Klebsiella, Enterobacter, Serratia, and Proteus, 71 strains of Pseudomonas aeruginosa, and 68 strains of enterococci by serial dilution and disk-diffusion tests employing 10(-3) dilutions of overnight cultures as inocula for both. Commercial 10-mug ampicillin and 50- and 100-mug carbenicillin disks, and freshly prepared 10-, 50-, and 75-mug ampicillin and 10- and 50-mug carbenicillin disks were used. Results were displayed as cumulative distribution curves for both minimal inhibitory concentrations and zone diameters, and as scattergrams for correlating them. Differences in susceptibility to the two antibiotics were small for Klebsiella, Enterobacter, and Serratia and large for the others. The freshly prepared and commercial disks of the same content gave comparable zones. There was good correlation of zone diameter with each disk and the minimal inhibitory concentration. Among the ampicillin disks tested, none was useful for Pseudomonas; with the other species, the 10-mug disk, as well as those with higher ampicillin content, could discriminate susceptible from resistant strains. However, only the 75-mug disk selected some Klebsiella strains susceptible to high concentrations. The 50- and 100-mug carbenicillin disks were equally discriminating for most strains, but the higher concentration was more selective for Klebsiella. The 10-mug carbenicillin disk was as effective as the 50- and 100-mug disks for discriminating among Enterobacter, Serratia, Pseudomonas, and Proteus, but not for Klebsiella or enterococci. The 10(-3) inoculum gave zone sizes considerably larger than those reported by other workers who used the standard Kirby-Bauer method.

Ampicillin↗
PubMed1992

Routine procedures for isolation and identification of enterococci and fecal streptococci.

Over the past 6 years, a revised classification of the streptococci and enterococci, based primarily on molecular techniques such as 16S rRNA sequencing and DNA-DNA hybridization, emerged. However, little attention was placed on routine physiological tests that could be used in food and clinical laboratories to differentiate between species of a new genus, Enterococcus, and fecal Streptococcus spp. The purpose of this study was to devise a convenient and reliable system to identify enterococci and fecal streptococci by using conventional procedures. Fifty-nine strains of 13 Enterococcus spp., including the type strains and many strains used by previous investigators, were characterized by using conventional tube tests, the API Rapid Strep system, and MicroScan Pos ID panels. Results were compared with each other and with previously published results. A comparison of conventional tube tests versus published tube test results yielded 17 discrepancies. Although not all tests were done with each of the three systems, 28 discrepancies between results obtained with the API system and those obtained with conventional tube tests were found. There were 24 discrepancies between results obtained with the MicroScan Pos ID panel and those obtained with conventional tube tests. There were 12 discrepancies between the results with the API Rapid Strep system and those with the MicroScan Pos ID panels. We devised flow charts of key tests that might be used to identify cultures without resorting to nucleic acid analysis and other labor- and equipment-intensive analyses.

Diagnostic Tests, Routine↗
PubMed2004

Persistence of vancomycin-resistant enterococci in New Zealand broilers after discontinuation of avoparcin use.

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was > or =256 microg/ml for 98.7% of isolates, and the avilamycin MIC was > or =8 microg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.

Animal Husbandry↗
PubMed2004

Occurrence, structure, and mobility of Tn1546-like elements in environmental isolates of vancomycin-resistant enterococci.

The occurrence, structure, and mobility of Tn1546-like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored vanA. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as Enterococcus faecium (n = 13), E. casseliflavus (n = 6), E. mundtii (n = 3), E. faecalis (n = 3), and E. durans (n = 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn1546-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn1546 in the prototype strain E. faecium BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for E. casseliflavus strains. Indistinguishable Tn1546-like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to E. faecium BM4105 was demonstrated for E. faecalis, E. mundtii, and E. durans. This study indicates that VRE, including species other than E. faecium and E. faecalis, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn1546-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.

Animals↗
PubMed1962

Coliforms, enterococci, thermodurics, thermophiles, and psychrophiles in untreated farm pond waters.

Untreated waters from ten farm ponds located in central, north central, southeastern, and southwestern Ohio were examined for numbers of coliforms, enterococci, thermodurics, thermophiles, and psychrophiles. The median population densities per 100 ml water for all ponds were: coliforms, 23; enterococci, 3.6; thermodurics, 6,000; thermophiles, 450; psychrophiles, 1,000. The results indicate that these farm pond waters were only lightly polluted and suggest that farm ponds, properly maintained, are a source of raw water of high bacteriological quality, requiring a minimum of treatment to be made suitable for domestic and livestock purposes.

Enterococcus↗
PubMed1968

Comparison of the action of ampicillin and benzylpenicillin on enterococci in vitro.

The bactericidal activity of benzylpenicillin and ampicillin on 21 strains of enterococci was evaluated and compared to the activity of these drugs in combination with streptomycin (20 mug/ml). On a weight basis, ampicillin was about twice as effective as benzylpenicillin. Neither of the drugs was rapidly and completely bactericidal for any of the 21 strains of enterococci when used alone. The addition of streptomycin greatly enhanced the early bactericidal rate achieved with any given amount of either penicillin and permitted the elimination of viable organisms in vitro. These results suggest that, for the time being, combined antibiotic therapy might be desirable in enterococcus endocarditis and that ampicillin, although more effective than benzylpenicillin, should not be relied upon as a single drug in that disease.

Ampicillin↗
PubMed1971

Medium-dependent activity of gentamicin sulfate against enterococci.

Routine disc diffusion susceptibility tests (Bauer-Kirby technique), employing 5% sheep blood-Mueller-Hinton agar and 10-mug gentamicin sulfate discs, disclosed that a significant number of clinical enterococcal isolates were sensitive to the antibiotic, as also revealed by the agar dilution technique. With few exceptions, the isolates proved resistant to this antibiotic when tested for susceptibility in Brain Heart Infusion and Trypticase soy broth or agar. The addition of 5% sheep blood to Trypticase soy and Brain Heart Infusion agars resulted in markedly enhanced activity of the antibiotic, indicating medium-dependent activity of gentamicin against enterococci. Human serum and urine failed to support optimal growth of enterococci. Thus, it was not possible to correlate the activity of gentamicin in any of the media examined with that in serum or urine.

Agar↗
PubMed1972

Improved procedure for identification of group D enterococci with two new media.

With the use of Pfizer selective enterococcus medium as a screening process, tyrosine decarboxylase (TD) medium and D-broth were devised for additional confirmation of the identification of group D enterococci. The semisolid TD medium was used in a method similar to inoculating a motility tube. TD activity was indicated by clarification of the medium near the line of inoculation because the insolubility of tyrosine provided an otherwise milky suspension. D-broth was used to show the tolerance of organisms to 6.5% NaCl at pH 8.2. In addition, enterococcal species (except Streptococcus durans) could also be demonstrated by the acid formation due to the fermentation of mannitol or arabinose. With this improved system, about 97% of enterococci could be accurately identified and confirmed.

Arabinose↗
PubMed1974

Improved isolation and differentiation of enterococci in cheese.

Further documentation of an enterococcus selective differential (ESD) medium was obtained in isolations from eight different cheeses. An improved differentiation of tetrazolium salt (2, 3, 5-triphenyl tetrazolium hydrochloride [TTC])-reducing strains of Streptococcus faecalis from TTC-nonreducing or TTC-faintly-reducing Streptococcus faecium was attained. The sensitivity of the medium was evaluated in comparison with that of KF streptococcal, Pfizer selective enterococcus (PSE), the medium of Reinbold, Swern, and Hussong (RSH), and the medium of Saraswat, Clark, and Reinbold (SCR). Selective counts, rate of colony formation, and ease of isolation and differentiation of colonies were examined. The specificity of the medium was also investigated. ESD supported the fastest rate of growth and the maximum size of colonies; counts in this medium were in most cases possible with 17 h of incubation, whereas the other media required 24 to 48 h. A presumptive identification of 1,077 isolates by four biochemical tests disclosed that SCR, RSH, and ESD selected high, comparable percentages of strains that approximated most closely the typical description of enterococci (66, 60.1, and 58%, respectively). Low percentages (21.1 and 30.7%) were yielded by KF and PSE. The utility of ESD for a rapid, presumptive identification of enterococci was confirmed by serological and biochemical testing of color TTC-differentiated colonies isolated from 18 cheeses.

Cell Count↗
PubMed1971

Mechanism of resistance to antibiotic synergism in enterococci.

Enterococci exhibit two types of resistance to streptomycin. Moderately high-level resistance is observed in most naturally occurring strains and can be overcome by simultaneous exposure to penicillin. In addition, very high-level resistance is found in those strains against which penicillin plus streptomycin fail to produce synergism in vitro. To study the mechanism of streptomycin resistance in enterococci, ribosomes from a wild-type strain and from a highly streptomycin-resistant mutant were isolated, characterized, and studied in an in vitro amino acid incorporation system. The ribosomes from the organism with moderately high-level streptomycin resistance were sensitive to streptomycin in vitro, suggesting that this type of resistance is caused by failure of streptomycin to reach the ribosomes. Very high-level resistance (and lack of penicillin-streptomycin synergism), on the other hand, appears to be due to ribosomally mediated streptomycin resistance.

Amino Acids↗
PubMed1997

Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin.

Analyses of the peptidoglycan nucleotide precursor contents of enterococci and staphylococci treated with ramoplanin, tunicamycin, or vancomycin were carried out by high-pressure liquid chromatography coupled with mass spectrometry (MS). In all cases, a sharp increase in the UDP-N-actetylmuramoyl-pentapeptide or -pentadepsipeptide pool was observed. Concomitantly, new peptidoglycan nucleotide peptides of higher molecular masses with hexa- or heptapeptide moieties were identified: UDP-MurNAc-pentapeptide-Asp or pentadepsipeptide-Asp in enterococci and UDP-MurNAc-pentapeptide-Gly or -Ala and UDP-MurNAc-pentapeptide-Gly-Gly or -Ala-Gly in staphylococci. These new compounds are derivatives of normal UDP-MurNAc-pentapeptide or -pentadepsipeptide precursors with the extra amino acid(s) linked to the lysine epsilon-amino group as established by various analytical procedures (MS, MS-MS fragmentation, chemical analysis, and digestion with R39 D,D carboxypeptidase). Except for tunicamycin-treated cells, it was not possible to ascertain whether these unusual nucleotides were formed by direct addition of the amino acids to UDP-MurNAc-pentapeptide (or -pentadepsipeptide) or whether they arose by reverse reactions from lipid I intermediates to which the amino acids had been added.

Anti-Bacterial Agents↗
PubMed1963

FOOD-POISONING POTENTIAL OF THE ENTEROCOCCI.

Deibel, R. H. (American Meat Institute Foundation, Chicago, Ill.), and J. H. Silliker. Food-poisoning potential of the enterococci. J. Bacteriol. 85:827-832. 1963.-A total of 23 enterococcus strains were fed to two and sometimes three human volunteers in an effort to elicit food-poisoning symptoms. Each culture was consumed after it was grown in whole sterile milk or on the surface of commercially sterile ham slices. Six strains of Streptococcus faecalis var. liquefaciens were consumed after complete liquefaction of gelatin. In addition, strains of S. faecalis were consumed after having been grown in media which altered the energy metabolism (arginine, gluconate, malate, and pyruvate). In no instance were any of the above conditions of growth conducive to the production of food-poisoning symptoms in the volunteers. Moreover, no evidence was found to indicate that either the age of the culture or the disruption of the cell was a factor in the production or release of a toxic principle. It would appear that until the environmental conditions (if any) for food poisoning are defined the evidence obtained suggests that the association of enterococci and food poisoning is questionable.

Animals↗
PubMed1963

SURVEY OF THE BACTERIOCINES OF ENTEROCOCCI.

Brock, Thomas D. (Indiana University, Bloomington), Barbara Peacher, and Deborah Pierson. A survey of the bacteriocines of enterococci. J. Bacteriol. 86:702-707. 1963.-A survey has been made of bacteriocine production by a wide variety of well-characterized strains of group D streptococci. On the basis of spectrums and sensitivity to chloroform, heat, and proteolytic enzymes, five distinct bacteriocines can be defined. Type 1 is produced by all Streptococcus zymogenes (S. faecalis var. zymogenes) strains, is active against a wide variety of gram-positive bacteria, and is also a hemolysin. Type 2 is produced by some S. liquefaciens (S. faecalis var. liquefaciens) strains, and acts on many enterococci as well as on certain other lactic acid bacteria. Type 3 is produced by certain strains of both S. faecalis and S. faecium, and inhibits a wide variety of group D streptococci, but is inactive against all other lactic acid bacteria tested except Leuconostoc citrovorum. Type 4 is produced by certain S. faecium strains and resembles in certain ways the type 3 activity, but differs from it in other ways. Type 5 has been found to be produced by only one proteolytic strain of S. zymogenes, and this bacteriocine has a very narrow spectrum. The strain that produces this bacteriocine also produces type 1 activity. No strain is sensitive to a bacteriocine of the type it produces.

Bacillus cereus↗
PubMed1963

PHYSIOLOGY OF THE ENTEROCOCCI AS RELATED TO THEIR TAXONOMY.

Deibel, R. H. (American Meat Institute Foundation, Chicago, Ill.), Donald E. Lake, and C. F. Niven, Jr. Physiology of the enterococci as related to their taxonomy. J. Bacteriol. 86:1275-1282. 1963-Studies on a collection of enterococci isolated from diverse sources have confirmed the existence of two distinct species, namely, Streptococcus faecalis and Streptococcus faecium. In contrast with S. faecium, S. faecalis characteristically ferments melezitose, sorbitol, glycerol (anaerobically), citrate, and gluconate; fails to ferment arabinose and melibiose; has strong reducing capacities as demonstrated by prompt reduction of litmus in milk or tetrazolium in an agar medium; initiates growth on an agar medium containing 0.04% potassium tellurite; does not require folic acid for growth; and does not produce strong greening in blood agar. S. liquefaciens and S. zymogenes differ from S. faecalis only in their proteolytic capacities and, therefore, deserve only varietal status. Some S. faecalis and S. faecium strains are proteolytic when tested on agar media. S. durans differs from S. faecium only in its inability to ferment arabinose and mannitol, and the failure of most strains to ferment sucrose. Therefore, it should be considered as a variety of S. faecium. It is recommended that the term "enterococcus" be used only when referring to S. faecalis and S. faecium, and their respective varieties.

Arabinose↗
PubMed1981

Methodological variation in antibiotic synergy tests against enterococci.

Thirty-two human isolates of enterococci were tested for antibiotic synergy by using penicillin and one of six aminoglycosides. Three methods were used: synergy screen, microdilution checkerboard, and time-kill curves. The synergy screen accurately predicted synergy for gentamicin-penicillin combinations, and this synergy was later confirmed by time-kill curves. The microdilution checkerboard method suffered from inherent variation, and agreement with time-kill curves ranged from 92% (twofold reduction in minimum inhibitory concentration) to 4.2% (fourfold reduction in minimum inhibitory concentration). We suggest that enterococci be screened for synergy (i.e., presence or absence of high-level resistance) by using the criterion of growth or no growth in the presence of 2,000 microgram of an aminoglycoside per ml. The microdilution checkerboard test for synergy is not recommended.

Aminoglycosides↗
PubMed1983

Rapid identification of enterococci.

Enterococci were identified in 4 h with bile esculin agar and the Autobac system (General Diagnostics, Warner-Lambert Co., Morris Plains, N.J.), which was used to incubate and monitor salt broth for growth. Of 86 group D streptococci tested, 41 enterococci grew in salt broth and were bile esculin positive within 4 h; none of 45 group D nonenterococcal streptococci exhibited growth in the salt broth, and only 38 of them were bile esculin positive within 4 h.

Agar↗