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PubMed1997

Incidence and detection of multi-drug-resistant enterococci in Dublin hospitals.

In February 1994, an outbreak of vancomycin-resistant Enterococcus faecium (VREM) occurred in the oncology unit of a Dublin hospital. Between February and July 1994, VREM was isolated from 18 patients, one staff member and 14 environmental sites within the unit. The isolates also had high-level aminoglycoside and penicillin resistance. Three pulsed-field gel electrophoresis (PFGE) types were identified, two of them from multiple patients and environmental sites. Plasmid typing allowed subdivision of PFGE types. A retrospective study of enterococci isolated from blood cultures between January 1991 and January 1994 showed that, before the outbreak, fewer than 2% of isolates were vancomycin-resistant but that the incidence of high-level gentamicin resistance had increased from 17% to 60% and ampicillin resistance from 22% to 51%. Among clinically significant non-blood-culture enterococci isolated between September and December 1993, fewer than 1% were vancomycin-resistant, 13% were ampicillin-resistant and 44% highly gentamicin-resistant. None produced beta-lactamase. High-content gentamicin disks (120 micrograms) and low-content vancomycin disks (5 micrograms) allowed simple, reliable detection of resistant enterococci. MICs of vancomycin and teicoplanin determined by agar dilution and E-test agreed well, but values tended to be slightly lower by E-test.

Ampicillin Resistance↗

PubMed2001

EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Aminoglycosides↗

PubMed2003

Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens.

Synercid, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 A resolution in the absence of ligands, at 2.8 A resolution bound to dalfopristin, and at 3.0 A resolution in the presence of acetyl-coenzyme A. Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle. Inactivation of dalfopristin involves acetyl transfer from acetyl-coenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >105-fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid resistance.

Acetyltransferases↗

PubMed2003

Heterogeneity of macrolide-lincosamide-streptogramin B resistance phenotypes in enterococci.

We determined the macrolide resistance phenotypes of 241 clinical isolates of erythromycin-resistant enterococci (MICs, > or = 1 microg/ml), including 147 Enterococcus faecalis strains and 94 Enterococcus faecium strains, collected from a hospital in Seoul, Korea, between 1999 and 2000. By the erythromycin (40 micro g)-josamycin (100 microg) double-disk test, 93 strains were assigned to the constitutive macrolide, lincosamide, and streptogramin B (MLS(B)) resistance (cMLS(B)) phenotype, and the remaining 148 strains were assigned to the inducible MLS(B) resistance (iMLS(B)) phenotype. Of the strains with the iMLS(B) phenotype, 36 exhibited a reversibly inducible MLS(B) (riMLS(B)) phenotype, i.e., blunting of the erythromycin zone of inhibition, which indicates that the 16-membered-ring macrolide josamycin is a more effective inducer than the 14-membered-ring macrolide erythromycin. Sequence analysis of the regulatory regions of the erm(B) genes from all of the strains exhibiting the riMLS(B) phenotype revealed not only erm(Bv) [where v represents variant; previously erm(AMR)] (n = 13), as reported previously, but also three kinds of erm(B) variants, which were designated erm(Bv1) (n = 17), erm(Bv2) (n = 3), and erm(Bv3) (n = 3), respectively. In lacZ reporter gene assays of these variants, the 16-membered-ring macrolide tylosin had stronger inducibility than erythromycin at > or = 0.1 microg/ml. These findings highlight the versatility of erm(B) in induction specificity.

Anti-Bacterial Agents↗

PubMed2004

Efficacy of 10 different cleaning processes in a washer-disinfector for flexible endoscopes.

Successful cleaning of medical devices, such as flexible endocopes, has been recognized to be of major importance for effective processing. Washer-disinfectors (WD) are considered to be an important step in this direction. The cleaning process in WD, however, has only been partially assessed regarding its effectiveness, and therefore to study this in more detail, tests were carried out, according prEN ISO 15883, using transparent teflon tubes as test pieces (length 2 m). For each experiment three test pieces were contaminated with the 'German test soil' containing Enterococcus faecium in blood, two for the test and one as a control (no automatic cleaning). Automatic cleaning was performed with a Wassenburg WD 440. Ten cleaning agents were used. In addition the process was carried out with water alone. After automated cleaning, test pieces were assessed visually (four categories, range: very poor to excellent visible cleanliness) and microbiologically [log(10) reduction factor (RF)]. Each experiment was repeated three times. Using the WD water gave excellent visible cleanliness with a mean RF of 1.1+/-0.6. The same excellent visible cleanliness was obtained with seven cleaning processes: deconex 23 Neutrazym, Helimatic Cleaner enzymatic, Korsolex-Endo-Cleaner, Labomat E, neodisher mediclean, Thermosept ER, and Thermoton NR. Worse visible cleanliness was found with three cleaning processes: Olympus ETD Cleaner and neodisher FE led to adequate visible cleanliness, and the cleaning process with neodisher medizym led to poor visible cleanliness. Six cleaning processes reduced the test organism by RF>or=3, i.e. the reduction was significantly higher than after cleaning with water alone. No significant difference between use of water alone and the cleaning process was found with three cleaning processes: Olympus ETD Cleaner, neodisher mediclean, and Thermosept ER (range RF: 0.8-1.8; P > 0.05). The cleaning process with neodisher medizym yielded a significantly lower mean RF (P = 0.039) in comparison with water treatment alone. Both visible cleanliness and mean RF, varied indicating that the choice of cleaning process had a major impact on the overall result.

Decontamination↗

PubMed2006

Efficacy of daptomycin in the treatment of experimental endocarditis due to susceptible and multidrug-resistant enterococci.

OBJECTIVES: Daptomycin was tested in vitro and in rats with experimental endocarditis against the ampicillin-susceptible and vancomycin-susceptible Enterococcus faecalis JH2-2, the vancomycin-resistant (VanA type) mutant of strain JH2-2 (strain JH2-2/pIP819), and the ampicillin-resistant and vancomycin-resistant (VanB type) Enterococcus faecium D366. METHODS: Rats with catheter-induced aortic vegetations were treated with doses simulating intravenously kinetics in humans of daptomycin (6 mg/kg every 24 h), amoxicillin (2 g every 6 h), vancomycin (1 g every 12 h) or teicoplanin (12 mg/kg every 12 h). Treatment was started 16 h post-inoculation and continued for 2 days. RESULTS: MICs of daptomycin were 1, 1 and 2 mg/L, respectively, for strains JH2-2, JH2-2/pIP819 and D366. In time-kill studies, daptomycin showed rapid (within 2 h) bactericidal activity against all strains. Daptomycin was highly bound to rat serum proteins (89%). In the presence of 50% rat serum, simulating free concentrations, daptomycin killing was maintained but delayed (6-24 h). In vivo, daptomycin treatment resulted in 10 of 12 (83%), 9 of 11 (82%) and 11 of 12 (91%) culture-negative vegetations in rats infected with strains JH2-2, JH2-2/pIP819 and D366, respectively (P < 0.001 compared to controls). Daptomycin efficacy was comparable to that of amoxicillin and vancomycin for susceptible isolates. Daptomycin, however, was significantly (P < 0.05) more effective than teicoplanin against the glycopeptide-susceptible strain JH2-2 and superior to all comparators against resistant isolates. CONCLUSIONS: These results support the use of the newly proposed daptomycin dose of 6 mg/kg every 24 h for treatment of enterococcal infections in humans.

Amoxicillin↗

PubMed2001

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish.

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.

Aeromonas↗

PubMed2005

Characterization of bacteriocins produced by lactic acid bacteria isolated from spoiled black olives.

Bacteriocin-producing strains of Lactobacillus plantarum ST23LD and ST341LD, Enterococcus faecium ST311LD and Leuconostoc mesenteroides subsp. mesenteroides ST33LD were isolated from the brine of spoiled black olives. The bacteriocins produced by all four strains inhibited the growth of Gram-positive bacteria (E. faecalis, L. casei and Streptococcus pneumoniae), but also Escherichia coli and Pseudomonas aeruginosa. Strain ST23LD produced two bacteriocins (ST23LDa and ST23LDb of approximately 3.0 and 14.0 kDa, respectively), with a combined maximum level of activity of 25,600 AU/ml after 18 h of growth. The same level of activity was recorded for bacteriocin ST341LD (approximately 3.0 kDa), but after 16 h. Bacteriocins ST311LD (ca. 2.3 kDa) and ST33LD (ca. 2.7 kDa) were produced at much lower levels (6400 AU/ml), and only after 20 h of growth. Bacteriocin activity was destroyed after treatment with proteolytic enzymes and Triton X, but not when treated with alpha-amylase, SDS, Tween 20, Tween 80, urea and EDTA, or when heated for 20 min at 121 degrees C. Addition of bacteriocins ST23LD, ST341LD and ST311LD to cells of Lactobacillus casei LHS in logarithmic phase resulted in growth inhibition for one hour, followed by a slight increase in optical density over the next seven hours. Bacteriocin ST33LD also inhibited the growth of strain LHS, but to a lesser extent. Bacteriocins ST23LD, ST341LD and ST33LD remained at the same level of activity for 6 h at pH<4.0. However, the activity of bacteriocin ST311LD decreased by 50% within 2 h at pH 4.4. The possibility of the bacteriocin adsorbing to the producer cell and proteolytic degradation is unlikely.

Bacteria↗

PubMed1998

In vivo characterization of the type A and B vancomycin-resistant enterococci (VRE) VanRS two-component systems in Escherichia coli: a nonpathogenic model for studying the VRE signal transduction pathways.

Escherichia coli reporter strains modeling the high-level type A and B vancomycin resistances of Enterococcus faecium BM4147 and Ent. faecalis have been developed to study the respective VanR-VanS two-component regulatory systems. PvanH-, PvanRa-, PvanY-, and PvanRb-lacZ fusions report on expression from the vancomycin-resistant enterococci promoters of the type A vanRSHAXYZ and type B vanRSYWHBX gene clusters. These strains also express from single-copy chromosomal genes vanRa, vanRb, or vanRSb behind their respective promoter (PvanRa or PvanRb) or vanSa or vanSb behind the rhamnose-inducible PrhaB. Results show that activation (phosphorylation) of the response regulator VanRa by its sensor kinase VanSa leads to transcriptional activation of both PvanH and PvanRa. Additionally, VanRb activates its cognate promoters PvanY and PvanRb, although this occurs only in the absence of VanSb and presumably is caused by VanRb phosphorylation by an unidentified endogenous E. coli kinase. Thus, VanSb interferes with activation of VanRb, probably by acting as a phospho-VanRb phosphatase. Although both VanRa and VanRb activate their cognate promoters, neither activates the heterologous PvanR, PvanH, or PvanY, arguing against the interchangeability of type A and B two-component regulatory switches in vancomycin-resistant enterococci. VanRa also is activated by the nonpartner kinase PhoR. Because this occurs in the absence of its inducing signal (Pi limitation), PhoR autophosphorylation apparently is regulated in vivo. Furthermore, the activation of VanRa caused by cross talk from PhoR in the absence of a signal allows distinction of cross talk from crossregulation as the latter, but not the former, responds to environmental cues.

Anti-Bacterial Agents↗

PubMed1997

The VanS sensor negatively controls VanR-mediated transcriptional activation of glycopeptide resistance genes of Tn1546 and related elements in the absence of induction.

Transposon Tn1546 from Enterococcus faecium BM4147 encodes a histidine protein kinase (VanS) and a response regulator (VanR) that regulate transcription of the vanHAX operon encoding a dehydrogenase (VanH), a ligase (VanA), and a D,D-dipeptidase (VanX). These last three enzymes confer resistance to glycopeptide antibiotics by production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate. Transcription of vanS and the role of VanS in the regulation of the vanHAX operon were analyzed by inserting a cat reporter gene into vanS. Transcription of cat and vanX was inducible by glycopeptides in partial diploids harboring vanS and vanS(omega)cat but was constitutive in strains containing only vanS(omega)cat. Promoters P(R) and P(H), located upstream from vanR and vanH, respectively, were cloned into a promoter probing vector to study transactivation by chromosomally encoded VanR and VanS. The promoters were inactive in the absence of vanR and vanS, inducible by glycopeptides in the presence of both genes, and constitutively activated by VanR in the absence of VanS. Thus, induction of the vanHAX operon involves an amplification loop resulting from binding of phospho-VanR to the P(R) promoter and increased transcription of the vanR and vanS genes. Full activation of P(R) and P(H) by VanR was observed in the absence of VanS, indicating that the sensor negatively controls VanR in the absence of glycopeptides, presumably by dephosphorylation. Activation of the VanR response regulator in the absence of VanS may involve autophosphorylation of VanR with acetyl phosphate or phosphorylation by a heterologous histidine protein kinase.

Anti-Bacterial Agents↗

PubMed1999

The influence of bacterial inoculants on the microbial ecology of aerobic spoilage of barley silage.

The aerobic decomposition of barley silage treated with two inoculants (LacA and LacB) containing mixtures of Lactobacillus plantarum and Enterococcus faecium was investigated over a 28-day period. Initially, yeast and bacterial populations were larger in silage inoculated with LacA than in silage treated with LacB or water alone (control). Differences in the succession of yeasts in silage treated with LacA were observed relative to the other two treatments. From silage treatment with LacA, Issatchenkia orientalis was the most prevalent yeast taxon over all of the sample times, and the filamentous fungus Microascus brevicaulis was also frequently isolated at later sample dates (> or = 14 days). In contrast, Saccharomyces exiguus was the most prominent yeast recovered from silage treated with LacB and water alone on days 2 and 4, although it was supplanted by I. orientalis at later sample times. Successional trends of bacteria were similar for all three treatments. Lactobacillus spp. were initially the most prevalent bacteria isolated, followed by Bacillus spp. (primarily Bacillus pumilus). However, the onset of Bacillus spp. prominence was faster in LacA silage, and Klebsiella planticola was frequently recovered at later sample times (> or = 14 days). More filamentous fungi were recovered from LacA silage on media containing carboxylmethylcellulose, pectin, or xylan. The most commonly isolated taxa were Absidia sp., Aspergillus flavus, Aspergillus fumigatus, Byssochlamys nivea, Monascus ruber, Penicillium brevicompactum, Pseudoallescheria boydii, and M. brevicaulis. The results of this study indicated that the two bacterial inoculants incorporated into barley at the time of ensilage affected the microbial ecology of silage decomposition following exposure to air. However, neither of the microbial inoculants effectively delayed aerobic spoilage of barley silage, and the rate of decomposition of silage treated with one of the inoculants (LacA) was actually enhanced.

Aerobiosis↗

PubMed1996

Comparative assessment of bacterial inoculation and propionic acid treatment of aerobic stability and microbial populations of ensiled high-moisture ear corn.

High-moisture ear corn (HMEC) was untreated, treated with propionic acid (PA), or inoculated with a mixture of Lactobacillus plantarum and Enterococcus faecium and allowed to ensile in laboratory silos for 0, 7, 21, 42, 138, or 202 d. The silages were evaluated for fermentation quality, microbial populations, and aerobic stability. In all treatments, silage pH declined rapidly within 7 d, but the rate of decline seemed greatest with the inoculum. The lactic acid content of inoculated HMEC was higher (P < .05) than that of control of PA-treated HMEC. Regardless of treatment, the population of lactic acid bacteria (LAB) increased (P < .1) up to 7 to 21 d of fermentation then declined; LAB counts decreased (P < .05) up to 42 d in control and PA-treated silage but continued to decline until 138 d for inoculated silage. Yeast and mold counts tended to decrease up to 42 d of ensiling then decreased (P < .05) as fermentation progressed. Between 138 and 202 d of ensiling, the control silage showed a marked increase (P < .10) in pH and yeast and mold populations, providing evidence of secondary fermentation; PA treatment and bacterial inoculation prevented secondary fermentation. Inoculation tended to reduce estimates of sample temperature for silage stored for 138 d and exposed to air, but not for the corresponding silage stored for 202 d. Treatment with PA prevented the loss (P > .05) of acetic acid and the rise (P > .05) in pH during air exposure of the 138-d silage; both control and PA-treated silage showed an increase (P < .05) in yeast and mold populations, but the increments were 38% and 23%, respectively. Compared with PA, the relative efficacy of inoculation in improving aerobic spoilage of HMEC depended on the period of silo storage and the criterion used to assess aerobic stability.

Acetates↗

PubMed2000

The effect of a probiotic milk product on plasma cholesterol: a meta-analysis of short-term intervention studies.

INTRODUCTION: Certain fermented dairy milk products may have beneficial effects on plasma cholesterol levels. However, a number of studies have produced conflicting results as to whether dietary supplementation by a probiotic dairy product containing the bacteria culture Causido(R) reduces plasma cholesterol. OBJECTIVE: To conduct a meta-analysis of intervention studies to evaluate the effect of the Causido(R) culture on plasma total cholesterol and low-density lipoprotein (LDL)-cholesterol. THE PROBIOTIC MILK PRODUCT: The yoghurt product Gaio(R) is fermented with Causido(R), composed of one strain of Enterococcus faecium (human species) with the proposed cholesterol-lowering effect, and two strains of Streptococcus thermophilus. STUDY INCLUSION AND DATA EXTRACTION: Six studies were identified from a literature search and from the yoghurt producer. All studies met the inclusion criteria. Summary data for plasma concentrations of total cholesterol and LDL-cholesterol were extracted from the original publications or by personal request to the authors. Data from 4-8 weeks of treatment duration was used. STATISTICAL ANALYSIS: We performed a traditional meta-analysis where mean differences between intervention and control of the pre-post changes in total cholesterol and LDL-cholesterol were calculated, as well as 95% confidence intervals (CIs). RESULTS: In the six studies included in the meta-analysis, the Gaio(R) interventions produced changes in total cholesterol above those of the control groups ranging from -0.02 to -1.02 mmol/l and in LDL-cholesterol ranging from -0.02 to -1.15 mmol/l. After inclusion of an open-label study, the meta-analysis of the double-blind studies showed that Gaio(R) as compared to the control group changed total cholesterol by -0.22 mmol/l (95% CI: -0.35 to -0.08, P<0.01) and LDL-cholesterol by -0.20 mmol/l (95% CI: -0.33 to -0.06, P<0.005). The outcome was essentially the same if all studies were included. CONCLUSIONS: The present meta-analysis of controlled short-term intervention studies shows that the fermented yoghurt product produced a 4% decrease in total cholesterol and a 5% decrease in LDL-cholesterol when the open-label study is excluded. To demonstrate sustained effects on blood lipids, long-term studies are required. SPONSORSHIP: MD Foods A/S, Denmark.

Adult↗

PubMed1998

Diversity of VanA glycopeptide resistance elements in enterococci from humans and nonhuman sources.

Elements mediating VanA glycopeptide resistance in 106 diverse enterococci from humans and nonhuman sources were compared with the prototype VanA transposon, Tn1546, in Enterococcus faecium BM4147. The isolates included 64 from individual patients at 15 hospitals in the United Kingdom (isolated between 1987 and 1996) and 42 from nonhuman sources in the United Kingdom (27 from raw meat, 7 from animal feces, and 8 from sewage). VanA elements were assigned to 24 groups (designated groups A to X) with primers that amplified 10 overlapping fragments of Tn1546. Ten groups of elements were found only in human enterococci, eight groups of elements were unique to nonhuman strains, and six groups of elements were common in enterococci from all sources. Elements indistinguishable from Tn1546 (group A) were observed more frequently in enterococci from nonhuman sources (34 versus 9%) but were identified in enterococci that caused outbreaks in hospital patients between 1987 and 1995. The most common group found in human enterococci (group H; 33%) was rarely observed in enterococci from other sources (5%). Group H elements differed from Tn1546 in three regions and included a novel insertion sequence, designated IS1542, between orf2 and vanR. The VanA elements of 14 other groups had a similar insertion at this position and/or distinct insertions at other positions. We conclude that VanA elements in enterococci are heterogeneous, although all show regions of homology with Tn1546. Furthermore, the elements most common among the human and nonhuman enterococci studied were different. This approach may be useful for monitoring the evolution of VanA resistance and may also be applicable in local "snapshot" epidemiological studies. However, as transposition events involving insertion sequences accounted for the differences observed between several groups, the stability of the elements must be assessed before their true epidemiological significance can be determined.

Animals↗

PubMed1994

[Further studies on the falsification of the steam resistance of bioindicators by superheating].

As a supplement to a preceding paper (Zbl. Hyg., 194 (1993). 369-279) the resistance of bioindicators has been investigated which differed only in the material of which the carriers are made (filter paper and glass fibre fleece, respectively). The conditions of the tests were such that further evidence could be expected for the fact that the characteristic values of bioindicators can be falsified by superheating of the carrier and its envelope. As a test organism Enterococcus faecium has been used. To avoid side effects, the bacteria have been dried on to the carriers from a suspension in water distillators. The exposition basket of the resistometer has been loaded with 3 rows of 15 indicators (disc shaped; diameter: 6 mm), arranged parallel. At a temperature of 68 degrees C the dependency of (relative) frequency of indicators having surviving test organisms capable of multiplying on exposure time to saturated steam has been determined. Free exposed indicators made of filter paper required considerably longer exposure periods than indicators made of glass fibre fleece to change from "(nearly) all the indicators have test organisms capable of multiplying" to "(nearly) all the indicators are free from test organisms capable of multiplying". The exposure time to free 50% of carriers made of filter paper from test organisms capable of multiplying (t50%) amounted to 44.1 minutes. The frequency of indicators free from test organisms was higher at the ends than in the middle of the exposed rows. When such indicators had been wetted before exposure, they showed a t50% value of 2.3 minutes, and the frequency of indicators free from surviving test organisms was distributed over the exposed indicators evenly. Free exposed indicators made of glass fibre fleece showed a t50% value of 4.4 minutes. The frequency of indicators free from test organisms was evenly distributed. Wetting of the indicators before exposure changed t50% value only slightly. When indicators made of glass fibre fleece had been exposed between two layers of filter paper they showed a very high t50% value (47.5 minutes), and the frequency of indicators free from test organisms was unevenly distributed. Indicators being sterile could be found mainly at the ends of the exposed rows. In an envelope of parchment paper bioindicators made of glass fibre fleece showed considerably higher characteristic values than when free exposed. Superheating of the carriers amounted to about 3 Kelvin.(ABSTRACT TRUNCATED AT 400 WORDS)

Enterococcus faecium↗

PubMed2005

Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli.

We assessed the sensitivities and specificities of three previously described PCR primers on enrichment broth cultures of feces for the accurate detection of fecal carriage of vancomycin-resistant enterococci (VRE). In addition, we investigated specimens that were vanB PCR positive but VRE culture negative for the presence of other vanB-containing pathogens. Feces from 59 patients (12 patients carrying vanB Enterococcus faecium strains and 47 patients negative for VRE carriage) were cultured for 36 h in aerobic brain heart infusion (BHI) broth, anaerobic BHI (AnO(2)BHI) broth, or aerobic Enterococcosel (EC) broth. DNA was extracted from the cultures and tested for the presence of vanB by using the PCR primers of Dutka-Malen et al. (S. Dutka-Malen, S. Evers, and P. Courvalin, J. Clin. Microbiol. 33:24-27, 1995), Bell et al. (J. M. Bell, J. C. Paton, and J. Turnidge, J. Clin. Microbiol. 36:2187-2190, 1998), and Stinear et al. (T. P. Stinear, D. C. Olden, P. D. R. Johnson, J. K. Davies, and M. L. Grayson, Lancet 357:855-856, 2001). The sensitivity (specificity) of PCR compared with the results of culture on BHI, AnO(2)BHI, and EC broths were 67% (96%), 50% (94%), and 17% (100%), respectively, with the primers of Dutka-Malen et al.; 92% (60%), 92% (45%), and 92% (83%), respectively, with the primers of Bell et al.; and 92% (49%), 92% (43%), and 100% (51%) respectively, with the primers of Stinear et al. The primers of both Bell et al. and Stinear et al. were significantly more sensitive than those of Dutka-Malen et al. in EC broth (P = 0.001 and P < 0.001, respectively). The poor specificities for all primer pairs were due in part to the isolation and identification of six anaerobic gram-positive bacilli, Clostridium hathewayi (n = 3), a Clostridium innocuum-like organism (n = 1), Clostridium bolteae (n = 1), and Ruminococcus lactaris-like (n = 1), from five fecal specimens that were vanB positive but VRE culture negative. All six organisms were demonstrated to contain a vanB gene identical to that of VRE. VanB-containing bowel anaerobes may result in false-positive interpretation of PCR-positive fecal enrichment cultures as VRE, regardless of the primers and protocols used.

Bacteria, Anaerobic↗

PubMed2002

Effect of inoculants on whole-crop barley silage fermentation and dry matter disappearance in situ.

Whole-crop barley harvested at the mid-dough stage was ensiled in 3-L laboratory silos either directly (at 30.7% DM) or after wilting (37.8% DM), and with or without application of Inoculant A, B, or D. Each inoculant contained multiple strains of Lactobacillus plantarum and Enterococcus faecium. Two silos per treatment were opened on d 1, 3, 7, 15, and 47 for silage analysis. Wilted silages had higher (P < 0.05) pH than unwilted silages, and Inoculant B (unwilted crop) and Inoculants A, B, and D (wilted crop) decreased (P < 0.05) silage pH compared to the controls. Reducing sugars concentration was 36% lower (P < 0.05), on average, in the wilted than in the unwilted silages. Silages prepared with Inoculants B or D had lower (P < 0.05) concentrations of reducing sugars than the control. Wilting of the barley crop further increased the difference between inoculated and control silages (wilting x inoculation P < 0.05). Neither wilting nor silage inoculants affected concentrations of nonprotein N, ammonia N, or free amino acid N in silage. Wilting did not affect the concentration of lactic acid bacteria (LAB) in the silages. Across DM levels, inoculant-treated silages had larger (P < 0.01) populations of LAB than did the uninoculated controls (7.1 x 10(9) vs 2.3 x 10(9) cfu/g silage DM). Wilting resulted in lesser (P < 0.05) silage lactic acid concentration than the directly ensiled crop. At both DM levels, lactic acid concentration was higher (P < 0.001) in inoculated silages than in controls. The in situ soluble and potentially degradable fractions of silage DM were not affected by wilting or inoculant treatment. The rate of degradation of the potentially degradable silage DM was 35% lower (P < 0.05) in wilted than in unwilted silage. As a result, the calculated effective degradability of silage DM was lower (P < 0.001) in wilted than in unwilted silage. Inoculants did not influence the rate of degradation or effective degradability of silage DM in the rumen. Whole-crop barley ensiled at approximately 30% DM (without wilting) contained higher concentrations of soluble sugars and lactic acid and had higher ruminal degradability of DM than wilted silage (38% DM). Although inoculants did not improve DM degradability of barley silage, lower terminal pH and increased concentrations of lactic acid may improve aerobic stability upon feed-out.

Animals↗

PubMed2004

Molecular mechanism of the enterococcal aminoglycoside 6'-N-acetyltransferase': role of GNAT-conserved residues in the chemistry of antibiotic inactivation.

The Gram-positive pathogen Enterococcus faecium is intrinsically resistant to aminoglycoside antibiotics due to the presence of a chromosomally encoded aminoglycoside 6'-N-acetyltransferase [AAC(6')-Ii]. This enzyme is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily and is therefore structurally homologous to proteins that catalyze acetyl transfer to diverse acyl-accepting substrates. This study reports the investigation of several potential catalytic residues that are present in the AAC(6')-Ii active site and also conserved in many GNAT enzymes. Site-directed mutagenesis of Glu72, His74, Leu76, and Tyr147 with characterization of the purified site mutants gave valuable information about the roles of these amino acids in acetyl transfer chemistry. More specifically, steady-state kinetic analysis of protein activity, solvent viscosity effects, pH studies, and antibiotic resistance profiles were all used to assess the roles of Glu72 and His74 as potential active site bases, Tyr147 as a general acid, and the importance of the amide NH group of Leu76 in transition-state stabilization. Taken together, our results indicate that Glu72 is not involved in general base catalysis, but is instead critical for the proper positioning and orientation of aminoglycoside substrates in the active site. Similarly, His74 is also not acting as the active site base, with pH studies revealing that this residue must be protonated for optimal AAC(6')-Ii activity. Mutation of Tyr147 was found not to affect the chemical step of catalysis, and our results were not consistent with this residue acting as a general acid. Last, the amide NH group of Leu76 is implicated in important interactions with acetyl-CoA and transition-state stabilization. In summary, the work described here provides important information regarding the molecular mechanism of AAC(6')-Ii catalysis that allows us to contrast our findings with those of other GNAT proteins and to demonstrate that these enzymes use a variety of chemical mechanisms to accelerate acyl transfer.

Acetyltransferases↗