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Development of a meropenem susceptibility disc: drug content, preliminary interpretive and quality control criteria and potency bioassay.

Laboratory studies were conducted to develop an appropriate susceptibility disc for meropenem. Laboratory-prepared paper discs containing 5, 10, and 20 micrograms of meropenem were investigated. Agar dilution MICs and agar disc diffusion tests were performed with 367 rapidly growing clinical isolates by methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Agar disc diffusion tests with 10 micrograms meropenem discs acceptably defined organisms as susceptible or resistant. The preliminary interpretive zone size criteria for 10 micrograms meropenem discs were: susceptible, greater than or equal to 14 mm, and resistant, less than or equal to 10 mm, by application of the NCCLS imipenem MIC interpretive susceptible and resistant breakpoints to the meropenem data. A total of 270 agar disc diffusion assays were performed with each of four ATCC reference strains, using prototype discs (Becton Dickinson (BBL] containing 10 micrograms meropenem. The preliminary quality control limits for agar disc diffusion tests with these discs are: Escherichia coli ATCC 25922, 28-32 mm; Pseudomonas aeruginosa ATCC 27853, 26-31 mm; Staphylococcus aureus ATCC 25923, 33-39 mm; and Enterococcus faecalis ATCC 29212, 19-23 mm. Further studies are necessary to re-evaluate both the preliminary interpretive and quality control criteria before definitive limits can be established. A reproducible, large plate bioassay with Enterococcus faecalis ATCC 29212 was developed to monitor meropenem disc potency.

Bacteria↗

Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus.

Enterococcus faecium T8, isolated from vaginal secretions of children with human immunodeficiency virus, produces a class IIa sec-dependent bacteriocin that is structurally different from three other class IIa sec-dependent bacteriocins, i.e., enterocin P and an enterocin P-like bacteriocin, produced by Enterococcus faecium, and bacteriocin 31, produced by Enterococcus faecalis, and from a class III bacteriocin produced by E. faecalis. The genes encoding the bacteriocin, immunity protein, mobilization protein, and relaxase nuclease are located on a 7-kb plasmid. Bacteriocin T8 has a molecular mass of 5.1 kDa based on its DNA sequence, similar to the 5.0 kDa recorded for bacteriocin 31 but larger than the 4.6 kDa reported for enterocin P. At the amino acid level, bacteriocin T8 is 69% homologous to bacteriocin 31 and 47% homologous to enterocin P. Bacteriocin T8 is active against E. faecalis isolated from patients diagnosed with vaginosis, against Lactobacillus sakei, and against a Propionibacterium sp. The peptide is heat stable (60 min at 100 degrees C) and remains active in phosphate buffer from pH 4.0 to 10.0. The mode of activity is bactericidal, as determined with E. faecalis.

Amino Acid Sequence↗

Frequency of isolation and antimicrobial resistance of gram-negative and gram-positive bacteria from patients in intensive care units of 25 European university hospitals participating in the European arm of the SENTRY Antimicrobial Surveillance Program 1997-1998.

A total of 3,981 isolates from patients treated at intensive care units were collected in 25 European university hospitals during 1997 and 1998 as part of the SENTRY Antimicrobial Surveillance Program. Overall, the most important species isolated were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, coagulase-negative staphylococci (CNS), Enterobacter spp., Haemophilus influenzae, Streptococcus pneumoniae, and Enterococcus faecalis. Thirty-nine percent of all Staphylococcus aureus isolates were resistant to oxacillin. All Staphylococcus aureus isolates were fully susceptible to linezolid and vancomycin. Moreover, all CNS isolates were susceptible to vancomycin and minocycline. All Enterococcus faecalis isolates were susceptible to vancomycin, and 99% of these isolates were also susceptible to ampicillin. The antimicrobial agents most effective against Pseudomonas aeruginosa isolates were amikacin, piperacillin/tazobactam, meropenem, and cefepime, with 87, 85, 84, and 83% of isolates being susceptible, respectively. Escherichia coli isolates were fully susceptible to carbapenems, and at least 99% of these isolates were susceptible to ceftriaxone, cefepime, and amikacin. The Enterobacter spp. were also highly susceptible to carbapenems, amikacin, and cefepime, with 99, 97, and 96% of isolates being susceptible, respectively. Haemophilus influenzae was susceptible to most of the antibiotics tested. Only 68% of the pneumococcal isolates were fully susceptible to penicillin, yet 100% were susceptible to a number of fluoroquinolones and vancomycin. There are still sufficient treatment options for patients infected with the most important bacterial species involved in infections in intensive care units. However, the situation for patients with Pseudomonas aeruginosa infections is critical.

Anti-Bacterial Agents↗

In vitro susceptibility of vancomycin-resistant enterococci (VRE) to fosfomycin.

We evaluated the in vitro activity of fosfomycin against 75 clinical isolates of vancomycin-resistant enterococci (VRE). Using the NCCLS breakpoint for susceptibility of urinary tract isolates to fosfomycin (MIC > or = 256), 51 out of 52 Enterococcus faecium and all Enterococcus faecalis isolates tested were susceptible or intermediate to fosfomycin.

Anti-Bacterial Agents↗

Synthesis and antimicrobial activity of substituted benzimidazole, benzothiazole and imidazole derivatives.

New benzimidazole, benzothiazole and imidazole derivatives were synthesized by reacting electron-rich olefins (5, 23 and 29) with appropriate reagents. The compounds synthesized were identified by 1H, 13C-NMR, FT-IR and mass spectroscopic techniques and micro analysis. All new and related compounds were evaluated for their in vitro antimicrobial activity against different bacteria. The compounds 17, 18, 19, 20, 21, 22 and 24 were found very effective to inhibit the growth of Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 29213) at minimum inhibitory concentrations (MICs) of 25, 25, 12.5, 50, 25, 50 and 50 micrograms/ml, respectively. The compounds 4, 10a, 10c, 16, 25, 26 and 31 were significantly effective against Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 29213) with MIC values of 100-200 micrograms/ml. None of the compounds proved to be effective against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) in the concentrations studied.

Anti-Bacterial Agents↗

Source of enterococci in a farmhouse raw-milk cheese.

Enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. Their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. The source of enterococci in raw-milk cheese is unknown. In the present study, an epidemiological approach with pulsed-field gel electrophoresis (PFGE) was used to type 646 Enterococcus strains which were isolated from a Cheddar-type cheese, the milk it was made from, the feces of cows and humans associated with the cheese-making unit, and the environment, including the milking equipment, the water used on the farm, and the cows' teats. Nine different PFGE patterns, three of Enterococcus casseliflavus, five of Enterococcus faecalis, and one of Enterococcus durans, were found. The same three clones, one of E. faecalis and two of E. casseliflavus, dominated almost all of the milk, cheese, and human fecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination, suggesting that a niche where enterococci could grow was present and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human feces are due to consumption of the cheese. Cow feces were not considered the source of enterococci in the cheese, as Enterococcus faecium and Streptococcus bovis, which largely dominated the cows' intestinal tracts, were not found in either the milk or the cheese.

Animals↗

Comparative activity of linezolid against staphylococci and enterococci isolated in Italy.

The activity of linezolid, a new oxazolidinone, was tested against 862 Gram-positive cocci isolated in Italy and compared with the activities of 12 antibiotics. Overall, MIC90s for linezolid (2-4 mg/L) indicated an in vitro activity comparable to that of vancomycin in methicillin-resistant Staphylococcus aureus (4 mg/L), S. epidermidis (2 mg/L) and methicillin-susceptible strains. Enterococcus faecalis strains were susceptible to linezolid (MIC90 2-4 mg/L), glycopeptides and beta-lactams. In E. faecium, only glycopeptides (MIC90 2 mg/L) and linezolid (MIC90 2 mg/L) were active. Linezolid was the only drug active against two strains of Enterococcus showing a VanA phenotype. Owing to its antibacterial profile, linezolid represents a promising drug for the treatment of Gram-positive infections.

Acetamides↗

Penetration into tissues of various drugs active against gram-positive bacteria.

Gram-positive bacteria are the most important pathogens causing hospital- and community-acquired infections. We therefore reviewed the penetration of various antibiotics active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus into tissues, where staphylococcal infections are common. Rifampicin reaches heart valve concentrations of 65% of the simultaneous serum levels. At 8 h after administration blood and tissues concentrations of rifampicin exceeded the MIC90 values for S. aureus as well as for S. epidermidis. After a 2-g intravenous bolus injection of flucloxacillin heart valve concentrations exceeded MIC values for staphylococci for more than 8 h whereas subcutaneous and muscle concentrations declined within the same time to undetectable levels. The MIC90 values of vancomycin for S. epidermidis and Enterococcus faecalis are 2.0 and 4.0 mg/l respectively and for S. aureus 1.0 mg/l. This concentration is reached in subcutaneous tissue, heart valves and muscle for at least 4-6 h after administration of 15 mg/kg, however the corresponding value for Enterococcus faecalis in heart valve is maintained only for 3-4 h. After two and three dose regimens of teicoplanin serum and bone levels were significantly higher than fat levels, exceeding the MIC90 values for S. aureus, S. epidermidis and E. faecalis. The ratio of tissue concentration of teicoplanin to serum concentrations was 11% for fat and 65% for bone.

Anti-Bacterial Agents↗

Peritoneal infection in acute intermittent peritoneal dialysis.

A prospective study was done to evaluate the incidence and microbiological trend of peritoneal infection in patients undergoing acute intermittent peritoneal dialysis (PD). Complete sterile procedure was ensured and at the completion of the procedure PD fluid was sent for bacteriological culture, sensitivity, and total and differential cell count. During the period September 2000 to February 2001 a total of 100 patients were evaluated. Male female ratio was 72:28. Mean age was 43.17 +/- 17.2 years. In 26 patients cyclers were used. Bacterial culture was positive in total of 30 cases (30%). Gram positive, Gram negative and mixed infection was found in 10%, 15%, and 5% respectively. Number of exchanges (31.61 +/- 7.7 vs. 31.3 +/- 6, p = 0.8) were similar and number of repositioning was significantly more in the infected group (23.3% vs. 11.4%, p < 0.01). Total cell count was significantly higher in infected group (274.3 +/- 502 vs. 31.25 +/- 79.34, p < 0.01). Among Gram +ve organisms Staphylococcus was found in 7, Enterococcus faecalis in 4 and Coryne bacterium sps. in 2 cases. Among Gram -ve organisms, E. coli was found in 4, Enterobacter in 3, Klebsiella 1, Pseudomonas 1, Acinetobacter arinatus 5, Acinetobacter baumani 3, and Citrobacter freundii 3. Mixed flora comprised of Enterococcus faecalis 3, Enterobacter 1, Staphlococcus 1, E. coli 3, Citrobacter 1, Acinobacter baumani 1. Although with the cyclers using collapsible bags, staphylococcus was not isolated, the total incidence of infection (11/26 cases) was not decreased with the use of cyclers. We conclude that in acute intermittent peritoneal dialysis the incidence of bacterial infection is 30% with preponderance of Gram -ve over Gram +ve organisms and organism of fecal origin being commoner than those of skin origin. Use of cycler-assisted over manual PD do not improve the incidence of infection. Repositioning of the stiff catheter significantly increases the incidence of infection.

Adult↗

Prevalence in Singapore of enterococci with high-level resistance to aminoglycosides and comparison of three methods for their detection.

The first study of the prevalence in Singapore of enterococci with high-level resistance to aminoglycosides has been made in the National University Hospital. From March to June 1992, 225 isolates were studied by means of three methods: (1) use of brain-heart infusion (BHI) agar incorporating the antibiotic; (2) microdilution of the antibiotic in broth; and (3) use of high content antibiotic discs. The BHI agar was the reference method. Of the total, 49 (22%) isolates were resistant to gentamicin, 86 (38%) to streptomycin, 82 (36%) to kanamycin and 93 (41%) to one or more of the agents. Results of the disc diffusion method correlated completely with the BHI agar method. The broth microdilution method showed a 100, 90 and 96% sensitivity in relation to gentamicin, streptomycin and kanamycin, respectively, and a specificity of 100% for all three aminoglycosides. Statistical significance (P < 0.01 to P < 0.001) was demonstrated in (1) a greater incidence of enterococci with high-level resistance to aminoglycosides among blood isolates; (2) gentamicin resistance predicting kanamycin high-level resistance; and (3) more resistance to gentamicin, streptomycin and kanamycin among Enterococcus faecium isolates compared to those of Enterococcus faecalis. Beta-lactamase activity was not detected.

Aminoglycosides↗

The vanB gene confers various levels of self-transferable resistance to vancomycin in enterococci.

Thirty-nine strains of Enterococcus faecium and Enterococcus faecalis resistant to vancomycin and susceptible to teicoplanin on disk susceptibility testing (phenotypic class B) were isolated in 15 hospitals in Europe and the United States. The MICs of vancomycin for these strains ranged from 4 to 1024 micrograms/mL. Part of the vancomycin resistance gene vanB from E. faecalis V583 hybridized with a single but variably sized HindIII-KpnI fragment of total DNA from all 39 strains. This indicates that a single class of resistance determinants accounts for the VanB phenotype. No hybridization was detected with DNA from intrinsically resistant Enterococcus gallinarum or Enterococcus casseliflavus. Hybridization with DNA from enterococcal strains susceptible to or with acquired resistance to vancomycin and teicoplanin was not observed. The genes conferring resistance to vancomycin were self-transferable to other Enterococcus strains in 14 of the 39 strains. It thus appears that vanB confers various levels of conjugative vancomycin resistance in enterococci.

Bacterial Proteins↗

Proficiency of determination of vancomycin susceptibility in enterococci by clinical laboratories in Taiwan.

Eighty clinical microbiology laboratories in Taiwan were evaluated for proficiency in the determination of vancomycin susceptibility of enterococci. Each laboratory was given 1 vancomycin-sensitive isolate and 3 vancomycin-resistant enterococci (VRE) isolates to determine the levels of vancomycin resistance. Among a total of 240 tests performed, 153 (63.8%) correctly determined the levels of vancomycin resistance of the survey isolates. Seventy eight (98%) of the 80 laboratories accurately identified the high level of vancomycin resistant isolates [Enterococcus faecalis; minimum inhibitory concentration (MIC) >256 microg/mL]. Seventy two laboratories (90%) correctly determined the level of vancomycin resistance of another VRE with a vancomycin MIC of 64 microg/mL. Only 3 of the 80 laboratories correctly determined the intermediate-level vancomycin resistant isolates (Enterococcus casseliflavus; MIC = 8 microg/mL). Eight laboratories reported the vancomycin-susceptible isolate as being vancomycin-resistant or of intermediate susceptibility. This survey demonstrated that clinical microbiology laboratories in Taiwan are proficient in detecting high-level but not low-level VRE, suggesting a need to improve their proficiency in VRE detection.

Enterococcus↗

Aetiology of catheter-associated bacteriuria in Lagos University Teaching Hospital.

A prospective study to determine the causative agents of catheter-related bacteriuria was carried out on 99 patients with median age of 55 years, on admission in the Oncology, Paediatric Surgery and Obstetric and Gynaecology wards of the Lagos University Teaching Hospital. There was significant bacteriuria in seventy-one (72%) of the 99 urine samples. All catheter urine samples were contaminated by day 5 Gram-negative bacilli were predominant (89.3%) of which Pseudomonas aeruginosa (26.2%) was the most common, while Escherichia coli and Enterobacter aerogenes accounted for 15.5% each, closely followed by Proteus mirabilis (14.3%) and Klebsiella pneumoniae (9.5%). The Gram positive isolates Enterococcus faecalis (8.3%) and Staphylococcus saprophyticus (2.4%) made up only 10.7% of isolates. Most of the isolates from catheterised patients were resistant to the common antibiotics such as ampicillin, cotrimoxazole, gentamicin and ceftriaxone with high susceptibility to ofloxacin and ceftazidime though the isolates of Enterococcus faecalis was also resistant to ofloxacin. However, isolates of S. saprophyticus were still susceptible to the penicillins and tetracycline.

Adolescent↗

In vitro activity of LY333328, a new glycopeptide, against extracellular and intracellular vancomycin-resistant enterococci.

The objectives of the study were to observe the activity of LY333328, a new semisynthetic glycopeptide, compared to that of vancomycin against six strains of Enterococcus faecium and Enterococcus faecalis, including four vancomycin-resistant strains. Bacteria ingested by polymorphonuclear leukocytes (PMN) as well as extracellular bacteria were studied using a colony count method. The activity against intracellular bacteria was tested with the drugs present in the extracellular medium, as well as after preincubating the PMN and removal of the drugs. LY333328 is active against the tested enterococci, regardless of their susceptibility to vancomycin, with MICs of 1-2 mg/l. It is bacteriostatic against extracellular enterococci at concentrations of 2 microg/ml and above regardless of their resistance to vancomycin. After 4 h incubation at 10 MIC, vancomycin-resistant strains of E. faecium and E. faecalis located intracellularly were reduced by 55% and 90%, respectively. Even after preincubation and removal of the drug, LY333328 had an effect at 10 MIC with a 20-30% reduction in the inoculum. The results suggest that in contrast to vancomycin, LY333328 is active against intracellular vancomycin-resistant enterococci, particularly E. faecalis, even after removal of the extracellular drug.

Anti-Bacterial Agents↗

High level aminoglycoside resistance in enterococci isolated from hospitalized patients.

Drug resistance in enterococci isolated from hospitalized patients is reported. Out of 421 strains tested, 45 (10.7%) showed high level aminoglycoside resistance (HLAR). At the species level, 8.2 per cent of Enterococcus faecalis and 33.3 per cent of E. faecium were HLAR. While all the strains of E. faecalis were sensitive to vancomycin, one strain of E. faecium was vancomycin resistant.

Anti-Bacterial Agents↗

Multicentre evaluation of the in vitro activity of linezolid in the Western Pacific.

Multiresistance to antimicrobial agents is common in staphylococci and pneumococci isolates in the Western Pacific region. The activity of linezolid, a new oxazolidinone, was evaluated against a spectrum of Gram-positive species collected in the region. Eighteen laboratories from six countries in the Western Pacific examined the linezolid susceptibility of 2143 clinical isolates of Staphylococcus aureus, coagulase-negative staphylococci (CoNS) and Enterococcus spp. using broth microdilution or disc diffusion methodology. For Streptococcus pneumoniae (n = 351) and other streptococci (n = 83), Etest (AB Biodisk, Solna, Sweden) strips were used. Results were compared with other common and important antimicrobials. Linezolid-resistant strains were not detected among streptococci or staphylococci, including a significant proportion of S. aureus strains that were multiresistant. Almost all enterococci, including 14 vancomycin-resistant Enterococcus faecium, were linezolid susceptible. A small proportion of enterococci (0.8%) were intermediate to linezolid, and one strain of Enterococcus faecalis had a zone diameter of 20 mm (resistant). The linezolid MIC ranges (MIC(90)) of those strains tested by broth microdilution or Etest were: 1-4 mg/L (2 mg/L) for S. aureus, 0.5-4 mg/L (2 mg/L) for CoNS, 0.5-4 mg/L (2 mg/L) for Enterococcus spp., 0.12-2 mg/L (1 mg/L) for S. pneumoniae and 0.25-2 mg/L (1 mg/L) for Streptococcus spp. There was no difference in linezolid susceptibility between countries or between multiresistant and susceptible strains of each species monitored.

Acetamides↗

Binding of extracellular matrix proteins by enterococci.

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of 125I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of 125I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (Mr 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface.

Ammonium Sulfate↗

Postoperative enterococcal infection after treatment of complicated intra-abdominal sepsis.

BACKGROUND: The prevalence of enterococcal isolation and factors associated with postoperative enterococcal infection remain ill defined. METHODS: A prospective longitudinal observational study was conducted of consecutive patients with a first episode of intra-abdominal infection and a positive microbiological culture who did or did not develop a postoperative septic complication involving enterococci. The prevalence of initial enterococcal isolation was determined for each focus of infection. Postoperative enterococcal infections were related to whether appropriate (piperacillin--tazobactam), suboptimal (carbapenems) or inappropriate (cefotaxime plus metronidazole) antienterococcal therapy had been administered empirically. RESULTS: Enterococci were isolated in 42 (21 per cent) of the 200 patients investigated. The isolation rates were 11 per cent for community-acquired peritonitis, 50 per cent for postoperative peritonitis and 23 per cent for intra-abdominal abscesses of both origins. No enterococci were isolated from 49 patients with perforated appendicitis. Independent factors for postoperative enterococcal infection were type of intra-abdominal infection (P = 0.006), Acute Physiology And Chronic Health Evaluation (APACHE) II score greater than 12 (P = 0.04) and inappropriate empirical antibiotic cover (P = 0.05). Postoperative enterococcal infections were associated with a high mortality rate (21 versus 4 per cent; P < 0.0007). CONCLUSION: Enterococci are frequently isolated from intra-abdominal infections of non-appendiceal origin and are often involved in postoperative infectious complications, particularly peritonitis. Empirical antibiotic therapy covering Enterococcus faecalis should be contemplated in some circumstances.

Abdominal Abscess↗