Publications by authors named "Frank Moller Aarestrup"

37 Publications

Danish Whole-Genome-Sequenced and Samples Fit into Globally Prevalent Clades.

J Fungi (Basel) 2021 Nov 12;7(11). Epub 2021 Nov 12.

Division for Global Surveillance, National Food Institute, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.

and are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the population in the country. The majority of the 30 isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all isolates to be susceptible to both azoles and echinocandins. Two isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species.
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http://dx.doi.org/10.3390/jof7110962DOI Listing
November 2021

Prevalence and genomic analysis of ESBL-producing Escherichia coli in retail raw meats in Singapore.

J Antimicrob Chemother 2021 02;76(3):601-605

Nanyang Technological University Food Technology Centre (NAFTEC), 62 Nanyang Drive, 637459, Singapore.

Objectives: To determine the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets.

Methods: A total of 634 raw meat (chicken, pork and beef) samples were collected from markets in Singapore during June 2017-October 2018. The samples were enriched overnight and then incubated on Brilliance™ ESBL Agar. Presumptive ESBL isolates were confirmed using the double-disc synergy test. Confirmed ESBL-producing E. coli were sent for WGS and bioinformatic analysis was performed.

Results: The prevalence of ESBL-producing E. coli in chicken, pork and beef meats was 51.2% (109/213), 26.9% (58/216) and 7.3% (15/205), respectively. A total of 225 ESBL-producing E. coli were isolated from 184 samples. β-Lactam resistance genes were detected in all isolates. After β-lactam resistance genes, the most common antimicrobial resistance genes detected were aminoglycoside resistance genes (92.4%). One hundred and seventy-two (76.4%), 102 (45.3%) and 52 (23.1%) isolates carried blaCTX-M genes, blaTEM genes and blaSHV genes, respectively. blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Colistin resistance genes (including mcr-1, mcr-3 and mcr-5) were found in 15.6% of all isolates.

Conclusions: This study indicates that ESBL-producing E. coli are widely found in retail raw meats, especially chicken, in Singapore. Occurrence of MDR (resistance to at least three classes of antimicrobial) and colistin resistance genes in retail raw meat suggests potential food safety and public health risks.
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http://dx.doi.org/10.1093/jac/dkaa461DOI Listing
February 2021

Host Resistance, Genomics and Population Dynamics in a Enteritidis and Phage System.

Viruses 2019 02 22;11(2). Epub 2019 Feb 22.

Department of Biological Sciences, Universidad de Los Andes, 111711 Bogotá, Colombia.

Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of serovar Enteritidis and San23 phage. Samples were taken for 12 days every 48 h. Bacteria and phage samples were collected; and isolated bacteria from each time point were challenged against phages from previous, contemporary, and subsequent time points. The phage plaque tests, with the genomics analyses, showed a mutational asymmetry dynamic in favor of the bacteria instead of antagonistic coevolution. This is important for future phage-therapy applications, so we decided to explore the population dynamics of under different conditions: pressure of one phage, a combination of phages, and phages plus an antibiotic. The data from cultures with single and multiple phages, and antibiotics, were used to create a mathematical model exploring population and resistance dynamics of under these treatments, suggesting a nonlethal, growth-inhibiting antibiotic may decrease resistance to phage-therapy cocktails. These data provide a deep insight into bacterial dynamics under different conditions and serve as additional criteria to select phages and antibiotics for phage-therapy.
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http://dx.doi.org/10.3390/v11020188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410252PMC
February 2019

Dissection of the antimicrobial and hemolytic activity of Cap18: Generation of Cap18 derivatives with enhanced specificity.

PLoS One 2018 31;13(5):e0197742. Epub 2018 May 31.

National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark.

Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against Y. ruckeri, A. salmonicida, S. Typhimurium and L. lactis as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against S. Typhimurium, Y. ruckeri, A. salmonicida, E. coli, P. aeruginosa, L. lactis, L. monocytogenes and E. faecalis. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197742PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978884PMC
December 2018

SCCFinder, a Web-Based Tool for Typing of Staphylococcal Cassette Chromosome in Using Whole-Genome Sequence Data.

mSphere 2018 Jan-Feb;3(1). Epub 2018 Feb 14.

Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Copenhagen, Denmark.

Typing of methicillin-resistant (MRSA) is important in infection control and surveillance. The current nomenclature of MRSA includes the genetic background of the strain determined by multilocus sequence typing (MLST) or equivalent methods like typing and typing of the mobile genetic element staphylococcal cassette chromosome (SCC), which carries the or gene. Whereas MLST and typing are relatively simple, typing of SCC is less trivial because of its heterogeneity. Whole-genome sequencing (WGS) provides the essential data for typing of the genetic background and SCC, but so far, no bioinformatic tools for SCC typing have been available. Here, we report the development and evaluation of SCCFinder for characterization of the SCC element from WGS data. SCCFinder is able to identify all SCC element types, designated I to XIII, with subtyping of SCC types IV (2B) and V (5C2). SCC elements are characterized by two different gene prediction approaches to achieve correct annotation, a Basic Local Alignment Search Tool (BLAST)-based approach and a -mer-based approach. Evaluation of SCCFinder by using a diverse collection of clinical isolates ( = 93) showed a high typeability level of 96.7%, which increased to 98.9% upon modification of the default settings. In conclusion, SCCFinder can be an alternative to more laborious SCC typing methods and is freely available at https://cge.cbs.dtu.dk/services/SCCmecFinder. SCC in MRSA is acknowledged to be of importance not only because it contains the or gene but also for staphylococcal adaptation to different environments, e.g., in hospitals, the community, and livestock. Typing of SCC by PCR techniques has, because of its heterogeneity, been challenging, and whole-genome sequencing has only partially solved this since no good bioinformatic tools have been available. In this article, we describe the development of a new bioinformatic tool, SCCFinder, that includes most of the needs for infection control professionals and researchers regarding the interpretation of SCC elements. The software detects all of the SCC elements accepted by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, and users will be prompted if diverging and potential new elements are uploaded. Furthermore, SCCFinder will be curated and updated as new elements are found and it is easy to use and freely accessible.
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http://dx.doi.org/10.1128/mSphere.00612-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812897PMC
February 2018

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.

Sci Rep 2017 05 25;7(1):2398. Epub 2017 May 25.

Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology, University of Copenhagen, 1356, Copenhagen K, Denmark.

Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8-100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.
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http://dx.doi.org/10.1038/s41598-017-02724-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445084PMC
May 2017

MGmapper: Reference based mapping and taxonomy annotation of metagenomics sequence reads.

PLoS One 2017 3;12(5):e0176469. Epub 2017 May 3.

Department of Bio and Health Informatics, Technical University of Denmark, Kongens Lyngby, Denmark.

An increasing amount of species and gene identification studies rely on the use of next generation sequence analysis of either single isolate or metagenomics samples. Several methods are available to perform taxonomic annotations and a previous metagenomics benchmark study has shown that a vast number of false positive species annotations are a problem unless thresholds or post-processing are applied to differentiate between correct and false annotations. MGmapper is a package to process raw next generation sequence data and perform reference based sequence assignment, followed by a post-processing analysis to produce reliable taxonomy annotation at species and strain level resolution. An in-vitro bacterial mock community sample comprised of 8 genuses, 11 species and 12 strains was previously used to benchmark metagenomics classification methods. After applying a post-processing filter, we obtained 100% correct taxonomy assignments at species and genus level. A sensitivity and precision at 75% was obtained for strain level annotations. A comparison between MGmapper and Kraken at species level, shows MGmapper assigns taxonomy at species level using 84.8% of the sequence reads, compared to 70.5% for Kraken and both methods identified all species with no false positives. Extensive read count statistics are provided in plain text and excel sheets for both rejected and accepted taxonomy annotations. The use of custom databases is possible for the command-line version of MGmapper, and the complete pipeline is freely available as a bitbucked package (https://bitbucket.org/genomicepidemiology/mgmapper). A web-version (https://cge.cbs.dtu.dk/services/MGmapper) provides the basic functionality for analysis of small fastq datasets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176469PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415185PMC
September 2017

A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds.

J Antimicrob Chemother 2017 02 8;72(2):385-392. Epub 2016 Nov 8.

Research Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs Lyngby, Denmark

Objectives: Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring.

Methods: We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing.

Results: Metagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment.

Conclusions: We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.
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http://dx.doi.org/10.1093/jac/dkw415DOI Listing
February 2017

Bacterial whole genome-based phylogeny: construction of a new benchmarking dataset and assessment of some existing methods.

BMC Genomics 2017 01 5;18(1):19. Epub 2017 Jan 5.

Center for Biological Sequence Analysis, DTU Bioinformatics, Technical University of Denmark, Kongens Lyngby, Denmark.

Background: Whole genome sequencing (WGS) is increasingly used in diagnostics and surveillance of infectious diseases. A major application for WGS is to use the data for identifying outbreak clusters, and there is therefore a need for methods that can accurately and efficiently infer phylogenies from sequencing reads. In the present study we describe a new dataset that we have created for the purpose of benchmarking such WGS-based methods for epidemiological data, and also present an analysis where we use the data to compare the performance of some current methods.

Results: Our aim was to create a benchmark data set that mimics sequencing data of the sort that might be collected during an outbreak of an infectious disease. This was achieved by letting an E. coli hypermutator strain grow in the lab for 8 consecutive days, each day splitting the culture in two while also collecting samples for sequencing. The result is a data set consisting of 101 whole genome sequences with known phylogenetic relationship. Among the sequenced samples 51 correspond to internal nodes in the phylogeny because they are ancestral, while the remaining 50 correspond to leaves. We also used the newly created data set to compare three different online available methods that infer phylogenies from whole-genome sequencing reads: NDtree, CSI Phylogeny and REALPHY. One complication when comparing the output of these methods with the known phylogeny is that phylogenetic methods typically build trees where all observed sequences are placed as leafs, even though some of them are in fact ancestral. We therefore devised a method for post processing the inferred trees by collapsing short branches (thus relocating some leafs to internal nodes), and also present two new measures of tree similarity that takes into account the identity of both internal and leaf nodes.

Conclusions: Based on this analysis we find that, among the investigated methods, CSI Phylogeny had the best performance, correctly identifying 73% of all branches in the tree and 71% of all clades. We have made all data from this experiment (raw sequencing reads, consensus whole-genome sequences, as well as descriptions of the known phylogeny in a variety of formats) publicly available, with the hope that other groups may find this data useful for benchmarking and exploring the performance of epidemiological methods. All data is freely available at: https://cge.cbs.dtu.dk/services/evolution_data.php .
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http://dx.doi.org/10.1186/s12864-016-3407-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217230PMC
January 2017

A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance.

PLoS One 2016 21;11(6):e0157718. Epub 2016 Jun 21.

Department of Systems Biology, Technical University of Denmark, Kemitorvet Building 208, 2800 Kgs. Lyngby, Denmark.

Recent advances in whole genome sequencing have made the technology available for routine use in microbiological laboratories. However, a major obstacle for using this technology is the availability of simple and automatic bioinformatics tools. Based on previously published and already available web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services. The reported results enable a rapid overview of the major results, and comparing that to the previously found results showed that the platform is reliable and able to correctly predict the species and find most of the expected genes automatically. In conclusion, a combined bioinformatics platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 and it is the intention that it will continue to be expanded with new features as these become available.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157718PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915688PMC
July 2017

Fatal Septicemia Linked to Transmission of MRSA Clonal Complex 398 in Hospital and Nursing Home, Denmark.

Emerg Infect Dis 2016 May;22(5):900-2

We describe 2 fatal cases of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 septicemia in persons who had no contact with livestock. Whole-genome sequencing of the isolated MRSA strains strongly suggest that both were of animal origin and that the patients had been infected through 2 independent person-to-person transmission chains.
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http://dx.doi.org/10.3201/eid2205.151835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861525PMC
May 2016

Consolidating and Exploring Antibiotic Resistance Gene Data Resources.

J Clin Microbiol 2016 Apr 27;54(4):851-9. Epub 2016 Jan 27.

Laboratory of Medical Microbiology, Vaccine & Infectious Disease Institute, Universiteit Antwerpen, Antwerp, Belgium

The unrestricted use of antibiotics has resulted in rapid acquisition of antibiotic resistance (AR) and spread of multidrug-resistant (MDR) bacterial pathogens. With the advent of next-generation sequencing technologies and their application in understanding MDR pathogen dynamics, it has become imperative to unify AR gene data resources for easy accessibility for researchers. However, due to the absence of a centralized platform for AR gene resources, availability, consistency, and accuracy of information vary considerably across different databases. In this article, we explore existing AR gene data resources in order to make them more visible to the clinical microbiology community, to identify their limitations, and to propose potential solutions.
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http://dx.doi.org/10.1128/JCM.02717-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809949PMC
April 2016

Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria.

PLoS One 2015 11;10(12):e0144611. Epub 2015 Dec 11.

National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, 2860, Søborg, Denmark.

Analysis Of A Selected Set Of Antimicrobial Peptides: The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alternative to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have suggested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attachment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared the potency of Cap18, Cap11, Cap11-1-18m2, Cecropin P1, Cecropin B, Bac2A, Bac2A-NH2, Sub5-NH2, Indolicidin, Melittin, Myxinidin, Myxinidin-NH2, Pyrrhocoricin, Apidaecin and Metalnikowin I towards Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas salmonicida, Listeria monocytogenes, Campylobacter jejuni, Flavobacterium psychrophilum, Salmonella typhimurium and Yersinia ruckeri by minimal inhibitory concentration (MIC) determinations. Additional characteristics such as cytotoxicity, thermo and protease stability were measured and compared among the different peptides. Further, the antimicrobial activity of a selection of cationic AMPs was investigated in various E. coli LPS mutants.

Cap18 Shows A High Broad Spectrum Antimicrobial Activity: Of all the tested AMPs, Cap18 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria. In addition, Cap18 is highly thermostable and showed no cytotoxic effect in a hemolytic assay, measured at the concentration used. However, Cap18 is, as most of the tested AMPs, sensitive to proteolytic digestion in vitro. Thus, Cap18 is an excellent candidate for further development into practical use; however, modifications that should reduce the protease sensitivity would be needed. In addition, our findings from analyzing LPS mutant strains suggest that the core oligosaccharide of the LPS molecule is not essential for the antimicrobial activity of cationic AMPs, but in fact has a protective role against AMPs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144611PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684357PMC
June 2016

Biocide Susceptibility of Staphylococcus aureus CC398 and CC30 Isolates from Pigs and Identification of the Biocide Resistance Genes, qacG and qacC.

Microb Drug Resist 2015 Oct 1;21(5):527-36. Epub 2015 Jun 1.

1 Division for Epidemiology and Microbial Genomics, National Food Institute, Technical University of Denmark , Lyngby, Denmark .

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA), in particular clonal complex (CC) 398, is increasingly found in livestock. Recently, MRSA CC30 was identified in Danish pigs. We determined the susceptibility of porcine S. aureus isolates of CC398 and CC30 to disinfectants used in pig farming (benzalkonium chloride, hydrogen peroxide, formaldehyde, sodium hypochlorite, and caustic soda). Furthermore, efflux pump activity, antimicrobial resistance profiles, hemolysis properties, and the presence of toxic shock syndrome toxin-1 (TSST-1) and Panton-Valentine Leukocidin (PVL)-encoding virulence factors were investigated.

Methods: Susceptibilities to biocides and antimicrobial agents of 79 porcine S. aureus isolates were determined by the microdilution method. Isolates comprised 21 methicillin-sensitive S. aureus (MSSA) and 40 MRSA isolates belonging to CC398 and 13 MSSA and 5 MRSA isolates belonging to CC30. The presence of quaternary ammonium compound (QAC) resistance efflux pumps was analyzed using an ethidium bromide accumulation assay. The presence of qac resistance genes in active efflux pump positive isolates was determined by whole-genome sequencing data. All isolates were screened for lukPV and tst genes with PCR, and hemolytic activities were determined using an agar plate assay.

Results: S. aureus isolates did not show reduced susceptibility to the biocides tested. However, the QAC resistance gene, qacG, was detected in three MRSA CC30 isolates and the qacC in one MRSA CC30 isolate. CC30 isolates were generally more susceptible to non-beta-lactam antibiotics than CC398. Isolates generally had low hemolytic activity and none encoded PVL or TSST-1.

Conclusion: The presence of qac genes in European porcine S. aureus isolates and in livestock-associated MRSA CC30 is for the first time described in this study. This finding is concerning as it ultimately may compromise disinfection with QACs and thereby contribute to the selection and spread of MRSA CC30.
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http://dx.doi.org/10.1089/mdr.2014.0215DOI Listing
October 2015

Identification of a Pseudomonas aeruginosa co-producing NDM-1, VIM-5 and VIM-6 metallo-β-lactamases in Denmark using whole-genome sequencing.

Int J Antimicrob Agents 2015 Mar 5;45(3):324-5. Epub 2014 Dec 5.

Division for Epidemiology and Microbial Genomics, Technical University of Denmark, Lyngby, Denmark.

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http://dx.doi.org/10.1016/j.ijantimicag.2014.11.004DOI Listing
March 2015

[Pasteurella multocida septicaemia and pneumonia after chemotherapy in a patient who had no known contact with animals].

Ugeskr Laeger 2014 Dec;176(25A)

Mikrobiologisk Afdeling Midt-Vest, Regionshospitalet Herning, Gl. Landevej 61, 7400 Herning.

Pasteurella multocida inhabits the upper respiratory tract of many animals. It can cause skin and soft tissue infections in humans, usually in association with animal bites. We present a case of a 66-year-old chemotherapy-induced immunocompromised patient with lung cancer, who was treated for pneumonia and septicaemia due to P. multocida. There was no anamnestic contact with animals, which underlines the fact that immunocompromised patients can suffer from serious systemic infections due to P. multocida - even with no known animal contact.
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December 2014

In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing.

Antimicrob Agents Chemother 2014 Jul 28;58(7):3895-903. Epub 2014 Apr 28.

Danish Technical University, National Food Institute, Division for Epidemiology and Microbial Genomics, Lyngby, Denmark

In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49 S. Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.
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http://dx.doi.org/10.1128/AAC.02412-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068535PMC
July 2014

PathogenFinder--distinguishing friend from foe using bacterial whole genome sequence data.

PLoS One 2013 28;8(10):e77302. Epub 2013 Oct 28.

Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

Although the majority of bacteria are harmless or even beneficial to their host, others are highly virulent and can cause serious diseases, and even death. Due to the constantly decreasing cost of high-throughput sequencing there are now many completely sequenced genomes available from both human pathogenic and innocuous strains. The data can be used to identify gene families that correlate with pathogenicity and to develop tools to predict the pathogenicity of newly sequenced strains, investigations that previously were mainly done by means of more expensive and time consuming experimental approaches. We describe PathogenFinder (http://cge.cbs.dtu.dk/services/PathogenFinder/), a web-server for the prediction of bacterial pathogenicity by analysing the input proteome, genome, or raw reads provided by the user. The method relies on groups of proteins, created without regard to their annotated function or known involvement in pathogenicity. The method has been built to work with all taxonomic groups of bacteria and using the entire training-set, achieved an accuracy of 88.6% on an independent test-set, by correctly classifying 398 out of 449 completely sequenced bacteria. The approach here proposed is not biased on sets of genes known to be associated with pathogenicity, thus the approach could aid the discovery of novel pathogenicity factors. Furthermore the pathogenicity prediction web-server could be used to isolate the potential pathogenic features of both known and unknown strains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077302PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810466PMC
August 2014

Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins.

Appl Environ Microbiol 2012 Aug 8;78(16):5948-55. Epub 2012 Jun 8.

Area Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybridizations were performed with 18 representative S. aureus strains, and a high number of plasmids of different sizes and organizations were detected.
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http://dx.doi.org/10.1128/AEM.00870-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406130PMC
August 2012

In silico prediction of human pathogenicity in the γ-proteobacteria.

PLoS One 2010 Oct 27;5(10):e13680. Epub 2010 Oct 27.

Center for Biological Sequence Analysis, Technical University of Denmark, Kongens Lyngby, Denmark.

Background: Although the majority of bacteria are innocuous or even beneficial for their host, others are highly infectious pathogens that can cause widespread and deadly diseases. When investigating the relationships between bacteria and other living organisms, it is therefore essential to be able to separate pathogenic organisms from non-pathogenic ones. Using traditional experimental methods for this purpose can be very costly and time-consuming, and also uncertain since animal models are not always good predictors for pathogenicity in humans. Bioinformatics-based methods are therefore strongly needed to mine the fast growing number of genome sequences and assess in a rapid and reliable way the pathogenicity of novel bacteria.

Methodology/principal Findings: We describe a new in silico method for the prediction of bacterial pathogenicity, based on the identification in microbial genomes of features that appear to correlate with virulence. The method does not rely on identifying genes known to be involved in pathogenicity (for instance virulence factors), but rather it inherently builds families of proteins that, irrespective of their function, are consistently present in only one of the two kinds of organisms, pathogens or non-pathogens. Whether a new bacterium carries proteins contained in these families determines its prediction as pathogenic or non-pathogenic. The application of the method on a set of known genomes correctly classified the virulence potential of 86% of the organisms tested. An additional validation on an independent test-set assigned correctly 22 out of 24 bacteria.

Conclusions: The proposed approach was demonstrated to go beyond the species bias imposed by evolutionary relatedness, and performs better than predictors based solely on taxonomy or sequence similarity. A set of protein families that differentiate pathogenic and non-pathogenic strains were identified, including families of yet uncharacterized proteins that are suggested to be involved in bacterial pathogenicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013680PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965111PMC
October 2010

Decreased susceptibility to zinc chloride is associated with methicillin resistant Staphylococcus aureus CC398 in Danish swine.

Vet Microbiol 2010 May 31;142(3-4):455-7. Epub 2009 Oct 31.

National Food Institute, Technical University of Denmark, Copenhagen V, Denmark.

A total of 31 MRSA and 60 MSSA isolated from different swine farms in Denmark were examined for their susceptibility to zinc chloride, erythromycin, penicillin and tetracycline, as well as their spa-type. mecA positive isolates were examined for their SCCmec type. The isolates were assigned to a CC-type based on their spa-type and supportive multi-locus sequence typing (MLST). No difference in susceptibility to erythromycin, penicillin or tetracycline could be observed between methicillin resistant and susceptible isolates of CC398. Twenty-three (74%) of the MRSA CC398 isolates had reduced susceptibility to ZnCl(2) (MIC 4-12 mM), whereas all MSSA had MICs from 0.5 to 2 mM. Thirty MRSA, including all 23 zinc resistant isolates harboured SCCmec type V. This provides biological evidence to suggest that the use of zinc compounds may be partly implicated in the emergence of some MRSA clones among swine in Denmark.
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http://dx.doi.org/10.1016/j.vetmic.2009.10.021DOI Listing
May 2010

The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions.

FEMS Microbiol Lett 2009 Oct 22;299(1):53-9. Epub 2009 Jul 22.

Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.

The objective of this study was to investigate the influence of stressful growth conditions on the fitness cost of antimicrobial resistance in Escherichia coli BJ4 caused by chromosomal mutations and plasmid acquisition. The fitness cost of chromosomal streptomycin resistance increased significantly when the bacteria were grown under all stress conditions tested, while the cost in 1/3 Luria-Bertani was not significantly changed in a streptomycin+rifampicin mutant. The increase in the fitness cost depended in a nonregular manner on the strain/stress combination. The fitness cost of plasmid-encoded resistance on R751 did not differ significantly, and was generally less under stressful growth conditions than in rich media. The fitness cost associated with R751 with the multiple drug resistance cassette from Salmonella Typhimurium DT104 increased significantly only under stressful conditions at low pH and at high-salt concentrations. Strains with an impaired rpoS demonstrated a reduced fitness only during growth in a high-salt concentration. In conclusion, it was demonstrated that bacterial fitness cost in association with antimicrobial resistance generally increases under stressful growth conditions. However, the growth potential of bacteria with antimicrobial resistances did not increase in a straightforward manner in these in vitro experiments and is therefore probably even more difficult to predict in vivo.
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http://dx.doi.org/10.1111/j.1574-6968.2009.01734.xDOI Listing
October 2009

Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations in quinolone-resistant Escherichia coli isolated from humans and swine in Denmark.

Microb Drug Resist 2008 Jun;14(2):163-9

Research Group for Antimicrobial Resistance and Molecular Epidemiology, Department for Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark, Copenhagen V, Denmark.

Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were investigated in 124 Escherichia coli isolated from humans (n=85) and swine (n=39) in Denmark. The collection included 59 high-level CIP-resistant isolates (MIC >or= 4) from human (n=51) and pig origin (n=8) and 65 low-level CIP-resistant isolates (MIC >or= 0.125) from human (n=34) and pig origin (n=31). Resistance by target modification was screened by PCR amplification and sequencing of the quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC, and parE. QRDR mutations occurred in all except two isolates (98%). All high-level CIP-resistant E. coli had one or two mutations in gyrA in combination with mutations in parC or parE. Mutations in parC and parE were only found in combination with gyrA mutations, and no mutations were observed in gyrB. Efflux pump mechanisms were detected in 10 human (11.8%) and 29 porcine (74.4%) isolates by an efflux pump inhibitor (EPI) agar dilution assay. The aac(6')-Ib-cr gene mediating resistance by enzymatic modification was found in 12 high-level CIP-resistant human isolates. The qnrA and qnrS genes conferring quinolone resistance by target protection were detected in two human low-level CIP-resistant isolates that did not display NAL resistance. As expected, target mutation in QRDRs was the most prevalent mechanism of quinolone resistance. This mechanism was complemented by efflux mechanisms in most porcine isolates. Transferable resistance by target protection or enzymatic modification was less common (10%) and restricted to human isolates.
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http://dx.doi.org/10.1089/mdr.2008.0821DOI Listing
June 2008

Ten years of antimicrobial susceptibility testing of Salmonella from Danish pig farms.

J Antimicrob Chemother 2008 Aug 7;62(2):360-3. Epub 2008 May 7.

National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.

Objectives: This study analysed the trends in antimicrobial resistance in Salmonella serovars and phage types from pigs in Denmark from 1997 to 2006.

Methods: Salmonella isolates collected through the Salmonella surveillance programme in pigs were serotyped and phage-typed, and susceptibilities to the following antimicrobials were determined: ampicillin, chloramphenicol, gentamicin, nalidixic acid, colistin, streptomycin, sulphonamide, tetracycline and trimethoprim.

Results: No significant development of resistance occurred within the most important serovars, except Salmonella Typhimurium. A major decrease in Salmonella Typhimurium DT12 occurred from 46.5% in 1998 to 16.8% in 2006 while DT120, DT170 and DT104 increased. Throughout the study period, 80.9% of the DT12 isolates remained susceptible to the antimicrobials tested despite an increase in antimicrobial consumption in pigs during the period. In DT120, DT170 and DT104, only 20.1%, 33.1% and 23.0%, respectively, remained fully susceptible.

Conclusions: The results support that the use of antimicrobial agents might select for multiple resistant clones and that this might be the driver of changes in antimicrobial resistance within a serovar, rather than an emergence of resistance within clones. The results of this study also support that susceptible serovars only slowly become resistant to the antimicrobials tested.
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http://dx.doi.org/10.1093/jac/dkn196DOI Listing
August 2008

Copper resistance in Enterococcus faecium, mediated by the tcrB gene, is selected by supplementation of pig feed with copper sulfate.

Appl Environ Microbiol 2006 Sep;72(9):5784-9

Danish Institute for Food and Veterinary Research, Bülowsvej 27, 1790 Copenhagen V, Denmark.

The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes in the enterococcal species E. mundtii, E. casseliflavus, and E. gallinarum for the first time.
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http://dx.doi.org/10.1128/AEM.02979-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563648PMC
September 2006

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry.

J Antimicrob Chemother 2006 May 24;57(5):832-9. Epub 2006 Mar 24.

Danish Institute for Food and Veterinary Research, 1790 Copenhagen V, Denmark.

Objectives: To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark.

Methods: A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates.

Results: In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements.

Conclusions: This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.
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http://dx.doi.org/10.1093/jac/dkl069DOI Listing
May 2006

Pheno- and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin.

Vet Microbiol 2006 Jun 13;115(1-3):163-72. Epub 2006 Mar 13.

Division of Food Hygiene and Bacteriology, Swedish University of Agricultural Sciences, Uppsala, Sweden.

The purpose of this study was to improve our knowledge concerning the epidemiology and strain diversity of Staphylococcus epidermidis isolated from bovine milk in commercial dairy herds. A total of 341 S. epidermidis isolates obtained from cows' milk (317), farmers (17) and patients (7) were characterized. Of these 105 isolates were from cows' milk in two farms, where also 17 isolates were sampled from farmers. The remaining 212 isolates from cows' milk were from 170 farms. All isolates were examined by antimicrobial susceptibility, whereas 202 were examined by pulsed-field gel electrophoresis (PFGE) and 122 by ribotyping. PFGE showed single patterns in the human strains with one exception; one strain was categorised as the same clone as four of the milk strains. PFGE divided 73 of the milk strains into 62 different patterns. The PFGE method had high discriminatory power and shows that many different S. epidermidis types exist in milk samples. Antibiotic resistance patterns matched the SmaI profiles closely in the two herds, but poorly in the routinely collected milk samples. Isolates from herd 1 showed one to five patterns, depending on the typing method used. Isolates from the milker's skin showed one pattern, which was identical to the most common pattern found in the milk isolates. Isolates from herd 2 showed three to four patterns, two of these being identical to skin isolates from the milker. As dairy cows are not a natural host for S. epidermidis the results suggest a human source of these udder infections.
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http://dx.doi.org/10.1016/j.vetmic.2006.01.013DOI Listing
June 2006

Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci.

J Antimicrob Chemother 2006 Mar 31;57(3):450-60. Epub 2006 Jan 31.

Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, 4 Stigbøjlen, DK-1870 Frederiksberg C., Denmark.

Objectives: To elucidate the diversity and evolutionary history of plasmid- and chromosomally-located blaZ, to detect indications of frequent exchange of blaZ between human and bovine staphylococci and to estimate the frequency of transfer of blaZ between coagulase-negative staphylococci (CoNS) and Staphylococcus aureus of bovine origin.

Methods: blaZ was detected in 143 strains of penicillin-resistant S. aureus and CoNS from five Danish cattle herds (n = 25/23), random CoNS isolates from Denmark (n = 37), a collection of S. aureus from six different countries (n = 52), humans in Denmark (n = 3) and beta-lactamase control strains (n = 3). The sequence was determined in 105 strains and compared to published sequences by pairwise and multiple alignments. Maximum likelihood analysis was performed including bootstrap analysis. Parsimony, neighbour joining and consensus comparisons were performed for recombination. The localization of blaZ was determined by Southern blotting in 108 isolates.

Results: All penicillin-resistant strains carried blaZ and showed a similar organization of blaR1 and blaZ. The blaZ gene was localized to a plasmid in only 16 of the resistant strains. Sixty-nine sequences representing 105 isolates and sequences retrieved from public databases were compared. A phylogenetic tree showed that blaZ exists in three evolutionary lines: one group was of plasmid origin, one group was of chromosomal origin and one intermediate group. Sixty-nine sequence types were demonstrated. They translated into 11 BlaZ protein types. The major types all contained strains of both human and bovine origin, and more than one Staphylococcus species, demonstrating a shared gene pool. In a comparison of S. aureus and CoNS obtained from five Danish cattle herds, the same type of blaZ was only detected in one case.

Conclusions: Results indicated a separate evolution for plasmid- and chromosomally-encoded blaZ. Although a common gene pool seems to exist among staphylococci, exchange of blaZ between strains and species is judged to be an extremely rare event.
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http://dx.doi.org/10.1093/jac/dki492DOI Listing
March 2006

Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark.

Microb Drug Resist 2004 ;10(4):334-40

Danish Institute for Food and Veterinary Research, DK-1790 Copenhagen V, Denmark.

The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla (TEM-1), of which bla (TEM-1b) was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla (TEM-1a) (eight E. coli, one Salmonella) and bla (TEM-1c) (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE beta-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla (TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two new variants of bla (TEM) were detected, which have been designated bla (TEM-127) and bla (TEM-128). In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum beta-lactams.
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http://dx.doi.org/10.1089/mdr.2004.10.334DOI Listing
March 2005
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