A PCR assay for rapid detection of vancomycin-resistant enterococci. - Related Documents




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596801.0000A PCR assay for rapid detection of vancomycin-resistant enterococci. Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.200212007446
597410.9999Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus. As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.201021083822
597220.9998Method of Selection of Bacteria Antibiotic Resistance Genes Based on Clustering of Similar Nucleotide Sequences. A new method for selection of bacterium antibiotic resistance genes is proposed and tested for solving the problems related to selection of primers for PCR assay. The method implies clustering of similar nucleotide sequences and selection of group primers for all genes of each cluster. Clustering of resistance genes for six groups of antibiotics (aminoglycosides, β-lactams, fluoroquinolones, glycopeptides, macrolides and lincosamides, and fusidic acid) was performed. The method was tested for 81 strains of bacteria of different genera isolated from patients (K. pneumoniae, Staphylococcus spp., S. agalactiae, E. faecalis, E. coli, and G. vaginalis). The results obtained by us are comparable to those in the selection of individual genes; this allows reducing the number of primers necessary for maximum coverage of the known antibiotic resistance genes during PCR analysis.201729063318
597330.9998DNA microarray detection of antimicrobial resistance genes in diverse bacteria. High throughput genotyping is essential for studying the spread of multiple antimicrobial resistance. A test oligonucleotide microarray designed to detect 94 antimicrobial resistance genes was constructed and successfully used to identify antimicrobial resistance genes in control strains. The microarray was then used to assay 51 distantly related bacteria, including Gram-negative and Gram-positive isolates, resulting in the identification of 61 different antimicrobial resistance genes in these bacteria. These results were consistent with their known gene content and resistance phenotypes. Microarray results were confirmed by polymerase chain reaction and Southern blot analysis. These results demonstrate that this approach could be used to construct a microarray to detect all sequenced antimicrobial resistance genes in nearly all bacteria.200616427254
599240.9998Emergence of Enterococcus gallinarum carrying vanA gene cluster displaying atypical phenotypes. Motile enterococci such as Enterococcus gallinarum has the ability to acquire and transfer antibiotic resistance genes to other enterococci. Even though infections caused by E. gallinarum are rare, the discovery of this bacteria in food sources and in clinical environments is disturbing. Here, we report the isolation and identification of E. gallinarum from the wound of a hospital in-patient. The isolate was identified using 16S rDNA sequencing. Isolate 146 harboured the vanA and vanC1 gene clusters, was vancomycin-susceptible, and displayed resistance to ampicillin, penicillin, erythromycin and teicoplanin. This isolate also showed intermediate resistance to linezolid and sequencing of the 23S rRNA peptidyl transferase region did not unveil any known mutations associated to the conferment of linezolid resistance. The presence of vanA did not confer resistance to vancomycin. Structural analyses into the Tn1546 transposon carrying the vanA gene revealed distinct genetic variations in the vanS, vanY and vanS-vanH intergenic region that could be associated to the atypical antibiotic resistance phenotypes of isolate 146. Finding from this study are suggestive of the occurrence of interspecies horizontal gene transfer and that similarities in genotypic characteristic may not necessarily correlate with actual antibiotic resistance pattern of E. gallinarum.201633579083
583350.9998Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing. Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.200717141897
590360.9997Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. In a previous study, the authors isolated lactic acid bacteria from breast milk of healthy mothers. Since some of the identified isolates belonged to the species Enterococcus faecium, the objective of this work was to evaluate their safety. The enterococcal strains were screened by polymerase chain reaction (PCR) and Southern hybridization for the presence of virulence determinants. The potential of the strains to acquire plasmids by conjugation was investigated by screening for genes involved in conjugation processes. Parallel, phenotypic assays were performed. Presence of genes conferring resistance to vancomycin was assessed by PCR. PCR amplifications and Southern hybridizations revealed that all the strains were clear of the majority of potential virulence determinants. None of the strains showed gelatinase activity, hemolysin production, or aggregation phenotype, and none carried the vanA or vanB genes. These findings suggest that milk of healthy mothers may be a source of avirulent E faecium isolates to the newborns.200515886339
549870.9997The prevalence of multidrug resistance in Staphylococcus hominis isolated from clinical materials. The treatment of infections caused by Staphylococcus hominis remains a challenge, mainly due to the increasing resistance of these bacteria to antibiotics. The aim of the study was to determine antibiotic resistance in 62 strains S. hominis isolated from clinical materials, and to identify the molecular basis of resistance to antibiotics. Forty-six strains were both methicillin-resistant and harbored the mecA gene. Twenty-three of these strains had mec complex A and ccr complex AB1. Such a combination of the mec and ccr complexes does not correspond to any cassettes that have been demonstrated so far. However, over 80% of the tested strains were multidrug-resistant, of which as many as 12 were resistant to at least seven antibiotics. More than a half of strains harbored the tetK, acc(6')-Ie aph(2''), and ant(4')-I genes. erm(C) was the most common resistant gene to antibiotics from the MLS group. Two strains had as many as five antibiotic resistance genes from the tested groups (erm(C), msr(A), msr(B), mph(C), lnu(A)). The presence of the vga gene encoding resistance to streptogramins A was detected in one strain. All of strains were sensitive to vancomycin. However, 11 of them had reduced sensitivity to this antibiotic and eight of them were characterized by a heterogeneous resistance profile to this antibiotic. Our results clearly shows increasing threat of S. hominis caused by their multi-resistance. Moreover, these bacteria can constitute a reservoir of resistance genes for more pathogenic bacteria.202539747570
583280.9997New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.200415528678
597190.9997Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. In this study the feasibility of 50- and 60-mer oligonucleotides in microarray analysis for the detection and identification of antibiotic resistance genes in various Salmonella strains was assessed. The specificity of the designed oligonucleotides was evaluated, furthermore the optimal spotting concentration was determined. The oligonucleotide microarray was used to screen two sets of Salmonella strains for the presence of several antibiotic resistance genes. Set 1 consisted of strains with variant Salmonella Genomic Island 1 (SGI1) multidrug resistance (MDR) regions of which the antibiotic resistance profiles and genotypes were known. The second set contained strains of which initially only phenotypic data were available. The microarray results of the first set of Salmonella strains perfectly matched with the phenotypic and genotypic information. The microarray data of the second set were almost completely in concordance with the available phenotypic data. It was concluded that the microarray technique in combination with random primed genomic labeling and 50- or 60-mer oligonucleotides is a powerful tool for the detection of antibiotic resistance genes in bacteria.200515823391
5969100.9997Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.200515872258
5773110.9997LBJMR medium: a new polyvalent culture medium for isolating and selecting vancomycin and colistin-resistant bacteria. BACKGROUND: Multi-drug resistant bacteria are a phenomenon which is on the increase around the world, particularly with the emergence of colistin-resistant Enterobacteriaceae and vancomycin-resistant enterococci strains. The recent discovery of a plasmid-mediated colistin resistance with the description of the transferable mcr-1 gene raised concerns about the need for an efficient detection method for these pathogens, to isolate infected patients as early as possible. The LBJMR medium was developed to screen for all polymyxin-resistant Gram-negative bacteria, including mcr-1 positive isolates, and vancomycin-resistant Gram-positive bacteria. RESULTS: The LBJMR medium was developed by adding colistin sulfate salt at a low concentration (4 μg/mL) and vancomycin (50 μg/mL), with glucose (7.5 g/L) as a fermentative substrate, to a Purple Agar Base (31 g/L). A total of 143 bacterial strains were used to evaluate this universal culture medium, and the sensitivity and specificity of detection were 100% for the growth of resistant strains. 68 stool samples were cultured on LBJMR, and both colistin-resistant Gram-negative and vancomycin-resistant Gram-positive strains were specifically detected. CONCLUSIONS: The LBJMR medium is a multipurpose selective medium which makes it possible to identify bacteria of interest from clinical samples and to isolate contaminated patients in hospital settings. This is a simple medium that could be easily used for screening in clinical microbiology laboratories.201729169321
5693120.9997Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broad range of gram-negative bacterial pathogens. A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.201323129055
5814130.9997Role of CRISPR-Cas system on antibiotic resistance patterns of Enterococcus faecalis. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in Enterococcus faecalis. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among E. faecalis isolates. A total of 151 isolates of E. faecalis were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant E. faecalis strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive strains. These data indicate a negative correlation between CRISPR-cas loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.202134321002
2441140.9997Phenotypic and molecular assessment of antimicrobial resistance profile of airborne Staphylococcus spp. isolated from flats in Kraków. Bacteria of the genus Staphylococcus were isolated from air sampled from living spaces in Kraków (Poland). In total, 55 strains belonging to the genus Staphylococcus were isolated from 45 sites, and 13 species of coagulase-negative staphylococci were identified. The species composition of studied airborne microbiota contains Staphylococcus species that are rarely infectious to humans. Most commonly isolated species comprised S. hominis and S. warneri. The disk-diffusion tests showed that the collected isolates were most frequently resistant to erythromycin. The PCR technique was employed to search for genes conferring the resistance in staphylococci to antibiotics from the group of macrolides, lincosamides and streptogramins. The analyzed Staphylococcus isolates possessed simultaneously 4 different resistance genes. The molecular analysis with the use of specific primers allowed to determine the most prevalent gene which is mphC, responsible for the resistance to macrolides and for the enzymatic inactivation of the drug by phosphotransferase. The second most often detected gene was msrA1, which confers the resistance of staphylococci to macrolides and is responsible for active pumping of antimicrobial particles out of bacterial cells.201728955110
5970150.9997DNA microarray for detection of macrolide resistance genes. A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria.200616723563
5502160.9997Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk. The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.201930928272
3659170.9997Resistance to vancomycin and teicoplanin: an emerging clinical problem. Vancomycin and teicoplanin are glycopeptides active against a wide range of gram-positive bacteria. For 30 years following the discovery of vancomycin in 1956, vancomycin resistance was not detected among normally susceptible bacteria recovered from human specimens. Since 1986, however, bacteria resistant to vancomycin or teicoplanin or both have been described. Strains of the genera Leuconostoc, Lactobacillus, Pediococcus, and Erysipelothrix seem inherently resistant to glycopeptides. Species and strains of enterococci and coagulase-negative staphylococci appear to have acquired or developed resistance. There are at least two categories of glycopeptide resistance among enterococci, characterized by either high-level resistance to vancomycin (MIC, greater than or equal to 64 mg/liter) and teicoplanin (MIC, greater than or equal to 8 mg/liter) or lower-level vancomycin resistance (MIC, 32 to 64 mg/liter) and teicoplanin susceptibility (MIC, less than or equal to 1 mg/liter). The two categories appear to have similar resistance mechanisms, although genetic and biochemical studies indicate that they have arisen independently. Among coagulase-negative staphylococci, strains for which vancomycin MICs are up to 20 mg/liter or teicoplanin MICs are 16 to 32 mg/liter have been reported, but cross-resistance between these glycopeptides varies. The selective advantage accorded to glycopeptide-resistant bacteria and the observation that high-level resistance in enterococci is transferable suggest that such resistance may be expected to increase in incidence. Clinicians and microbiologists need to be aware of this emerging problem.19902143434
6065180.9997Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.200312810293
5901190.9997Identification and characterization of vancomycin-resistant Enterococcus species frequently isolated from laboratory mice. To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics.201425077759