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PubMed2005

Engulfed pathogen-induced apoptosis in haemocytes of giant freshwater prawn, Macrobrachium rosenbergii.

Haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, were investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated haemocytes of M. rosenbergii were cultured at a ratio of 1:50 haemocytes to pathogen with the yeast Debaryomyces hansenii, the bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25 degrees C for 2 h, followed by washing to remove free particles. At least 200 haemocytes were counted to determine the phagocytosis rate, and the results showed that haemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing haemocytes displayed morphological changes characteristic of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation and fragmentation of nuclei. This pathogen-induced apoptosis was further confirmed by DNA laddering and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end-labelling) assays. Neither haemocytes treated with latex beads nor uninfected haemocytes (control group) showed signs of apoptosis after 48 h in culture.

Aeromonas hydrophila↗

PubMed2006

Adhesion of vancomycin-resistant enterococcus to human intestinal mucus.

The intestinal mucus layer provides a potential niche for colonization by vancomycin-resistant Enterococcus faecium (VREF). We therefore examined the ability of six VREF strains to adhere to human intestinal mucus and determined binding kinetics. Four of six (67%) VREF strains demonstrated significant adhesion to immobilized intestinal mucus compared with a Salmonella typhimurium-negative control strain, but the level of adherence was low compared with Lactobacillus rhamnosus GG. Binding kinetics studies demonstrated that the maximum number of these four VREF strains that could adhere to a unit surface area of immobilized mucus was similar to or higher than the maximum number of L. rhamnosus GG that could adhere; however, L. rhamnosus GG demonstrated 20- to 130-times higher affinity than the VREF strains. These results demonstrate that VREF strains may adhere to human intestinal mucus and suggest that L. rhamnosus GG might be able to displace VREF strains.

Bacterial Adhesion↗

PubMed1999

Clinical and molecular biological analysis of a nosocomial outbreak of vancomycin-resistant enterococci in a neonatal intensive care unit.

Vancomycin-resistant enterococci (VRE) have emerged as important nosocomial pathogens since 1988. We report here an outbreak of VRE between April 1997 and May 1997 in our neonatal intensive care unit (NICU). All isolates from four patients were identified as Enterococcus faecium positive and were resistant to vancomycin and teicoplanin. All of the patients with VRE were isolated for at least 5 d after admission to the unit and the positive cultures lasted between 13 and 31 d. There were no cases of sepsis or mortality in the patients with VRE. Two cases had previously received vancomycin therapy. All isolates were shown to have the vanA gene and had the same band pattern on repetitive PCR. After the four episodes, all equipment used to care for the patients were decontaminated and the staff engaged in therapy used disposable gloves and gowns. There were no more episodes. However, the NICU is no longer a safety area with regards to vancomycin-resistant enterococcal infection.

Anti-Bacterial Agents↗

PubMed2000

Molecular characterization of the vanD gene cluster and a novel insertion element in a vancomycin-resistant enterococcus isolated in Canada.

A single vanD-containing Enterococcus faecium strain (N97-330) was isolated in Canada. The vanD-containing region was cloned and sequenced. Although the proteins have more than 96% identity to a previously described vanD region in BM4339, the vanS(D) gene contains a frameshift mutation that leads to a predicted truncated protein. Furthermore, sequence analysis of the ddl gene revealed the presence of an IS982-like element (ISEfm1) which interrupted the D-Ala-D-Ala ligase. This suggested the constitutive expression of the vanD operon, which was confirmed. Pulsed-field gel electrophoresis fingerprinting demonstrated that BM4339 was not related to N97-330 (>15 band differences). Both strains contained multiple copies of the IS982-like element.

Amino Acid Sequence↗

PubMed2006

Predicting the thermal inactivation of bacteria in a solid matrix: simulation studies on the relative effects of microbial thermal resistance parameters and process conditions.

A combined mathematical model for predicting heat penetration and microbial inactivation in a solid body heated by conduction was tested experimentally by inoculating agar cylinders with Salmonella typhimurium or Enterococcus faecium and heating in a water bath. Regions of growth where bacteria had survived after heating were measured by image analysis and compared with model predictions. Visualisation of the regions of growth was improved by incorporating chromogenic metabolic indicators into the agar. Preliminary tests established that the model performed satisfactorily with both test organisms and with cylinders of different diameter. The model was then used in simulation studies in which the parameters D, z, inoculum size, cylinder diameter and heating temperature were systematically varied. These simulations showed that the biological variables D, z and inoculum size had a relatively small effect on the time needed to eliminate bacteria at the cylinder axis in comparison with the physical variables heating temperature and cylinder diameter, which had a much greater relative effect.

Agar↗

PubMed1999

Cell-wall determinants of the bactericidal action of group IIA phospholipase A2 against Gram-positive bacteria.

We have shown previously that a group IIA phospholipase A2 (PLA2) is responsible for the potent bactericidal activity of inflammatory fluids against many Gram-positive bacteria. To exert its antibacterial activity, this PLA2 must first bind and traverse the bacterial cell wall to produce the extensive degradation of membrane phospholipids (PL) required for bacterial killing. In this study, we have examined the properties of the cell-wall that may determine the potency of group IIA PLA2 action. Inhibition of bacterial growth by nutrient deprivation or a bacteriostatic antibiotic reversibly increased bacterial resistance to PLA2-triggered PL degradation and killing. Conversely, pretreatment of Staphylococcus aureus or Enterococcus faecium with subinhibitory doses of beta-lactam antibiotics increased the rate and extent of PL degradation and/or bacterial killing after addition of PLA2. Isogenic wild-type (lyt+) and autolysis-deficient (lyt-) strains of S. aureus were equally sensitive to the phospholipolytic action of PLA2, but killing and lysis was much greater in the lyt+ strain. Thus, changes in cell-wall cross-linking and/or autolytic activity can modulate PLA2 action either by affecting enzyme access to membrane PL or by the coupling of massive PL degradation to autolysin-dependent killing and bacterial lysis or both. Taken together, these findings suggest that the bacterial envelope sites engaged in cell growth may represent preferential sites for the action and cytotoxic consequences of group IIA PLA2 attack against Gram-positive bacteria.

Anti-Bacterial Agents↗

PubMed2001

Vancomycin resistance reversal in enterococci by flavonoids.

The development of clinical vancomycin-resistant strains of enterococci (VRE) is a major cause for concern. Here we show that a combination of galangin or 3,7-dihydroxyflavone with vancomycin may be used to sensitize resistant strains of Enterococcus faecalis and Enterococcus faecium to the level of vancomycin-sensitive strains. Minimum inhibitory concentrations (MICs) and viable counts were determined in Iso-sensitest broth using a microtitre method. MICs of vancomycin against 67% of resistant clinical isolates and a type strain of enterococci were lowered from > 250 microg mL(-1) to < 4 microg mL(-1) in the presence of galangin (12.5 microg mL(-1)) or 3,7-dihydroxyflavone (6.25 microg mL(-1)). Viable counts for type culture E. faecalis ATCC 51299 showed the flavonoids alone significantly lowered numbers of colony forming units (CFUs). CFUs were maintained at low levels (< 10(3) CFU mL(-1)) for 24 h by vancomycin/flavone combinations. This combinational action in reversing vancomycin resistance of enterococci highlights novel drug targets and has importance in the design of new therapeutic regimes against resistant pathogens.

Anti-Bacterial Agents↗

PubMed2002

Treatment of life-threatening multiresistant staphylococcal and enterococcal infections in patients with end-stage renal failure with quinupristin/dalfopristin: preliminary report.

BACKGROUND: Life-threatening infections with multiresistant gram-positive bacteria are increasing. Treatment with quinupristin/dalfopristin (Q-D) has turned out to be effective against such resistant pathogens. PATIENTS AND METHODS: We report on treatment of six patients on dialysis (four with additional liver injury) and of one renal graft recipient with normal renal function who had severe infections caused by multiresistant Staphylococus epidermidis (1/7), methicillin-resistant Staphylococcus aureus (4/7) and vancomycin-resistant Enterococcus faecium (2/7). RESULTS: Six out of seven patients were cured by therapy with Q-D in adjusted doses lasting for 10 to 34 days. Pharmacokinetics of Q-D and its metabolites were determined and remained within the therapeutic range, despite a modest increase of all compounds at the presumed steady state. The concentrations of the metabolites of Q-D were clearly lower than the parent drugs, including those of quinupristin-conjugated derivatives, which has not been reported previously. CONCLUSION: These preliminary results suggest that: a) neither quinupristin nor dalfopristin or its metabolites accumulated despite the long duration of treatment; b) no adjustment of the standard dosage regimen (three times 7.5 mg/kg/day) is necessary in end-stage renal disease.

Aged↗

PubMed2006

Evaluation of the effectiveness of an enzymatic cleaner and glutaraldehyde-based disinfectant for chemothermal processing of flexible endoscopes in washer-disinfectors in accordance with prEN ISO 15 883.

BACKGROUND AND STUDY AIMS: This study evaluated the effectiveness of the cleaning process, the disinfection process, and a combination of the two in accordance with the new international standard, prEN ISO 15 883. MATERIALS AND METHODS: The cleaning process consisted of a 1-min prerinse at 20 degrees C, followed by a 5-min cleaning step at 45 degrees C (with an enzymatic cleaner, 0.5 %), followed by a 1-min interim rinse from 45 degrees C to 55 degrees C. The disinfection process consisted of a 1-min prerinse at 20 degrees C, followed by a 5-min disinfection step at 55 degrees C (with a glutaraldehyde-based disinfectant, 1 %), followed by two final rinses of 1 min each at 55 degrees C. Transparent test pieces were contaminated with a mixture of blood and ENTEROCOCCUS FAECIUM, and were assessed for visible cleanliness and microbial load. RESULTS: Cleaning alone, disinfection alone, and the combination of the two always led to visible cleanliness of all test pieces. The cleaning process revealed a mean reduction factor of > or = 4.6 (n = 6); the disinfection process revealed a mean reduction factor of > or = 9.0 (n = 6), and the combination of the two was found to reduce the test organism in the WD440 by 9.0 +/- 0.2 log (10) steps (n = 12) and in the AdaptaScope by 9.3 +/- 0.4 log (10) steps (n = 5). CONCLUSIONS: Overall, the entire process was found to be very effective and compatible for reprocessing flexible endoscopes in washer-disinfectors. No visible residual blood was found, despite the use of glutaraldehyde in the disinfection phase. These findings once again emphasize the importance of effective cleaning for the overall results when reprocessing flexible endoscopes.

Disinfectants↗

PubMed2005

A novel peptidoglycan cross-linking enzyme for a beta-lactam-resistant transpeptidation pathway.

The beta-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the beta-lactam ring and the d-alanyl(4)-d-alanine(5) extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in abeta-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldt(fm), catalyzes in vitro the cross-linking of peptidoglycan subunits in a beta-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldt(fm) for the l-lysyl(3)-d-alanine(4) peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic beta-lactams in contrast to d-alanyl(4)-d-alanine(5) in the pentapeptide donors required for dd-transpeptidation. Ldt(fm) homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.

Alanine↗

PubMed1999

Outcome of a screening program for vancomycin-resistant enterococci in a hospital in Victoria.

OBJECTIVE: To screen for faecal colonisation with vancomycin-resistant enterococci (VRE) among potentially at-risk patients. DESIGN: Infection control screening program. SETTING: Monash Medical Centre (a tertiary care hospital), Melbourne, Victoria, in the seven months from June 1997. PATIENTS: Patients in the Renal, Oncology and Intensive Care (ICU) Units. MAIN OUTCOME MEASURES: Presence of VRE in a rectal swab or faecal specimen taken at admission and at regular intervals during inpatient stay; presence of vancomycin-resistance genes (vanA, vanB and vanC) assessed by polymerase chain reaction (PCR); genetic clonality of isolates assessed by pulsed-field gel electrophoresis (PFGE). RESULTS: 574 patients (356 renal, 134 ICU and 84 oncology) were screened; 12 were colonised with VRE--nine renal inpatients, two having peritoneal dialysis or incentre haemodialysis, and one ICU patient. Nine isolates were Enterococcus faecalis (seven positive for vanB and two negative for all three resistance genes) and three were Enterococcus faecium (all positive for vanB). Eight were high-level gentamicin resistant. PFGE suggested genetic clonality between the index isolate and five other isolates from renal patients. No specific clinical practice was associated with VRE colonisation. Attempts to clear rectal carriage with oral ampicillin/amoxycillin or bacitracin were of limited success. Although antibiotic prescribing in the Renal Unit was generally consistent with defined protocols, use of vancomycin and third-generation cephalosporins has been further restricted. CONCLUSIONS: Renal inpatients in our institution appear most at risk of VRE colonisation (4.6% overall) and therefore of VRE infection. Routine screening, especially of potentially high-risk patients, should be considered in major Australian hospitals.

Adult↗

PubMed1996

Influence of oral glycopeptides on the fecal flora of human volunteers: selection of highly glycopeptide-resistant enterococci.

Changes in fecal flora were evaluated in 22 healthy volunteers administered oral vancomycin or teicoplanin in 1989-1991 in Belgium. Evaluation of 5 colonies per subject revealed no glycopeptide-resistant enterococci in the predominant flora before glycopeptide administration; however, large numbers (mostly Enterococcus faecium) emerged by the end of the study in 14 (64%) of the subjects. Pediococci and lactobacilli also increased in number. In 1992, 40 healthy volunteers and 33 cancer patients were evaluated by plating stool samples directly onto selective media containing vancomycin; low numbers of vancomycin-resistant enterococci (< 50 cfu/g) were found in 11 (28%) of the 40 and 4 (12%) of the 33 samples, respectively. DNA restriction fragment length polymorphism analysis showed that most isolates were different, but all contained vanA in Tn1546-like elements. These results indicate that vanA and Tn1546-like elements were common in Belgium as early as 1989 and that community-based individuals in that location likely form a major reservoir for glycopeptide-resistant enterococci.

Adult↗

PubMed1998

Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates.

Enterococci with resistance to glycopeptides have recently emerged in Australia. We developed multiplex PCR assays for vanA, vanB, vanC1, and vanC2 or vanC3 in order to examine the genetic basis for vancomycin resistance in Australian isolates of vancomycin-resistant Enterococcus faecium and E. faecalis (VRE). The predominant genotype from human clinical E. faecium isolates was vanB. The PCR van genotype was consistent with the resistance phenotype in all but six cases. One vanA E. faecalis isolate had a VanB phenotype, one vanB E. faecium isolate had a VanA phenotype, and four E. faecalis isolates were consistently negative for vanA, vanB, vanC1, and vanC2 or vanC3, even though they exhibited a VanB phenotype. These four isolates were subsequently examined for the presence of vanD by published methods and were found to be negative. No vancomycin-susceptible strains produced a PCR product. On the basis of our findings the epidemiology of VRE in Australia appears to be different from that in either the United States or Europe. Our multiplex PCR assays gave a rapid and accurate method for determining the genotype and confirming the identification of glycopeptide-resistant enterococci. Rapid and accurate methods are essential, because laboratory-based surveillance is critical in programs for the detection, control, and prevention of the transmission of glycopeptide-resistant enterococci.

Anti-Bacterial Agents↗

PubMed2002

Risk factors for development of vancomycin-resistant enterococcal bloodstream infection in patients with cancer who are colonized with vancomycin-resistant enterococci.

Vancomycin-resistant Enterococcus faecium (VRE) is a common nosocomial isolate, especially among patients with cancer. VRE infections have substantial attributable mortality among patients with cancer. The purpose of this study was to identify risk factors for developing bloodstream infection with VRE in patients with cancer who are colonized with VRE. VRE colonization was prospectively identified in 197 patients with cancer during 4-year period, of whom 179 (91%) had complete records for evaluation. Of these 179 patients, 24 (13.4%) developed hospital-acquired VRE bloodstream infections. Risk factors for VRE bloodstream infection included vancomycin use (relative risk [RR], 1.98; 95% confidence interval [CI], 1.25-3.14), diabetes mellitus (RR, 3.91; 95% CI, 1.20-12.77), gastrointestinal procedures (RR, 4.56; 95% CI, 1.05-19.7), and acute renal failure (RR, 3.10; 95% CI, 1.07-8.93). Strategies for preventing VRE bloodstream infection in VRE-colonized patients with cancer should include limiting vancomycin use and, perhaps, gastrointestinal procedures.

Bacteremia↗

PubMed2003

In vitro activities of linezolid combined with other antimicrobial agents against Staphylococci, Enterococci, Pneumococci, and selected gram-negative organisms.

The activities of linezolid, an oxazolidinone antibacterial agent active against gram-positive organisms, alone and in combination with 35 antimicrobial agents were tested in vitro against methicillin-sensitive (n = 1 to 2 strains) and methicillin-resistant (n = 8 to 10) Staphylococcus aureus strains; vancomycin-sensitive (n = 6) and vancomycin-resistant (n = 6 to 8) Enterococcus faecalis strains; vancomycin-sensitive (n = 5) and vancomycin-resistant (n = 6) Enterococcus faecium strains; penicillin-sensitive (n = 2 to 5), penicillin-intermediate (n = 5 to 6), and penicillin-resistant (n = 5 to 6) Streptococcus pneumoniae strains; Escherichia coli (n = 6); and Klebsiella pneumoniae (n = 6). The fractional inhibitory concentration indices of linezolid in combination with other antimicrobial agents for the organisms tested were generated on checkerboard broth microdilution plates prepared by a semiautomated method. Of 1,380 organism-drug combinations, 1,369 (99.2%) combinations of linezolid with 28 antimicrobial drugs were indifferent, 9 combinations (0.65%) of linezolid with 6 drugs (amoxicillin, erythromycin, imipenem, sparfloxacin, teicoplanin, and tetracycline) were synergistic, and 2 combinations (0.15%) of linezolid with 2 drugs (ofloxacin and sparfloxacin) were antagonistic. Overall, the in vitro data demonstrated that linezolid combined with other antimicrobial agents primarily produces an indifferent response, with infrequent occurrences of synergism and antagonism.

Acetamides↗

PubMed1998

Enterococcal bacteremia after transjugular intrahepatic portosystemic shunts (TIPS).

The objective of this study was to analyze a series of patients with Enterococcus faecium infection following transjugular intrahepatic portosystemic shunts (TIPS) in order to define the risk factors, outcome, and role of treatment including hepatic transplantation. This study is a case series from a tertiary referral center for liver transplantation. The medical records of four patients referred to one teaching hospital in San Francisco between 1990 and 1995 for evaluation or management of Enterococcal infection following TIPS were reviewed. A review of the microbiology records of all 314 patients who underwent TIPS at that institution and a MEDLINE search were performed to assess whether any other cases existed. The effect of therapy on survival was assessed, in particular, the repeated use of TIPS and prolonged courses of antibiotics. All four patients had thrombosis of their TIPS at the time of diagnosis of enterococcal bacteremia. All were treated with prolonged courses of intravenous antibiotics. One patient had echocardiographic evidence of subacute bacterial endocarditis with chronic aortic insufficiency. In all cases, liver transplantation was contraindicated in the acute setting because of uncontrolled endovascular infection. Two of four patients survived; these were the only two patients who had had a successful repeat TIPS. Enterococcal bacteremia is a rare complication following TIPS but carries a high mortality. It usually occurs in the setting of technically difficult TIPS with shunt thrombosis. Management should be focused on long term antibiotics and attempts at reestablishment of portal decompression with another TIPS. Liver transplantation should not be considered until the infection is cleared. Prophylaxis for Enterococcus species should be considered in technically difficult or unsuccessful TIPS.

Adult↗

PubMed2006

Development of a competitive exclusion product for poultry meeting the regulatory requirements for registration in the European Union.

Competitive exclusion treatment is able to increase the pathogen colonization resistance of day-old chicks by applying probiotic bacteria stabilizing the indigenous microflora. In order to develop a safe microbial feed additive, various bacterial strains were isolated out of the gastrointestinal tract of healthy chickens. One hundred twenty-one representatives were selected based on differences in whole-cell protein patterns and screened for antagonistic properties. Five effective strains (Pediococcus acidilactici, Enterococcus faecium, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, and Lactobacillus salivarius ssp. salivarius) exhibited in vitro the ability to inhibit a range of common pathogens and were evaluated with regard to the risks associated with genetic transfer of antibiotic resistances from animals to humans via the food chain. The probiotic strains were sensitive to several clinically effective antibiotics, though some of them showed single resistances. None of the vancomycin-resistant (R) strains carried the enterococcal vanA gene. Two tetracycline R strains were shown to harbor a tet(M)-associated resistance. The strains contained no extrachromosomal DNA and were not able to transfer the resistance by means of conjugation. On basis of the collected data the presence of easy transferable resistances was excluded and the chicken strains were considered to be suitable for the use as feed additive.

Agriculture↗

PubMed2004

International dissemination of antibiotic resistant strains of bacterial pathogens.

The increasing incidence of methicillin and multiple resistant Staphylococcus aureus (MRSA) in nosocomial infections is mainly associated with a wide, international dissemination of well defined clonal lineages (epidemic MRSA) which are clearly different from community acquired MRSA by molecular typing patterns and structure of the staphylococcal cassette chromosome containing the mecA gene. Although belonging to a definite subpopulation within the species Enterococcus faecium, hospital associated vancomycin resistant isolates also containing the esp gene have very likely evolved by acquisition of glycopeptide resistance gene clusters at different occasions and at different times by a susceptible already disseminated clonal lineage. There is obviously a continuous selection of new types of extended spectrum beta-lactamases in enterobacteriaceae and also horizontal spread of bla-genes. Intrahospital dissemination of particular strains has often been observed, however, an international dissemination until now has been described only for Salmonella enterica serovar Typhimurium producing the CTX-M-3 enzyme. Multiresistant isolates of Salmonella enterica serovar Typhimurium phage type DT104 harbour a multiresistant gene cluster with resistance genes from taxonomically more unrelated species (tetG, floR, bla(PSE1)). Although in vitro transduction has been demonstrated, this gene cluster has only rarely been reported from isolates exhibiting other phage patterns of the same serovar or from other serovars of S. enterica.

Animals↗