COMPARISON BETWEEN AUTOMATED SYSTEM AND PCR-BASED METHOD FOR IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF CLINICAL Enterococcus spp

Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci.     

Emergence of unusual species of enterococci causing infections,South India

Background  

Enterococci tend to be one of the leading causes of nosocomial infections, with E. faecalis and E. faecium accounting up to 90% of the clinical isolates. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any healthcare facility. Hence this work was undertaken to study the prevalence of unusual species of enterococci causing human infections, in a tertiary care hospital in South India.     

In vitro Assessment of the Host Response against Enterococcus faecalis Used in Probiotic Preparations

Abstract Along with other lactic acid bacteria, enterococci are used in food products and as health promoting agents. The safety of these products must be ensured, because they contain potentially pathogenic microorganisms. Here we present an in vitro opsonophagocytic assay that closely mimics the protective human immune response to Enterococcus faecalis and Enterococcus faecium. A collection of closely related E. faecalis isolates used as probiotics showed different susceptibilities to opsonic killing, suggesting that some of these isolates possess a capsule while other do not. This information may be helpful in assessing the safety of a given bacterial isolate used and could detect likely enterococcal candidates for probiotic preparations. This paper is dedicated to the founders of the Walter Marget Foundation, D. Adam and F. Daschner, in gratitude for their support of the training in infectious diseases.     

Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS

Objective To examine the clonal diversity of vancomycin‐resistant enterococci (VRE). Methods A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed‐field gel electrophoresis (PFGE) typing. Results Forty‐nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC ≥ 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, D_i = 0.93) and belonged to 24 PhP‐types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C→A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. Conclusion The isolated clinical VRE strains were highly diverse in Tehran.     

In Vitro Activity of LY333328, a New Glycopeptide, against Extracellular and Intracellular Vancomycin-Resistant Enterococci

Summary The objectives of the study were to observe the activity of LY333328, a new semisynthetic glycopeptide, compared to that of vancomycin against six strains of Enterococcus faecium and Enterococcus faecalis, including four vancomycin-resistant strains. Bacteria ingested by polymorphonuclear leukocytes (PMN) as well as extracellular bacteria were studied using a colony count method. The activity against intracellular bacteria was tested with the drugs present in the extracellular medium, as well as after preincubating the PMN and removal of the drugs. LY333328 is active against the tested enterococci, regardless of their susceptibility to vancomycin, with MICs of 1–2mg/l. It is bacteriostatic against extracellular enterococci at concentrations of 2 μg/ml and above regardless of their resistance to vancomycin. After 4 h incubation at 10 MIC, vancomycin-resistant strains of E. faecium and E. faecalis located intracellularly were reduced by 55% and 90%, respectively. Even after preincubation and removal of the drug, LY333328 had an effect at 10 MIC with a 20–30% reduction in the inoculum. The results suggest that in contrast to vancomycin, LY333328 is active against intracellular vancomycin-resistant enterococci, particularly E. faecalis, even after removal of the extracellular drug. Received: December 15, 1999 · Revision accepted: May 5, 2000     

Two Novel Lytic Bacteriophages Infecting Enterococcus spp. Are Promising Candidates for Targeted Antibacterial Therapy

The rapid emergence of antibiotic resistance is of major concern globally. Among the most worrying pathogenic bacteria are vancomycin-resistant enterococci. Phage therapy is a highly promising method for controlling enterococcal infections. In this study, we described two virulent tailed bacteriophages possessing lytic activity against Enterococcus faecalis and E. faecium isolates. The SSsP-1 bacteriophage belonged to the Saphexavirus genus of the Siphoviridae family, and the GVEsP-1 bacteriophage belonged to the Schiekvirus genus of Herelleviridae. The genomes of both viruses carried putative components of anti-CRISPR systems and did not contain known genes coding for antibiotic-resistance determinants and virulence factors. The conservative arrangement of protein-coding sequences in Saphexavirus and Schiekvirus genomes taken together with positive results of treating enterococcal peritonitis in an animal infection model imply the potential suitability of GVEsP-1 and SSsP-1 bacteriophages for clinical applications.     

Vancomycin-resistant enterococci among haemodialysis patients in Portugal: Prevalence and molecular characterization of resistance,virulence and clonality

Introduction

Vancomycin-resistant enterococci (VRE) among haemodialysis patients has increased rapidly and, to date, there is no report of this incidence in Portugal.

Methods

A total of 121 faecal samples were collected from haemodialysis patients, and then tested for VRE. Antimicrobial resistance, virulence and multilocus sequence typing (MLST) were studied.

Results

VRE prevalence was 3.3%. Three VRE isolates, Enterococcus faecium, Enterococcus faecalis and Enterococcus raffinosus, were multi-resistant and vanA-positive. E. faecium and E. faecalis belonged to CC17 and CC2, respectively.

Conclusion

Haemodialysis patients in Portugal are colonized with virulent, multi-resistant enterococci from high-risk clonal complexes, representing a public health concern.     

Recurrent septicemias withEnterococcus faecium

Summary A 17-year-old male patient with extrahepatic biliary atresia underwent an orthotopic liver transplantation in September 1994. In blood cultures drawn in November and (6 weeks later) December 1994, from bile secretions in May 1995, stool in June 1995 an wound abscess in August 1995, ampicillin-resistantEnterococcus faecium was isolated. Pulsed-field gel electrophoresis demonstrated the clonal identity of the isolates. To our knowledge, repeated infections with the sameE. faecium strain over a period of 9 months have not been described before. As multiple-resistant enterococci may colonize and reinfect liver transplant recipients for such a long time, preoperative antibiotic therapy should be administered cautiously in order not to select these organisms.     

Risk Factors for Infective Endocarditis in Patients with Enterococcal Bacteremia: A Case-Control Study

Abstract.Background: Based on previous studies, enterococcal infective endocarditis (IE) is considered a unimicrobial, community-acquired disease of older Caucasian men.Patients and Methods: We evaluated the relationship between enterococcal bacteremia and IE by comparing clinical and demographic characteristics of all cases of enterococcal IE within an 8-year period (n = 41) with controls randomly chosen from patients with enterococcal bacteremia without IE.Results: By univariate and multivariable analyses, the presence of a prosthetic valve (PV) and infection with Enterococcus faecalis were significantly associated with IE, while age, gender, race, polymicrobial infection and community-acquired infection were not. Almost an equal number of women and men had enterococcal IE. Cases of enterococcal IE were commonly nosocomial (39%) and polymicrobial (17%).Conclusions: Enterococcal endocarditis can no longer be considered exclusively a unimicrobial, community-acquired disease of Caucasian men. Instead, our data suggest that the presence of a PV and infection by E. faecalis are associated with an increased risk for IE.     

Risk Factors for Acute Cholangitis Caused by Enterococcus faecalis and Enterococcus faecium

Background/AimsAcute cholangitis (AC) is a potentially life-threatening bacterial infection, and timely antimicrobial treatment, faster than that achieved with bacterial cultures, is recommended. Although the current guidelines refer to empirical antimicrobial treatment, various kinds of antimicrobial agents have been cited because of insufficient analyses on the spectrum of pathogens in AC. Enterococcus spp. is one of the most frequently isolated Gram-positive bacteria from the bile of patients with AC, but its risk factors have not been extensively studied. This study aimed to analyze the risk factors of AC caused by Enterococcus faecalis and Enterococcus faecium.MethodsPatients with AC who were hospitalized in a Japanese tertiary center between 2010 and 2015 were retrospectively analyzed. Patients’ first AC episodes in the hospital were evaluated.ResultsA total of 266 patients with AC were identified. E. faecalis and/or E. faecium was isolated in 56 (21%) episodes of AC. Prior endoscopic sphincterotomy (EST), the presence of a biliary stent, prior cholecystectomy, and past intensive care unit admission were more frequently observed in AC patients with E. faecalis and/or E. faecium than in those without such bacteria. Prior EST was identified as an independent risk factor for AC caused by E. faecalis and/or E. faecium in the multivariate analysis.ConclusionsGiven the intrinsic resistance of E. faecalis and E. faecium to antibiotics, clinicians should consider empirical therapy with anti-enterococcal antibiotics for patients with prior EST.     

Species prevalence and antimicrobial susceptibility of enterococci isolated in a tertiary care hospital of North India

The present prospective study was carried out to determine the species distribution and antimicrobial susceptibilities of enterococci isolated from clinical samples in a tertiary care hospital of North India. Enterococcus species isolated from blood, urine, pus, sterile fluids and the hospital environment from October 2003 to January 2004 were identified by standard biochemical tests. Antimicrobial susceptibility testing was performed by the disk diffusion method as per NCCLS guidelines. Out of a total of 105 Enterococcus species recovered during the study period, E. faecium (42.90%) and E. faecalis (40.00%) constituted the predominant isolates. Enterococcus faecium was the commonest blood culture isolate while E. faecalis predominated pus and urine samples. Other species isolated were E. mundtii, E dispar, E. durans, E. avium, E. raffinosus and E. gallinarum. High-level aminoglycoside resistance was detected in 73.3% of isolates. Resistance to vancomycin, teicoplanin and linezolid was not detected. Prevalence of a wide variety of Enterococcus species in clinical samples together with their variable antimicrobial susceptibility patterns emphasizes the need for routinely carrying out detailed speciation and in vitro susceptibility testing of enterococcal isolates in the clinical bacteriology laboratory.     

Typing of Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Strains in a Cohort of Patients in an Italian Intensive Care Unit

Abstract Background: Vancomycin-resistant enterococci (VRE) have become a major cause of nosocomial infections. The increase of vancomycin-resistant Enterococcus faecium (VR-Efm) in an intensive care unit (ICU) of an Italian university hospital from 2003 through 2004, led us to evaluate the phenotypic and genetic features of these strains. The prevalence of different bacterial species in this ICU is described. The antibiotic resistance profiles of VR-Efm strains, their van-genotype and pulsed-field gel electrophoresis (PFGE) profiles were also analyzed. Materials and Methods: From January 2003 to December 2004, VR-Efm strains were collected from several biological samples. Bacteria were identified using standard biochemical reactions and automated systems. Antibiotic susceptibility was evaluated by disk diffusion and microdilution methods. Resistance to glycopeptides was confirmed by the E test. Vancomycin-resistant genotypes (vanA, vanB) were identified by PCR. Strains were typed by PFGE. Results: Fifty E. faecium strains were isolated from a total of 700 patients. Of these, 26 were vancomycin-resistant and were isolated from 26 different patients. We also found one strain with resistance to linezolid. The vanA genotype was identified in 20/26 strains and vanB in the remaining strains. A major pulsed-field cluster (“A”) was identified. In this cluster, 14 strains were identified (A1–A14) and 25 out of 26 VR-Efm belonged to it. Only one strain showed a different pattern (strain type “B”). All isolates with the vanA genotype belonged to cluster “A”, therefore five out of six isolates with the vanB genotype belonged to cluster A. The only strain with type B pattern was the vanB genotype. Conclusions: Isolation of VR-Efm was very frequent (52%) in our cohort of patients and the vanA genotype was the most frequent (77%). We found 25 out of 26 VR-E. faecium strains to be epidemiologically related by PFGE (cluster A). Strains with distinct genotypes shared closely related PFGE profiles. The occurrence of one major cluster among patients of a single unit indicated intra-facility VRE transmission.     

Epidemiology of Enterococcal Bacteremia in a Referral Center for Hepatobiliary Diseases

Summary Background: Enterococcus faecium (EFM) and Enterococcus faecalis (EFL) account for most infections which predominantly originate in the abdomen or the urinary tract. The objectives of this study were to compare the riskfactors associated with EFM and EFL bacteremia Patients and Method: Retrospective study of 64 EFL and 27 EFM bacteremia cases that occurred between January 1993 and December 1996 in a referral center for hepatobiliary diseases. Results: Univariate predictors of EFM bacteremia, compared to EFL, were an orthoptic liver transplantation (OLT), use of steroids, admission in the hepatology service, a central vascular catheter and an abdominal source. Forward regression models identified OLT as the only independent risk factor for EFM bacteremia (odds ratio, OR = 4.320; p = 0.0064), and septic shock as the only predictor of a fatal enterococcal bacteremia (OR = 13.152; p = 0.0003). Molecular typing of EFM isolates identified four small nosocomial clusters (of two to seven patients each) of EFM bacteremia, involving primarily patients admitted to the invasive care unit or on the hepatology ward. Conclusion: Strategies are needed to prevent enterococcal bacteremia in patients with severe liver disease, especially those undergoing OLT. Received: February 8, 2000 · Revision accepted: August 16, 2000     

IS element IS<Emphasis Type="Italic">16</Emphasis> as a molecular screening tool to identify hospital-associated strains of <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

Background  

Hospital strains of Enterococcus faecium could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, these techniques are laborious, time-consuming and cost-intensive. Our aim was to identify hospital E. faecium strains and differentiate them from colonizing and animal variants by a simple, inexpensive and reliable PCR-based screening assay. We describe here performance and predictive value of a single PCR detecting the insertion element, IS16, to identify hospital E. faecium isolates within a collection of 260 strains of hospital, animal and human commensal origins.     

Harnessing bacteriocin biology as targeted therapy in the GI tract

Recently, our laboratory demonstrated that bacteriocins produced by commensal enterococci provide an advantage in niche maintenance in the highly competitive environment of the gastrointestinal (GI) tract. Bacterial production of bacteriocins is a conserved defense strategy to help establish an ecological niche. Bacteriocin-encoding genes in enterococci are often carried on mobile genetic elements, including conjugative plasmids, enabling the transfer of such traits to other community members in a shared niche. Use of a novel mouse model for enterococcal colonization of the GI tract allowed us to investigate enterococcal dynamics and the role of enterococcal bacteriocins in the mouse GI tract. We examined the role of bacteriocin-21, carried on the pPD1 plasmid, in enterococcal colonization of the gut. We discovered that Enterococcus faecalis (EF) harboring pPD1 effectively colonizes the GI tract by using Bac-21 to eliminate its competition. In our study, we also present evidence for active conjugation in the GI tract, a strategy EF uses to enhance the number of bacteriocin producers in a given niche and eliminate bacteriocin-susceptible populations. Using an engineered strain of EF that is capable of producing Bac-21 but impaired in its conjugation ability, we were able to reduce pre-existing colonization by vancomycin-resistant enterococci in the mouse gut. Thus, our results suggest a novel therapeutic strategy to de-colonize antibiotic-resistant enterococci from the GI tract of patients and thereby prevent the emergence of resistant enterococcal infections that are otherwise difficult, or impossible, to treat.     

Virulence of Enterococcus isolates collected in Lower Silesia (Poland)

148 enterococcal strains: E.faecalis (108), E.faecium (35), E.gallinarum (3), E.casseliflavus (1) and E.durans (1) from various clinical specimens were investigated for their ability to adhere to Caco-2 and HEp-2 cell lines, and also for the presence of the esp gene, biofilm formation, production of haemolysins, DNAse and lipase. Several types of enterococcal adhesion to both cell lines were noted. An aggregative adherence was the most frequent among E.faecalis and E.faecium isolates. Other species presented various adhesive types. The occurrence of virulence factors in the whole group of strains was as follows: esp gene in 53.4%, biofilm in 45.3%, haemolysins in 15.5%, DNAse in 12.2% and lipase in 33.1% of enterococcal isolates. It appears that the adherence of the enterococci studied was not significantly associated with the presence of virulence factors.     

Interaction of Fibronectin and Aggregation Substance Promotes Adherence of Enterococcus faecalis to Human Colon

This in vitro study investigates the interaction between aggregation substance (AS), a virulence factor of Enterococcus faecalis, and colonic mucosal fibronectin in normal colon and colon from patients with Crohn's disease. Fibronectin was found to be overexpressed in Crohn's disease compared to normal colon. Compared to E. faecalis OG1X:pAM944 (AS-negative), E. faecalis OG1X:pAM721 (expressing AS) showed a significantly enhanced adhesion to human colonic mucosa in normal colon and in colon from patients with Crohn's disease. Double-staining of fibronectin and AS-positive enterococci showed that colocalization of bacteria and fibronectin was significantly more frequent in Crohn's disease than in normal colon. Preincubation of bacteria with soluble fibronectin caused a significant reduction in the adherence to fibronectin. In conclusion, the interaction between AS and fibronectin plays is an important factor that mediates adhesion of Enterococcus faecalis to colonic mucosa. This might be one of the mechanisms responsible for bacterial translocation of Enterococcus faecalis.     

Endocarditis due to group D streptococci. Comparison of disease caused by streptococcus bovis with that produced by the enterococci

The group D streptococci include the nonenterococcal Streptococcus bovis in addition to the classic enterococci. Endocarditis due to Strep. bovis has received little previous attention in the medical literature. A review of all cases of group D streptococcal endocarditis seen at the Massachusetts General Hospital between 1964 and 1973 revealed 14 cases caused by Strep. bovis and 15 by enterococci. There were only minor differences in the clinical presentations of endocarditis caused by these two groups of organisms. Although it contains the group D antigen Strep. bovis behaved like Strep. viridans in producing endocarditis. Moreover, the strains of Strep. bovis in this study were much more susceptible to penicillin than the enterococci. Therapy of severe enterococcal infections requires penicillin plus an aminoglycoside antibiotic whereas the present study strongly suggests that penicillin alone is adequate therapy for endocarditis due to Strep. bovis.     

Enterococcus faecium AND Enterococcus faecalis IN BLOOD OF NEWBORNS WITH SUSPECTED NOSOCOMIAL INFECTION

Enterococci are Gram-positive cocci saprophyte of the human gastrointestinal tract, diners who act as opportunistic pathogens. They can cause infections in patients hospitalized for a long time or who have received multiple antibiotic therapy. Enterococcus faecalis and Enterococcus faecium are the most common species in human infections. To evaluate the possibility of rapid detection of these species and their occurrence in the blood of newborns with suspected nosocomial infection, blood samples were collected from 50 newborns with late infections, admitted to the Neonatal Care Unit of the University Hospital Federal de Mato Grosso do Sul (UFMS-HU), from September 2010 to January 2011. The samples were subjected to conventional PCR and real time PCR (qPCR) to search for Enterococcus faecium and Enterococcus faecalis, respectively. The PCR results were compared with respective blood cultures from 40 patients. No blood cultures were positive for Enterococci, however, eight blood samples were identified as genomic DNA of Enterococcus faecium by qPCR and 22 blood samples were detected as genomic DNA of Enterococcus faecalis by conventional PCR. These findings are important because of the clinical severity of the evaluated patients who were found positive by conventional PCR and not through routine microbiological methods.     

An analysis of Group D streptococci recovered from cancer patients

Summary A total of 4,166 (17.4%) strains of Group D streptococci were recovered from 27,474 surveillance samples taken from the nose, throat, gingiva, axilla, and rectum of patients at a cancer center. An additional 2,217 strains were recovered from 7,106 urine samples and 58 strains from 5,286 blood samples. Approximately 11% of gingival cultures yielded Group D streptococci.Streptococcus faecalis was most prevalent, followed byStreptococcus bovis, Streptococcus faecium, Streptococcus avium, Streptococcus equinus, andStreptococcus durans. Six of the 11 premortem blood cultures positive forS. faecalis grew other organisms as well, chiefly gram negative bacilli. The sevenS. faecalis recovered from postmortem bloods all occurred together with gram-negative bacilli or yeasts. Abscesses and lesions had recovery rates of 7.9% and 13.2%, respectively.S. faecium was the Group D streptococcus resistant to the most antibiotics andS. bovis was the most susceptible. Although most strains were only moderately susceptible to penicillin by disc susceptibility tests, minimum inhibitory concentrations of representative strains were in the susceptible range. Ampicillin susceptibilities ranged from 71% to 93%. About half of theS. faecium andS. avium strains were resistant to carbenicillin, but the other species were more susceptible. Vancomycin, furadantin, and chloramphenicol all had 82% susceptibilities to Group D streptococci. The widely divergent antibiotic susceptibilities of the Group D streptococci makes their specific susceptibility tests an essential part of optimal patient care.

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Analyse der aus Krebspatienten isolierten Streptokokken der Gruppe D

Zusammenfassung 27 474 Probeabstriche aus Nase, Schlund, Zahnfleisch, Achselhöhle und Rektum von Patienten eines Krebszentrums ergaben die Isolation von insgesamt 4166 (= 17,4%) Streptokokkenstämmen der Gruppe D. Weitere 2217 Stämme wurden aus 7106 Harnproben und 58 Stämme aus 5286 Blutproben isoliert. Annähernd 11% der Zahnfleischkulturen ergaben Streptokokken der Gruppe D.Streptococcus faecalis überwog, gefolgt vonS. bovis, S. faecium, S. avium, S. equinus undS. durans. Sechs von elf prämortalen,S. faecalis-positiven Blutkulturen zeigten auch Wachstum anderer Organismen, insbesondere gramnegativer Bakterien. Sieben postmortale Blutproben ergabenS. faecalis zusammen mit gramnegativen Bakterien oder Hefen. Bei Abszessen und Verletzungen betrugen die Isolate 7,9% bzw. 13,2%. Als Streptococcus der Gruppe D erwies sichS. faecium gegenüber den meisten Antibiotika resistent undS. bovis am empfindlichsten. Obwohl sich im Blättchentest die meisten Stämme nur als mäßig Penicillin-empfindlich erwiesen, lag die minimale Hemmkonzentration repräsentativer Stämme im empfindlichen Bereich. Die Empfindlichkeit gegen Ampicillin schwankte zwischen 71 und 93%. Etwa die Hälfte der Stämme vonS. faecium undS. avium waren gegen Carbenicillin resistent, doch waren die anderen Spezies empfindlicher. Bei Vancomycin, Furadantin und Chloramphenicol lag die Empfindlichkeit von Streptokokken der Gruppe D über 82%. Die breite Streuung der Empfindlichkeit von Streptokokken der Gruppe D läßt ihre spezifische Testung zum wesentlichen Bestandteil optimaler Patientenbetreuung werden.