Clinical Isolates Of Enterococcus Faecalis Research Articles

Mechanisms of linezolid resistance in staphylococci and enterococci isolated from two teaching hospitals in Shanghai, China.

BackgroundLinezolid is one of the most effective treatments against Gram-positive pathogens. However, linezolid-resistant/intermediate strains have recently emerged in worldwide. The purpose of this study was to analyse the prevalence and resistance mechanisms of linezolid-resistant/intermediate staphylococci and enterococci in Shanghai, China.ResultsThirty-two linezolid-resistant/intermediate strains, including 14 Staphylococcus capitis, three Staphylococcus aureus, 14 Enterococcus faecalis and one Enterococcus faecium clinical isolates, were collected in this study which displayed linezolid MICs of 8 to 512 μg/ml, 8–32 μg/ml, 4–8 μg/ml and 4 μg/ml, respectively. All linezolid-resistant S. capitis isolates had a novel C2131T mutation and a G2603T mutation in the 23S rRNA region, and some had a C316T (Arg106Cys) substitution in protein L4 and/or harboured cfr. Linezolid-resistant S. aureus isolates carried a C389G (Ala130Gly) substitution in protein L3, and/or harboured cfr. The cfr gene was flanked by two copies of the IS256-like element, with a downstream orf1 gene. Linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. The semi-quantitative biofilm assay showed that 14 linezolid-resistant E. faecalis isolates produced a larger biofilm than linezolid-susceptible E. faecalis strains. Transmission electron microscopy showed the cell walls of linezolid-resistant/intermediate strains were thicker than linezolid-susceptible strains.ConclusionOur data indicated that major resistance mechanisms, such as mutations in 23S rRNA and ribosomal proteins L3 and L4, along with cfr acquisition, played an important role in linezolid resistance. Secondary resistance mechanisms, such as biofilm formation and cell wall thickness, should also be taken into account.

The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles.

Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18–24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL-1 of vancomycin and 8 μg mL-1 of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

Evaluation of Enterococcus faecalis clinical isolates with ‘penicillin-resistant, ampicillin-susceptible’ phenotype as reported by Vitek-2 Compact system

It has been recently reported that ampicillin susceptibility cannot accurately predict piperacillin and imipenem susceptibilities in penicillin-resistant, ampicillin-susceptible (Pen-R, Amp-S) Enterococcus faecalis isolates, contrary to the current Clinical and Laboratory Standards Institute (CLSI) recommendations. This has important therapeutic implications. Such isolates were noted after the use of Vitek-2 Compact system AST-GP67 susceptibility cards in a Singapore general hospital and they were increasing in numbers. The primary aim of this study was to evaluate these clinical isolates against microbroth dilution (MBD) technique and other commonly used antimicrobial susceptibility test (AST) methods for penicillin and ampicillin. The secondary aim was to evaluate whether ampicillin susceptibility could indeed be a reliable surrogate marker for piperacillin and imipenem susceptibilities in E. faecalis isolates that were confirmed Pen-R, Amp-S.From 2009 to 2013, a total of 49 isolates (5%) of 983 non-duplicate E. faecalis tested by Vitek-2 displayed the 'Pen-R, Amp-S' phenotype in a general hospital in Singapore. These were tested against MBD which was the reference method, Etest and disc diffusion for penicillin and ampicillin. Susceptibilities to piperacillin and imipenem were also tested using MBD. In addition, β-lactamase production test was performed. Forty E. faecalis isolates with penicillin-susceptible, ampicillin-susceptible (Pen-S, Amp-S) phenotype were included for comparative purposes.The categorical agreement rate was 100% for all AST methods in ampicillin reporting for the 'Pen-R, Amp-S' group of E. faecalis isolates. However, a large number of isolates (46 isolates, 93.9%) fell into the major error category for penicillin testing by the Vitek-2 system. Penicillin minimum inhibitory concentrations (MICs) generated by the Vitek-2 system for the majority of these isolates were two doubling dilutions higher compared to those obtained by the reference test. The Etest method correlated well with the MBD method. Thirty-two isolates (65.3%) were in categorical agreement with the MBD method when tested by the disc diffusion method for penicillin. Only three E. faecalis isolates (6.1%) were confirmed to have the uncommon penicillin resistance phenotype, with two of them showing resistance to piperacillin and intermediate to imipenem. β-lactamase production test was negative for all isolates. Among the Pen-S, Amp-S E. faecalis isolates, the categorical agreement was 100% for penicillin and ampicillin in all the tested methods.Enterococcus faecalis with 'Pen-R, Amp-S' phenotype reported by the Vitek-2 system using AST-GP67 susceptibility cards must be confirmed with a reference test, the Etest method being a good alternative. The Vitek-2 system generated higher penicillin MIC readings compared to MBD in this study. The actual prevalence of this uncommon penicillin resistance phenotype in E. faecalis was found to be low in this institution. More studies are required to confirm the reliability of ampicillin as a surrogate marker for piperacillin and imipenem susceptibilities in these isolates.

Identification of a Novel Clone, ST736, among Enterococcus faecium Clinical Isolates and Its Association with Daptomycin Nonsusceptibility

Resistance to daptomycin in enterococcal clinical isolates remains rare but is being increasingly reported in the United States and worldwide. There are limited data on the genetic relatedness and microbiological and clinical characteristics of daptomycin-nonsusceptible enterococcal clinical isolates. In this study, we assessed the population genetics of daptomycin-nonsusceptible Enterococcus faecium (DNSE) clinical isolates by multilocus sequence typing (MLST) and whole-genome sequencing analysis. Forty-two nonduplicate DNSE isolates and 43 randomly selected daptomycin-susceptible E. faecium isolates were included in the analysis. All E. faecium isolates were recovered from patients at a tertiary care medical center in suburban New York City from May 2009 through December 2013. The daptomycin MICs of the DNSE isolates ranged from 6 to >256 μg/ml. Three major clones of E. faecium (ST18, ST412, and ST736) were identified among these clinical isolates by MLST and whole-genome sequence-based analysis. A newly recognized clone, ST736, was seen in 32 of 42 (76.2%) DNSE isolates and in only 14 of 43 (32.6%) daptomycin-susceptible E. faecium isolates (P < 0.0001). This report provides evidence of the association between E. faecium clone ST736 and daptomycin nonsusceptibility. The identification and potential spread of this novel E. faecium clone and its association with daptomycin nonsusceptibility constitute a challenge for patient management and infection control at our medical center.

Diverse outcomes of Photodynamic Antimicrobial Chemotherapy on five Enterococcus faecalis strains

In the present study, the effectiveness of Photodynamic Antimicrobial Chemotherapy (PACT) was evaluated on planktonic cells and biofilms of five Enterococcus faecalis clinical isolates. Planktonic cells and biofilms of E. faecalis E2, E3, ER3/2s, OS16 and AA-OR34 were grown in SDMY medium plus 0.4% glucose. Approximately 5.0×10(7)CFU planktonic cells and 24h biofilms were subjected to PACT using the combination of Light Emitting Diodes (LEDs, Biotable(®)) and Photogem(®). The metabolic activity of bacterial cells was evaluated by a resazurin assay. Biomass values of the biofilms were determined by a crystal violet assay. Compared to the water-treated control group, gradual increases of light energy led to greater reduction of metabolic activity of planktonic cells and biofilms of E. faecalis when the combination of LEDs and Photogem(®) was applied. Photogem(®) alone significantly reduced the metabolic activity of planktonic cells, whereas LEDs or Photogem(®) alone did not result in biofilm viability changes. PACT yielded similar antimicrobial outcomes on planktonic cells of all tested E. faecalis strains, whereas biofilms of E. faecalis E3, ER3/2s and OS16 were more resistant to PACT than biofilms of E. faecalis E2 and AA-OR34. The efficacy of PACT on E. faecalis biofilms was strain dependent. PACT demonstrated its potential as an adjuvant antimicrobial treatment by killing of E. faecalis planktonic and biofilm cells.

Is the mazEF toxin-antitoxin system responsible for vancomycin resistance in clinical isolates of Enterococcus faecalis?

The current study was conducted to investigate the relationship between vancomycin-resistant Enterococcus faecalis (VRE) and the presence of mazEF toxin-antitoxin (TA) system, which may be useful as target for novel antimicrobial therapy concepts. The susceptibility of E. faecalis was determined by MIC, and the presence of the mazEF TA system was evaluated by PCR. Among 200 E. faecalis isolates 39.5% showed resistance to vancomycin (VRE), while 60.5% were susceptible strains (VSE). The mazEF TA system was positive in all VRE isolates (100%), but less prevalent (38/121, 31.4%) among the 121 VSE strains. In conclusion, our study demonstrated a positive relationship between the presence of vancomycin resistance and mazEF TA system. This observation may introduce therapeutic options against a novel antimicrobial target in enterococci.

Effects of bacterial products on the activity of odontoblast‐like cells and their formation of type 1 collagen

To study how products released from different bacteria in a deep carious lesion affect the metabolic activity of odontoblast-like cells and their ability to produce the major organic component of dentine, collagen 1. MDPC-23 cells were exposed to supernatants from biofilm cultures of strains isolated from the deepest part of a carious lesion as well as from a clinical isolate of Enterococcus faecalis. Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were used for comparison. Cell activity was assessed using an methyl-thiazolyl-diphenyl tetrazolium bromide (MTT) assay, and collagen 1 levels were determined by ELISA. The lesion microflora was dominated by Lactobacillus spp. Neither extracellular products from the isolates nor LPS affected the activity of the MDPC-23 cells, whereas extracellular products from E. faecalis and LTA significantly reduced total cell activity (P < 0.01). Enterococcus faecalis had an inhibitory effect upon collagen 1 production by the cells, whereas no such effect or even a slight stimulatory effect was seen for the isolates from the deep carious lesion. These studies indicate that culture supernatants from E. faecalis reduced the metabolic activity of odontoblast-like cells as shown using the MTT assay. No effect was seen for supernatants from biofilms of bacteria recovered from a deep carious lesion. Different bacteria varied in their effects upon collagen 1 production suggesting that the nature of the bacterial species in a carious lesion may have a direct influence upon the ability of the odontoblasts to produce tertiary dentine.

Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases.

Abundance and diversity of plasmid-associated genes among clinical isolates of Enterococcus faecalis

Enterococcus faecalis, a normal compound of the human intestinal microbiome, plays an important role in hospital-acquired infections. Plasmids make a significant contribution to the acquisition of the novel traits such as antimicrobial resistance and virulence by this pathogen. The study investigated the plasmid content and the diversity of plasmid-associated genes in a group of 152 hospital isolates of E. faecalis. The majority of plasmids visualized by pulsed-field gel electrophoresis of S1 nuclease-digested DNA fell into the range of 50–100kb. PCR-based screening allowed detection of genes of the rep1pIP501, rep2pRE25, rep4pMBB1, rep6pS86, rep7pT181, rep8pAM373, rep9pAD1/pTEF2/pCF10, rep10pIM13 and rep13pC194 families in 29 different combinations. The par and ω-ε-ζ plasmid stabilization systems were ubiquitous (45 isolates, 29.6% and 88 isolates, 57.9%, respectively), while the axe–txe system was not found. The asa1 gene homologues encoding aggregation substance characteristic for the pAD1 and related group of pheromone-responsive plasmids were present in 106 isolates. A variety of sequence variants, including novel ones, of genes associated with pheromone-responsive plasmids, such as rep8pAM373, rep9pAD1/pTEF2/pCF10, par, and asa1 were observed. In conclusion, there is a big and only partially characterized pool of diverse plasmids in clinical E. faecalis.

P116 Enterococcus faecalis and Enterococcus faecium clinical isolates carrying the vanA gene but susceptible to vancomycin

P116 Enterococcus faecalis and Enterococcus faecium clinical isolates carrying the vanA gene but susceptible to vancomycin

Transferable Plasmid-Mediated Resistance to Linezolid Due to cfr in a Human Clinical Isolate of Enterococcus faecalis

Nonmutational resistance to linezolid is due to the presence of cfr, which encodes a methyltransferase responsible for methylation of A2503 in the 23S rRNA. The cfr gene was first described in animal isolates of staphylococci, and more recently, it has been identified in Staphylococcus aureus from human clinical infections, including in an outbreak of methicillin-resistant S. aureus. In enterococci, cfr has been described in an animal isolate of Enterococcus faecalis from China. Here, we report an isolate of linezolid-resistant E. faecalis (603-50427X) recovered from a patient in Thailand who received prolonged therapy with the antibiotic for the treatment of atypical mycobacterial disease. The isolate lacked mutations in the genes coding for 23S rRNA and L3 and L4 ribosomal proteins and belonged to the multilocus sequence type (MLST) 16 (ST16), which is commonly found in enterococcal isolates from animal sources. Resistance to linezolid was associated with the presence of cfr on an ~97-kb transferable plasmid. The cfr gene environment exhibited DNA sequences similar to those of other cfr-carrying plasmids previously identified in staphylococci (nucleotide identity, 99 to 100%). The cfr-carrying plasmid was transferable by conjugation to a laboratory strain of E. faecalis (OG1RF) but not to Enterococcus faecium or S. aureus. The cfr gene was flanked by IS256-like sequences both upstream and downstream. This is the first characterization of the potential horizontal transferability of the cfr gene from a human linezolid-resistant isolate of E. faecalis.

Molecular screening of virulence genes in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in Northwest Iran

Purpose: The present study screened clinical isolates of Enterococcus faecalis and Enterococcus faecium to determine the prevalence of high-level gentamicin-resistant enterococci and the potential virulence genes among them. Materials and Methods: Clinical enterococcal isolates were obtained from three university teaching hospitals in Northwest Iran. Isolated enterococci were identified phenotypically followed by antibiotic susceptibility testing. Multiplex PCR was performed for the detection of genus, species-specific targets, gentamicin resistance, and potential virulence genes. Results: Of 220 enterococcal isolates, 133 (60.45%) isolates were identified as high-level gentamicin-resistant. Of these isolates, 79 (59.4%) and 54 (40.6%) were E. faecalis and E. faecium, respectively. All high-level gentamicin-resistant strains carried aac(6′)Ie-aph(2″)Ia. Of 220 isolates, 65.9% were positive for gelE, and 55%, 53.6%, 51.8%, and 49.5% of isolates were positive for cpd, asa1, ace, and esp, respectively. Phenotypically detected β-haemolytic strains (19.54%) were found to possess cylLlsMAB. Conclusion: The study revealed that high-level gentamicin-resistance was related to the presence of aac(6′)Ie-aph(2″)Ia. Isolated enterococci harboured potential virulence determinants, which were more common among E. faecalis than among E. faecium strains.

The Enterococcus faecalis Exoproteome: Identification and Temporal Regulation by Fsr

Analysis of the culture supernatant exoproteins produced by two PFGE clusters of high-level gentamicin and ciprofloxacin-resistant clinical isolates of Enterococcus faecalis from the UK and Ireland revealed two distinct protein profiles. This grouping distinguished OG1RF and GelE metalloprotease-expressing isolates from JH2-2 and other GelE-negative isolates. The integrity of the fsrABDC operon was found to determine the exoproteome composition, since an fsrB mutant of strain OG1RF appeared very similar to that of strain JH2-2, and complementation of the latter with the fsrABDC operon produced an OG1RF-like exoproteome. The proteins present in the supernatant fraction of OG1RF were separated using 2D gels and identified by mass spectrometry and comprised many mass and pI variants of the GelE and SprE proteases. In addition cell wall synthesis and cell division proteins were identified. An OG1RF fsrB mutant had a distinct exoprotein fraction with an absence of the Fsr-regulated proteases and was characterised by general stress and glycolytic proteins. The exoproteome of the OG1RF fsrB mutant resembles that of a divIVA mutant of E. faecalis, suggestive of a stress phenotype.

Novel Vancomycin-Resistance Transposon, Plasmid Replicon Types, and Virulence Factors of Vancomycin-ResistantEnterococciin Zhejiang, China

Forty-seven vancomycin-resistant Enterococcus (VRE) strains were isolated from clinical samples in 13 Zhejiang hospitals and fecal samples from ICU patients in a large teaching hospital in China. No VRE isolates were detected in healthy human subjects. CC17 was the main clonal complex in clinical Enterococcus faecium isolates but not in isolates from healthy human subjects. Novel vancomycin-resistance transposons were detected among VRE strains. This is the first report demonstrating insertion of tnpA and fosB genes in the vanRS-vanH intergenic region of Tn1546 leading to coresistance to vancomycin and fosfomycin. The four plasmid replicon types (pRUM, pRE25, pEF418, and pB82) were more common in VRE isolates, suggesting their association with vancomycin resistance and nosocomial transmission. The prevalence rate of vancomycin-resistant Staphylococcus aureus-related Inc18-like plasmid, pIP501, in VRE was 21.3%. The prevalence of the esp gene among VRE isolates was high (76.6%). In several VRE strains, the esp and hyl genes were cotransferred with the vanA gene by conjugation. Although the frequency of VRE is low in Chinese hospitals, its association with virulence determinants, the vancomycin-resistance transposon with other resistance gene insertions or plasmids may lead to multidrug resistance and the evolution of pathogenic VRE.

Virulence properties of a peptide hemolysin produced by &amp;lt;i&amp;gt;Enterococcus faecalis&amp;lt;/i&amp;gt;

The characterization and prevalence of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated. Recently, we described the production of a heat-stable hemolysin by clinical isolates of Enterococcus faecalis cultived in BHI-GA (BHI with glucose and L-arginine). Now, we purified the hemolysin from the culture supernatant by ultra-filtration (PM-10 membrane) and ethanol extraction followed by chromatography in a mBondapak C18 and Superdex Peptide columns. The hemolytic activity was not affected by the proteolytic enzymes. Cholesterol, phospholipids, EDTA and also bivalent ions did not inhibit the hemolytic activity. Among the various carbohydrates, only dextran 4 protected the erythrocytes against lyse. Scanning electron microscopy showed that lyse of erythrocytes occured at once after the exposure to the hemolysin. The mito-chondrial activity and the cell membrane integrity were significantly affected by the hemolysis, within 20 min of exposure and caused apoptosis after 12 h incubation, 51.92% in HeLa and 68% in HEp-2 cells, analyzed by flow cytometry. These results suggest that the heat-stable pore forming hemolysin might be a putative virulence factor in enterococci infections.

Antibacterial Activity of EndoSequence Root Repair Material and ProRoot MTA against Clinical Isolates of Enterococcus faecalis

Introduction Endodontic repair materials such as mineral trioxide aggregate (MTA) are used for various endodontic procedures. An alternative material to MTA with purportedly improved handling properties is EndoSequence Root Repair Material, which is available as premixed putty (ESP) or syringeable paste (ESS) and is described as possessing antibacterial activity during its setting reaction due to its highly alkaline pH. The aim of this in vitro study was to determine whether ESP and ESS possess antibacterial properties against a collection of Enterococcus faecalis strains recovered from root canal infections. The hypotheses tested were that (1) ESP and ESS possess antibacterial activity during their setting reaction, (2) there is no difference between ESP, ESS, and MTA in antibacterial activity, and (3) E. faecalis strains isolated from root canals differ in susceptibility to the materials. Methods The direct contact test was used. ESP, ESS, and white MTA were preincubated at 37°C in >95% humidity for 30 minutes and 24 hours before 1-hour direct contact exposure to E. faecalis strains (n = 10). Absence of antibacterial carryover effect from the materials to the bacterial cultures was confirmed. Log 10 viable counts were compared by using analysis of variance with significance level at P ≤ .05. Results Combining data for all strains, the mean (± standard deviation) log 10 viable counts for ESP (4.55 ± 0.85), ESS (4.5 ± 0.95), and MTA (4.12 ± 1.26) were significantly lower than for untreated controls (7.40 ± 0.33) ( P < .0001). The reduction in viable counts ranged from 1.86 ± 0.24 to 4.78 ± 0.42, with no statistically significant differences between the materials or preincubation periods. One strain was significantly more susceptible than 4 other strains. Conclusions ESP, ESS, and MTA had similar antibacterial efficacy against clinical strains of E. faecalis. Clinical strains varied in their susceptibility to the root repair materials.

Gelatinase-Associated Phenotypes and Genotypes Among Clinical Isolates of Enterococcus faecalis in Poland

Enterococcus faecalis is an important nosocomial pathogen causing serious invasive infections. One of the virulence factors of this pathogen, gelatinase GelE, is a protease whose gene expression is regulated by the Fsr quorum sensing system. In this study, we used a well-characterized collection of 153 clinical E. faecalis isolates to investigate the distribution of genes involved in gelatinase expression. Although 140 isolates (91% of the group) harbored the gelE gene, only 81 isolates (53%) produced active gelatinase. The gelatinase-negative phenotype was found in several unrelated clones, and appeared to be caused by various genetic events. Isolates of the hospital-adapted clonal complex 2 (CC2) and of CC40 were uniformly gelatinase-positive, while all the CC87 isolates contained the 23.9 kb deletion encompassing most of the fsr locus and were gelatinase-negative. No significant differences among isolates of different clinical origin and gelatinase activity or presence of the fsr genes were found with the exception of isolates from cerebrospinal fluid, which were more often gelatinase-positive than colonizing isolates.

Comparative study among clinical and commensal isolates of Enterococcus faecalis for presence of esp gene and biofilm production

Because of increasing difficulty in treating enterococcal infections, effort is being devoted to understanding factors that are responsible for causing nosocomial infection, with a focus toward targeting these factors with new therapeutics. Evidence has emerged that the esp genemediates biofilm formation in vitro, which helps the organism colonize and cause infection. This study was conducted over a four-year period in a tertiary-care hospital. There were 200 clinical pathogenic strains isolated from nosocomial infections and 100 commensals from stool specimens of healthy individuals. The study compared the production of biofilm and detection of the esp gene among clinical and commensal isolates. Among 200 clinical isolates of Enterococcus faecalis 65 (32.5%) isolates were positive for biofilm production and 60 (30%) for the esp gene by PCR. Among 100 commensal isolates, 16(8%) and 14 (7%) were positive for biofilm formation and the esp gene, respectively. Five clinical and two commensal isolates produced biofilm without any amplification of the esp gene. The study shows a significant difference in production of biofilm and presence of the esp gene between clinical and commensal isolates (P < 0.002). Therefore, it can be concluded that biofilm production has an important role in causing nosocomial infection. Although detection of the esp gene correlates with biofilm production, it may not be the only factor determining the formation of biofilm since few isolates produced biofilm without the esp gene. Strains isolated from indwelling medical devices showed high production of biofilm and esp gene.

Molecular analysis of linezolid resistance in clinical Enterococcus faecium isolates by polymerase chain reaction and pyrosequencing

Resistance to linezolid has been associated with a G2576T mutation in the 23S rRNA gene. Clinical isolates of linezolid-sensitive and linezolid-resistant vancomycin-resistant Enterococcus faecium of a liver transplant patient have been analysed for the G2576T mutation by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), conventional sequencing and pyrosequencing. A clear association between the number of mutated 23S rRNA genes and the level of linezolid resistance has been demonstrated. Linezolid susceptibility re-emerged after cessation of linezolid therapy; however, the re-initiation of linezolid therapy resulted in the re-emergence of linezolid-resistant strains. Pyrosequencing rapidly detected the number of mutated alleles and is superior to conventional PCR-RFLP for the detection of heterozygous mutations.

Comparative Study for the Presence of Enterococcal Virulence Factors Gelatinase, Hemolysin and Biofilm Among Clinical and Commensal Isolates of Enterococcus Faecalis

Background:Biofilm production, gelatinase and hemolysin are the potential virulence factors of Enterococci. Gelatinase and hemolysin producing strains of Enterococcus Faecalis have been shown to cause severe infections in animal models. Biofilm production has been shown to enhance the persistence of E. faecalis in urinary bladder and other medical indwelling devices infections.Aims:To compare the presence of gelatinase, hemolysin and biofilm formation among clinical and commensal isolates and to study the co-relation between virulence factors with respect to different clinical specimens.Settings and Design:During the study period of 2 years from July 2004 to July 2006, 200 clinical isolates from nosocomial infections and 100 commensal isolates of E. faecalis were taken for the study.Materials and Methods:The clinical and commensal isolates were tested for the presence of gelatinase, hemolysin and biofilm and compared. The presence of these virulence factors among different clinical isolates was also studied.Statistical Analysis:Chi-square and likelihood ratio analysis were carried out using SSPS version 5.1 software.Results:The clinical isolates produced 39, 16.5 and 32.5% of gelatinase, hemolysin and biofilm, respectively, as compared to 31, 19 and 16% produced by the commensal isolates, respectively. Endotracheal tube infection, urinary tract infection, umbilical catheter tip infected isolates produced 60.8, 86.6 and 100% biofilm, respectively.Conclusion:Significant difference in the production of biofilm (P<0.001) was noted between clinical and commensal isolates. Organism isolated from medically indwelling devices produced high amount of biofilm, confirming its role in colonization and causing nosocomial infections.