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PubMed2003

Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration.

Covalently linked to vancomycin (Van), chemically stable and highly magnetic anisotropic FePt magnetic nanoparticles (3-4 nm) become water-soluble and capture vancomycin-resistant enterococci (VRE) and other Gram-positive bacteria at concentrations approximately 10(1) cfu/mL via polyvalent ligand-receptor interactions. When a pyramidal end of a magnet "focuses" the nanoparticles into approximately 1 mm(2) area, the bacteria can be observed by an optical microscope and further identified by electron micrograph (EM). Compared to the conventional use of magnetic particles (with the sizes of 1-5 microm) in biological separation or drug delivery, magnetic nanoparticles, combined with specific receptor-ligand interactions, promise a sensitive and rapid protocol to detect pathogens.

Alloys↗
PubMed2003

Multivalent antibiotics via metal complexes: potent divalent vancomycins against vancomycin-resistant enterococci.

Dimers of vancomycin (Van), linked by a rigid metal complex, [Pt(en)(H(2)O)(2)](2+), exhibit potent activities (MIC approximately 0.8 mug/mL, approximately 720 times more potent than that of Van itself) against vancomycin-resistant enterococci (VRE). The result suggests that combining metal complexation and receptor/ligand interaction offers a useful method to construct multivalent inhibitors.

Anti-Bacterial Agents↗
PubMed2000

Detection of vancomycin resistance genes combined with typing of Enterococci by means of multiplex PCR amplification and multiple primer DNA sequencing.

A multiplex PCR assay for the detection of vancomycin resistance (van) genes in enterococci was established. Primers targeting the 16S rRNA gene were included in the reaction mixture. Multiple-primer DNA sequencing of the PCR products provided species identification through partial nucleotide sequences of 16S rRNA genes, as well as confirmation of the correct identification of vanA, vanB, vanC-1, and vanC-2/3 genotypes. Thirty-nine enterococcal clinical isolates and type strains were examined for the presence of vancomycin resistance determinants. Twelve other isolates from a clinical reference collection (some of them having vanA, vanB, vanC-1, or vanC-2/3 genotypes) were used as controls. Hybridization and partial DNA sequence analysis of multiplex PCR products revealed that none of the clinical isolates had a vanA genotype and only one had a vanB genotype. vanC-1 was found in three clinical isolates, and vanC-2/3 in one. Results obtained with the reference and type strains were in agreement with earlier results.

Bacterial Typing Techniques↗
PubMed2003

Genetic characterization of glycopeptide-resistant enterococci of human and animal origin from mixed pig and poultry farms.

Glycopeptide resistant enterococci (GRE) isolated from animals and humans were characterised using both AFPL typing and genetic characterisation of the glycopeptide resistance transposon Tn1546. All isolates were collected in 1997 when the glycopeptide avoparcin was still being used as growth promoter. All investigated animal isolates were from mixed pig and poultry farms in the Netherlands and the human isolated from the farmers of these farms. A total of 24 isolates were investigated. AFLP and Tn1546 typing revealed that both pig and poultry related enterococcal and vanA transposon genotypes were found among the human isolates indicating spread of glycopeptide resistance from both pig and poultry to the farmers. These findings contradict previous finding that showed that GRE recovered from the general population were genotypically undistinguishable from GRE isolated from pigs but are in line with other studies that demonstrated spread of GRE from poultry to farmers in poultry farms.

Agriculture↗
PubMed1996

Nine episodes of CPD-associated peritonitis with vancomycin resistant enterococci.

Nine episodes of chronic peritoneal dialysis (CPD)-associated peritonitis with vancomycin resistant enterococci (VRE) were described between November 1993 and February 1996 in our dialysis unit. During the time period, 216 patients were treated for 227 episodes of peritonitis. Of the patients developing peritonitis with VRE the mean age +/- SD was 56.3 +/- 9.7 years. There were 5 females, 4 males, 5 Caucasians and 4 African-Americans. Diabetes mellitus, cardiovascular disease and gastrointestinal disease were present in 7, 8 and 7 of the 9 patients with VRE peritonitis, respectively. Patients were maintained on CPD therapy for an average of 29.9 +/- 19.2 patient months before developing VRE peritonitis. The prior rate of CPD associated peritonitis in the patients developing VRE peritonitis was significantly higher than the rate noted in the CPD patients not developing peritonitis with VRE (1 episode in 6.3 patient months vs. 1 episode in 12.5 patient months, P < 0.05). All 9 patients had used vancomycin in the six months prior to the development of VRE peritonitis and 78% had used a cephalosporin. The antimicrobial therapy used to eradicate peritonitis with VRE varied among the 9 patients with chloramphenicol used in 4 patients. The Tenckhoff catheter was removed in 6 of the 9 patients and was successfully reinserted in one patient. The catheter was not removed in 3 patients and 2 of these patients expired. Five of the 9 patients expired while being treated for VRE, 2 transferred to hemodialysis and 2 continued CPD therapy. VRE peritonitis is a major concern for patients maintained on CPD therapy. Future studies are needed with case controls to determine the significance of prior vancomycin and cephalosporin therapy, fecal VRE carriage and certain demographic data on the acquisition of VRE peritonitis. Furthermore, the optimal therapy and outcome may be better clarified through such a review.

Adult↗
PubMed2000

Antimicrobial susceptibilities of enterococci isolated from faeces of broiler and layer chickens.

Enterococci were isolated from faecal droppings of chickens in broiler and layer farms and the susceptibilities to nine therapeutic antimicrobial agents and six growth-promoting antibiotics were determined by the agar dilution method. Resistance to therapeutic antimicrobial agents such as ampicillin, clindamycin, erythromycin, streptomycin, tetracycline or tylosin was more frequent in enterococcal isolates from broiler farms than in those from layer farms. Resistance to ofloxacin was rare, occurring in only one (0.7%) of the Enterococcus faecium isolates from broiler farms. Resistance to growth-promoting antibiotics such as avilamycin, salinomycin and virginiamycin was common among isolates from broiler farms. Of the E. faecium isolates from broiler farms, 12.4% were resistant to avilamycin and 27.4% were resistant to virginiamycin. Resistance to salinomycin was detected in all enterococcal species, ranging from 12.4% of E. faecium isolates to 50% of E. hirae isolates.

Animal Husbandry↗
PubMed2000

A survey of the enterococci isolated from an artisanal Italian goat's cheese (semicotto caprino).

Enterococci were isolated from semicotto caprino cheese, a traditional cheese produced in Southern Italy: they were a significant part of the microbial population of this cheese, confirming the importance of the presence of these micro-organisms during cheese-making and ripening. They were also identified and studied for their phenotypic and genotypic characteristics: Enterococcus faecalis and Ent. faecium were the most frequently isolated species, followed by Ent. durans, Ent. hirae and Ent. gallinarum. None of the isolates showed lipolytic activity, whereas they were characterized by a relevant proteolytic activity as well as an antagonistic activity towards Listeria innocua. One strain of Ent. gallinarum showed a low-level resistance to vancomycin, while six out of the 79 Ent. faecalis strains possessed beta-haemolysis reaction. The highest acidifying potential in skim milk was obtained by Ent. faecalis isolates. Thirty enterococcal strains representative of the different species at different ripening times were analysed by means of RAPD-PCR, and revealed species-specific profiles for all the considered species.

Animals↗
PubMed2000

Continuing high prevalence of VanA-type vancomycin-resistant enterococci on Norwegian poultry farms three years after avoparcin was banned.

Avoparcin was used as a feed additive in Norwegian broiler and turkey production from 1986 until 1995. It was banned due to the selection of VanA-type vancomycin-resistant enterococci (VRE) in animal husbandry and to reduce the potential for human exposure to VRE. The aim of the present study was to investigate the prevalence of VRE carriage in Norwegian poultry farmers and their poultry three years after avoparcin was banned. Corresponding faecal samples from poultry and humans on farms where avoparcin had previously been used (exposed farms, n = 73) and farms where avoparcin had never been used (unexposed farms, n = 74) were analysed for the presence of VRE. For each farm, one sample from the poultry house and one sample from the farmer were obtained. VRE were isolated from 72 of 73 (99%) and eight of 74 (11%) poultry samples from exposed and unexposed farms, respectively. VRE were isolated from 13 of 73 (18%) and one of 74 (1%) farmer samples from exposed and unexposed farms, respectively. All VRE isolates were highly resistant to vancomycin and possessed the vanA gene, as shown by PCR. The high prevalence of VRE is in accordance with previous Norwegian studies, and shows a remarkable stability of the VanA resistance determinant in an apparently non-selective environment.

Animal Husbandry↗
PubMed2002

Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese.

AIMS: To assess the intraspecific genetic diversity of lactococci and enterococci isolated from 24-h, 1- and 2-month-old home-made Pecorino Sardo ewes' milk cheese. METHODS AND RESULTS: Two molecular techniques, plasmid profiling and pulsed-field gel electrophoresis, were used in order to type the isolates at strain level. The present study revealed that the lactococcal and enterococcal microbial populations of home-made Pecorino Sardo cheese were complex, not only 24 h after manufacture, but also after 1 and 2 months of ripening. The genetic diversity at subspecies level ranged from 58 to 80% during the three periods examined. The study also showed that the strains that dominated in the first stage of ripening were not necessarily predominant in the later periods. A high number of strains isolated at 24 h were still present in the mature cheese, but many of the genotypes were only found in the cheese after 1 or 2 months. CONCLUSIONS: The results showed a high intraspecific genetic diversity in the natural microbial population colonizing home-made Pecorino Sardo cheese. Two molecular techniques are necessary for a thorough and precise typing at strain level in order to better distinguish between closely related isolates and between isolates that probably belong to the same clonal lineage. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic complexity observed in the present study is of particular relevance in the preservation of the natural microflora of traditional Protected Designation of Origin raw milk cheeses, as well as in the selection of new starter strains for the dairy industry.

Animals↗
PubMed2003

Isolation and biochemical characterisation of enterocins produced by enterococci from different sources.

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.

Animals↗
PubMed2002

Nurses' experience with vancomycin-resistant enterococci (VRE).

The emergence and spread of resistant organisms, in particular vancomycin-resistant enterococci (VRE), is an issue facing all staff in acute hospitals. This study explored how nurses coped with the responsibility of halting further spread of this organism during an outbreak. VRE-positive patients were cohorted with nurses who cared for them in an endeavour to contain the spread of VRE. The majority of nurses found the situation extremely stressful because of the need to act as 'gatekeepers' responsible for educating and monitoring the practices of staff and visitors. The nurses reported that they felt they were inadequately supported, were blamed for the outbreak, and that they had an increased workload as they took on duties of other staff. The results reinforce the need for a multidisciplinary team approach to education and control of VRE, more support for nursing staff cohorted with VRE-positive patients, and stringent adherence to infection control measures by all hospital staff.

Communicable Disease Control↗
PubMed1997

Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB-type enterococci.

The vanB gene cluster mediates glycopeptide resistance by production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac) instead of D-Ala-D-Ala found in susceptible enterococci. Synthesis of D-Ala-D-Lac and hydrolysis of D-Ala-D-Ala is controlled by the VanR(B)S(B) two-component regulatory system that activates transcription of the resistance genes in response to vancomycin but not to teicoplanin. Two substitutions (A3C-->G or D168-->Y) in the VanS(B) sensor kinase resulted in induction by teicoplanin, indicating that the N-terminal domain of the protein was involved in glycopeptide sensing. A substitution (T237-->K) located in the vicinity of the putative autophosphorylation site of VanS(B) (H233) was associated with a constitutive phenotype and affected a conserved residue known to be critical for the phosphatase activity of related kinases. A mutant producing an impaired host D-Ala:D-Ala ligase required vancomycin for growth, since D-Ala-D-Lac was only produced under inducing conditions. The ddl and vanS(B) mutations, alone or in combination, resulted in various resistance phenotypes that were determined by the amount of D-Ala-D-Ala and D-Ala-D-Lac incorporated into peptidoglycan precursors under different inducing conditions.

Anti-Bacterial Agents↗
PubMed1998

Requirement of the VanY and VanX D,D-peptidases for glycopeptide resistance in enterococci.

Transposon Tn 1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D-peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D-alanyl-D-lactate (D-Ala-D-Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N-terminal membrane anchor of the protein. The enzyme was a Zn2+-dependent D,D-carboxypeptidase that cleaved the C-terminal residue of peptidoglycan precursors ending in R-D-Ala-D-Ala or R-D-Ala-D-Lac but not the dipeptide D-Ala-D-Ala. The specificity constants kcat/Km were 17- to 67-fold higher for substrates ending in the R-D-Ala-D-Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP-MurNAc-tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high-level glycopeptide resistance in a medium supplemented with D-Ala. The enzyme could not replace the VanX D,D-dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D-Ala:D-Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D-carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D-Ala-containing medium. Thus, VanX and VanY had non-overlapping functions involving the hydrolysis of D-Ala-D-Ala and the removal of D-Ala from membrane-bound lipid intermediates respectively.

Anti-Bacterial Agents↗
PubMed1998

Occurrence of bacteriocin production among environmental enterococci.

The occurrence of enteroccoci in cattle dung water from the basins of 25 cattle farms of 15 northeastern Slovakia districts was screened as well as bacteriocin production among selective enterococcal isolates. The average total count of enterococci detected reached 5.0 x 10(3) cfu ml-1. Enterococcus faecium was the predominant species (25%) followed by Ent. casseliflavus (19.2%), Ent. faecalis (9.6%), Ent. avium and Ent. durans (1.9%). The antagonistic activity of isolates showed a mainly antilisterial effect. Enterococcus faecalis V24 strain produced a heat stable, largely hydrophobic antimicrobial substance with best production in the range pH 4 to 7 and with a strong inhibitory effect even against Gram-negative bacteria.

Animals↗
PubMed2001

Vancomycin-resistant enterococci colonization in patients at seven hemodialysis centers.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are increasing in prevalence at many institutions, and are often reported in dialysis patients. We studied the prevalence of and risk factors for VRE at seven outpatient hemodialysis centers (three in Baltimore, MD, USA, and four in Richmond, VA, USA). METHODS: Rectal or stool cultures were performed on consenting hemodialysis patients during December 1997 to April 1998. Consenting patients were recultured during May to July 1998 (median 120 days later). Clinical and laboratory data and functional status (1 to 10 scale: 1, normal function; 9, home attendant, not totally disabled; 10, disabled, living at home) were recorded. RESULTS: Of 478 cultures performed, 20 (4.2%) were positive for VRE. Among the seven centers, the prevalence of VRE-positive cultures varied from 1.0 to 7.9%. Independently significant risk factors for a VRE-positive culture were a functional score of 9 to 10 (odds ratio 6.9, P < 0.001), antimicrobial receipt within 90 days before culture (odds ratio 6.1, P < 0.001), and a history of injection drug use (odds ratio 5.4, P = 0.004). CONCLUSIONS: VRE-colonized patients were present at all seven participating centers, suggesting that careful infection-control precautions should be used at all centers to limit transmission. In agreement with previous studies, VRE colonization was more frequent in patients who had received antimicrobial agents recently, underscoring the importance of judicious antimicrobial use in limiting selection for this potential pathogen.

Cross Infection↗
PubMed2002

Patient colonization and environmental contamination by vancomycin-resistant enterococci in a rehabilitation facility.

OBJECTIVES: To determine the frequency of environmental contamination in patient and common-use rooms and patient colonization by vancomycin-resistant enterococci (VRE). DESIGN: Cross-sectional study. SETTING: A 146-bed rehabilitation facility. PARTICIPANTS: Rectal cultures were collected from 74 (80%) of 93 patients. Environmental cultures were obtained from surfaces in 15 patient rooms (5 floors) and common-use areas on 8 floors. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Gastrointestinal colonization of patients and environmental contamination of surfaces by VRE. RESULTS: VRE was detected from 13 (18%) of 74 patients and 32 (10%) of 319 surfaces. The frequency of positive environmental cultures varied by location; cultures were more likely to be positive in patient rooms (15%), followed by common areas on patient floors (9%) and common areas separate from patient floors (1.3%). Surfaces were more likely to be positive in rooms with a VRE-colonized patient (24%), compared with rooms in which patient colonization status was unknown (13%, P=.13) or the patient was not colonized (0%, P=.002). Surfaces were more likely to be contaminated in a room that housed an incontinent compared with continent patients (22% vs 7%, P=.01). CONCLUSIONS: Although environmental contamination by VRE was common in patient rooms, contamination of common-use areas separate from patient floors was infrequent. Despite use of common-use areas by colonized patients, isolation practices at this facility appear to have minimized environmental surface contamination in these areas.

Cross-Sectional Studies↗
PubMed1998

Detection of vancomycin-resistant enterococci colonization in a children's hospital.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens in many hospitals. The true prevalence of VRE in pediatric hospitals is not known. METHODS: A surveillance study was performed at a pediatric tertiary care medical center by using vancomycin-containing screening media. RESULTS: Six children (of 112 screened) were found to be colonized with VRE. Colonized patients had a history of receiving broad-spectrum antimicrobial agents. CONCLUSION: In the absence of VRE infections, surveillance studies can help determine the extent of VRE colonization and support infection control measures.

Anti-Bacterial Agents↗