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Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium.

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.

Alanine↗

Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium.

The peptidoglycan structure of in vitro selected ampicillin-resistant mutant Enterococcus faecium D344M512 and of the susceptible parental strain D344S was determined by reverse phase high performance liquid chromatography and mass spectrometry. The muropeptide monomers were almost identical in the two strains. The substantial majority (99.3%) of the oligomers from the susceptible strain D344S contained the usual d-alanyl --> d-asparaginyl (or d-aspartyl)-l-lysyl cross-link (d-Ala --> d-Asx-l-Lys) generated by beta-lactam-sensitive DD-transpeptidation. The remaining oligomers (0.7%) were produced by beta-lactam-insensitive LD-transpeptidation, because they contained l-Lys --> d-Asx-l-Lys cross-links. The muropeptide oligomers of the ampicillin-resistant mutant D344M512 contained only these l-Lys --> d-Asx-l-Lys cross-links indicating that resistance was due to the bypass of the beta-lactam-sensitive DD-transpeptidation reaction. The discovery of this novel resistance mechanism indicates that DD-transpeptidases cannot be considered anymore as the sole essential transpeptidase enzymes.

Amino Acid Sequence↗

Catabolite repression in Enterococcus faecalis.

Metabolism of citrate, pyruvate and sugars by Enterococcus faecalis E-239 and JH2-2 and an isogenic, catabolite derepressed mutant of JH2-2, strain CL4, was investigated. The growth rates of E. faecalis E-239 on citrate and pyruvate were 0.58 and 0.63 h(-1), respectively, indicating that both acids were used as energy sources. Fructose and glucose prevented the metabolism of citrate until all the glucose or fructose had been metabolised. Diauxie growth was not observed but growth on glucose and fructose was much faster than on citrate. In contrast, citrate was co-metabolized with galactose or sucrose and pyruvate with glucose. When glucose was added to cells growing on citrate, glucose metabolism began immediately but inhibition of citrate utilisation did not begin for approximately 1.5 h. Growth rates of E. faecalis JH2-2 and its isogenic, catabolite derepressed mutant, strain CL4, on citrate, were 0.41 and 0.36 h(-1), respectively. The catabolite derepressed mutant was able to co-metabolise citrate and glucose at all concentrations of glucose tested (3-25 mM), while its parent, could only metabolise citrate once all the glucose had been consumed. In strains JH2-2 and E-239, the growth rate on citrate decreased as the glucose concentration increased and, in 25 mM glucose, consumption of citrate was inhibited for several hours after glucose had been consumed. These results indicate that catabolite repression by glucose and fructose occurs in enterococci.

Carbohydrate Metabolism↗

Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German university pediatric hospital.

Nosocomial Infections caused by vancomycin-resistant enterococci (VRE) are an emerging threat to critically ill patients. At the University Hospital Eppendorf, VRE were isolated from 38 patients between August 1993 and April 1997, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 Enterococcus faecium isolates from patients of the Department of Pediatrics were identical or closely related, and that isolates from three additional patients of the same department were possibly related. All of these isolates were of vanA genotype. They were resistant to glycopeptides, ampicillin, ciprofloxacin, clindamycin, and erythromycin. Most isolates displayed high-level resistance to gentamicin, but all remained susceptible to quinupristin/dalfopristin. Implementation of stringent hand disinfection and environmental disinfection policies, as well as measures for patient isolation contained this first outbreak of VRE at a German Children's hospital, which emphasizes the importance of hygienic measures for the control of nosocomial spread of these organisms.

Child, Preschool↗

Changes in arterial blood pressure in hypertensive rats caused by long-term intake of milk fermented by Enterococcus faecalis CECT 5728.

We have evaluated the changes in arterial blood pressure caused in spontaneously hypertensive rats (SHR) by long-term intake of an Enterococcus faecalis CECT 5728-fermented milk with significant angiotensin-converting enzyme (ACE)-inhibitory activity. After being weaned, male 3-week-old SHR were randomized into five groups. Until the 20th week of life, rats in each group were given one of the following drinking fluids: tap water (negative control 1), a fermented milk without ACE-inhibitory activity (negative control 2), captopril (100 mg/kg) (positive control), the E. faecalis CECT 5728-fermented milk that had significant ACE-inhibitory activity, or Ca-enriched E. faecalis CECT 5728-fermented milk. Animals in the different groups were then given tap water as drinking fluid from the 20th to 25th week of life. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured weekly in the rats, from the 6th to 25th week of life, by the tail-cuff method. A definite decrease in SBP and DBP could be observed in the rats treated with captopril and also in the rats that received the E. faecalis CECT 5728-fermented milks. The greatest antihypertensive effect was observed when the pharmacological treatment was administered. The effect of the Ca-enriched fermented milk was slightly more accentuated and more constant than the effect of the E. faecalis CECT 5728-fermented milk that had not been enriched in Ca. SBP and DBP increased in the treated SHR when the corresponding antihypertensive treatment was removed. Fermentation of milk with E. faecalis CECT 5728 may therefore be a successful strategy to produce a functional food with antihypertensive activity.

Angiotensin-Converting Enzyme Inhibitors↗

Impact on human intestinal microflora of an Enterococcus faecium probiotic and vancomycin.

The aims of this study were to evaluate the impact of a fermented milk product containing viable Enterococcus faecium on human intestinal microflora and to evaluate any risk of development of vancomycin-resistant enterococci (VRE). Twenty Danish and 20 Swedish healthy volunteers were given 150 ml of the fermented milk product once daily, equivalent to a daily dose of 4.5 x 10(9) to 7.5 x 10(9) CFU E. faecium, for 10 d. Half of the volunteers also received 125 mg vancomycin orally q.i.d. for 10 d. Faecal samples were collected on day 0 before intake, on day 10 directly after end of intake and on day 31, 3 weeks after the end of the experiment. There was a significant increase in the total number of enterococci on day 10 (p < 0.01) in the group receiving only the E. faecium supplement, but 3 weeks later the level was as before intake. In the vancomycin group, the total number of enterococci was reduced on day 10 (p < 0.01) but had increased on day 31 (p < 0.01) in relation to day 0. In none of the Swedish and 4 of the Danish volunteers, VRE were sporadically detected, but without relation to intake of the probiotic or vancomycin. In healthy young Danish individuals the VRE carrier rate tended to be higher than previously found.

Adult↗

Antimicrobial susceptibility of vancomycin-resistant Enterococcus faecium: potential utility of fosfomycin.

The increasing prevalence of vancomycin-resistant enterococcal (VRE) infections has necessitated a search for drugs that are effective in treating these infections, and a need to determine whether currently available antimicrobials are effective. 75 consecutive clinical isolates of vancomycin-resistant Enterococcus faecium (VRE faecium) (40 blood and 35 urine isolates) isolated over 1 y at the Cleveland Clinic Foundation were tested for susceptibility to linezolid, quinupristin-dalfopristin, fosfomycin and nitrofurantoin using the Etest. The minimum inhibitory concentrations were read independently by 3 observers and compared, and a final reading was obtained by predetermined criteria. The proportion of isolates susceptible to linezolid, quinupristin-dalfopristin, fosfomycin and nitrofurantoin was 100%, 98.7%, 98.7% and 78.7%, respectively. No single isolate was resistant to more than 1 of the 4 drugs tested. Etest presented significant unexpected difficulties in testing for VRE faecium susceptibility to nitrofurantoin. Fosfomycin may be a useful alternative to linezolid and quinupristin-dalfopristin in the treatment of VRE infections in certain clinical situations, e.g. uncomplicated urinary tract infections. In addition, the use of fosfomycin could limit the use of newer agents, thus reducing the chance of development of further resistance in the enterococci.

Anti-Bacterial Agents↗

Outbreak of vancomycin resistant enterococcus in a hematology/oncology unit in a Korean University Hospital, and risk factors related to patients, staff, hospital care and facilities.

A vancomycin-resistant Enterococcus (VRE) outbreak occurred at a hematology/oncology (HO) service in a tertiary acute care hospital, 1 January to 30 April 2002. This study was undertaken to identify risk factors, and to establish relations between seasonal factors and this VRE outbreak. Every patient (n = 82) and health care worker (HCW) (n = 31) at the HO unit was screened by rectal swab and stool culture during the VRE outbreak. Environmental surface culture (n = 35) and pulsed-field gel electrophoresis (PFGE) of VRE isolates were preformed. Among a total of 82 patients, 17 had VRE. All VRE isolates were E. faecium, the 6 studied further were Van A with identical PFGE patterns. Cultures of surfaces and HCWs were negative for VRE. Prolonged hospital stay (p = 0.02), male gender (p = 0.02), and care in a 6-bed room (p < 0.01) were risk factors for acquisition of VRE. An association with the VRE outbreak period was found again for long hospital stay, but also for the antibiotic usage pattern, frequent surgery and a high proportion of new inexperienced staff (52% vs 4% before the outbreak (p < 0.01, Fisher's exact test). Sudden personnel turnover in high-risk units should be avoided.

Adolescent↗

Study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis in a flock of brown layer chickens.

Enterococcus faecalis-related amyloid arthropathy was diagnosed in a sample of birds from a flock of brown layer parent chickens aged 57 weeks. E. faecalis was isolated from amyloidotic knee joints and from blood samples from lame birds. From this flock a smaller one was kept for production purposes to study the vertical transmission of arthropathic and amyloidogenic E. faecalis. Three batches of non-soiled and thoroughly disinfected eggs, to prevent egg-shell contamination, were collected for 6 weeks and submitted for incubation every 2 weeks. No joint pathology was found in the offspring chicks (n = 458) monitored for joint disease until 16 or 21 weeks of age. Fresh and candled eggs (infertiles and dead embryos) obtained at day 18 of the incubation period had negative results at bacteriological examination of the yolk sac, except one non-hatched egg (late embryonal death in shell) from which E. faecalis was isolated. Genotyping by pulsed-field gel electrophoresis (PFGE) of all E. faecalis isolates obtained from blood and joints (except one) of the parent birds and of the non-hatched egg after Sma I digestion showed that all isolates were genetically closely related or identical to a known arthropathic and amyloidogenic E. faecalis strain. In this study, vertical transmission of E. faecalis, although it may occur on a small scale as shown by PFGE, did not seem to play a significant role. Conversely, the chronicity of the condition and the development of an immune response may have affected the efficiency of its transmission.

Amyloidosis↗

Biofilm formation by Enterococcus faecalis on intraocular lens material.

PURPOSE: To compare biofilm formation by Enterococcus faecalis on different intraocular lens (IOL) materials. METHODS: E. faecalis biofilms were cultivated on disks of IOL material (silicone, PMMA [polymethylmethacrylate], or acrylic). Biofilms were stained with crystal violet (CV), which served as an index of biofilm formation. The bacterial population was enumerated after biofilm homogenization. Biofilms were also examined by confocal microscopy. RESULTS: At 24 hr, there was no significant difference in biofilm formation, or the population within biofilms, among the three materials tested. However, after 48 and 72 hr, it was observed that silicone supported the least amount of biofilm formation (p < 0.05); biomass on PMMA and acrylic IOLs continued to increase, with acrylic demonstrating the greatest amount of stainable biofilm (p < 0.0005). The population on PMMA was significantly greater than the other IOL materials (p < 0.005). Confocal microscopy confirmed the lack of biomass on silicone. CONCLUSIONS: Among three IOL materials, E. faecalis biofilms formed more readily on PMMA and acrylic compared to silicone.

Acrylic Resins↗

Molecular epidemiology of unilateral amyloid arthropathy in broiler breeders associated with Enterococcus faecalis.

Although symmetrical polyarticular amyloidosis has been described extensively in brown layers, spontaneous unilateral amyloid arthropathy has not been described previously in chickens. Birds from nine flocks of broiler parent stock (PS) had unilateral lameness associated with severe swelling of the left hock joint and the caudal aspect of the metatarsus. Gross pathology was restricted to the left hock joint and the left digital flexor tendons in almost all cases, suggesting an association with administration of Marek's disease vaccine. Amyloid deposits were found in 83% (25/30) of affected joints by histological examination of Congo red stained sections. Systemic amyloidosis, involving mainly the liver and spleen, was found in 59% (10/17) of birds. Enterococcus faecalis was isolated from joints in 77% (23/30) of cases and Staphylococcus aureus was isolated from the joint in one case (1/30). Thirty-five E. faecalis isolates from joints, tendons and blood samples from birds in five affected PS flocks were compared using pulsed-field gel electrophoresis (PFGE) to separate genomic fragments after digestion with SmaI. All but one isolate had identical or closely related restriction endonuclease digestion (RED) patterns that were very similar to a known arthropathic and amyloidogenic E. faecalis isolate. A further 30 E. faecalis isolates from seven grandparent stock (GPS) flocks and two isolates from two unaffected PS flocks of the same genetic background were analysed by PFGE. Among these isolates, 11 originating from four GPS flocks had RED patterns identical to or closely related to the reference amyloid-inducing strain. Moreover, one E. faecalis isolate from amyloidotic joints of brown layers housed in California, USA was included in the analysis and appeared to be identical to the reference strain. This study showed that the E. faecalis isolates involved in these outbreaks of unilateral amyloid arthropathy in broiler breeders belonged to the same clone as that responsible for outbreaks in brown layers.

Amyloidosis↗

Investigations of Enterococcus faecalis-induced bacteraemia in brown layer pullets through different inoculation routes in relation to the production of arthritis.

In the present aerosol experiment, assessment of the respiratory tract of 1-day-old birds as a natural route of infection for induction of Enterococcus faecalis bacteraemia and arthritis was performed. Second, the severity and type of arthritis produced through intramuscular infection in two different inoculation sites (musculus pectoralis versus musculus gastrocnemius) was studied. Third, the resulting bacteraemia was assessed qualitatively and quantitatively in relation to the occurrence of arthritis. Exposure of 1-day-old brown layer pullets to aerosolized E. faecalis with an estimated uptake of 10(4) to 10(5) colony forming units per chick resulted in bacteraemia; however, joint lesions were not induced. In contrast, 3/10 birds inoculated intratracheally with 10(8) colony forming units developed both bacteraemia and arthritis. This suggests the occurrence of a dose effect and a role for the respiratory tract as a natural infection route in young chickens. In both intramuscularly inoculated groups the incidence of arthritis was 10/10 birds and 9/10 birds, respectively. Birds inoculated in the m. pectoralis developed symmetric polyarthritis, which harmonizes with haematogenous colonization of joints. In contrast, m. gastrocnemius-inoculated chicks mostly had asymmetric (poly)arthritis of the injected leg and varus deformation of the contralateral leg, suggesting predominantly local spread. The qualitative and quantitative results of bacteriology of blood samples show that arthritis develops in those groups with the highest number of bacteraemic birds and the highest median bacterial colony forming units per millilitre of blood during the first 24 to 36 h after treatment.

Aerosols↗

Aerosolization of Mycoplasma synoviae compared with Mycoplasma gallisepticum and Enterococcus faecalis.

In order to study the airborne transmission of an arthropathic strain of Mycoplasma synoviae, preliminary aerosol experiments were performed. They were conducted in duplicate in an empty isolator (1.3 m3) to assess the yield and viability of M. synoviae with time compared with Mycoplasma gallisepticum and Enterococcus faecalis. After aerosol generation air samples were taken with two different devices using gelatine or cellulose nitrate filters. There was no difference between the devices, but cellulose nitrate filters yielded very low bacterial counts. The aerosolized dose per isolator for M. synoviae was 3.4 x 10(10) colony-forming units (cfu), for M. gallisepticum was 2.6 x 10(10) cfu and for E. faecalis was 3 x 10(10) cfu. Immediately after aerosolization, concentrations of about 10(6) to 10(7) cfu/m3, 10(7) to 10(8) cfu/m3 and 10(8) to 10(9) cfu/m3 air of M. synoviae, M. gallisepticum and E. faecalis were found, respectively. At 25 min M. synoviae concentrations dropped below the detection level (<4 x 10(4) cfu), while 10(5) to 10(6) and 10(8) to 10(9) cfu were found for M. gallisepticum and E. faecalis, respectively. The average M. synoviae concentration during the experiment was estimated at 10(2) to 10(3) cfu/l. The M. gallisepticum and E. faecalis aerosol generated an average of approximately 10(3) to 10(4) cfu/l air and 10(5) to 10(6) cfu/l air, respectively. Thus mycoplasma and E. faecalis aerosols were successfully generated despite considerable initial loss as measured by culture. The loss was greater in the mycoplasma aerosols, especially those of M. synoviae.

Aerosols↗

Presence and mechanism of macrolide-lincosamide resistance in Enterococcus columbae strains belonging to the intestinal flora of pigeons.

Faecal samples from 50 pigeons all originating from different lofts were screened for the presence of macrolide and lincosamide (ML)-resistant isolates of Streptococcus gallolyticus and Enterococcus columbae by plating the samples onto selective media. Sixty-eight ML-resistant E. columbae strains were recovered from the faecal samples of 29 animals. Two of these samples also harboured ML-resistant S. gallolyticus strains. The erm(B) gene was detected in 58 E. columbae and in five S. gallolyticus isolates. Four of these E. columbae isolates also carried the mef(A) gene. Five E. columbae strains possessed the mef(A) gene in the absence of erm(B). On the basis of the sequence of the complete erm(B) gene, 10 E. columbae isolates clustered together in six groups. In two of these isolates, the erm(B) gene sequence was identical to that of S. gallolyticus strains, indicating that exchange of resistance genes might occur between pathogenic and non-pathogenic bacterial species belonging to the pigeon's intestinal flora.

Animals↗

Reproduction of sepsis and endocarditis by experimental infection of chickens with Streptococcus gallinaceus and Enterococcus hirae.

This study describes experimental infections in 4-week-old chickens inoculated intravenously with approximately 10(8) colony-forming units Streptococcus gallinaceus strain CCUG 42692T (C13156) or Enterococcus hirae strain DSM 20160 (C17410). Birds were necropsied following death and obvious clinical signs of disease or were euthanized weekly after infection for up to 4 weeks. At necropsy, lesions included splenomegaly, hepatomegaly, valvular and/or mural endocarditis. Cardiac lesions included focal necrotizing myocarditis and/or yellow-white vegetative valvular endocarditis or greyish proliferations associated with the mitral valves in 35% (6/20) and 79% (19/24) of birds infected with S. gallinaceus and in 20% (4/20) and 55% (12/22) of birds infected with E. hirae via the brachial and jugular veins, respectively. S. gallinaceus was reisolated from heart valves in 45% (9/20) and 75% (18/24) and E. hirae in 35% (7/20) and 73% (16/22) after inoculation via brachial and jugular veins, respectively. Both challenge strains were also isolated from liver, spleen, bone marrow and hock joints. A significant difference between the infections with the two strains was seen only with reisolation of E. hirae from hock joints (P < 0.007). Significant differences were apparent between the two inoculation routes only with E. hirae, where infection via the jugular vein was associated with higher culture positive isolations from the heart (P = 0.029), bone marrow (P = 0.002) and hock joints (P < 0.001) compared with the brachial vein. Birds injected with sterile phosphate-buffered saline were negative for culture of the challenge strains and no lesions were observed in these controls. The results confirm that both S. gallinaceus and E. hirae can cause endocarditis in experimentally infected chickens.

Animals↗

Antibiotic resistance of Enterococcus spp. isolated from wastewater and sludge of poultry slaughterhouses.

Antibiotimicrobial resistance was investigated in 537 Enterococcus spp. isolates recovered from 22 samples of crude inflow, treated effluent and sludge collected in wastewater treatment plants of eight poultry slaughterhouses of Portugal. No significant differences (P > 0.05) were found in the resistance to each antimicrobial agent with regards to the origin of the sample (inflow, sludge and effluent). Many of the isolates displayed resistance to tetracycline (85.7%), erythromycin (45.7%), nitrofurantoin (34.0%) and rifampicin (17.8%). Resistance was also observed, but to a lesser extent, to ciprofloxacin (10.2%), ampicillin (8.0%), chloramphenicol (4.6%), vancomycin (0.9%) and gentamicin (0.4%). Resistance to three or more antimicrobial classes was present in 37.1% of the isolates. Wastewater treatment resulted in viable enterococci decrease between less than 1 log and 4 log; nevertheless, more than 4.4 x 10(5) colony forming units (CFU)/100 mL were present in the outflow of the plants and thus resistant enterococci are not prevented from reaching the general environment.

Abattoirs↗

Inhibition of vaginal lactobacilli by a bacteriocin-like inhibitor produced by Enterococcus faecium 62-6: potential significance for bacterial vaginosis.

OBJECTIVE: Bacterial vaginosis (BV) is characterized by a shift in vaginal tract ecology, which includes a decrease in the concentration and/or prevalence of facultative lactobacilli. Currently, mechanisms which could account for the disappearance of lactobacilli are not well understood. The objective of this study was to determine whether vaginal streptococci/enterococci can produce bacteriocin-like inhibitors antagonistic to vaginal lactobacilli. METHODS: Seventy strains of vaginal streptococci or enterococci were tested for antagonistic activities against vaginal lactobacilli using the deferred antagonism technique. RESULTS: One strain, Enterococcus faecium 62-6, which strongly inhibited growth of lactobacilli was selected for further characterization. The spectrum of inhibitory activity of strain 62-6 included Gram-positive organisms from the vaginal environment, although native lactobacilli from the same host were resistant to inhibitor action. Following growth in MRS broth the strain 62-6 inhibitor was shown to be heat- (100 degrees C, 30 minutes), cold- (4 degrees C, less than 114 days) and pH- (4-7) stable. The sensitivity of inhibitor-containing supernatants to pepsin and alpha-chymotrypsin suggested an essential proteinaceous component. The inhibitor was sensitive to lipase but resistant to lysozyme. Dialysis of inhibitor-containing culture supernatants suggested a molecular mass greater than 12,000 Da. All physicochemical properties were consistent with its classification as a bacteriocin-like inhibitor. Kinetic assays demonstrated a sharp onset of inhibitor production coinciding with a concentration of 62-6 of 10(7) cfu/ml, suggesting that production may be regulated by quorum sensing. CONCLUSIONS: These results may have clinical significance as a novel mechanism to account for the decline of vaginal Lactobacillus populations and contribute to both the establishment and recurrence of BV.

Bacterial Adhesion↗