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High occurrence of esp among ampicillin-resistant and vancomycin-susceptible Enterococcus faecium clones from hospitalized patients.

The ability to colonize patients successfully may be essential for the emergence and spread of resistant nosocomial strains. We determined the presence of Esp, a surface protein involved in colonization ability in Enterococcus faecalis, in 96 Enterococcus faecium isolates from hospitalized patients (77 PFGE clones), 33 faecal isolates from healthy volunteers (32 clones) and 20 environmental isolates (20 clones). Esp was found significantly more often in E. faecium isolated from hospitalized patients than in isolates from the community setting (26% versus 6%, P < 0.01) and was significantly more common among ampicillin-resistant than among ampicillin-susceptible strains (37% versus 4%, P < 0.001), regardless of the isolation site. The frequency of the esp gene in the hospital clearly correlates with antibiotic-resistant E. faecium clones. This observation indicates that antibiotic-resistant variants may frequently arise under antibiotic selective pressure among esp-positive clones reaching ecological abundance in the nosocomial habitat.

Ampicillin Resistance↗

The vanC-3 vancomycin resistance gene cluster of Enterococcus flavescens CCM 439.

Enterococcus flavescens CCM 439 is phenotypically similar to Enterococcus casseliflavus; it possesses intrinsic low-level resistance to vancomycin and has the VanC phenotype. The complete vanC-3 vancomycin resistance gene cluster was cloned and sequenced, and found to contain five open reading frames. These encoded five proteins that displayed a high degree of amino acid identity to the proteins of the vanC-2 cluster of E. casseliflavus. The serine racemases displayed the lowest degree of identity (97%), whereas the response regulators VanR(C-2) and VanR(C-3) were 100% identical. Long-PCR-RFLP analysis of the vanC-3 and vanC-2 gene clusters distinguished E. flavescens CCM 439 from E. casseliflavus ATCC 25788 due to the absence of a single EcoRV restriction endonuclease site from the E. flavescens gene cluster. However, the lack of nucleotide divergence between the sequences of the vanC-2 and vanC-3 clusters casts doubt on the validity of E. flavescens and E. casseliflavus being classed as distinct species.

Bacterial Proteins↗

Molecular and clinical epidemiology of vancomycin-resistant Enterococcus faecalis.

OBJECTIVES: With the recent emergence of vancomycin-resistant (VR) Staphylococcus aureus, subsequent to the suggested transfer of the vanA resistance gene from Enterococcus faecalis, we sought to determine risk factors for acquisition of VR E. faecalis and to evaluate the molecular epidemiology of this less-prevalent and less-studied species of VR enterococcus. METHODS: We compared clinical isolates of VR E. faecalis from 71 patients, collected over 12 years in a large community teaching hospital, with isolates from 126 patients with vancomycin-susceptible E. faecalis. RESULTS: Risk factors for VR E. faecalis acquisition by multivariate analysis were nursing home residence (P = 0.0005), haemodialysis (P = 0.009), decubitus ulcers (P = 0.03) and receipt of parenteral vancomycin (P = 0.0002). Twenty-one percent of VR E. faecalis demonstrated vanA and 79% vanB resistance. The number of VanA isolates increased over time. Molecular analysis showed vanA or vanB in multiple PFGE groups. CONCLUSIONS: The results of this study suggest gene dissemination among some isolates and intra-hospital spread of other isolates. The risk factors identified clearly suggest that VR E. faecalis is a nosocomial pathogen and should be considered in infection control practices. Further surveillance of VR E. faecalis is warranted, due to the potential spread of vancomycin resistance among enterococci and staphylococci.

Confidence Intervals↗

A comparison of the antimicrobial efficacy of three calcium hydroxide formulations on human dentin infected with Enterococcus faecalis.

This study compared the antibacterial efficacy of three different formulations of calcium hydroxide by using human dentin specimens that were infected with Enterococcus faecalis. After exposure to three forms of calcium hydroxide (calcium hydroxide mixed with distilled water, calcium hydroxide mixed with 0.2% chlorhexidine, and calcium hydroxide mixed with camphorated paramonochlorophenol) for 7 days, dentin powder from the infected specimens was obtained and assessed for bacterial quantity by spectrophotometry. It was found that calcium hydroxide mixed with camphorated paramonochlorophenol killed all of the Enterococcus faecalis inside the dentinal tubules. This result was better than that obtained with calcium hydroxide mixed with distilled water or with 0.2% chlorhexidine (p < 0.05). Calcium hydroxide mixed with distilled water and calcium hydroxide mixed with 0.2% chlorhexidine were ineffective against these bacteria.

Anti-Infective Agents, Local↗

Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat-killed microbial whole cells.

The antibacterial activity of chlorhexidine digluconate and iodine potassium iodide on Enterococcus faecalis A197A was tested in the presence of dentin, dentin matrix, dentin pretreated by EDTA and citric acid, collagen, and heat-killed cells of Enterococcus faecalis and Candida albicans. Medications were preincubated for 1 h with each of the potential inhibitors and tested for their antibacterial activity against E. faecalis, strain A197A. Surviving bacteria were sampled after 1 and 24 h of incubation. Dentin matrix and heat-killed microbial cells were the most effective inhibitors of chlorhexidine, whereas dentin pretreated by citric acid or EDTA showed only slight inhibition. Dentin and skin collagen showed some inhibition at 1 h but not after 24 h. Iodine potassium iodide was effectively inhibited by dentin, dentin matrix, and heat-killed microbial cells. Skin collagen and dentin pretreated by EDTA or by citric acid showed little or no inhibitory effect on iodine potassium iodide. Different components of dentin are responsible for the divergent patterns of inhibition of the antibacterial activity of chlorhexidine digluconate and iodine potassium iodide. Chemical treatment of dentin before applying the medication into the root canal may alter the antibacterial effect of the medication.

Candida albicans↗

Transmission of vancomycin-resistant enterococcus in a neonatal intensive care unit.

An outbreak of vancomycin-resistant Enterococcus transmission in our neonatal intensive care unit persisted despite strict contact precautions and cohorting of colonized neonates and their caregivers. After terminal cleaning was performed, incubators were found to harbor the neonates' clone of vancomycin-resistant Enterococcus. Transmission ceased after cohorting of incubators, suggesting the effectiveness of infection control measures targeting environmental contamination.

Disease Outbreaks↗

Inducer expulsion and the occurrence of an HPr(Ser-P)-activated sugar-phosphate phosphatase in Enterococcus faecalis and Streptococcus pyogenes.

Inducer expulsion, a phenomenon in which rapidly metabolizable sugars cause cytoplasmic dephosphorylation and efflux of pre-accumulated sugar-phosphates (sugar-P), has been documented for Streptococcus pyogenes, Streptococcus bovis, and Lactococcus lactis, but not for other Gram-positive bacteria. Using intact cells and membrane vesicles, we show that Enterococcus faecalis exhibits both inducer exclusion and inducer expulsion, and that the latter phenomenon is dependent on the metabolite-activated ATP-dependent HPr(Ser) kinase that phosphorylates Ser-46 in HPr of the phosphotransferase system. A small, heat-stable, membrane-associated, HPr(Ser-P)-activated sugar-P phosphatase (Pase II), previously identified only in Lc. lactis, is shown to be present in extracts of Enterococcus faecalis and Streptococcus pyogenes but not in those of Staphylococcus aureus, Streptococcus mutans, Streptococcus salivarius, or Bacillis subtilis, organisms that do not exhibit the inducer expulsion phenomenon. Further, Lactobacillus brevis, an organism that exhibits inducer expulsion by a different mechanism, also apparently lacks Pase II. The results reveal that Pase II is present in those organisms that exhibit the coupled sugar-P hydrolysis/expulsion mechanism but not those that lack this mechanism. They provide correlative evidence that Pase II initiates inducer expulsion in species of enterococci, streptococci and lactococci.

Biological Transport↗

Enterococcus faecalis divIVA: an essential gene involved in cell division, cell growth and chromosome segregation.

Enterococcus faecalis divIVA (divIVAEf) is an essential gene implicated in cell division and chromosome segregation. This gene was disrupted by insertional inactivation creating E. faecalis JHSR1, which was viable only when a wild-type copy of divIVAEf was expressed in trans, confirming the essentiality of the gene. The absence of DivIVAEf in E. faecalis JHSR1 inhibited proper cell division, which resulted in abnormal cell clusters possessing enlarged cells of altered shape instead of the characteristic diplococcal morphology of enterococci. The lower viability of the divIVAEf mutant is caused by improper nucleoid segregation and impaired septation within the numerous cells generated in each cluster. Overexpression of DivIVAEf in Escherichia coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain could be used as an indicator for functionality of DivIVAEf. A Bacillus subtilis divIVA mutant was not complemented by DivIVAEf, indicating that this protein does not recognize DivIVA-specific target sites in B. subtilis, or that it does not interact with other proteins of the cell division machinery of this micro-organism. DivIVAEf also failed to complement a Streptococcus pneumoniae divIVA mutant, supporting the phylogenetic distance between Enterococcus and Streptococcus. Our results indicate that DivIVA is a species-specific multifunctional protein implicated in cell division and chromosome segregation in E. faecalis.

Bacterial Proteins↗

Antimicrobial susceptibility of Enterococcus faecalis isolated from canals of root filled teeth with periapical lesions.

AIM: To test, in vitro, the susceptibility to different antibiotics of Enterococcus faecalis isolates from canals of root filled teeth with periapical lesions. METHODOLOGY: Twenty-one E. faecalis isolates, from canals of root filled teeth with persisting periapical lesions, were tested for their antibiotic susceptibilities. The following antibiotics were used: benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, erythromycin, azithromycin, vancomycin, chloramphenicol, tetracycline, doxycycline, ciprofloxacin and moxifloxacin. Minimal inhibitory concentrations (MICs) for the antimicrobial agents were determined using the E-test System (AB BIODISK, Solna, Sweden), and the E. faecalis strains classified as susceptible or resistant according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS). The strains were also tested for beta-lactamase production with nitrocefin (Oxoid, Basingstoke, UK). RESULTS: All strains were susceptible to penicillins in vitro, however, the MICs of amoxicillin and amoxicillin-clavulanic acid (MIC(90) = 0.75 microg mL(-1)) were lower than for benzylpenicillin (MIC(90) = 3.0 microg mL(-1)). All strains studied were also susceptible to vancomycin and moxifloxacin, whilst 95.2% were susceptible to chloramphenicol. Amongst the isolates, 85.7% were susceptible to tetracycline and doxycycline and 80.9% to ciprofloxacin. The MIC of erythromycin ranged from 0.38 to >256 microg mL(-1); only 28.5% of the strains were susceptible (MIC < or = 0.5 microg mL(-1)). Limited susceptibility was also observed with azithromycin which was active against only 14.2% of isolates. No strains produced beta-lactamase. CONCLUSION: Enterococcus faecalis isolates were completely susceptible, in vitro, to amoxicillin, amoxicillin-clavulanic acid, vancomycin and moxifloxacin. Most isolates were susceptible to chloramphenicol, tetracycline, doxycycline or ciprofloxacin. Erythromycin and azithromycin were least effective.

Anti-Bacterial Agents↗

An in vitro evaluation of the ability of ozone to kill a strain of Enterococcus faecalis.

AIM: To evaluate the potential of ozone as an antibacterial agent using Enterococcus faecalis as the test species. METHODOLOGY: Ozone was produced by a custom-made bench top generator and its solubility in water determined by ultraviolet (258 nm) spectrophotometric analysis of solutions through which ozone was sparged for various time-periods. The antibacterial efficacy of ozone was tested against both broth and biofilm cultures. Ozone was sparged for 30, 60, 120 and 240 s, through overnight broth cultures of a strain of E. faecalis (E78.2) and compared with those that were centrifuged, washed and resuspended in water. Enterococcus faecalis (E78.2) biofilms were grown on cellulose nitrate membrane filters for 48 h and suspended in water through which ozone gas was sparged with stirring for 60, 120 and 240 s in a standard fashion. In a separate test, biofilms were also exposed to gaseous ozone. Sodium hypochlorite (NaOCl) was used as a positive control. All experiments were repeated four times. RESULTS: There were significant (P < 0.05) reductions of bacteria in the unwashed (2 log(10) reductions) and washed (5 log(10) reductions) broth cultures following 240 s applications. Biofilms incubated for 240 s with ozonated water showed no significant reduction in cell viability attributable to ozone alone, whereas with NaOCl no viable cells were detected over the same time. Gaseous ozone applied for 300 s had no effect on these biofilms. CONCLUSIONS: Ozone had an antibacterial effect on planktonic E. faecalis cells and those suspended in fluid, but little effect when embedded in biofilms. Its antibacterial efficacy was not comparable with that of NaOCl under the test conditions used.

Anti-Bacterial Agents↗

Growth at high pH increases Enterococcus faecalis adhesion to collagen.

AIM: To evaluate the effect of growth at pH levels from 7.1 to 9.5 on the adherence of Enterococcus faecalis to bovine serum albumin (BSA) and collagen type I. METHODOLOGY: Enterococcus faecalis strain A197A was grown in broth of adjusted pHs varying between 7.1 and 9.5. Aliquots of bacterial suspensions were added to wells coated either with BSA or with collagen type I. Bacteria adhering to the surfaces were stained with crystal violet. Spectrophotometric measurements of the dissolved stain were used to assess the number of bacteria adhering to the surfaces. The data obtained were analysed using the Kolmogorov-Smirnov test, Levene's test and Student's t-test, with alpha = 0.05 as the level for statistical significance. RESULTS: The adhesion of E. faecalis to BSA-coated surfaces decreased inversely with alkalinity of the growth medium. The pH 7.1-grown bacteria bound to BSA significantly more than the other BSA groups. On the contrary, the adhesion to collagen type I-coated surfaces of bacteria grown at pH 8.0 and 8.5 was significantly greater than for those grown at pH 7.1. CONCLUSIONS: A minor increase in pH up to 8.5, which may be a consequence of insufficient treatment with alkaline medicaments such as calcium hydroxide, increases the collagen-binding ability of E. faecalis, in vitro. This can be a critical mechanism by which E. faecalis predominates in persistent endodontic infections.

Animals↗

Recovery of Enterococcus faecalis after single- or multiple-visit root canal treatments carried out in infected teeth ex vivo.

AIM: To assess the presence of Enterococcus faecalis after root canal treatment in single or multiple visits in an ex vivo model. METHODOLOGY: Forty-five premolar teeth were infected ex vivo with E. faecalis for 60 days. The canals were then prepared using a crowndown technique with System GT and Gates-Glidden burs and irrigated with 2% chlorhexidine gel. The specimens were divided into five groups (G1, G2, G3, G4 and G5) according to the time elapsed between chemical-mechanical preparation and root canal filling, the irrigant solution used and the use or nonuse of a calcium hydroxide intra-canal medicament. The teeth were then root-filled and incubated for 60 days at 37 degrees C. Dentine chips were removed from the canal walls with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing Brain-Heart Infusion broth. These samples were placed onto agar plates and colony forming units were counted after 24 h at 37 degrees C. Data were ranked and analysed using the Kruskal-Wallis statistical test. RESULTS: Enterococcus faecalis was recovered from 20% (three of 15 specimens) of G1 (chlorhexidine irrigation and immediate root filling in a single visit), 25% (four of 15 specimens) of G2 (chlorhexidine irrigation and filling after 14 days use of a calcium hydroxide dressing in multiple visits), 40% (two of five specimens) of G3 (chlorhexidine irrigation and filling after 7 days), 60% (three of five specimens) of G4 (saline irrigation and filling after 7 days) and from 100% (five of five specimens) of G5 (saline irrigation and immediate filling without sealer). CONCLUSIONS: Neither single- nor multiple-visit root canal treatment ex vivo, eliminated E. faecalis completely from dentinal tubules. Up to 60 days after root filling, E. faecalis remained viable inside dentinal tubules. When no sealer was used, E. faecalis presented a higher growth rate.

Anti-Infective Agents, Local↗

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean.

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.

Amino Acid Sequence↗

Outbreak of vancomycin-resistant Enterococcus spp. in an Italian general intensive care unit.

Following the identification of two clinical isolates of vancomycin-resistant enterococci (VRE) from intensive care unit (ICU) patients, a surveillance programme detected that six of eight ICU patients were colonised by VRE. Standard epidemic control measures were instituted in the ICU. During a 16-month period, 13 (2.5%) of 509 ICU patients had VRE-positive swabs upon admission, and 43 (8.7%) of 496 VRE-negative patients were colonised by VRE in the ICU. Patients who acquired VRE in the ICU had a longer ICU stay (p < 0.0001). No other statistically significant differences were demonstrated. Two patients had documented infection (infection/colonisation index, 3.6%; overall VRE infection frequency, 0.4%), but both recovered and were discharged. VRE colonisation did not increase the mortality rate. Automated ribotyping identified three clusters containing, respectively, the first 52 Enterococcus faecium isolates, two Enterococcus faecalis isolates, and two further isolates of E. faecium. Multilocus sequence typing demonstrated that two E. faecium isolates representative of the two ribotypes belonged to sequence types 78 and 18, and that these two isolates belonged to the epidemic lineage C1, which includes isolates with a wide circulation in northern Italy. The outbreak was controlled by continuous implementation of the infection control programme, and by the opening of a new unit with an improved structural design and hand-washing facilities.

Adult↗

Bacteriocin production by Enterococcus faecium NA01 from 'wara'--a fermented skimmed cow milk product from west Africa.

An Enterococcus faecium strain from Nigerian fermented skimmed cow milk ('wara') produced bacteriocin inhibitory towards Lactobacillus, Enterococcus and Listeria strains. The bacteriocin (designated enterocin 01) was inactivated by proteases, heat-stable at 100 degrees c and active at pH 2.0-6.0. The Ent. faecium isolate harboured plasmids of ca 36.3 and 23.1 kb. Curing experiments with ethidium bromide resulted in a bacteriocin-negative mutant which had not lost immunity to the bacteriocin. Slight differences in plasmid profiles between wild-type and mutant indicated a possible plasmid-coded bacteriocin production.

Africa↗

Study of two bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis.

AIMS: The antimicrobial activity of two plasmid-borne bacteriocins produced by Enterococcus casseliflavus IM 416K1 and Ent. faecalis IM 388C and their mating transferability were studied. METHODS AND RESULTS: Both bacteriocins showed antibacterial activity against taxonomically related micro-organisms and Listeria monocytogenes but differ for heat sensitivity, antimicrobial titre, molecular size and class of affiliation. The transferability by mating of the antibacterial properties from producers to Enterococcus faecalis JH2-2 revealed that the bacteriocin-phenotype was linked in both strains to genes located on a 34 MDa plasmid. This result was confirmed by loss of antibacterial activity and immunity after curing treatment. CONCLUSIONS: Restriction analysis has shown a different profile of the two conjugative plasmids. Enterocin 416K1 and Enterocin 388C could represent natural antilisterial agents to use in food technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The transferability of the 34 MDa conjugative plasmids might be considered a possibility for the study of bacteriocins expression in bacterial hosts different from the native strains.

Anti-Bacterial Agents↗

Durancin L28-1A, a new bacteriocin from Enterococcus durans L28-1, isolated from soil.

AIMS: To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil. METHODS AND RESULTS: Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant. CONCLUSIONS: Enterococcus durans L28-1 produced a new bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.

Amino Acid Sequence↗

Vancomycin-resistant Enterococcus faecium in a long-term care facility.

OBJECTIVE: To describe the epidemiology and natural history of colonization with vancomycin-resistant Enterococcus faecium (VREF) in a long-term care facility. DESIGN: All patients in whom VREF was isolated were followed prospectively, with rectal swab cultures at 2-week intervals, until discharge, death, or clearance of VREF. Clearance was defined as two consecutive negative cultures. In addition, three prevalence surveys were conducted of all patients in residence on one 34-bed intermediate care ward. SETTING: A 400-bed, long-term care Veterans Affairs facility. PARTICIPANTS: Thirty-six patients colonized with VREF. RESULTS: Vancomycin-resistant Enterococcus faecium was identified in 24 of the 36 patients at the time of transfer from an acute care facility. Seventeen patients had concomitant methicillin-resistant Staphylococcus aureus, and seven patients had a recent history of Clostridium difficile-associated diarrhea. VREF in these patients persisted for a median of 67 days after identification. Treatment of VREF colonization with antimicrobials was associated with prolongation of colonization. Serial surveillance of the 34-bed ward found stable rates of colonization, with only three documented instances of VREF acquisition. During 2.5 years of surveillance for infection, a single case of bacteremia occurred in a patient in whom colonization with VREF could not be demonstrated by rectal swab culture. No infections occurred in patients colonized with VREF. CONCLUSIONS: Long-term care patients have protracted carriage of VREF. Most will improve over time; however, receipt of antimicrobial therapy is associated with prolongation of VREF carriage. The risk of VREF infection is low in this population. When there are appropriate contact precautions, patient to patient transmission occurs at a low rate. These observations can be used to design a practical infection control strategy for long-term care facilities.

Adult↗