When was vre discovered

These data contributed to this study in terms of mapping the development of the outbreak i. Genetic characterization of the isolates by the PFGE was used for epidemiological characterization of the outbreak.

Contact tracing and screening revealed a total of 89 cases before the outbreak came to an end. All isolates were E. The negative predictive value of the vanA part of the PCR test was Distribution of patients positive for VRE was highest in September with 33 positive isolates. By July , no more positive samples were found among the screened samples. All 86 outbreak isolates were phenotypically and genotypically resistant to glycopeptide antibiotics.

All isolates were also resistant to ampicillin. Pulsotype 1. The index case strain ID 1 is highlighted with a yellow box. The pulsotypes are assigned by PFGE in a. The largest cluster cluster 1 consisted of 56 isolates The predominant pulsotype 1. The second most dominant pulsotype 1. Several isolates were indistinguishable from each other and hence clonally identical.

The second cluster cluster 2 showed a distinct pattern, compared to cluster 1, and consisted of 21 isolates The predominant pulsotype in the second group 2.

Monthly distribution of cluster 1 and cluster 2 related strains. Strains unrelated to the two main clusters are not included in the chart. Most of the isolates in cluster 1 were found during screening September and October , concurrent with the decline of positive isolates related to strains in cluster 1, cluster 2 related strains were introduced in November All isolates were identified as E.

Pulsotypes are indicated with colors; green and blue correspond to isolates in cluster 1, while red and pink correspond to isolates in cluster 2. Unrelated strains are illustrated colorless, except for the index case strain in yellow color. Three isolates 3. Cases were reported from more than ten different wards out of a total of 23, including both medical and surgical specialties. Retrospective review of hospital admissions showed that many patients were admitted to multiple wards during their hospital stays.

A total of 40 VRE-carrying patients were identified in the first two months of the outbreak August and September Seven of the remaining 12 patients were indirectly associated with one of these three hospital wards. They had been hospitalized together with a VRE-positive patient with previous stay at one of these wards during July-October, however at the time the VRE-positive diagnosis was not yet stated.

In particular, in March , four isolates with pulsotype 1. Review of their medical records showed that patients numbered 78—80 were hospitalized simultaneously in March at the same ward, and that patient number 78 had previously been hospitalized at one of the three index wards on two occasions in July , before the primary outbreak was discovered.

None of the 21 isolates belonging to cluster 2 were isolated from patients that had been admitted to any of the index outbreak wards in the three first months of the outbreak. Before proven positive, 15 of these patients had tested negative in earlier screening tests conducted after the peak of the outbreak. Spread of VRE is a serious issue in many hospitals. Rapid and sensitive microbial detection is important to limit the spread of infection, and determination of epidemiological relatedness is necessary for investigation of contamination patterns of resistant bacteria, and for outbreak management.

Genotyping results are, however, often available only far into the outbreak, or even after the situation has ended. In this study, we describe epidemiological relatedness of Vancomycin-resistant E. From screening conducted throughout all hospital wards, the majority of patients with VRE strains in PFGE cluster 1 were hospitalized within the three consecutive months July, August and September , in only three of the 23 hospital wards the three index wards. In addition, a few isolates belonging to cluster 1 were detected in patients hospitalized together in March , of which one had previously been hospitalized in one of the index wards July Likely, this patient carried the infection at that time, and initiated the small outbreak when rehospitalized March In conclusion, spread of isolates belonging to cluster 1 seems to mainly have taken place in three hospital wards Gastric surgery, Infectious diseases, and Pulmonary diseases during a three-month period starting from July , before the outbreak was recognized.

After the discovery of the first VRE isolate mid-August , 49 isolates were identified by the end of October, 42 belonging to cluster 1 and seven sporadic cases. Following initiation of infection control measures throughout September, a continuous and rapid decreasing incidence could be seen, proving that the increased focus on infection control and the measures taken were highly effective in limiting the outbreak. The importance of hand hygiene and other infection control issues in limiting the spread of hospital bacteria is also well documented in previous studies 27 , 28 , 29 , The majority of isolates belonging to cluster 2 were from patients who had not been hospitalized in any of the three index outbreak wards in the three summer months of , and most of these patients had screened negative in the early phase of the outbreak.

The better understanding of the outbreak dynamic obtained with PFGE genotyping results underscores the great importance of the implemented infection control measures.

The second outbreak did not spread as much as the first, probably because effective infection control measures were already established. An interesting finding in this study is the fact that the first index case isolate, which was a clinical isolate and the reason why screening was initiated, was unrelated to any of the two outbreak clusters, and hence not actually linked to the large outbreak in the three index wards caused by cluster 1 VRE strains.

Thus the outbreak was discovered by coincidence, which underscores the need for systematic screening of potentially at-risk patients, including all patients transferred from hospitals abroad or from central large hospitals, for early detection of hospital outbreaks. As is common in outbreaks with VRE, the majority of VRE-positive patients in our study was colonized with the bacteria, but did not have an infection. The strains found unrelated to both cluster 1 and 2, including the index case isolate, may have originated from patients colonized with VRE prior to the outbreak.

It is unknown how long VRE is carried, but colonization could persist from weeks to months 31 , 32 , 33 , hence colonized patients will occasionally be admitted to hospitals and other health care institutions.

However, three of the nine unrelated isolates were found in patients who had screened negative for VRE in an early phase of the outbreak, and thus the place and time for their colonization remains unknown. The negative predictive value of the test was excellent, and isolation precautions could therefore be terminated when a negative test result was recorded. The method had, however, a poor specificity and a high false positive rate for the vanB gene, also described by others 34 , The reason is most likely the frequent occurrence of this gene in various other bacterial species in the human gut.

Due to the low resolution within the two main clusters, and the inconsistent distribution between strains unrelated to the two main clusters, the method will have limitations in small local epidemiological studies, with limited allelic variation between isolates. Since real-time PCR technology is available in most routine laboratories, the method could, however, serve as a good choice for long-term studies of VRE isolates or for international comparison studies between hospitals, where the allelic variations at the SNP sites are greater.

HRM analysis incorporates not only information from each key SNP, but also from neighboring SNPs, and this information can improve the discriminatory power In addition, the method is promising for antibiotic resistance detection 38 , and is shown to be able to distinguish between vanB resistant and sensitive E.

Moreover, within each cluster, only a low or medium correlation between the two methods was found. It is however important to take into account that different typing techniques measure different cellular properties Discrimination at the strain level is influenced by minor changes in the mass spectra, which highlights the importance of applying similar culture conditions i. The major advantage with MALDI-TOF MS is its wide use as a routine microbial identification technique in hospitals worldwide, and typing information is thus readily available during an outbreak.

Studies have suggested that the method could serve as a first-line typing tool for investigation of possible hospital outbreaks of microorganisms such as E.

Although PFGE is a cumbersome method, it has up to recently been considered the gold-standard in epidemiological investigation of hospital outbreak situations due to the high resolution and ability to discriminate strains with minor genetic changes This is especially important in typing hospital associated strains where the rate of recombination is assumed to be relatively low compared to the high rate of recombination found elsewhere 52 , In addition, all three methods identified the index case strain as unrelated to the two main outbreak strains.

The major drawback with PFGE is the time consuming laboratory protocol which may hamper or delay the discovery of an outbreak. The DNA restriction patterns may differ slightly due to technicalities and technician performances, and even if the band pattern analysis is performed by data software, subjective investigation by trained personnel is often necessary.

Moreover, single variations can occur at restriction sites and lead to more than one band shift which may affect the epidemiological investigation Whole genome sequencing WGS of bacterial pathogens via Next Generation Sequencing NGS technologies has emerged as a powerful tool for determining the relatedness of bacterial isolates in outbreak situations 54 , 55 , WGS analysis of entire genomes provides markedly higher resolution than those of conventional methods, and full genetic information can be obtained.

Investigations of relatedness can be based on entire genome sequences, or e. Powerful NGS pipelines with reference genomes, e. While WGS show many advantages over standard microbiological methods, it is not yet widely implemented in routine hospital diagnostics.

Notable challenges have included bioinformatics workflow, costs, manpower, laboratory infrastructure, and quality control, but the technology is continuously developing towards more simple and affordable solutions 59 , Moreover, our comparison of genotyping and clustering methods showed that despite lower resolution than PFGE, readily available MALDI-TOF MS data may be of great importance for infection control and prevention if routinely used in real-time microbiological surveillance in health care institutions.

The experimental data that support the findings of this study are available from the corresponding author, A. R, upon reasonable request.

Medical Records that support the epidemiological finding of this study are not publicly available due to privacy restrictions. Fisher, K. The ecology, epidemiology and virulence of Enterococcus. Hidron, A. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, — Infec Control Hosp Epidemiol.

Article Google Scholar. Leclercq, R. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med. Sahm, D. In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrob Agents Chemother. Cattoir, V. Twenty-five years of shared life with vancomycin-resistant enterococci: is it time to divorce?

J Antimicrob Chemother. Sievert, D. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, — Infect Control Hosp Epidemiol.

Article PubMed Google Scholar. Walsh, C. Molecular mechanisms that confer antibacterial drug resistance. Hollenbeck, B. Intrinsic and acquired resistance mechanisms in enterococcus. Arthur, M. This approach significantly decreases the high innovation cost and time normally associated with bringing a new drug to the clinic [ 19 ].

Using this approach, we recently discovered ebselen, a multifunctional organoselenium molecule in clinical trials, it possesses a potent antibacterial activity against important Gram-positive bacterial pathogens including VRE [ 20 , 21 ]. Ebselen is being evaluated for various applications including cancer, cardiovascular disorders and kidney disorders [ 22 , 23 ]. The activity of ebselen was established against a wide range of microbes including several staphylococcus strains, Escherichia coli , Bacillus subtilis , Helicobacter pylori , Candida albicans and Aspergillus niger [ 24 ].

Despite its known antimicrobial activity, the potential to use ebselen as a decolonizing agent against VRE has not been investigated. The antibacterial activity of ebselen, linezolid, vancomycin, and ramoplanin was evaluated against 27 strains of enterococci from humans and animals. Most of the tested strains, Table 1 , were resistant to vancomycin. Utilizing the broth microdilution assay, ebselen was found to possess potent antibacterial activity against all the tested isolates Table 1.

After confirming the potent antibacterial activity of ebselen, we sought to investigate if ebselen is bacteriostatic or bactericidal against VRE. Linezolid exhibited a similar pattern of activity to ebselen at both concentrations. In contrast, ramoplanin was found to exhibit rapid bactericidal action, completely reducing the burden of VRE to zero after four hours at both test concentrations. Recognizing the great propensity of enterococci to develop resistance to antibacterial agents [ 25 ], we were curious to test whether or not VRE can develop resistance to ebselen.

To investigate this point, ebselen was evaluated via a multi-step resistance selection experiment against vancomycin-resistant E. As depicted in Fig 2 , E. Similar effects were observed with linezolid and ramoplanin only one-fold increase in MIC.

In contrast, resistance to gentamicin emerged rapidly. After the second passage, the MIC of gentamicin increased seven-fold. Although there was 1-fold increase in the MICs of Linezolid and ramoplanin, unlike ebselen, they did not cross the 4-fold cutoff limit that distinguishes sensitivity from resistance [ 26 ]. The MIC of all the test agents was determined daily for 14 passages to test for the development of resistance increase in MIC to the tested isolate.

A 4-fold increase in the MIC is indicative of resistance formation. We next moved to investigate if ebselen would be capable of interfering with a key virulence factor, biofilm formation, important for GIT colonization by VRE.

Interestingly, ebselen was found to exhibit a concentration-dependent inhibition of VRE biofilm formation. Linezolid, in contrast, was only effective at inhibiting biofilm formation at 0. A Biofilm inhibition activity of ebselen.

The biofilm mass OD was measured after staining with crystal violet and destaining with ethanol. B Biofilm eradication activity of ebselen. Supra-inhibitory concentrations of the drugs were then added and incubated with the bacterial biofilm for additional 24 hours before the biofilm density was measured OD by crystal violet staining.

We also investigated the ability of ebselen to disrupt mature, adherent VRE biofilm. Ebselen was superior to all other tested drugs in eradicating established VRE biofilm. Guided by the protocol of Ubeda et al. Both ebselen 0. Ebselen continued to reduce the burden of VRE by 2. This was similar to the result obtained for ramoplanin which reduced the burden of VRE relative to untreated mice in fecal samples by 2.

Infected mice were orally treated with ebselen 0. One group was left untreated. Fecal samples were freshly collected from mice in each group on days 0, 5, 10, 15, and 20 post treatment. In addition to examining the presence of VRE in stool samples of infected mice, the burden of VRE present in the cecum and ileum of mice was determined.

One day after the final treatment was administered, mice were humanely euthanized and the cecum and ileum were aseptically removed and homogenized to determine viable bacterial CFU. Similar to results obtained from the fecal samples, ebselen and ramoplanin significantly diminished the burden of VRE in both the cecal and ileal contents Fig 5.

Ebselen reduced the burden of VRE by 0. Ramoplanin generated a 2. Cecum and ileum contents were collected one day after the last treatment was administered day 21 of experiment. No significant difference was found between ebselen-treated and ramoplanin-treated groups.

The challenge of multidrug-resistant enterococcal infection continues to pose a threat to patients in healthcare facilities. Due to their broad tissue tropism, enterococci can infect a wide variety of human organs. Enterococci, principally E. Treatment of enterococcal infections has become increasingly challenging given the remarkable ability of enterococci to develop resistance to antibacterial agents [ 3 , 25 ].

The emergence of clinical isolates of E. Though newer antibiotics such as linezolid have become the mainstays of treatment for VRE infections, these antibiotics are not immune to resistance development. As highlighted in a recent study by Bi et al , enterococci exhibiting resistance to linezolid represents an emerging problem globally [ 28 ]. Interestingly, the ability of enterococci to develop resistance against antibiotics is more prominent in strains of E.

Given the potential challenge of treating VRE infections once they arise, alternative approached to combating infection are needed. One of the leading events that heralds enterococcal infections is gastrointestinal colonization. Enterococci normally reside in the human GIT as a member of the gut microflora.

In normal settings, the population of enterococci remains in balance with the other members of the healthy bacterial consortium of the gut.

However, administration of broad-spectrum antibiotics can disrupt the integrity of the normal gut flora leading to diminished ability to resist enterococcal overgrowth including strains of antibiotic-resistant enterococci. This GIT colonization has two major consequences: infection of the colonized individual and cross-transmission of enterococci to other patients residing within the same healthcare facility [ 15 , 29 — 31 ].

Important qualities to seek in a decolonizing agent for VRE include potent antibacterial activity against VRE, stability to resistance development, safety to humans, and efficacy to decrease the burden of VRE in the intestinal tract.

To date, no agent exists that possesses all of these qualities. Thus there remains a need to identify and develop new decolonizing agents effective against VRE. Ebselen is an organoselenium compound that is being investigated for the treatment of various conditions and has been proven to be safe for human use [ 33 , 34 ].

Herein, the capability of ebselen to serve as a novel decolonizing agent against VRE was investigated. Initially, the antibacterial activity of ebselen was evaluated against more than 20 clinical isolates of VRE. It is important to highlight that ebselen was effective against both E. Likewise, ebselen was active against both VRE and vancomycin-sensitive strains. When evaluated against VRE in a time-kill assay, ebselen was found to exert a bacteriostatic activity, similar to linezolid.

Although bacteria are more likely to develop resistance against bacteriostatic drugs [ 40 ], no change in MIC for ebselen was observed in a multi-step resistance selection experiment. This is similar to a previous report where resistant mutants to ebselen could not be isolated for other Gram-positive bacteria, including S. In this manner, both the establishment of new colonization and the level of colonization of those already colonized could be minimized. Multidrug-resistant enterococci continue to pose problems in U.

The best available evidence suggests that the emergence and spread of these pathogens are promoted by poor infection control techniques and by antibiotic selective pressure. Antibiotic selective pressure favoring the emergence and spread of VRE may involve more than simply the extent of vancomycin use.

Specifically, extended-spectrum cephalosporins and similarly active beta-lactams and drugs with potent activity against anaerobes appear to predispose to VRE colonization and infection. On the other hand, data from animal models suggest that the cephalosporins predispose to establishment of VRE colonization through their potent activity against many bacteria and essential lack of activity against ampicillin-resistant enterococci.

On the other hand, antianaerobic antibiotics appear to favor persistence of high levels of VRE colonization through their activity against competing flora. A more detailed understanding of the impact of different antibiotics on the upper and lower gastrointestinal flora will be an important step in controlling the emergence and spread of VRE.

His primary research interests are in the mechanisms of antimicrobial resistance and resistance transfer in enterococci and the evolution of extended-spectrum beta-lactamases in gram-negative bacilli. Table of Contents — Volume 7, Number 2—April Please use the form below to submit correspondence to the authors or contact them at the following address:.

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Facebook Twitter LinkedIn Syndicate. Table 1 Table 2. Article Metrics. Abstract Vancomycin and ampicillin resistance in clinical Enterococcus faecium strains has developed in the past decade. Ampicillin Resistance.

Vancomycin Resistance. Figure Figure. Risk Factors for Multidrug-Resistant Enterococci. Nonglycopeptide Antibiotics and VRE. Major trends in the microbial etiology of nosocomial infection. Am J Med. An overview of nosocomial infections, including the role of the microbiology laboratory.

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Arch Intern Med. Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin. J Bacteriol. Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC in Enterococcus faecium and Enterococcus faecalis.

J Clin Invest. Evidence of incorporation of the chromosomal-lactamase gene of Enterococcus faecalis CH19 into a transposon derived from staphylococci.

Antimicrob Agents Chemother. Isolation of a beta-lactamase-producing, aminoglycoside-resistant strain of Enterococcus faecium. J Infect Dis. J Gen Microbiol. Genetic linkage and co-transfer of a novel, vanB-encoding transposon Tn and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate.

Identification of a genetic element psr which negatively controls expression of Enterococcus hirae expression. Evidence that the PBP 5 synthesis repressor psr of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia.

N Engl J Med. Glycopeptide resistance in enterococci. Trends Microbiol. The vanB gene of vancomycin-resistant Enterococcus faecalis V is structurally related to genes encoding D-ala: D-ala ligases and glycopeptide-resistance proteins VanA and VanC.

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