IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium

<p>Abstract</p> <p>Background</p> <p>Hospital strains of <it>Enterococcus faecium </it>could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, these techniques are laborious, time-consuming and cost-intensive. Our aim was to identify hospital <it>E. faecium </it>strains and differentiate them from colonizing and animal variants by a simple, inexpensive and reliable PCR-based screening assay. We describe here performance and predictive value of a single PCR detecting the insertion element, IS<it>16</it>, to identify hospital <it>E. faecium </it>isolates within a collection of 260 strains of hospital, animal and human commensal origins.</p> <p>Methods</p> <p>Specific primers were selected amplifying a 547-bp fragment of IS<it>16</it>. Presence of IS<it>16 </it>was determined by PCR screenings among the 260 <it>E. faecium </it>isolates. Distribution of IS<it>16 </it>was compared with a prevalence of commonly used markers for hospital strains, <it>esp </it>and <it>hyl</it>_<it>Efm</it>. All isolates were typed by MLST and partly by PFGE. Location of IS<it>16 </it>was analysed by Southern hybridization of plasmid and chromosomal DNA.</p> <p>Results</p> <p>IS<it>16 </it>was exclusively distributed only among 155 invasive strains belonging to the clonal complex of hospital-associated strains ("CC17"; 28 MLST types) and various vancomycin resistance genotypes (<it>van</it>A/B/negative). The five invasive IS<it>16</it>-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS<it>16 </it>was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS<it>16</it>-positive human commensal isolates revealed MLST types belonging to the clonal complex of hospital-associated strains (CC17). The values predicting a hospital-associated strain ("CC17") deduced from presence and absence of IS<it>16 </it>was 100% and thus superior to screening for the presence of <it>esp </it>(66%) and/or <it>hyl</it>_{<it>Efm </it>}(46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS<it>16</it>.</p> <p>Conclusions</p> <p>This simple screening assay for insertion element IS<it>16 </it>is capable of differentiating hospital-associated from human commensal, livestock- and food-associated <it>E. faecium </it>strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given <it>E. faecium </it>isolate identified within the nosocomial setting.</p

IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium

<p>Abstract</p> <p>Background</p> <p>Hospital strains of <it>Enterococcus faecium </it>could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, these techniques are laborious, time-consuming and cost-intensive. Our aim was to identify hospital <it>E. faecium </it>strains and differentiate them from colonizing and animal variants by a simple, inexpensive and reliable PCR-based screening assay. We describe here performance and predictive value of a single PCR detecting the insertion element, IS<it>16</it>, to identify hospital <it>E. faecium </it>isolates within a collection of 260 strains of hospital, animal and human commensal origins.</p> <p>Methods</p> <p>Specific primers were selected amplifying a 547-bp fragment of IS<it>16</it>. Presence of IS<it>16 </it>was determined by PCR screenings among the 260 <it>E. faecium </it>isolates. Distribution of IS<it>16 </it>was compared with a prevalence of commonly used markers for hospital strains, <it>esp </it>and <it>hyl</it>_<it>Efm</it>. All isolates were typed by MLST and partly by PFGE. Location of IS<it>16 </it>was analysed by Southern hybridization of plasmid and chromosomal DNA.</p> <p>Results</p> <p>IS<it>16 </it>was exclusively distributed only among 155 invasive strains belonging to the clonal complex of hospital-associated strains ("CC17"; 28 MLST types) and various vancomycin resistance genotypes (<it>van</it>A/B/negative). The five invasive IS<it>16</it>-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS<it>16 </it>was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS<it>16</it>-positive human commensal isolates revealed MLST types belonging to the clonal complex of hospital-associated strains (CC17). The values predicting a hospital-associated strain ("CC17") deduced from presence and absence of IS<it>16 </it>was 100% and thus superior to screening for the presence of <it>esp </it>(66%) and/or <it>hyl</it>_{<it>Efm </it>}(46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS<it>16</it>.</p> <p>Conclusions</p> <p>This simple screening assay for insertion element IS<it>16 </it>is capable of differentiating hospital-associated from human commensal, livestock- and food-associated <it>E. faecium </it>strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given <it>E. faecium </it>isolate identified within the nosocomial setting.</p

Intra- and Interspecies Genomic Transfer of the Enterococcus faecalis Pathogenicity Island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transfered. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens

Structure of the Full-Length Major Pilin from Streptococcus pneumoniae: Implications for Isopeptide Bond Formation in Gram-Positive Bacterial Pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili

Sewage-based surveillance of antibiotic resistance 2020 – 2021. Antibiotic-resistant bacteria in the population measured in sewage

Het RIVM heeft in 2020 en 2021 in rioolwater twee soorten bacteriën gemeten die resistent zijn tegen antibiotica. Het gaat om CPE (carbapenemase-producerende Enterobacterales) en colistine-resistente E.coli (MCR-EC). De resultaten zijn vergeleken met rioolwatermetingen uit 2016. Op deze manier wordt duidelijk of het aantal mensen in Nederland die deze bacteriën bij zich draagt verandert. Het RIVM meet deze bacteriën omdat ze een bedreiging zijn voor de volksgezondheid. Ze veroorzaken namelijk ziekten die moeilijk te behandelen zijn met antibiotica. De bacteriën zijn vooral gevaarlijk voor mensen met een kwetsbare gezondheid. De meeste gezonde mensen worden niet ziek van de bacterie. Door regelmatig het rioolwater te meten wordt duidelijk of de overheid genoeg doet om de verspreiding onder de bevolking te beperken. Allebei de soorten bacteriën zijn in heel Nederland gevonden. De aantallen CPE in rioolwater zijn ongeveer hetzelfde als in 2016. De aantallen colistine-resistente E. coli-bacteriën konden met dit onderzoek niet precies worden gegeven. Het lijkt er daarmee op dat de coronamaatregelen, zoals handen wassen en minder reizen, de verspreiding van deze bacteriën niet hebben verminderd. Dit vermoeden kan niet hard worden gemaakt omdat er geen metingen zijn gedaan vlak voordat de corona-epidemie begon. Om inzicht te krijgen hoe de aantallen zich verder ontwikkelen is het belangrijk in rioolwater te blijven meten. Metingen in rioolwater vullen de bestaande onderzoeken bij mensen naar de verspreiding van resistente bacteriën, goed aan. Voor dit onderzoek zijn de concentraties in het rioolwater van 76 zuiveringsinstallaties onderzocht. Deze installaties lagen verspreid over heel Nederland, in stedelijke en landelijke gebieden. De onderzochte bacteriën zitten in ontlasting van mensen en dus in het rioolwater. Ze komen ook via reizigers uit bijvoorbeeld Zuid-Europa, Noord-Afrika en Zuidoost-Azië in Nederland terecht.In 2020 and 2021, the National Institute for Public Health and the Environment (RIVM) measured in sewage two strains of bacteria that are resistant to antibiotics. These are CPE (carbapenem-resistant Enterobacterales) and colistin-resistant E.coli (MCR-EC). The results were compared with sewage measurements from 2016, thereby shedding light on whether there has been any change in the number of people carrying these bacteria in the Netherlands. The RIVM measures these bacteria because they constitute a threat to public health, as they cause diseases that are difficult to treat with antibiotics. The bacteria are particularly dangerous to people who are considered clinically vulnerable. Most healthy people will not fall ill due to the bacteria. Regularly performing measurements on sewage makes it clear whether the government is doing enough to prevent the spread of the bacteria among the population. Both strains are found throughout the Netherlands. The level of CPE in sewage is approximately the same as it was in 2016. It was not possible to ascertain the precise level of colistin-resistant E. coli bacteria in this survey. It would appear that the coronavirus measures (e.g. handwashing and less travel) have not reduced the spread of these bacteria. It is impossible to corroborate this assumption as no measurements were done right before the coronavirus pandemic began. In order to gain insight into future development of the levels, it is important to continue carrying out measurements on sewage. Measurements in sewage nicely complement the existing studies on the spread of resistant bacteria being carried out among people. For the purposes of this survey, concentrations in sewage from 76 treatment plants were studied. These plants are situated throughout the Netherlands, in urban and rural areas. The bacteria studied are found in human stools, which is how they end up in sewage. They are also carried into the Netherlands by travellers from regions such as southern Europe, North Africa and Southeast Asia

Markers of epidemiological success of methicillin-resistant Staphylococcus aureus (MRSA) isolates in European populations

OBJECTIVES – Methicillin-resistant Staphylococcus aureus (MRSA) infections impose a considerable burden on health systems, yet there is remarkable variation in the global incidence and epidemiology of MRSA. The MACOTRA consortium aimed to identify bacterial markers of epidemic success of MRSA isolates in Europe using a representative MRSA collection originating from France, the Netherlands and the United Kingdom. METHODS – Operational definitions of success were defined in consortium meetings to compose a balanced strain collection of successful and sporadic MRSA isolates. Isolates were subjected to antimicrobial susceptibility testing and whole genome sequencing, genes were identified and phylogenetic trees constructed. Markers of epidemiological success were identified using genome-based time-scaled haplotypic density (THD) analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared to national MRSA incidence data. RESULTS - Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success, so country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In THD analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, while gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and beta-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. CONCLUSIONS - Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage to incidence of infection and successful clonal spread, which varied by country. Harmonized isolate collection, typing, resistance profiling and alignment with antimicrobial usage over time will aid comparisons and further support country-specific interventions to reduce MRSA burden